Identification of Tumor-Suppressive miRNA-1275 as a Novel Marker for Breast Cancer (BC) by MACE-Sequencing and RT-qPCR Techniques

Identification of Tumor-Suppressive miRNA-1275 as a Novel Marker for Breast Cancer (BC) by MACE-Sequencing and RT-qPCR Techniques | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Identification of Tumor-Suppressive miRNA-1275 as a Novel Marker for Breast Cancer (BC) by MACE-Sequencing and RT-qPCR Techniques Sevan Omer Majed, Suhad Asad Mustafa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-101189/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction Disruption of cellular processes in the breast by abnormally expressed miRNA is characterized to develop cancer. We aimed to determine the differential expression of coding and non-coding RNAs in formalin fixed paraffin embedded (FFPE) blocks of breast cancer (BC) tissue and normal adjacent tissue (NAT). Another aim is to determine differential expression of has-miR-1275 as novel biomarker for BC and identify its target genes using prediction sites and experimentally expression level of them via the MACE-sequencing technique. Methods MACE-sequencing technique was utilized to analyze differential expression of coding RNAs and small RNAs (sRNAs). Among small RNAs, miRNA-1275 expression was focused and confirmed using RT-qPCR technique in 20 Kurdish cases with BC . Moreover, clinical significance of miR- 1275 and its target genes was studied in a large number of patients with BC using the data obtained from The Cancer Genome Atlas database. Results The MACE-seq findings showed that 1400 sRNAs and 26843 coding RNAs were differentially expressed in FFPE of BC tissue compared to NAT. Among these sRNAs, miRNA-1275 expression was found to be decreased in BC tissue compared to NAT. The decreased expression level of which was then confirmed via RT-qPCR technique to farther prove in 20 Kurdish cases with BC . Furthermore, the correlation between the expression level of miRNA-1275 and clinical data were evaluated to be highly corrected in cases with BC (overall survival rate: P = 0.0401). However, putative target genes ( DVL3, PPP 2R2D, THSD4, CREB1, SYT7, and PRKACA) were computationally identified as direct targets of miRNA-1275 in several target predicted sites. Among coding RNAs, the expression level of these targets was increased in BC tissue compared to NAT. The levels of these targets were negatively associated with miRNA-1275 expression. Finally, the role of down-expressed miRNA-1275 and its targets in BC cells were identified to attenuated biological mechanisms; including cell growth, proliferation, movement, invasion, metastasis, and apoptosis. Conclusion down-expressed miR-1275 , a tumor suppressor, is as a novel biomarker for early detection of breast cancer. DVL3, PPP 2R2D, THSD4, CREB1, SYT7, and PRKACA are novely identified to be targeted by miR-1275 in BC cells. Cancer Biology Oncology Breast cancer miRNA miR-1275 and its target genes differential expression pathogenesis tumor suppressor Figures Figure 1 Figure 1 Figure 2 Figure 2 Figure 3 Figure 3 Figure 4 Figure 4 Figure 5 Figure 5 Figure 6 Figure 6 1. Introduction Breast malignant cell is a prominent type of cancers mostly diagnosed in females, and the second most frequent malignancy-associated deaths worldwide, especially in the US and Asian countries (1, 2). Approximately two million females are annually diagnosed and more than 620,000 deaths are newly recorded every year (3, 4). Frequently, BC is developed as a result of a genomic mutation. However, about 10% of BCs is inheritably come down from parents to their generations; whereas, more than 85% of BCs is developed in their lifetime (4, 5). Inherited abnormalities in TP53 and PTEN genes were studied to result in the high risk of the breast malignant cell progression (6, 7). Gene expression profiling has recently played a critical role in medicinal selection for BC subtypes. The analysis of BC gene expression can be used for molecular category of BC subtypes (8-10). Two studies reported that this classification facilitates the determination of the cure doses. The molecular subtypes of BC can be categorized into luminal-A, luminal-B (including HER2+ /-), HER2+, and triple negative (TN) (11, 12). These subtypes are pivotal for cure choice and are correlated to the biological characteristics of BC. MicroRNA (miRNA), which is a type of untranslated sRNAs, is synthesized from eukaryotic genomes, consisting of a single stranded RNA of about 19-22nt in length (13, 14). These short non-coding miRNAs are described as regulators of coding and non-coding (nc) RNAs in eukaryotic cells because they are involved in silencing RNA transcripts and in regulating the stability of their targeted mRNAs (14, 15). MiRNAs play also several regulatory roles in several cellular processes; cell development, proliferation, migration, invasion and death (16). Because more than 50% of RNA molecules has been detected to be controlled by miRNA, these mRNAs were damaged because of the effect of aberrant miRNA in malignant cells. MiRNAs in human malignancies were found to act as oncogenes or ant-oncogenes (tumor suppressors). Oncogenic miRNAs in tumor progress play a negative role in stimulating genes which regulate cell development and apoptosis process. Tumor suppressive miRNAs in human tumor have a key role in silencing genes which modulate cell development and apoptosis (17, 18). When normal cells do not undergo normal growth and apoptosis process, they normally cause tumor creation. Numerous recent experiments show that numerous miRNAs are directly implicated in modulating cell growth, proliferation as well as apoptosis (19, 20). They play major roles in the pathogenesis of a number of human malignancies; such as breast, colorectal cancer, lung, leukemia liver, and brain(16, 18). The miRNA expression level may be either down- or up-regulated in these cancers. Several molecular techniques, such as RNA sequencing, miRNA microarray, RT-PCR and northern blot are applied to determine the expression level of them. Numerous miRNAs have been recognized to be implicated in the pathogenesis of human breast cancer. It was found that the expression patterns of miR-145 , -125b , -155 , and -21 were significantly downregulated. In breast malignant cells, these miRNAs were observed to be associated with pathologic properties; cell proliferation, expression of progesterone and estrogen receptors (21). A recent study revealed that tumor suppressive miRNA-204-5p plays a key role in targeting several oncogenic genes which are closely connected to BC pathogenesis(20). Complete information on miR-1275 expression level and its targets in BC have not been available; whereas, the expression profile of which has be analyzed in some human cancer. A study reported that down-expressed miR-1275 leads to overexpression claudin11 in cancer stem cells or tumor-initiating cells (CSCs/TICs) (22). According to a study carried out on young women with BC, 6 miRNAs; including miR-1275, miR-1228*, miR-139 , miR-92b , miR-1207 , and miR-3196, were involved in the processes of cell movement, proliferation and invasion (23). It was also found that this miRNA was found to be downregulated in gastric cancer (24). Another study found that miRNA-1275 expression level was significantly abnormally deregulated in Ewing's Sarcoma (ES) (25). The significance of a large number of miRNAs have been reported to become an appropriate biomarker for human cancer diagnostics. However, the significance of miRNA-1275 in BC is not reported. The objective of this study was to determine the expression level of miRNA-1275 as a biomarker for BC diagnostics. Another objective is to identify the potential targeted genes of this miRNA. 2. Materials and Methods 2.1 Collection of FFPE-blocks of BC samples Formalin Fixed Paraffin Embedded (FFPE)-Block of 21 Kurdish cases with BC were collected at clinicopathological laboratories, called as Al Mufti and Luay. For each patient, two paraffin blocks (one adjacent normal tissue (NAT) and one breast cancer tissue) were collected. The normal tissues were histologically taken nearly 2 cm away from the tumor area. Clinical features of 21 cases were obtained using a questionnaire. The features were displayed in table 1. Permission was accomplished from all cases by signing the confirmed consent. any pre-operative chemotherapy or radiotherapy was taken by none of these cases 2.2 Generation of the mRNA and sRNA expression profiles for BC by MACE-sequencing To determine differential expression of protein-coding RNAs (mRNA transcripts) and non-coding RNAs (ncRNAs), analyzing two paraffin blocks (cancerous and normal) of a BC patient, Massive Analysis of cDNA Ends (MACE)-seq technique was performed (GenXpro GmbH, Frankfurt, Germany). cDNA synthesis, NGS-library and sequencing were subsequently prepared after isolation of sRNAs from mRNA transcripts. The raw sequencing data was bioinformatically analyzed and solved. The raw sequencing data is demultiplexed based on the different barcodes (GenXpro GmbH, Frankfurt, Germany). For removing adapter, the organized reads were cut out for high-quality sequences. Bowtie 2 tool was later used for aligning the sorted reads to the nominated reference sequences and annotating with corresponding properties. The numbers of aligned reads were subsequently normalized to account for distinct sequencing depths. Finally, the normalized and original read numbers were considered during statistical analysis. 2.3 Total RNA purification and cDNA synthesis miR-1275 was selected as a candidate from the MACE result and confirmed from 20 cases using Real Time-quantitative polymerase chain reaction (RT-qPCR) technique at Salahuddin University Research Center (SURC). Differential expression of miRNA-1275 was measured in 40 block specimens (20 adjacent normal tissues (NATs) and 20 breast cancer tissues). The total RNA molecules including miRNA were extracted using FFPE RNA/DNA Purification Plus kit (Cat. No. 54300, NORGEN BIOTEK CORP, Canada). Complementary DNA (cDNA) was synthesized using miRNA All-In-One cDNA Synthesis Kit (Cat. No. G898, abmgood company, US). 2.4 Total RNA purification and cDNA synthesis miR-1275 was selected as a candidate from the MACE result and confirmed from 20 cases using Real Time-quantitative polymerase chain reaction (RT-qPCR) technique at Salahuddin University Research Center (SURC). Differential expression of miRNA-1275 was measured in 40 block specimens (20 adjacent normal tissues (NATs) and 20 breast cancer tissues). The total RNA molecules including miRNA were extracted using FFPE RNA/DNA Purification Plus kit (Cat. No. 54300, NORGEN BIOTEK CORP, Canada). Complementary DNA (cDNA) was synthesized using miRNA All-In-One cDNA Synthesis Kit (Cat. No. G898, abmgood company, US). 2.5 Most common putative targeted genes regulated by miR-1275 Eleven databases were searched for finding the most common putative targets of miR-1275 (Table 3). Six putative targets ( DVL3, PPP 2R2D, THSD4, CREB1, SYT7, and PRKACA ) were determined to possess binding sequence to miR-1275 (Table 4) . , Graphpad prsim, version 8.0.1 was used to show the differential expression of these selected target genes were performed by MACE-seq. 2.6 Analysis of clinicopathological data associated with BC Association between miR-1275 and its target genes was computationally analyzed to determine the clinical significance using databases of cBioPortal ( http://www.cbioportal.org/ ) and OncoLnc ( http://www.oncolnc.org/ ). Clinical data and expression levels of the miR-1275 and its target genes gained from these sites and then were downloaded on 10 September 2020. Table 1. Clinicopathological features of 21 cases with BC. Cases Age Tumor Size Stage Lymph node metastasis Lymphatic Invasion Venous Invasion E.R. Pg.R. Her2 Ki-67 Technique Case1 74 4cm IIIA Yes 1 1 Negative Negative Negative 60 MACE-seq. Case2 35 4cm IIB Yes 1 0 Negative Negative Positive 35 RT-PCR Case3 64 4.5cm IIIA Yes 1 0 Positive Positive Negative 5 RT-PCR Case4 71 1.9cm I No 0 0 Positive Positive Negative Unavailable RT-PCR Case5 40 2.3cm IIB Yes 1 0 Positive Positive Negative 4 RT-PCR Case6 44 3.5cm IIIC Yes 1 0 Positive Positive Negative 13 RT-PCR Case7 53 2cm IIA No 1 1 Positive Negative Positive 20-30 RT-PCR Case8 46 1.8cm IIA Yes 1 1 Positive Negative Positive 10 RT-PCR Case9 33 1.4cm IIA No 0 0 Positive Positive Positive 8 RT-PCR Case10 49 5cm IIIC Yes 1 0 Negative Positive Negative 13 RT-PCR Case11 45 1.9cm IIIB No 0 0 Negative Negative Negative 80-90 RT-PCR Case12 64 1.5cm IIA No 0 0 Negative Negative Negative 80-90 RT-PCR Case13 49 1.7cm IIA No 0 1 Negative Negative Negative 70-80 RT-PCR Case14 35 1.3cm IIA No 0 0 Negative Negative Negative 90 RT-PCR Case15 52 3cm IIA No 0 0 Negative Negative Negative 60 RT-PCR Case16 18 2.5cm IIA No 0 0 Positive Negative Negative 50 RT-PCR Case17 63 1.5cm I No 0 0 Negative Negative Positive 12 RT-PCR Case18 19 2.5cm IIB Yes 1 0 Positive Positive Negative 24 RT-PCR Case19 30 1.3cm IIA No 1 0 Positive Positive Negative 15-20 RT-PCR Case20 45 1.5cm IIA No 0 0 Positive Positive Negative 15-20 RT-PCR Case21 44 6cm IIIC Yes 0 0 Positive Positive Positive 7 RT-PCR 3. Results 3.1 Construction of expression profile of sRNAs for BC by sRNA-sequencing SRNA sequencing was carried out to construct the differentia expression of non-coding RNAs in BC compared to NAT. Two small libraries were sequenced for two paraffin blocks (NAT and BC tissue). Histopathological properties of this specimen were shown in table 1 (Case1). By comparing non-coding RNA expression profiles of BC and NAT, 1400 sRNAs ( p <0.05) were filtered out by a SAM software. The raw data were then standardized and log2-transformed to show on a scatter plot (Fig. 1A). Among 1400 sRNAs, 723 non-coding RNAs were downregulated, but 678 sRNAs were upregulated. Each dot on the scatter plot represents the sRNA. Among 1400 sRNAs, 520 microRNAs were differentially expressed. 