Evaluation of Epigenetic Silencing of the miR-139-5p gene in the pathogenesis of colorectal cancer and its diagnostic biomarker capability in plasma samples

Evaluation of Epigenetic Silencing of the miR-139-5p gene in the pathogenesis of colorectal cancer and its diagnostic biomarker capability in plasma samples | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of Epigenetic Silencing of the miR-139-5p gene in the pathogenesis of colorectal cancer and its diagnostic biomarker capability in plasma samples Masoud Asefi, Nayebali Rezvani, Massoud Saidijam, Ali Reza Soltanian, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5527622/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 May, 2025 Read the published version in BMC Cancer → Version 1 posted 10 You are reading this latest preprint version Abstract Background : The pathogenesis of CRC requires primary genetic and epigenetic mechanisms including, methylation of CpG islands of the genes. In the current study, micro RNA-139-5p ( miR -139-5p) promoter methylated DNA was evaluated in tumor tissue and plasma samples from CRC affected patients. Methods: MiR -139-5p promoter methylation was investigated in 80 samples of tumoral tissue and healthy marginal tissue and the same number of plasma samples, using the MethyLight method. The miR-139-5p expression was assessed using the qPCR method. BT (Bioassay Technology) Elisa kit was applied to measure RAP-1b as a target gene of miR-139-5p. Results: Median PMR values of 12.4 (95% CI, 3.23-32.25) and 0.66 (95%CI, 0.51-1.0) were obtained from plasma samples of CRC patients and controls, sequentially. In plasma samples, the sensitivity and specificity of miR -139-5p promoter methylated marker were 75% and 92.5%, in the same order (AUC =0.958). Lower expression of miR-139-5p in plasma and tumor tissue of patients (P< 0.001) was shown. Also, a significant rise of RAP-1b protein concentration was observed in both mentioned specimens. Conclusion: Hyper-methylation of miR -139-5p could be suggested as high accuracy diagnostic biomarker for the detection of CRC in plasma samples. Biomarkers Colorectal Neoplasms DNA Methylation and human MIRN139 Figures Figure 1 Figure 2 Figure 3 1. Background The second and third most commonly diagnosed malignancy is attributed to colorectal cancer (CRC) in women and men, respectively, and the second leading cause of cancer death globally ( 1 , 2 ). There is a noticeable difference in CRC incidence and mortality ( 3 , 4 ). It is frequently diagnosed at advanced phases due to the limitations of current clinical screening methods ( 5 ). Relative CRC risk is specified by genetic potency and environmental influences, with age being the most imperative risk factor for sporadic CRC. Transformation of normal colonic mucosa into invasive cancer occurs through the accumulation of genetic and epigenetic changes ( 6 ). Despite the identifying of several molecular pathways intervened in the development of CRC, the detailed molecular pathogenesis of this cancer has not yet been clarified ( 7 , 8 ). MicroRNAs (miRNAs) with their target genes organize a very complex network in cells, and it is expected that abnormal expression of miRNAs interrupts this RNA system. Many surveys have revealed that deregulation of miRNAs act as tumor suppressors or oncogenes in CRC cells by targeting neoplasm-related genes ( 8 ). New advances in molecular biology have demonstrated the role of miRNAs in various biological and pathological functions including, down-regulation of tumor suppressor miRNAs in many cancers, particularly CRC ( 9 ). Epigenetic variations, in particular DNA methylation and histone modifications, perform major pathophysiological role in the beginning and progression of CRC ( 10 ). Covalent bonding of a methyl group to the 5-carbon status of the cytosine is widespread throughout the genome; however, when added to CpG in the promoter region, it generally results in lower expression of methylated gene. The mechanism of gene silencing by DNA methylation may be related to a barrier of the transcriptional machinery or modification in chromatin configuration or recruitment of repressors ( 11 ). Owing to the truth that methylation in a specific region of the genome occurs in the primary steps of tumorigenesis ( 12 ), and significantly, most cancer deaths are due to metastatic spread of tumor cells not due to the primary tumor ( 13 ), early diagnosis increases the chance of recovery and prevention of subsequent complications. MiR -139, located on 11q13.4 and is down-regulated in several cancers, exposes antioncogenic and anti-metastatic activity in human tumors( 14 , 15 ). MiR-139-5p, encoded within the second intron of the phosphodiesterase 2A gene (16), was recognized as, a tumor-suppressor miRNA, and is down regulated in a diversity of cancers, such as, gastric cancer and CRC( 17 , 18 ). The Ras family of low molecular weight GTP-binding proteins have been occupied in a numerous cellular processes, comprising differentiation and cell growth, intracellular vesicular transferring, nucleocytoplasmic transport, and cytoskeletal restructuring ( 19 ). Ras-Related Protein Rap-1b ( RAP- 1b ) is a low molecular mass GTP-binding protein (22-kDa) which is both a substrate for cAMP-dependent protein kinase and a member of the Ras superfamily ( 20 ). In previous studies, RAP-1b has been identified as one of the straight target of miR-139-5p ( 17 , 21 ). We aim to determine and evaluate the expression of miR-139-5p, RAP-1b protein, and the methylation condition of the miR -139-5p promoter region in plasma samples of CRC affected individuals, healthy controls and in tumor tissue and healthy near specimens. Furthermore, we examined the diagnostic value of these biomarkers and their relationship with clinical and pathological features. 2. Methods 2.1. Tissue and plasma samples A total of 80 plasma samples from CRC patients and healthy matched individuals, as well as 40 paired tumor and adjacent healthy tissues were provided in the present survey. The inclusion and exclusion criteria of clinical samples have been reported in our previous study ( 22 ). Written informed was signed by all participants. This survey authorized by the Ethics Committee of Hamadan University of medical sciences (IR.UMSHA.REC.1396.533). 2.2. Methylation Analysis using MethyLight method This section of the experiments has explained in detail in the previous article ( 22 ) and it is briefly as follows: The promoter area of the miR -139-5p gene was identified using the miR Start and Ensemble Databases. After that, the most specific primers and probes for fully methylated bisulfiteconverted DNA (one set for miR -139-5p gene and one for ALU-C4)were designed for a GC-rich area inside the promoter region using Beacon designer software (version 8.13) (Table 1 ). The probes and the primers were synthesized by the German company Metabion. Table 1 The primers and probes specifications. Primers and probes Sequence Tm o C Length bp ALU-C4 Forward primer 5'GGTTAGGTATAGTGGTTTATATTTGTAATTTTAGTA-3 66 36 ALU-C4 Reverse primer 5'ATTAACTAAACTAATCTTAAACTCCTAACCTCA-3' 65 33 ALU-C4 Probe 5'-CGCTAACCGAACGCAACGAC-3' 51 16 miR-139 Reverse Primer 5'-TTTCGTTTGAGGAATTGGATAATAGCG-3' 61.2 30 miR-139 Forward Primer 5'-6-FAM-ACCGACGAACGAACGAACGACTACGAAC-BHQ-1-3' 60 29 miR-139 Probe 5'-6-FAM-CCTAAACCACAAACAACCACGAACCTCGCC-BHQ-1-3' 68.9 30 Next, DNA of all specimens extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Germany, Cat.No:51104) ( 23 ) followed by bisulfite modification using an EpiTect Bisulfite kit (Qiagen.Cat.No.59104) in duplicate. Sodium bisulfite has no impression on methylated cytosine, but it causes deamination of non-methylated cytosine and modifies it to uracil. This difference is the basis for distinguishing methylated from non-methylated DNA ( 24 ). Finally, the qPCR reactions were done in 96well plates on an ABI 7500 RealTime PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in duplicate, using the EpiTect® MethyLight PCR + ROX Vial kit (Qiagen GmbH). ALU-C4, a consensus DNA sequence, was applied as the housekeeping gene for normalization. The percentage of methylated reference (PMR), the degree of methylation of every sample relatively to the entirely methylated control, of the samples, calculated using saleable methylated human bisulfite converted DNA (EpiTect PCR Control DNA; Qiagen GmbH), as an entirely methylated control. PMR, at a defined locus, was assessed using the subsequent equation : [(miR/ALU-C4) sample / (miR/ALU-C4) positive control ] x 100 ( 25 ). 