185 microRNAs were down-expressed, but 335 were overexpressed. The x-axis denotes the data of the NAT and y-axis denotes the data of the BC. Correlation plot was constructed to show expression levels of non-coding RNAs between BC and NAT (Fig. 1B). Blue color denoted the correlation of sRNAs between the BC and NAT. Heat map was designed to show 29 miRNAs which were markedly downregulated in BC compared to NAT (Fig. 1C). Table 2 shows the information on these 29 miRNAs which are notably downregulated. In this study, has - miR-1275 , which underlined with red color in heat map, was focused to identify sequence and expression level. 3.2 Confirmation of miR-1275 expression level by RT-qPCR The miR-1275 expression level in RNA-sequencing was observed to be significantly downregulated in BC tissue, as compared to NAT. The p. value of this miRNA was 0.614 (Fig. 2A). Then, this miRNA was selected to confirm. RT-qPCR machine was used to confirm the differential expression of miR-1275 in the laboratory. 40 FFPE blocks for 20 cases was used in this experiment, including 20 BC tissues and 20 normal tissues next to tumoral tissues (2cm away from tumoral tissues). The clinical properties of these cases were briefly explained in table 1. The miR-1275 expression level was detected to be markedly decreased in BC cells compared to adjacent normal cells and the P -value of this was 0.001** (Fig.2B). The mature sequence of which in the BC and NAT was made up of 17 nucleotides and also identical (Fig.2C). Then, Kaplan–Meier overall survival curve was designed to show the effect of miR-1275 expression on the prognosis of cases with BC. Data was used from The Cancer Genome Atlas (TCGA) database and analyzed. Kaplan–Meier overall survival curve displayed that cases were separated into two classes according to its expression. The decreased miR-1275 ( P -value=0.0401) was related to overall survival in cases with BC (Fig.2D). 3.3 Differentially expressed genes for adjacent normal and BC tissue by MACE-seq. Differentially expressed genes in BC and NATs was displayed (Fig. 3A). 26843 differentially expressed genes (P≤0.05) were filtered out by a SAM software. In order to show genes that were more significantly different in their expressions, 7041 genes were standardized and log2-transformed to show on a scatter plot. 3624 genes were significantly overexpressed and 3417 genes were significantly down-expressed in tumoral cells compared to adjacent normal cells. The P value for that was ranged from smaller (Blue) to greater (Red). Each point on the scatter plot represents the gene. the x-axis denotes the data of the NAT and y-axis denotes the data of the cancerous tissue. 3.4 Candidate target genes regulated in BC by miRNA-1275 Table 3 showed that eleven computational prediction programs were applied for discovering the strongest candidate genes possessed miR-1275 binding sites in the 3’ -UTR. Six predicted genes genes ( DVL3 , PPP 2R2D, THSD4, CREB1, SYT7, and PRKACA ) were selected to have binding site to miRNA-1275 . The information on these six predicted genes were summarized in table 4. Eleven databases showed that DVL3 and PPP2R2D possess the binding site to miRNA-1275 ; whereas, THSD4 , and CREB1 were confirmed in ten prediction programs to be targeted by miRNA-1275, but SYT7 and PRKACA were confirmed by six tools to be predicted targets. These putative target genes are important for biological analysis of the BC tissues because the over- or down-expression of which can play a damaging role in several cellular processes and contribute to the cancer progression and tumorigenesis. 3.5 Determination of expression level of candidate target genes by MACE-seq approach Among 3624 upregulated genes, the six predicted genes ( DVL3, PPP 2R2D, THSD4, CREB1, SYT7, and PRKACA ) were pointed and named in the BC cells as compared to NAT (Fig.3B). Then the differential expression of which and their binding sites to has-miR-1275 were shown in figure 4. SYT7 gene was more overexpressed in BC, as compared to PRKACA gene. Overexpression level of THSD54 gene was higher than the up-regulation of PPP2R2D and DVL 3 genes. CREB1 was upregulated but more over-expressed than ST73 gene. Table 5 demonstrates the information on P. value, False Discovering Rate (FDR), and Fold Change (FC) of these six predicted genes possessed miR-1275 binding sites in the 3’ -UTR. They were identified as potentially modulated by miR- 1275 using computational prediction databases and TCGA algorithm. Next, the relationship between the expression level of these 8 candidates and histopathological significance were examined based on data from TCGA database. Among 204 target genes, DVL3 : P =6.98E-04 , PPP2R2D : P =1.53E-03 , THSD4 : P =2.28E-12 , CREB1 : P =3.12E-01 , SYT7 : P =1.28E-48 , and PRKACA : P =5.68E-02 were markedly relationship with worse prognosis in cases with BC. The Kaplan–Meier overall survival curve analyses of cases with BC were designed to be separated into 2 classes according to their expression (Fig.5). 3.6 The role of miR-1275 by targeting selected putative genes in BC MiRNAs are implicated in silencing mRNA transcripts through matching or mismatching with target mRNAs. As hypothesis of microRNA biogenesis, major strands of miR-1275 come from miRNA duplex are joined into the RISC protein and modulate the mRNA transcripts, but minor strands are broken down and cannot modulate gene expression. miR-1275 can play an essential role in regulating several biological mechanisms; including cell growth, migration, differentiation, proliferation and apoptosis. In this study, the down-expression of which regulate a set of genes and regulators related with tumor development. Six genes ( DVL3, PPP 2R2D, THSD4, CREB1, SYT7, and PRKACA ) were detected to be over-expressed in BC cells. Fig.6 shows the relationship between miR-1275 and these target genes. miR-1275 in breast cancer promotes cancer cell proliferation, cell differentiation, tumor growth, invasion and migration and also inhibits apoptosis through several gene targets. PPP2R2D acts as a tumor suppressor in signaling pathway in BC and is negatively regulated by miR-1275 . The overexpression of which decreases AKT and RACK1 abilities. Then these regulators decrease cell survival and migration. DVL3 is implicated in the breast cancer pathways and negatively controlled by miR-1275 . The up-regulation of this gene increases the cancer cell proliferation, migration and invasion . The cancer cell proliferation ability is increased when miR-1275 becomes overexpressed. another target gene. CREB1 and PRKACA show also negative correlation with miR-1275 level . Whereas CREB1 was found to reduce apoptosis process and increase cell proliferation in breast cancer, PRKACA plays a key role in tumorigenesis and development of BC. However, the function of THSD4 and SYT7 , currently unidentified, may boost tumor growth in breast cancer. Table 2. Comparison of marked down-expressed miRNAs in BC with NAT miRNA miRBase accession Location Log2FC P . value FDR Hsa-miR-1 MIMAT0031892 20q13.33 -2.6291 0.3109 1.0 Hsa-miR-100-5p MIMAT0004512 11q24.1 -1.2349 0.2824 1.0 Hsa-miR-10b-5p MIMAT0000254 2q31.1 -1.2852 0.261 1.0 Hsa-miR-125a-5p MIMAT0000443 19q13.41 -1.4153 0.213 1.0 Hsa-miR-125b-5p MIMAT0000423 11q24.1 -2.0041 0.083 1.0 Hsa-miR-1275 MIMAT0005929 6p21.31 -1.4150 0.6 14 1.0 Hsa-miR-130a-3p MIMAT0004593 11q12.1 -2.0168 0.104 1.0 Hsa-miR-133a-5p MIMAT0026478 18q11.2 -2.3885 0.220 1.0 Hsa-miR-143-5p MIMAT0004599 5q32 -1.2823 0.308 1.0 Hsa-miR-204-5p MIMAT0000265 9q21.12 -4.0627 0.086 1.0 Hsa-miR-21-3p MIMAT0004494 17q23.1 -0.0365 0.995 1.0 Hsa-miR-214-3p MIMAT0000271 1q24.3 -0.8746 0.440 1.0 Hsa-miR-25-5p MIMAT0004498 7q22.1 -1.2630 0.793 1.0 Hsa-miR-30a-3p MIMAT0000088 6q13 -0.2358 0.838 1.0 Hsa-miR-30b-5p MIMAT0000420 8q24.22 -1.3254 0.245 1.0 Hsa-miR-30d-5p MIMAT0000245 8q24.22 -0.9730 0.389 1.0 Hsa-miR-374b-5p MIMAT0004955 Xq13.2 -0.2420 0.8435 1.0 Hsa-miR-410-5p MIMAT0026558 14q32.31 -0.6780 0.989 1.0 Hsa-miR-423-5p MIMAT0004748 17q11.2 -0.5727 0.614 1.0 Hsa-miR-451a MIMAT0001631 17q11.2 -0.41900 0.711 1.0 Hsa-miR-455-5p MIMAT0003150 9q32 -2.5081 0.305 1.0 Hsa-miR-505-5p MIMAT0004776 Xq27.1 -1.5956 0.611 1.0 Hsa-miR-532-3p MIMAT0004780 Xp11.23 -0.7496 0.612 1.0 Hsa-miR-624-5p MIMAT0003293 14q12 -0.0931 1.0 1.0 Hsa-miR-664a-3p MIMAT0005949 1q41 -0.9249 0.539 1.0 Hsa-miR-664b-5p MIMAT0022271 Xq28 -0.5081 0.899 1.0 Hsa-miR-92a-1-5p MIMAT0004507 13q31.3 -0.2085 0.972 1.0 Hsa-miR-934 MIMAT0004977 Xq26.3 -3.0931 0.417 1.0 Hsa-miR-99b-5p MIMAT0000689 19q13.41 -0.1667 0.883 1.0 Table 3. Brief information on target predicted databases was shown to find putative targets possessing binding sequence to miR-1275 . Target predicted sites Species Tool properties Website Websites miRTarBase Human, Mouse, Rat Conservation, seed location http://mirtarbase.mbc.nctu.edu.tw/php/index.php Target scan Human, Mouse, Fly, Fish, and Worm Conservation, seed location http://www.targetscan.org/ TargetMiner Human, Mouse, Rat, Fly Conservation, seed location https://www.isical.ac.in/~bioinfo_miu/TargetMiner.html MirTar2 Human, Mouse, rat, Dog and Chicken Conservation, seed location http://www.mirdb.org/ DIANA Any Conservation, seed match, and free energy http://www.microrna.gr/microT-CDS miRWalk Human, Mouse, and Rat Conservation, seed match and free energy http://mirwalk.uni-hd.de/ miRmap Human, Chimpanzee, Mouse, Rat, Cow,Chicken, Zebrafish, and Opossum Conservation, seed match, and free energy https://mirmap.ezlab.org/ RNA22 Human, Fruit Fly, Mouse, and Worm Seed match and free energy https://cm.jefferson.edu/rna22/ PicTar - Tools4miRs Human, Mouse, Rat, Fly Conservation, seed location https://tools4mirs.org/software/target_prediction/pictar/ mirPath Human, Mouse, D. melanogaster, C. elegans, R. norvegicus, D. rerio and G. gallus Conservation, seed match and free energy http://snf-515788.vm.okeanos.grnet.gr/index.php?r=mirpath/geneList Microrna. org Human, mouse, Fruit Fly, and rat Conservation, seed match, free energy http://www.microrna.org/ Table 4. candidate target genes possessing binding sequence to miR-1275 was shown. Target gene Ensemble ID Position on Chromosome No. of sites predicted the gene as miR-1275 target DVL3 ENST00000313143.3 3q27.1 11 PPP2R2D ENST00000422256.2 10q26 11 THSD4 ENST00000355327.3 15q23 10 CREB1 ENST00000432329.2 2q34 10 SYT7 ENST00000263846.4 11q12.2 6 PRKACA ENST00000308677.4 17q24.2 6 Table 5. Experimentally validated target genes of miR-1275 in BC Targets Gen ID Description P .value FDR Log2fc DVL3 ENSG00000161202 Dishevelled segment polarity protein 3 6.98E-04 3.48E-03 0.58975 PPP2R2D ENSG00000175470 Protein phosphatase 2, regulatory subunit B, delta 1.53E-03 6.95E-03 0.616981 THSD4 ENSG00000187720 Thrombospondin type 1 domain containing 4 2.28E-12 3.63E-11 0.938081 CREB1 ENSG00000118260 cAMP responsive element binding protein 1 3.12E-01 5.13E-01 0.269971 SYT7 ENSG00000011347 Synaptotagmin 7 1.28E-48 8.14E-47 1.84905 PRKACA ENSG00000072062 Protein kinase cAMP-activated catalytic subunit alpha 1.77E-02 5.68E-02 0.296759 4. Discussion There is evidence that a single microRNA modulates multiple protein-coding and non-coding genes in different ordinary cells and cancerous cells. In human cancer cells, new RNA groups can be detected using the specific nature of microRNA from relevant microRNA analysis. Multiple high-throughput approaches, such as DNA microarrays, MACE-sequencing, PCR-based arrays, and RNA-sequencing, are now available and have made microRNA expression profiles of BC, showing the irregular expression of numerous miRNAs (26-29). One approach to detect the most essential miRNA from numerous miRNAs is to detect differential expression of miRNAs which have been shown in numerous experiments. Several researches have reported that multiple miRNAs, such as miR-100, miR-107, miR-205-3p, miR-122 and miR-99a-5p , are continuously down-expressed and act as tumor-suppressive miRNA in BC cells (26, 28-33). In this study, these miRNAs were found to be downregulated in tumor cells but only miR-1275 was focused and their putative target genes were newly explored in paraffin embedded BC tissues. Several researches have reported that this miRNA is down-expressed in gastric and nasopharyngeal carcinoma and function as a tumor suppressor (34-36); whereas, this miRNA is overexpressed in non-small lung cell cancer, squamous carcinoma and chronic myelogenous leukemia (37-39). Some recent studies revealed that this downregulated miRNA was detected to have an essential effect on cancer cell proliferation, migration, invasion, metastasis, and angiogenesis through targeting multiple oncogenic genes HOXB5 , WNT7B and LncRNA HAND2-AS1(34, 36, 39). One previous study showed that miR-1275 regulates IGF1, NFIX , Claudin11 in very young women with BC (23). Whereas, down-expression of miR-1275 in all subtypes of paraffin BC tissues was not fully investigated. In this study, down-expression of miRNA was observed in all subtypes of paraffin embedded BC tissues of 21 cases with different ages. After that, the miR-1275 -modulated putative targets and their pathways were aimed to explain in the cells of BC. Six genes ( DVL3 , PPP2R2D , TSHD4 , CREB1 , SYT7 , and PRKACA ) were experimentally observed to be overexpressed in the cells of BC. Based on the databases of miRNA target prediction, they were selected and closely correlated with poor prognosis. Among these candidate genes, four genes ( PPP2R2D , DVL3 , and CREB1 ) were shown to be strongly targeted by the miR-1275 in the BC cells. Studies showed that these regulators were found to reduce cell survival and migration in cancer cells (40, 41). DVL3 is observed to be implicated in the breast cancer pathways (34) and negatively regulated by miR-1275 . The up-regulation of this gene can increase the cancer cell proliferation, migration and invasion in BC cells . The overexpression of which increase cancer cell proliferation ability (42). Another target gene, CREB1 and PRKACA show also negative correlation with miR-1275 level . Whereas CREB1 was found to reduce apoptosis process and increase cell proliferation in breast cancer (43), PRKACA plays a key role in tumorigenesis and development of BC (44). However, the role of THSD4 and SYT7 , currently unidentified, may enhance tumor growth in a variety of cancers, especially breast cancer (45, 46). 5. Conclusion In the present study, differential expression profiles of total mRNA transcripts and sRNAs were identified in BC paraffin tissue (NAT and tumoral tissue) by MACE- sequencing. Decreased miRNA-1275 expression develops breast cancer by increasing the activity of biological processes; such as growth, migration, invasion and metastasis. Upregulated miRNA-1275 prevented BC development by modulating direct expression of DVL3 , PPP2R2D , TSHD4 , CREB1 , SYT7 , and PRKACA . This is the first study revealing that miR-1275 function as a tumor-suppressive miRNA in BC cells, regulating numerous targets which were closely related with BC pathogenesis and oncogenesis. Abbreviations FFPE: Formalin Fixed Paraffin Embedded; BC: Breast Cancer; NAT: Normal adjacent tissue; sRNA: small RNA; MACE: Massive Analysis of cDNA Ends; E.R.: Estrogen Receptor; Pg. R.: Progesterone Receptor; HER2: Human epidermal growth factor receptor 2; TCGA: The Cancer Genome Atlas; FDR: False Discovering Rate; FC: Fold Change. Declarations Acknowledgement I wish to thank the patients and their families. I would like to express my specially thanks of gratitude to my supervisor for her frequent support and encouragement. I also offer my sincere appreciation for Al mufti and Luay laboratories which helped and supported me through collecting FFPE blocks of BC and clinical data. Author contribution Suhad and Sevan were responsible for the experimental design. They led to the execution of the experiments. Although analyses of MACE-sequencing and sRNA sequencing were done in Genxpro company, in Germany. Data analysis and bioinformatic tasks were done by Sevan. Sevan also discussed and interpreted the data. He also did the manuscript mapping and submission, but Suhad supervised the project. Funding This study was supported by Sevan Majed who is a Ph.D. student. Availability of data and materials Although row data of MACE-sequencing and sRNA may be available in the database of GenXpro, at https://genxpro.net/ , These data will be further studied for another research in the future. The findings described in this manuscript were provided by the Co-author. Ethics approval and consent to participate This study was followed and approved by Human Research Ethics Committee at Science College in Salahuddin University-Erbil (Approval no.4c/132). Informed consent was received from all patients in accordance with the requirements of the Human Research Ethics Committee. Consent for publication Not applicable. Competing interest The author announces no conflict of interest. Mr. Sevan is a teacher of Salahaddin University-Erbil, a subsidiary of ministry of higher education in Kurdistan region government (KRG). Author details 1 Biology Department, College of Education, Salahaddin University-Erbil. 2 Research Center, Molecular genetics lab, Salahaddin University-Erbil. References Control CfD, Prevention. Cancer survivors--United States, 2007. MMWR Morbidity and mortality weekly report. 2011;60(9):269. Jiang X, Tang H, Chen T. Epidemiology of gynecologic cancers in China. J Gynecol Oncol. 2018;29(1). Adhami M, Haghdoost AA, Sadeghi B, Malekpour Afshar R. Candidate miRNAs in human breast cancer biomarkers: a systematic review. Breast Cancer. 2018;25(2):198-205. Toda H, Seki N, Kurozumi S, Shinden Y, Yamada Y, Nohata N, et al. 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MiRNA-107 inhibits proliferation and migration by targeting CDK8 in breast cancer. International journal of clinical and experimental medicine. 2014;7(1):32-40. Wang B, Wang H, Yang Z. MiR-122 inhibits cell proliferation and tumorigenesis of breast cancer by targeting IGF1R. PloS one. 2012;7(10):e47053. Toda H, Seki N, Kurozumi S, Shinden Y, Yamada Y, Nohata N, et al. RNA‐sequence‐based microRNA expression signature in breast cancer: tumor‐suppressive miR‐101‐5p regulates molecular pathogenesis. Molecular oncology. 2020;14(2):426-46. Wang Y-W, Zhang W, Ma R. Bioinformatic identification of chemoresistance-associated microRNAs in breast cancer based on microarray data. Oncology reports. 2018;39(3):1003-10. Mei JW, Yang ZY, Xiang HG, Bao R, Ye YY, Ren T, et al. MicroRNA-1275 inhibits cell migration and invasion in gastric cancer by regulating vimentin and E-cadherin via JAZF1. BMC cancer. 2019;19(1):740. Sun KY, Peng T, Chen Z, Huang J, Zhou XH. MicroRNA-1275 suppresses cell growth, and retards G1/S transition in human nasopharyngeal carcinoma by down-regulation of HOXB5. J Cell Commun Signal. 2016;10(4):305-14. He J, Yu L, Wang CM, Zhou XF. MiR-1275 promotes non-small cell lung cancer cell proliferation and metastasis by regulating LZTS3 expression. European review for medical and pharmacological sciences. 2018;22(9):2680-7. Liu MD, Wu H, Wang S, Pang P, Jin S, Sun CF, et al. MiR-1275 promotes cell migration, invasion and proliferation in squamous cell carcinoma of head and neck via up-regulating IGF-1R and CCR7. Gene. 2018;646:1-7. Yang JR, Shi MX, Zeng Y. LncRNA HAND2-AS1 inhibits proliferation and promotes apoptosis of chronic myeloid leukemia cells by sponging with micRNA-1275. European review for medical and pharmacological sciences. 2019;23(5):2103-11. Kuo Y-C, Huang K-Y, Yang C-H, Yang Y-S, Lee W-Y, Chiang C-W. Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55α regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt. Journal of Biological Chemistry. 2008;283(4):1882-92. Kiely PA, O'Gorman D, Luong K, Ron D, O'Connor R. Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and β1 integrin to promote cell migration. Molecular and cellular biology. 2006;26(11):4041-51. Jiang H, Cheng L, Hu P, Liu R. MicroRNA‑663b mediates TAM resistance in breast cancer by modulating TP73 expression. Molecular medicine reports. 2018;18(1):1120-6. Friedrich M, Heimer N, Stoehr C, Steven A, Wach S, Taubert H, et al. CREB1 is affected by the microRNAs miR-22-3p, miR-26a-5p, miR-27a-3p, and miR-221-3p and correlates with adverse clinicopathological features in renal cell carcinoma. Scientific Reports. 2020;10(1):6499. Tang W, Li GS, Li JD, Pan WY, Shi Q, Xiong DD, et al. The role of upregulated miR-375 expression in breast cancer: An in vitro and in silico study. Pathology, research and practice. 2020;216(1):152754. Cohen H, Ben-Hamo R, Gidoni M, Yitzhaki I, Kozol R, Zilberberg A, et al. Shift in GATA3 functions, and GATA3 mutations, control progression and clinical presentation in breast cancer. Breast cancer research : BCR. 2014;16(6):464-. Liu X, Li C, Yang Y, Liu X, Li R, Zhang M, et al. Synaptotagmin 7 in twist-related protein 1-mediated epithelial – Mesenchymal transition of non-small cell lung cancer. EBioMedicine. 2019;46:42-53. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-101189","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":4192579,"identity":"71c047a5-1db5-4a96-a2d1-ae3813f2cc79","order_by":0,"name":"Sevan Omer Majed","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDADxgYGwwdg1gEGZgYGNuK0GBuQpgUIzCSI0iLf3mP4uYBhmzxze/O2yp9tDHJ8NxKYjSvKcGsxOHPGWHoGw23Dxp5jZbd52xiMJYFaEs+cw6NFIi1BmofhNmPjjByz24xtDIkbgFoONrbhcdj8Z8m/gVrsG+e/MSsEOqyeoBaGG8zHQLYkNs7gMWMAOizBAOQwfFoMziQfs+YxuJ3c2JNWLM1zTsJw5pmHzYYNePwi336w+TZPxW3bje2HN378UWYjz3c8+bBkA54Qg9rFwGDYAGaBooaxgZB6qHXEKRsFo2AUjIKRCAC1RVGUXet4wAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3636-7723","institution":"Salahaddin University- Hawler","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Sevan","middleName":"Omer","lastName":"Majed","suffix":""},{"id":4192580,"identity":"c440315b-cb58-4cf4-9926-0fd0cdf66855","order_by":1,"name":"Suhad Asad Mustafa","email":"","orcid":"","institution":"Salahaddin University- Hawler","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Suhad","middleName":"Asad","lastName":"Mustafa","suffix":""}],"badges":[],"createdAt":"2020-10-31 16:58:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-101189/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-101189/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":3409260,"identity":"27470efe-dd42-4778-843f-7c3c68e817ab","added_by":"auto","created_at":"2020-11-05 19:57:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":136799,"visible":true,"origin":"","legend":"Differential expression analysis of non-coding RNAs by MACE-sequencing. A The expression profile of 1400 sRNAs in BC compared to NAT is plotted. Red dots represent the sample sRNAs. A light green dot represents miRNA-1275 expression level in BC tissue compared to NAT. B Heat-map based clustering of several downregulated miRNAs in BC compared to NAT. Has-miRNA-1275 is underlined. C correlation between BC and NAT in differential expression of sRNAs.","description":"","filename":"Figure1.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/f1eca79754f94a6bd8c6e6a6.JPG"},{"id":3409254,"identity":"4d792dfd-8ad2-4817-947d-ab43f966332d","added_by":"auto","created_at":"2020-11-05 19:57:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":136799,"visible":true,"origin":"","legend":"Differential expression analysis of non-coding RNAs by MACE-sequencing. A The expression profile of 1400 sRNAs in BC compared to NAT is plotted. Red dots represent the sample sRNAs. A light green dot represents miRNA-1275 expression level in BC tissue compared to NAT. B Heat-map based clustering of several downregulated miRNAs in BC compared to NAT. Has-miRNA-1275 is underlined. C correlation between BC and NAT in differential expression of sRNAs.","description":"","filename":"Figure1.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/507ac72d1800ee1919cbac1b.JPG"},{"id":3409261,"identity":"d654f0aa-dfc1-4bab-8fc4-4717fb381d45","added_by":"auto","created_at":"2020-11-05 19:57:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72246,"visible":true,"origin":"","legend":"A Comparison of differential expression of miR-1275 by MACE-seq approach. B Comparison of differential expression of miR-1275 by RT-qPCR approach. C mature sequence of miR-1275 in NAT and BC was the same. D Kaplan–Meier overall survival curve designed to show the differential expression of miR-1275 related to overall survival in the patients with the BC. ","description":"","filename":"Figure2.