2.3. MiR-139-5p expression and measurement of RAP-1b Protein in Clinical Samples A favorgen miRNA (Favorgen, Taiwan, Cat.No:FAMIK002) and NucleoSpin® miRNA Plasma (Macherey-Nagel, Germany) extraction kits were applied to extract miRNAs from tissue and plasma samples, respectively. The extraction of miRNA from approximately 10 mg of tissue sample consists of four following steps: 1- Cell lysis 2- Binding of DNA to the silica column membrane, 3- Column washing, and 4- Dilution of DNA in buffer which in the first step ,tissue sample was ground into smaller pieces using a scalpel blade. Then the sample was transferred to a 1.5 ml microtube and the next steps were performed according to the kit instructions. The quality and quantity of RNA was evaluated using the nanodrop (ND-1000). BON miR Kit (Bon yakhteh, Inc) was applied to synthesis cDNA. Finally, qPCR was carried out to assess the expression of miR-139-5p supported by the BON miR SYBR®green PCR Kit (Bon yakhteh, Inc) in accordance with the company’s instructions. U6 as a housekeeping gene used to normalize data and the expression level was computed by the 2 (−ΔΔCT ) manner ( 26 ). ELISA kit (Cat.no:E2153Hu) was applied to measure RAP-1b Protein in an ELISA reader (Rayto 2100C). 2.4. Statistical analysis The Chi-square test was used to analyze descriptive data. The normality of data distribution evaluated by Kolmogorov-Smirnov test. Therefore, non-parametric Mann-Whitney, Kruskal-Wallis tests, Anova, and independent t-tests were used depending on the results of normality test. Using SPSS 16 software, P value < 0.05 was considered as the level of statistical meaningful. The specificity and sensitivity of studied biomarkers evaluated by Receiver Operating Characteristics (ROC) analysis using PMR, ΔCT, and RAP-1b quantities. In general, the area under the ROC curve (AUC) ≥ 0.8 is assumed acceptable and AUC ≥ 0.9 is interpreted excellent ( 27 ). 3. Results Forty tissue samples from CRC affected patients and 40 from marginal normal tissue (22 females and 18 males) with a mean age of (57.7 ± 17.2) and the same number of plasma samples from case group and healthy individuals ( 22 females and 18 males) with a matched ages as a control participants were included in the study. 3.1. Hyper methylation of miR -139-5p in CRC without any clinical and pathological association was observed Median PMR values of 0.78 (95% CI, 0.12–6.58) and 0.1 (95% CI, 0.01–0.29) were obtained in tumor and adjacent normal tissues, at the same order. The uppermost PMR value got from marginal normal tissues was assumed as a threshold level; specimen with a PMR > 5.1 were translated positively for methylation status (36/40, (90%)). As demonstrated in (Fig. 1 ), the PMR level was remarkably larger in the cancer tissues compared with that of the normal tissues (p < 0.0001). The median PMR levels of 12.4 (95% CI, 3.23–32.25) and 0.66 (95%CI, 0.51-1.0) in plasma samples from the CRC and the normal groups, were obtained, sequentially. Also, the maximum PMR value from the healthy plasma specimens was assumed as the threshold of methylation condition; thus, all samples with a PMR > 4.8 were supposed positively methylated (29/40, (72.5%)). The PMR values for CRC patients were noticeably greater in plasma samples in comparison with those of control subjects (p < 0.0001) (Fig. 1 ). Results from PMR values showed no statistically meaningful association between the tissue and plasma specimens and clinicopathological characteristics of CRC patients (Table 2 ). Figure 1 : Box plot of PMR level of the miR -139-5p gene in clinical samples. The median PMR values for CRC patients were higher in plasma samples in comparison with those of control persons (12.4 vs. 0.66, P < 0.0001) as well as in tissue specimens (0.78 vs. 0.1, p (< 0.0001). Table 2 Relationship between miR -139-5p methylation and clinicopathological specifications in clinical samples of case group. Tissue PMR Plasma PMR P-value Clinicopathological characteristic No. of patients Mean ± SD * P-value Mean ± SD Tumor type Adenocarcinoma 29 4.19 ± 7.98 0.353 a 17.22 ± 17.13 0.338 a Mucinous 11 12.89 ± 19.41 21.14 ± 18.22 Histological grading I 15 5.96 ± 10.07 0.490 c 21.74 ± 19.45 0.583 c II 21 7.98 ± 15.12 15.15 ± 13.99 III 4 1.61 ± 2.19 21.91 ± 25.87 Tumor location Distal 13 8.41 ± 17.99 0.732 a 13.03 ± 14.55 0.168 a Proximal 27 5.71 ± 9.21 20.83 ± 18.17 Tumor size <50 mm 21 7.89 ± 14.85 0.390 a 19.70 ± 19.33 0.452 a \(\:\ge\:50\:\varvec{m}\varvec{m}\) 19 5.14 ± 9.62 16.75 ± 15.09 TNM Stage I 13 4.83 ± 3.69 0.422 c 22.55 ± 16.46 0.348 c II 19 5.13 ± 9.01 14.47 ± 16.50 III 8 12.91 ± 21.99 20.49 ± 20.52 Lymph node involvement Yes 8 12.91 ± 21.99 0.209 a 20.49 ± 20.52 0.209 a No 32 5.01 ± 8.74 17.75 ± 16.71 Standard deviation, a: Mann-Whitney U two-tailed tests; b: ANOVA; c: Kruskal-Wallis * 3.2. Down-regulation of miR-139-5p and greater quantity of RAP-1b protein in both plasma and tissue samples without any association with clinicopathological parameters The expression of miR-139-5p in tumor tissue (p < 0.0001) and plasma of patients (p < 0.001) had a significant decrease, in comparison with the healthy participants (Fig. 2 ). There was no remarkable association between down regulation of miR-139-5p and any clinical and pathological characteristics (supplementary Table 1). Figure 2 : Overall 5.7 and 4.69 fold (2 −ΔΔCT ) lower expression of miR-139-5p in tissue and plasma specimens of CRC affected patients, respectively (Mean ± SEM, n = 40, p < 0.0001 and p < 0.001, respectively.) The concentration of RAP-1b, a valid target of miR-139-5p, was higher in CRC patients than in control groups. (Table 3 and supplementary Fig. 1). Also, the results revealed that there is no statistical meaningful association amongst RAP-1b concentration and clinicopathological characteristics (supplementary Table 2). Table 3 The greater quantity of RAP-1b protein in clinical samples of CRC patients Percentiles P-value Parameter No. of patients Minimum Maximum 25th 50th 75th Tissue RAP-1b(ng/l) Patient 15 29.00 85.00 41.00 51.00 64.30 < 0.0001 Control 15 9.50 36.80 11.80 21.00 29.00 Plasma RAP-1b(ng/l) Patient 30 3.10 93.00 14.53 32.67 40.25 0.004 Control 29 0.10 36.3 9.82 15.70 28.60 3.3. The results of diagnostic value of studied biomarkers using ROC curve analysis A high level of carcinoembryonic antigen (CEA) in plasma specimens of CRC subjects was observed in our previous work ( 22 ). As observed in Fig. 3 , the specificity and sensitivity of the miR -139-5p methylated biomarker in plasma is higher than CEA and FOBT tests. 4. Discussion MiRNAs have essential effects on gene expression and signaling pathways so that deregulated miRNAs could be related to different diseases, including CRC ( 28 ). In previous studies, miR-139-5p, as a tumor suppressor, has been identified as an influential factor in cancer diagnosis, prognosis, and treatment in various types of human cancers ( 29 , 30 ). Many molecular mechanisms lead to miRNA deregulation ( 31 ), including transcriptional tumor suppressor miRNAs silencing by special DNA methylation in cancer cells ( 32 ). Haiyan Guo et al. demonstrated that miR-139 decreases proliferation in CRC through targeting RAP-1b directly ( 33 ). The current research examined methylation pattern of miR -139-5p, its expression and target (RAP-1b) in both cancer and healthy groups to acquire more vision about the miR-139-5p function in CRC disease. This study showed that the methylation level of the promoter area of the miR -139-5p gene was significantly higher in the plasma and tissue specimens of CRC patients. On the other hand, the level of miR-139-5p significantly decreased in the tumoral group compared with the healthy group in both tissue and plasma samples. Also, a significant rise of RAP-1b protein was observed in mentioned clinical specimens of the patients group compared to those in the healthy participants (Table 3 ). Therefore, negative regulation of RAP-1b by underexpressed miR-138-5p occurs through epigenetic changes and RAP-1b is able to induce MAPK-dependent signaling cascade and cause cell growth and proliferation of CRC cells ( 33 , 34 ). The potential of miRNAs as a therapeutic target is being considered ( 35 ) to control this cascade such as miRNA mimics or demethylating agents like 5-azacytidine which can reactivate miRNA expression ( 36 , 37 ). For example, systemic or local application of intact miRNA molecules, miR-145, into mouse xenograft colon carcinoma reduces tumor growth, increases apoptosis, and simultaneously suppresses the c-Myc and ERK5 oncogenes ( 38 ). In line with our study, several studies had reported that RAP-1b is overexpressed in various cancers, such as thyroid cancer, hepatocellular carcinoma, and CRC, which in turn leading to proliferation and invasion of tumoral cells ( 39 – 41 ). Agree with our study, it was revealed that miR-139 is under expressed in CRC tissues. The outcomes verified that miR-139-5p reduces proliferation, invasion and migration by straightly targeting RAP-1b and tumor protein