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/d6ed3b9958405e8b53b856cb.JPG"},{"id":3409255,"identity":"13278458-d9d0-4ed3-a47c-1bc3a3d2fd08","added_by":"auto","created_at":"2020-11-05 19:57:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72246,"visible":true,"origin":"","legend":"A Comparison of differential expression of miR-1275 by MACE-seq approach. B Comparison of differential expression of miR-1275 by RT-qPCR approach. C mature sequence of miR-1275 in NAT and BC was the same. D Kaplan–Meier overall survival curve designed to show the differential expression of miR-1275 related to overall survival in the patients with the BC. ","description":"","filename":"Figure2.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/d3ee7d7693213bd4535dd3dc.JPG"},{"id":3409262,"identity":"57f56e69-56e5-4a4a-8b2a-974a9cbf3832","added_by":"auto","created_at":"2020-11-05 19:57:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":144203,"visible":true,"origin":"","legend":"A Scatter plot analysis of gene expression profile displays up- or down-regulation of genes in BC tissue, compared to NAT. Each point denotes the average value of one transcript in the experiment. The expression difference is taken account of significance for a Pvalue (0.05). B Outlined points and names denote the selected target genes for has-miR-1275.","description":"","filename":"Figure3.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/27d9209f1a19835192b6c2d9.JPG"},{"id":3409256,"identity":"9ae0ac51-9e55-4efc-adcf-15c1e9cba792","added_by":"auto","created_at":"2020-11-05 19:57:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":144203,"visible":true,"origin":"","legend":"A Scatter plot analysis of gene expression profile displays up- or down-regulation of genes in BC tissue, compared to NAT. Each point denotes the average value of one transcript in the experiment. The expression difference is taken account of significance for a Pvalue (0.05). B Outlined points and names denote the selected target genes for has-miR-1275.","description":"","filename":"Figure3.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/d8826d5298adde04ec85ccfd.JPG"},{"id":3409263,"identity":"ebf54c00-5b8d-4a39-bf7e-17fc5d704857","added_by":"auto","created_at":"2020-11-05 19:57:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":110256,"visible":true,"origin":"","legend":"Target genes and has-miR-1275 are combined in the seed region, including 6 to 8 nucleotides in the 5′ end, showing in red color. Differential expression level of target genes in BC compared to NAT.","description":"","filename":"Figure4.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/966867775886ba13b9b9f38f.JPG"},{"id":3409257,"identity":"4923b4dc-27cc-48c8-8c1a-a6133fa7ccde","added_by":"auto","created_at":"2020-11-05 19:57:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":110256,"visible":true,"origin":"","legend":"Target genes and has-miR-1275 are combined in the seed region, including 6 to 8 nucleotides in the 5′ end, showing in red color. Differential expression level of target genes in BC compared to NAT.","description":"","filename":"Figure4.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/cd2a4ff00853eeb87e6769a5.JPG"},{"id":3409264,"identity":"c037f3aa-9668-44a5-b575-44fa13d852cb","added_by":"auto","created_at":"2020-11-05 19:57:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":99867,"visible":true,"origin":"","legend":"An association between the expression levels of six genes (DVL3, PPP2R2D, TSHD4, CREB1, SYT7, and PRKACA) and histopathological significance was shown using data from TCGA database. The Kaplan–Meier overall survival curves show that patients with BC were separated into 2 classes according to their expression levels.","description":"","filename":"Figure5.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/84c58cebe456ed2c713644fb.JPG"},{"id":3409258,"identity":"cf1d7776-5d21-425f-92c2-297d47e73014","added_by":"auto","created_at":"2020-11-05 19:57:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":99867,"visible":true,"origin":"","legend":"An association between the expression levels of six genes (DVL3, PPP2R2D, TSHD4, CREB1, SYT7, and PRKACA) and histopathological significance was shown using data from TCGA database. The Kaplan–Meier overall survival curves show that patients with BC were separated into 2 classes according to their expression levels.","description":"","filename":"Figure5.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/fa3e5b78aec7272385ce7960.JPG"},{"id":3409265,"identity":"f62f19d0-3c22-4281-b1cd-8b1f3f3acf9c","added_by":"auto","created_at":"2020-11-05 19:57:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92246,"visible":true,"origin":"","legend":"miR-1275 putative targets and their roles in the BC.","description":"","filename":"Figure6.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/7f54bf5b1e5ceb1041301a64.JPG"},{"id":3409259,"identity":"e7bc904f-26ac-4d04-99b2-d83633e27004","added_by":"auto","created_at":"2020-11-05 19:57:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92246,"visible":true,"origin":"","legend":"miR-1275 putative targets and their roles in the BC.","description":"","filename":"Figure6.JPG","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/f7771d3a0407f339154fb78b.JPG"},{"id":13610958,"identity":"a5a52ec9-e709-4f35-a42e-62a297988add","added_by":"auto","created_at":"2021-09-17 06:26:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1566404,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-101189/v1/3a6f72d9-44ab-4f18-8e10-69912bf05452.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eIdentification of Tumor-Suppressive miRNA-1275 as a Novel Marker for Breast Cancer (BC) by MACE-Sequencing and RT-qPCR Techniques\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBreast malignant cell is a prominent type of cancers mostly diagnosed in females, and the second most frequent malignancy-associated deaths worldwide, especially in the US and Asian countries (1, 2). Approximately two million females are annually diagnosed and more than 620,000 deaths are newly recorded every year (3, 4). Frequently, BC is developed as a result of a genomic mutation. However, about 10% of BCs is inheritably come down from parents to their generations; whereas, more than 85% of BCs is developed in their lifetime (4, 5). Inherited abnormalities in \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003ePTEN\u003c/em\u003e genes were studied to result in the high risk of the breast malignant cell progression (6, 7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGene expression profiling has recently played a critical role in medicinal selection for BC subtypes. The analysis of BC gene expression can be used for molecular category of BC subtypes (8-10). Two studies reported that this classification facilitates the determination of the cure doses. The molecular subtypes of BC can be categorized into luminal-A, luminal-B (including HER2+ /-), HER2+, and triple negative (TN) (11, 12). These subtypes are pivotal for cure choice and are correlated to the biological characteristics of BC.\u003c/p\u003e\n\u003cp\u003eMicroRNA (miRNA), which is a type of untranslated sRNAs, is synthesized from eukaryotic genomes, consisting of a single stranded RNA of about 19-22nt in length (13, 14). These short non-coding miRNAs are described as regulators of coding and non-coding (nc) RNAs in eukaryotic cells because they are involved in silencing RNA transcripts and in regulating the stability of their targeted mRNAs (14, 15). MiRNAs play also several regulatory roles in several cellular processes; cell development, proliferation, migration, invasion and death (16). Because more than 50% of RNA molecules has been detected to be controlled by miRNA, these mRNAs were damaged because of the effect of aberrant miRNA in malignant cells.\u003c/p\u003e\n\u003cp\u003eMiRNAs in human malignancies were found to act as oncogenes or ant-oncogenes (tumor suppressors). Oncogenic miRNAs in tumor progress play a negative role in stimulating genes which regulate\u0026nbsp;cell development\u0026nbsp;and apoptosis process. Tumor suppressive miRNAs in human tumor have a key role in silencing genes which modulate cell development and apoptosis (17, 18). When normal cells do not undergo normal growth and apoptosis process, they normally cause tumor creation. Numerous recent experiments show that numerous miRNAs are directly implicated in modulating cell growth, proliferation as well as apoptosis (19, 20). They play major roles in the pathogenesis of a number of human malignancies; such as breast, colorectal cancer, lung, leukemia liver, and brain(16, 18). The miRNA expression level may be either down- or up-regulated in these cancers. Several molecular techniques, such as RNA sequencing, miRNA microarray, RT-PCR and northern blot are applied to determine the expression level of them.\u003c/p\u003e\n\u003cp\u003eNumerous miRNAs have been recognized to be implicated in the pathogenesis of human breast cancer. It was found that the expression patterns of \u003cem\u003emiR-145\u003c/em\u003e, \u003cem\u003e-125b\u003c/em\u003e,\u003cem\u003e -155\u003c/em\u003e, and \u003cem\u003e-21\u003c/em\u003ewere significantly downregulated. In breast malignant cells, these miRNAs were observed to be associated with pathologic properties; cell proliferation, expression of progesterone and estrogen receptors (21). A recent study revealed that tumor suppressive \u003cem\u003emiRNA-204-5p \u003c/em\u003eplays a key role in targeting several oncogenic genes which are closely connected to BC pathogenesis(20). Complete information on \u003cem\u003emiR-1275\u003c/em\u003e expression level and its targets in BC have not been available; whereas, the expression profile of which has be analyzed in some human cancer. A study reported that down-expressed \u003cem\u003emiR-1275\u003c/em\u003e leads to overexpression \u003cem\u003eclaudin11\u003c/em\u003e in cancer stem cells or tumor-initiating cells (CSCs/TICs) (22). According to a study carried out on young women with BC, 6 miRNAs; including \u003cem\u003emiR-1275, miR-1228*, miR-139\u003c/em\u003e, \u003cem\u003emiR-92b\u003c/em\u003e, \u003cem\u003emiR-1207\u003c/em\u003e, and \u003cem\u003emiR-3196,\u003c/em\u003e were involved in the processes of cell movement, proliferation and invasion (23). It was also found that this miRNA was found to be downregulated in gastric cancer (24). Another study found that \u003cem\u003emiRNA-1275\u003c/em\u003e expression level was significantly abnormally deregulated in Ewing's Sarcoma (ES) (25). The significance of a large number of miRNAs have been reported to become an appropriate biomarker for human cancer diagnostics. However, the significance of \u003cem\u003emiRNA-1275\u003c/em\u003e in BC is not reported. The objective of this study was to determine the expression level of \u003cem\u003emiRNA-1275\u003c/em\u003e as a biomarker for BC diagnostics. Another objective is to identify the potential targeted genes of this miRNA.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Collection of FFPE-blocks of BC samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormalin Fixed Paraffin Embedded (FFPE)-Block of 21 Kurdish cases with BC were collected at clinicopathological laboratories, called as Al Mufti and Luay. For each patient, two paraffin blocks (one adjacent normal tissue (NAT) and one breast cancer tissue) were collected. The normal tissues were histologically taken nearly 2 cm away from the tumor area. Clinical features of 21 cases were obtained using a questionnaire. The features were displayed in table 1. Permission was accomplished from all cases by signing the confirmed consent. any pre-operative chemotherapy or radiotherapy was taken by none of these cases\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Generation of the mRNA and sRNA expression profiles for BC by MACE-sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine differential expression of protein-coding RNAs (mRNA transcripts) and non-coding RNAs (ncRNAs), analyzing two paraffin blocks (cancerous and normal) of a BC patient, Massive Analysis of cDNA Ends (MACE)-seq technique was performed (GenXpro GmbH, Frankfurt, Germany). cDNA synthesis, NGS-library and sequencing were subsequently prepared after isolation of sRNAs from mRNA transcripts. The raw sequencing data was bioinformatically analyzed and solved. The raw sequencing data is demultiplexed based on the different barcodes (GenXpro GmbH, Frankfurt, Germany). \u0026nbsp;For removing adapter, the organized reads were cut out for high-quality sequences. Bowtie 2 tool was later used for aligning the sorted reads to the nominated reference sequences and annotating with corresponding properties. The numbers of aligned reads were subsequently normalized to account for distinct sequencing depths. Finally, the normalized and original read numbers were considered during statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Total RNA purification and cDNA synthesis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003emiR-1275\u003c/em\u003e was selected as a candidate from the MACE result and confirmed from 20 cases using Real Time-quantitative polymerase chain reaction (RT-qPCR) technique at Salahuddin University Research Center (SURC). Differential expression of \u003cem\u003emiRNA-1275 \u003c/em\u003ewas measured in 40 block specimens (20 adjacent normal tissues (NATs) and 20 breast cancer tissues). The total RNA molecules including miRNA were extracted using FFPE RNA/DNA Purification Plus kit (Cat. No. 54300, NORGEN BIOTEK CORP, Canada). Complementary DNA (cDNA) was synthesized using miRNA All-In-One cDNA Synthesis Kit (Cat. No. G898, abmgood company, US).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Total RNA purification and cDNA synthesis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003emiR-1275\u003c/em\u003e was selected as a candidate from the MACE result and confirmed from 20 cases using Real Time-quantitative polymerase chain reaction (RT-qPCR) technique at Salahuddin University Research Center (SURC). Differential expression of \u003cem\u003emiRNA-1275 \u003c/em\u003ewas measured in 40 block specimens (20 adjacent normal tissues (NATs) and 20 breast cancer tissues). The total RNA molecules including miRNA were extracted using FFPE RNA/DNA Purification Plus kit (Cat. No. 54300, NORGEN BIOTEK CORP, Canada). Complementary DNA (cDNA) was synthesized using miRNA All-In-One cDNA Synthesis Kit (Cat. No. G898, abmgood company, US).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Most common putative targeted genes regulated by \u003cem\u003emiR-1275\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEleven databases were searched for finding the most common putative targets of \u003cem\u003emiR-1275 \u003c/em\u003e(Table 3). Six putative targets (\u003cem\u003eDVL3,\u003c/em\u003e \u003cem\u003ePPP\u003c/em\u003e\u003cem\u003e2R2D,\u003c/em\u003e\u003cem\u003e THSD4, CREB1, SYT7, \u003c/em\u003eand\u003cem\u003e PRKACA\u003c/em\u003e) were determined to possess binding sequence to \u003cem\u003emiR-1275\u003c/em\u003e (Table 4)\u003cem\u003e.\u003c/em\u003e, Graphpad prsim, version 8.0.1 was used to show the differential expression of these selected target genes were performed by MACE-seq.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Analysis of clinicopathological data associated with BC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAssociation between \u003cem\u003emiR-1275 \u003c/em\u003eand its target genes was computationally analyzed to determine the clinical significance using databases of cBioPortal (\u003ca href=\"http://www.cbioportal.org/\"\u003ehttp://www.cbioportal.org/\u003c/a\u003e) and OncoLnc (\u003ca href=\"http://www.oncolnc.org/\"\u003ehttp://www.oncolnc.org/\u003c/a\u003e). Clinical data and expression levels of the \u003cem\u003emiR-1275 \u003c/em\u003eand its target genes gained from these sites and then were downloaded on 10 September 2020.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Clinicopathological features of 21 cases with BC.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 951px;\" border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCases\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003eAge\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003eTumor Size\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eStage\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eLymph node metastasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003eLymphatic Invasion\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003eVenous Invasion\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003eE.R.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePg.R.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eHer2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003eKi-67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eTechnique\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e4cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eMACE-seq.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e4cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e4.5cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e1.9cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003eUnavailable\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e2.3cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e3.5cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIIC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e2cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e20-30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e1.8cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e1.4cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e5cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIIC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e1.9cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIIB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e80-90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e1.5cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e80-90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e1.7cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e70-80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e1.3cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e3cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e2.5cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e1.5cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e2.5cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e1.3cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e15-20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e1.5cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e15-20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 63px;\"\u003e\n\u003cp\u003eCase21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 39px;\"\u003e\n\u003cp\u003e44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 59px;\"\u003e\n\u003cp\u003e6cm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 54px;\"\u003e\n\u003cp\u003eIIIC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 96px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 90px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 68px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 76px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 72px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89px;\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91px;\"\u003e\n\u003cp\u003eRT-PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Construction of expression profile of sRNAs for BC by sRNA-sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSRNA sequencing was carried out to construct the differentia expression of non-coding RNAs in BC compared to NAT. Two small libraries were sequenced for two paraffin blocks (NAT and BC tissue). Histopathological properties of this specimen were shown in table 1 (Case1). By comparing non-coding RNA expression profiles of BC and NAT, 1400 sRNAs (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) were filtered out by a SAM software. The raw data were then standardized and log2-transformed to show on a scatter plot (Fig. 1A). Among 1400 sRNAs, 723 non-coding RNAs were downregulated, but 678 sRNAs were upregulated. Each dot on the scatter plot represents the sRNA. Among 1400 sRNAs, 520 microRNAs were differentially expressed. 185 microRNAs were down-expressed, but 335 were overexpressed. The x-axis denotes the data of the NAT and y-axis denotes the data of the BC. Correlation plot was constructed to show expression levels of non-coding RNAs between BC and NAT (Fig. 1B). Blue color denoted the correlation of sRNAs between the BC and NAT. Heat map was designed to show 29 miRNAs which were markedly downregulated in BC compared to NAT (Fig. 1C). Table 2 shows the information on these 29 miRNAs which are notably downregulated. In this study, \u003cem\u003ehas\u003c/em\u003e-\u003cem\u003emiR-1275\u003c/em\u003e, which underlined with red color in heat map, was focused to identify sequence and expression level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Confirmation of \u003cem\u003emiR-1275\u003c/em\u003e expression level by RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003emiR-1275\u003c/em\u003e expression level in RNA-sequencing was observed to be significantly downregulated in BC tissue, as compared to NAT. The \u003cem\u003ep.\u003c/em\u003evalue of this miRNA was 0.614 (Fig. 2A).\u0026nbsp; Then, this miRNA was selected to confirm. RT-qPCR machine was used to confirm the differential expression of \u003cem\u003emiR-1275\u003c/em\u003e in the laboratory. 40 FFPE blocks for 20 cases was used in this experiment, including 20 BC tissues and 20 normal tissues next to tumoral tissues (2cm away from tumoral tissues). The clinical properties of these cases were briefly explained in table 1. The \u003cem\u003emiR-1275\u003c/em\u003e expression level was detected to be markedly decreased in BC cells compared to adjacent normal cells and the \u003cem\u003eP\u003c/em\u003e-value of this was 0.001** (Fig.2B). The mature sequence of which in the BC and NAT was made up of 17 nucleotides and also identical (Fig.2C). Then, Kaplan\u0026ndash;Meier overall survival curve was designed to show the effect of \u003cem\u003emiR-1275\u003c/em\u003e expression on the prognosis of cases with BC. Data was used from The Cancer Genome Atlas (TCGA) database and analyzed. Kaplan\u0026ndash;Meier overall survival curve displayed that cases were separated into two classes according to its expression. The decreased \u003cem\u003emiR-1275\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e-value=0.0401) was related to overall survival in cases with BC (Fig.2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Differentially expressed genes for adjacent normal and BC tissue by MACE-seq.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferentially expressed genes in BC and NATs was displayed (Fig. 3A). 26843 differentially expressed genes (P\u0026le;0.05) were filtered out by a SAM software. In order to show genes that were more significantly different in their expressions, 7041 genes were standardized and log2-transformed to show on a scatter plot. 3624 genes were significantly overexpressed and 3417 genes were significantly down-expressed in tumoral cells compared to adjacent normal cells. The \u003cem\u003eP \u003c/em\u003evalue for that was ranged from smaller (Blue) to greater (Red). Each point on the scatter plot represents the gene. the x-axis denotes the data of the NAT and y-axis denotes the data of the cancerous tissue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Candidate target genes regulated in BC by \u003cem\u003emiRNA-1275\u003c/em\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 3 showed that eleven computational prediction programs were applied for discovering the strongest candidate genes possessed \u003cem\u003emiR-1275\u003c/em\u003e binding sites in the 3\u0026rsquo; -UTR. Six predicted genes genes (\u003cem\u003eDVL3\u003c/em\u003e,\u003cem\u003e PPP\u003c/em\u003e\u003cem\u003e2R2D, THSD4, CREB1, SYT7, \u003c/em\u003eand\u003cem\u003e PRKACA\u003c/em\u003e) were selected to have binding site to \u003cem\u003emiRNA-1275\u003c/em\u003e. \u0026nbsp;The information on these six predicted genes were summarized in table 4. Eleven databases showed that DVL3 and \u003cem\u003ePPP2R2D\u003c/em\u003e possess the binding site to \u003cem\u003emiRNA-1275\u003c/em\u003e; whereas, \u003cem\u003eTHSD4\u003c/em\u003e, and \u003cem\u003eCREB1 \u003c/em\u003ewere confirmed in ten prediction programs to be targeted by \u003cem\u003emiRNA-1275, \u003c/em\u003ebut \u003cem\u003eSYT7 \u003c/em\u003eand \u003cem\u003ePRKACA \u003c/em\u003ewere confirmed by six tools to be predicted targets. These putative target genes are important for biological analysis of the BC tissues because the over- or down-expression of which can play a damaging role in several cellular processes and contribute to the cancer progression and tumorigenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Determination of expression level of candidate target genes by MACE-seq approach \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong 3624 upregulated genes, the six predicted genes (\u003cem\u003eDVL3,\u003c/em\u003e \u003cem\u003ePPP\u003c/em\u003e\u003cem\u003e2R2D,\u003c/em\u003e\u003cem\u003e THSD4, CREB1, SYT7, \u003c/em\u003eand\u003cem\u003e PRKACA\u003c/em\u003e) were pointed and named in the BC cells as compared to NAT (Fig.3B). Then the differential expression of which and their binding sites to \u003cem\u003ehas-miR-1275 \u003c/em\u003ewere shown in figure 4.\u0026nbsp; \u003cem\u003eSYT7\u003c/em\u003e gene was more overexpressed in BC, as compared to \u003cem\u003ePRKACA\u003c/em\u003e gene. Overexpression level of \u003cem\u003eTHSD54\u003c/em\u003e gene was higher than the up-regulation of \u003cem\u003ePPP2R2D \u003c/em\u003eand DVL\u003cem\u003e3\u003c/em\u003e genes. \u003cem\u003eCREB1\u003c/em\u003e was upregulated but more over-expressed than \u003cem\u003eST73\u003c/em\u003e gene. Table 5 demonstrates the information on \u003cem\u003eP.\u003c/em\u003evalue, False Discovering Rate (FDR), and Fold Change (FC) of these six predicted genes possessed \u003cem\u003emiR-1275\u003c/em\u003e binding sites in the 3\u0026rsquo; -UTR. They were identified as potentially modulated by \u003cem\u003emiR-\u003c/em\u003e1275 using computational prediction databases and TCGA algorithm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, the relationship between the expression level of these 8 candidates and histopathological significance were examined based on data from TCGA database. Among 204 target genes, \u003cem\u003eDVL3\u003c/em\u003e:\u003cem\u003e P\u003c/em\u003e=6.98E-04\u003cem\u003e,\u003c/em\u003e \u003cem\u003ePPP2R2D\u003c/em\u003e:\u003cem\u003e P\u003c/em\u003e=1.53E-03\u003cem\u003e, THSD4\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e=2.28E-12\u003cem\u003e, CREB1\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e=3.12E-01\u003cem\u003e, SYT7\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e=1.28E-48\u003cem\u003e, \u003c/em\u003eand\u003cem\u003e PRKACA\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e=5.68E-02 were markedly relationship with worse prognosis in cases with BC. The Kaplan\u0026ndash;Meier overall survival curve analyses of cases with BC were designed to be separated into 2 classes according to their expression (Fig.5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 The role of \u003cem\u003emiR-1275\u003c/em\u003e by targeting selected putative genes in BC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMiRNAs are implicated in silencing mRNA transcripts through matching or mismatching with target mRNAs. As hypothesis of microRNA biogenesis, major strands of \u003cem\u003emiR-1275\u003c/em\u003e come from miRNA duplex are joined into the RISC protein and modulate the mRNA transcripts, but minor strands are broken down and cannot modulate gene expression. \u003cem\u003emiR-1275\u003c/em\u003e can play an essential role in regulating several biological mechanisms; including cell growth, migration, differentiation, proliferation and apoptosis. In this study, the down-expression of which regulate a set of genes and regulators related with tumor development. Six genes (\u003cem\u003eDVL3,\u003c/em\u003e PPP\u003cem\u003e2R2D, THSD4, CREB1, SYT7, \u003c/em\u003eand\u003cem\u003e PRKACA\u003c/em\u003e) were detected to be over-expressed in BC cells. Fig.6 shows the relationship between \u003cem\u003emiR-1275 \u003c/em\u003eand these target genes. \u003cem\u003emiR-1275\u003c/em\u003e in breast cancer promotes cancer cell proliferation, cell differentiation, tumor growth, invasion and migration and also inhibits apoptosis through several gene targets. \u003cem\u003ePPP2R2D \u003c/em\u003eacts as a tumor suppressor in signaling pathway in BC and is negatively regulated by \u003cem\u003emiR-1275\u003c/em\u003e. The overexpression of which decreases \u003cem\u003eAKT\u003c/em\u003e and \u003cem\u003eRACK1\u003c/em\u003e abilities. Then these regulators decrease cell survival and migration. \u003cem\u003eDVL3\u003c/em\u003e is implicated in the breast cancer pathways and negatively controlled by \u003cem\u003emiR-1275\u003c/em\u003e. The up-regulation of this gene increases the cancer cell proliferation, migration and invasion\u003cem\u003e.\u003c/em\u003e The cancer cell proliferation ability is increased when \u003cem\u003emiR-1275\u003c/em\u003e becomes overexpressed. another target gene. \u003cem\u003eCREB1\u003c/em\u003e and \u003cem\u003ePRKACA\u003c/em\u003e show also negative correlation with \u003cem\u003emiR-1275\u003c/em\u003e level\u003cem\u003e.\u003c/em\u003e Whereas \u003cem\u003eCREB1\u003c/em\u003e was found to reduce apoptosis process and increase cell proliferation in breast cancer, \u003cem\u003ePRKACA\u003c/em\u003e plays a key role in tumorigenesis and development of BC. However, the function of \u003cem\u003eTHSD4\u003c/em\u003e and \u003cem\u003eSYT7\u003c/em\u003e, currently unidentified, may boost tumor growth in breast cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Comparison of marked down-expressed miRNAs in BC with NAT\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003emiRNA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cstrong\u003emiRBase accession \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e\u003cstrong\u003eLocation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e\u003cstrong\u003eLog2FC\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e. value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e\u003cstrong\u003eFDR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0031892\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e20q13.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-2.6291\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.3109\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-100-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004512\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e11q24.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-1.2349\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.2824\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-10b-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0000254\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e2q31.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-1.2852\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.261\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-125a-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0000443\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e19q13.41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-1.4153\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.213\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-125b-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0000423\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e11q24.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-2.0041\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.083\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-1275\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0005929\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e6p21.31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-1.4150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.6 14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-130a-3p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004593\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e11q12.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-2.0168\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.104\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-133a-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0026478\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e18q11.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-2.3885\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.220\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-143-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004599\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e5q32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-1.2823\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.308\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-204-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0000265\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e9q21.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-4.0627\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.086\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-21-3p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004494\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e17q23.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.0365\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.995\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-214-3p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0000271\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e1q24.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.8746\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.440\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-25-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004498\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e7q22.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-1.2630\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.793\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-30a-3p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0000088\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e6q13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.2358\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.838\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-30b-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eMIMAT0000420\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e8q24.22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-1.3254\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.245\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-30d-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0000245\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e8q24.22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.9730\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.389\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-374b-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004955\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003eXq13.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.2420\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.8435\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-410-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0026558\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e14q32.31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.6780\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.989\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-423-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004748\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e17q11.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.5727\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.614\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-451a\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0001631\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e17q11.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.41900\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.711\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-455-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0003150\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e9q32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-2.5081\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.305\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-505-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004776\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003eXq27.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-1.5956\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.611\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-532-3p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004780\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003eXp11.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.7496\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.612\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-624-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0003293\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e14q12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.0931\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-664a-3p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0005949\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e1q41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.9249\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.539\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-664b-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0022271\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003eXq28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.5081\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.899\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-92a-1-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004507\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e13q31.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.2085\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.972\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-934\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0004977\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003eXq26.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-3.0931\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.417\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"146\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHsa-miR-99b-5p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003e\u003cem\u003eMIMAT0000689\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e19q13.41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e-0.1667\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.883\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Brief information on target predicted databases was shown to find putative targets possessing binding sequence to \u003cem\u003emiR-1275\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\" width=\"147\"\u003e\n\u003cp\u003e\u003cstrong\u003eTarget predicted \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esites\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"120\"\u003e\n\u003cp\u003e\u003cstrong\u003eSpecies\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"147\"\u003e\n\u003cp\u003e\u003cstrong\u003eTool properties \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"307\"\u003e\n\u003cp\u003e\u003cstrong\u003eWebsite\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWebsites\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003emiRTarBase\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eHuman, Mouse, Rat\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eConservation, seed location\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"http://mirtarbase.mbc.nctu.edu.tw/php/index.php\"\u003ehttp://mirtarbase.mbc.nctu.edu.tw/php/index.php\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003eTarget scan \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eHuman, Mouse, Fly, Fish, and Worm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eConservation, seed location\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"http://www.targetscan.org/\"\u003ehttp://www.targetscan.org/\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003eTargetMiner\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eHuman, Mouse, Rat, Fly\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eConservation, seed location\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"https://www.isical.ac.in/~bioinfo_miu/TargetMiner.html\"\u003ehttps://www.isical.ac.in/~bioinfo_miu/TargetMiner.html\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003eMirTar2\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eHuman, Mouse, rat, Dog and Chicken\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eConservation, seed location\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"http://www.mirdb.org/\"\u003ehttp://www.mirdb.org/\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003eDIANA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eAny\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eConservation, seed match, and free energy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"http://www.microrna.gr/microT-CDS\"\u003ehttp://www.microrna.gr/microT-CDS\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003emiRWalk\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eHuman, Mouse, and Rat\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eConservation, seed match and free energy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"http://mirwalk.uni-hd.de/\"\u003ehttp://mirwalk.uni-hd.de/\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003emiRmap\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eHuman, Chimpanzee, Mouse, Rat, Cow,Chicken, Zebrafish, and Opossum\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eConservation, seed match, and free energy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"https://mirmap.ezlab.org/\"\u003ehttps://mirmap.ezlab.org/\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003eRNA22\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eHuman, Fruit Fly, Mouse, and Worm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eSeed match and free energy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"https://cm.jefferson.edu/rna22/\"\u003ehttps://cm.jefferson.edu/rna22/\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003ePicTar - Tools4miRs\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eHuman, Mouse, Rat, Fly\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eConservation, seed location\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"https://tools4mirs.org/software/target_prediction/pictar/\"\u003ehttps://tools4mirs.org/software/target_prediction/pictar/\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"108\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emirPath\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"15\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eHuman, Mouse, D. melanogaster, C. elegans, R. norvegicus, D. rerio and G. gallus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eConservation, seed match and free energy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"http://snf-515788.vm.okeanos.grnet.gr/index.php?r=mirpath/geneList\"\u003ehttp://snf-515788.vm.okeanos.grnet.gr/index.php?r=mirpath/geneList\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"108\"\u003e\n\u003cp\u003e\u003cstrong\u003eMicrorna. org\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"15\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eHuman, mouse, Fruit Fly, and rat\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eConservation, seed match, free energy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"341\"\u003e\n\u003cp\u003e\u003ca href=\"http://www.microrna.org/\"\u003ehttp://www.microrna.org/\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. candidate target genes possessing binding sequence to \u003cem\u003emiR-1275\u003c/em\u003e was shown.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003eTarget gene\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"174\"\u003e\n\u003cp\u003e\u003cstrong\u003eEnsemble ID\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"168\"\u003e\n\u003cp\u003e\u003cstrong\u003ePosition on Chromosome\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003e\u003cstrong\u003eNo. of sites predicted the gene as \u003cem\u003emiR-1275\u003c/em\u003e target\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDVL3\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"174\"\u003e\n\u003cp\u003eENST00000313143.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"168\"\u003e\n\u003cp\u003e3q27.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePPP2R2D\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"174\"\u003e\n\u003cp\u003eENST00000422256.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"168\"\u003e\n\u003cp\u003e10q26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTHSD4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"174\"\u003e\n\u003cp\u003eENST00000355327.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"168\"\u003e\n\u003cp\u003e15q23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCREB1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"174\"\u003e\n\u003cp\u003eENST00000432329.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"168\"\u003e\n\u003cp\u003e2q34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSYT7\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"174\"\u003e\n\u003cp\u003eENST00000263846.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"168\"\u003e\n\u003cp\u003e11q12.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePRKACA\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"174\"\u003e\n\u003cp\u003eENST00000308677.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"168\"\u003e\n\u003cp\u003e17q24.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5. Experimentally validated target genes of \u003cem\u003emiR-1275\u003c/em\u003e in BC\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u003cstrong\u003eTargets\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003e\u003cstrong\u003eGen ID\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e.value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e\u003cstrong\u003eFDR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e\u003cstrong\u003eLog2fc\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDVL3\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003e\u003ca href=\"http://www.ensembl.org/id/ENSG00000161202\"\u003eENSG00000161202\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003eDishevelled segment polarity protein 3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e6.98E-04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e3.48E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.58975\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePPP2R2D\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003eENSG00000175470\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003eProtein phosphatase 2, regulatory subunit B, delta\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e1.53E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e6.95E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.616981\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTHSD4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003eENSG00000187720\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003eThrombospondin type 1 domain containing 4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e2.28E-12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e3.63E-11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.938081\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCREB1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003e\u003ca href=\"http://www.ensembl.org/id/ENSG00000118260\"\u003eENSG00000118260\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003ecAMP responsive element binding protein 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e3.12E-01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e5.13E-01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.269971\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSYT7\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003e\u003ca href=\"http://www.ensembl.org/id/ENSG00000011347\"\u003eENSG00000011347\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003eSynaptotagmin 7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e1.28E-48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e8.14E-47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e1.84905\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePRKACA\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"137\"\u003e\n\u003cp\u003eENSG00000072062\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"198\"\u003e\n\u003cp\u003eProtein kinase cAMP-activated catalytic subunit alpha\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e1.77E-02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e5.68E-02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"69\"\u003e\n\u003cp\u003e0.296759\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThere is evidence that a single microRNA modulates multiple protein-coding and non-coding genes in different ordinary cells and cancerous cells. In human cancer cells, new RNA groups can be detected using the specific nature of microRNA from relevant microRNA analysis. Multiple high-throughput approaches, such as DNA microarrays, MACE-sequencing, PCR-based arrays, and RNA-sequencing, are now available and have made microRNA expression profiles of BC, showing the irregular expression of numerous miRNAs (26-29). One approach to detect the most essential miRNA from numerous miRNAs is to detect differential expression of miRNAs which have been shown in numerous experiments. Several researches have reported that multiple miRNAs, such as \u003cem\u003emiR-100, miR-107, miR-205-3p, miR-122\u003c/em\u003e and \u003cem\u003emiR-99a-5p\u003c/em\u003e, are continuously down-expressed and act as tumor-suppressive miRNA in BC cells (26, 28-33). In this study, these miRNAs were found to be downregulated in tumor cells but only \u003cem\u003emiR-1275 \u003c/em\u003ewas focused and their putative target genes were newly explored in paraffin embedded BC tissues. Several researches have reported that this miRNA is down-expressed in gastric and nasopharyngeal carcinoma and function as a tumor suppressor (34-36); whereas, this miRNA is overexpressed in non-small lung cell cancer, squamous carcinoma and chronic myelogenous leukemia (37-39). Some recent studies revealed that this downregulated miRNA was detected to have an essential effect on cancer cell proliferation, migration, invasion, metastasis, and angiogenesis through targeting multiple oncogenic genes \u003cem\u003eHOXB5\u003c/em\u003e, \u003cem\u003eWNT7B\u003c/em\u003e and \u003cem\u003eLncRNA\u003c/em\u003e HAND2-AS1(34, 36, 39). One previous study showed that \u003cem\u003emiR-1275 \u003c/em\u003eregulates \u003cem\u003eIGF1, NFIX\u003c/em\u003e, \u003cem\u003eClaudin11\u003c/em\u003e in very young women with BC (23). Whereas, down-expression of\u003cem\u003e miR-1275 \u003c/em\u003ein all subtypes of paraffin BC tissues was not fully investigated. In this study, down-expression of miRNA was observed in all subtypes of paraffin embedded BC tissues of 21 cases with different ages.\u003c/p\u003e\n\u003cp\u003eAfter that, the \u003cem\u003emiR-1275\u003c/em\u003e-modulated putative targets and their pathways were aimed to explain in the cells of BC. Six genes (\u003cem\u003eDVL3\u003c/em\u003e, \u003cem\u003ePPP2R2D\u003c/em\u003e, \u003cem\u003eTSHD4\u003c/em\u003e, \u003cem\u003eCREB1\u003c/em\u003e, \u003cem\u003eSYT7\u003c/em\u003e, and \u003cem\u003ePRKACA\u003c/em\u003e) were experimentally observed to be overexpressed in the cells of BC. Based on the databases of miRNA target prediction, they were selected and closely correlated with poor prognosis. Among these candidate genes, four genes (\u003cem\u003ePPP2R2D\u003c/em\u003e, \u003cem\u003eDVL3\u003c/em\u003e, and \u003cem\u003eCREB1\u003c/em\u003e) were shown to be strongly targeted by the \u003cem\u003emiR-1275\u003c/em\u003e in the BC cells. Studies showed that these regulators were found to reduce cell survival and migration in cancer cells (40, 41). \u003cem\u003eDVL3\u003c/em\u003e is observed to be implicated in the breast cancer pathways (34) and negatively regulated by \u003cem\u003emiR-1275\u003c/em\u003e. The up-regulation of this gene can increase the cancer cell proliferation, migration and invasion in BC cells\u003cem\u003e.\u003c/em\u003e The overexpression of which increase cancer cell proliferation ability (42). Another target gene, \u003cem\u003eCREB1\u003c/em\u003e and \u003cem\u003ePRKACA\u003c/em\u003e show also negative correlation with \u003cem\u003emiR-1275\u003c/em\u003e level\u003cem\u003e.\u003c/em\u003e Whereas \u003cem\u003eCREB1\u003c/em\u003e was found to reduce apoptosis process and increase cell proliferation in breast cancer (43), \u003cem\u003ePRKACA\u003c/em\u003e plays a key role in tumorigenesis and development of BC (44). However, the role of \u003cem\u003eTHSD4\u003c/em\u003e and \u003cem\u003eSYT7\u003c/em\u003e, currently unidentified, may enhance tumor growth in a variety of cancers, especially breast cancer (45, 46).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn the present study, differential expression profiles of total mRNA transcripts and sRNAs were identified in BC paraffin tissue (NAT and tumoral tissue) by MACE- sequencing. Decreased \u003cem\u003emiRNA-1275\u003c/em\u003e expression develops breast cancer by increasing the activity of biological processes; such as growth, migration, invasion and metastasis. Upregulated \u003cem\u003emiRNA-1275\u003c/em\u003e prevented BC development by modulating direct expression of \u003cem\u003eDVL3\u003c/em\u003e, \u003cem\u003ePPP2R2D\u003c/em\u003e, \u003cem\u003eTSHD4\u003c/em\u003e, \u003cem\u003eCREB1\u003c/em\u003e, \u003cem\u003eSYT7\u003c/em\u003e, and \u003cem\u003ePRKACA\u003c/em\u003e. This is the first study revealing that \u003cem\u003emiR-1275\u003c/em\u003e function as a tumor-suppressive miRNA in BC cells, regulating numerous targets which were closely related with BC pathogenesis and oncogenesis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFFPE: Formalin Fixed Paraffin Embedded; BC: Breast Cancer; NAT: Normal adjacent tissue; sRNA: small RNA; MACE: Massive Analysis of cDNA Ends; E.R.: Estrogen Receptor; Pg. R.: Progesterone Receptor; HER2: Human epidermal growth factor receptor 2; TCGA: The Cancer Genome Atlas; FDR: False Discovering Rate; FC: Fold Change.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI wish to thank the patients and their families. I would like to express my specially thanks of gratitude to my supervisor for her frequent support and encouragement. I also offer my sincere appreciation for Al mufti and Luay laboratories which helped and supported me through collecting FFPE blocks of BC and clinical data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuhad and Sevan were responsible for the experimental design. They led to the execution of the experiments. Although analyses of MACE-sequencing and sRNA sequencing were done in Genxpro company, in Germany. Data analysis and bioinformatic tasks were done by Sevan. Sevan also discussed and interpreted the data. He also did the manuscript mapping and submission, but Suhad supervised the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Sevan Majed who is a Ph.D. student.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough row data of MACE-sequencing and sRNA may be available in the database of GenXpro, at \u003ca href=\"https://genxpro.net/\"\u003ehttps://genxpro.net/\u003c/a\u003e, These data will be further studied for another research in the future. The findings described in this manuscript were provided by the Co-author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was followed and approved by Human Research Ethics Committee at Science College in Salahuddin University-Erbil (Approval no.4c/132). Informed consent was received from all patients in accordance with the requirements of the Human Research Ethics Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author announces no conflict of interest. Mr. Sevan is a teacher of Salahaddin University-Erbil, a subsidiary of ministry of higher education in Kurdistan region government (KRG).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eBiology Department, College of Education, Salahaddin University-Erbil. \u003csup\u003e2\u003c/sup\u003eResearch Center, Molecular genetics lab, Salahaddin University-Erbil.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eControl CfD, Prevention. Cancer survivors--United States, 2007. MMWR Morbidity and mortality weekly report. 2011;60(9):269.\u003c/li\u003e\n\u003cli\u003eJiang X, Tang H, Chen T. Epidemiology of gynecologic cancers in China. J Gynecol Oncol. 2018;29(1).\u003c/li\u003e\n\u003cli\u003eAdhami M, Haghdoost AA, Sadeghi B, Malekpour Afshar R. Candidate miRNAs in human breast cancer biomarkers: a systematic review. 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Breast cancer research : BCR. 2014;16(6):464-.\u003c/li\u003e\n\u003cli\u003eLiu X, Li C, Yang Y, Liu X, Li R, Zhang M, et al. Synaptotagmin 7 in twist-related protein 1-mediated epithelial \u0026ndash; Mesenchymal transition of non-small cell lung cancer. EBioMedicine. 2019;46:42-53.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, miRNA, miR-1275 and its target genes, differential expression, pathogenesis, tumor suppressor","lastPublishedDoi":"10.21203/rs.3.rs-101189/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-101189/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntroduction\u003c/p\u003e\u003cp\u003eDisruption of cellular processes in the breast by abnormally expressed miRNA is characterized to develop cancer. We aimed to determine the differential expression of coding and non-coding RNAs in formalin fixed paraffin embedded (FFPE) blocks of breast cancer (BC) tissue and normal adjacent tissue (NAT). Another aim is to determine differential expression of\u0026nbsp;\u0026nbsp;\u003cem\u003ehas-miR-1275\u0026nbsp;\u003c/em\u003e\u0026nbsp;as novel biomarker for BC and\u0026nbsp;identify its target genes using prediction sites and experimentally expression level of them via the MACE-sequencing technique.\u0026nbsp;\u003c/p\u003e\u003cp\u003eMethods\u003c/p\u003e\u003cp\u003eMACE-sequencing technique was utilized to analyze differential expression of coding RNAs and\u0026nbsp;\u0026nbsp;small RNAs (sRNAs). Among small RNAs,\u0026nbsp;\u0026nbsp;\u003cem\u003emiRNA-1275\u0026nbsp;\u003c/em\u003e\u0026nbsp;expression was focused and confirmed using RT-qPCR technique in 20 Kurdish cases with BC\u0026nbsp;\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u0026nbsp;Moreover, clinical significance of\u0026nbsp;\u0026nbsp;\u003cem\u003emiR-\u0026nbsp;\u003c/em\u003e1275 and its target genes was studied in a large number of patients with BC using the data obtained from The Cancer Genome Atlas database.\u003c/p\u003e\u003cp\u003eResults\u003c/p\u003e\u003cp\u003eThe MACE-seq findings showed that 1400 sRNAs and 26843 coding\u0026nbsp;RNAs were differentially expressed in FFPE of BC tissue\u0026nbsp;compared to NAT. Among these sRNAs,\u0026nbsp;\u0026nbsp;\u003cem\u003emiRNA-1275\u0026nbsp;\u003c/em\u003e\u0026nbsp;expression was found to be decreased in BC tissue compared to NAT. The decreased expression level of which was then confirmed via RT-qPCR technique to farther prove in 20 Kurdish cases with BC\u0026nbsp;\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u0026nbsp;Furthermore, the correlation between the expression level of\u0026nbsp;\u0026nbsp;\u003cem\u003emiRNA-1275\u0026nbsp;\u003c/em\u003e\u0026nbsp;\u0026nbsp;and clinical data were evaluated to be highly corrected in cases with BC (overall survival rate:\u0026nbsp;\u0026nbsp;\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026nbsp;= 0.0401). However, putative target genes (\u0026nbsp;\u003cem\u003eDVL3,\u0026nbsp;\u003c/em\u003e\u0026nbsp;PPP\u0026nbsp;\u003cem\u003e2R2D,\u0026nbsp;THSD4, CREB1, SYT7,\u0026nbsp;\u0026nbsp;\u003c/em\u003eand\u0026nbsp;\u003cem\u003e\u0026nbsp;PRKACA)\u0026nbsp;\u003c/em\u003e\u0026nbsp;were computationally identified as direct targets of\u0026nbsp;\u0026nbsp;\u003cem\u003emiRNA-1275\u0026nbsp;\u003c/em\u003e\u0026nbsp;in several target predicted sites. Among coding\u0026nbsp;RNAs, the expression level of these targets was\u0026nbsp;increased in BC tissue\u0026nbsp;compared to NAT. The levels of these targets were negatively associated with\u0026nbsp;\u003cem\u003emiRNA-1275\u0026nbsp;\u003c/em\u003e\u0026nbsp;expression. Finally, the role of down-expressed\u0026nbsp;\u0026nbsp;\u003cem\u003emiRNA-1275\u0026nbsp;\u003c/em\u003e\u0026nbsp;and its targets in BC cells were identified to attenuated biological mechanisms; including cell growth, proliferation, movement, invasion, metastasis, and apoptosis.\u003c/p\u003e\u003cp\u003eConclusion\u003c/p\u003e\u003cp\u003edown-expressed\u0026nbsp;\u0026nbsp;\u003cem\u003emiR-1275\u0026nbsp;\u003c/em\u003e, a tumor suppressor, is as a novel biomarker for early detection of breast cancer.\u0026nbsp;\u0026nbsp;\u003cem\u003e\u0026nbsp;DVL3,\u0026nbsp;\u003c/em\u003e\u0026nbsp;PPP\u0026nbsp;\u003cem\u003e2R2D,\u0026nbsp;THSD4, CREB1, SYT7,\u0026nbsp;\u0026nbsp;\u003c/em\u003eand\u0026nbsp;\u003cem\u003e\u0026nbsp;PRKACA\u0026nbsp;\u003c/em\u003e\u0026nbsp;are novely identified to\u0026nbsp;be\u0026nbsp;targeted by\u0026nbsp;\u0026nbsp;\u003cem\u003emiR-1275\u0026nbsp;\u003c/em\u003e\u0026nbsp;\u0026nbsp;in BC cells.\u003c/p\u003e","manuscriptTitle":"Identification of Tumor-Suppressive miRNA-1275 as a Novel Marker for Breast Cancer (BC) by MACE-Sequencing and RT-qPCR Techniques","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-05 19:57:42","doi":"10.21203/rs.3.rs-101189/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c3f34b44-9402-46c1-bfe6-34e6d548a92e","owner":[],"postedDate":"November 5th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":983514,"name":"Cancer Biology"},{"id":983515,"name":"Oncology"}],"tags":[],"updatedAt":"2020-11-05T19:57:44+00:00","versionOfRecord":[],"versionCreatedAt":"2020-11-05 19:57:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-101189","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-101189","identity":"rs-101189","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}