D52, manifesting miR-139 as a putative tumor suppressor miRNA in patients with CRC( 33 , 42 ). Also, miR-139-5p might be suggested as a biomarker to predict the transformation of inflammatory bowel disease to CRC ( 43 ). Furthermore, we realized that methylation and expression levels of miR-139-5p as well as its target, RAP-1b protein, are not correlated with clinicopathological parameters, including histological grade, tumor stage, tumor size, lymph node metastasis, tumor type, age, gender, and distant metastasis, because on one hand, all eligible untreated patients, pathologically confirmed CRC, irrespective of sex, age, tumor type and location, histological grading, TNM staging, etc. were included in the study and the control group was matched with the patient group by age and sex ( 22 ) ; therefore, the number of patients was divided between the mentioned subgroups, including different grades and different stages, etc. ,and on the other hand we were not able to increase the number of samples in the study subgroups because of limitation of financial resources. In our opinion, it is better to calculate the sample size for subgroups to assess the relationship between the level of gene expression as well as gene methylation with clinical and pathological features of patients. Another suggestion in future surveys is that considering the larger sample size to provide a more appropriate distribution of cases between subgroups. To the best of our knowledge, although the methylation of miR -139-5p in CRC disease has not been yet evaluated, a decrease in methylation-based expression has been reported for other miRNAs such as miR-148a, is hypermethylated in tissue of 65% of CRC patients with no meaningful relationship with clinicopathological specifications ( 44 ) ; and other researches have illustrated that DNA methylation has an essential impression on deregulation of some miRNAs, consist of miR-125 and 34 in CRC, and accelerating the tumor development ( 45 , 46 ). The use of diagnostic tests to determine the presence or absence of a suspected disease is critical in clinical situations. The ideal test is defined by a sensitivity and specificity of 100% which is rare in the clinical setting, because sensitivity and specificity decrease as each of them increases. Also, an ideal test has a ROC curve with AUC = 1.0, but in general, AUC ≥ 0.8 is assumed suitable and AUC ≥ 0.9 is interpreted excellent ( 27 ). Graphical plot of diagnostic classification, ROC curve, presented a great AUC of 0.767, 0.958, and 0.718 of miR-139-5p, PMR value, and RAP-1b protein for plasma specimens, in the same order (Fig. 3 ). These results suggest the ability of these biomarkers to recognize CRC disease from healthful persons. To our knowledge, there are no surveys have previously assessed the diagnostic value and methylation pattern of miR -139-5p and its expression in plasma specimens. Therefore, our results compared to other miRNA , s methylation modes and other classes of molecular biomarkers. Using conventional methylation-specific PCR (MSP), the sensitivity of 76.8% and a specificity of 93.6% for stool miR -34a methylation test were reported ( 47 ), although compared to quantitative MSP, the mentioned technique is not quantitative and has lower accuracy for detection of CRC. Also, compared to the sensitivity (55%) and the specificity (82.5%) of plasma level of methylated miR -138-5p ( 22 ), the present study has higher diagnostic value with an excellent AUC. There are different classes of molecular biomarkers by source or by type including traditional or new kind. Measurement of gene expression at mRNA level and DNA methylation in plasma belongs to genomic new biomarkers while protein detection is traditional ( 48 ). Jelski Wojciech et al. reported diagnostic value of aldehyde dehydrogenase isoenzyme I (ADH I) in the serum of patients with CRC with the sensitivity and specificity of 76% and 82%, respectively ( 49 ). Thus, compared to traditional type of biomarkers, the diagnostic value of miR -139-5p promoter methylated marker is more favorable (the sensitivity was 75%, and specificity was 92.5% with AUC = 0.958). Because methylation changes befalls in the primitive stages of tumor formation ( 12 ), perhaps it is possible to get an admissible clinical sensitivity by evaluating this methylation marker in the biological samples including, plasma and serum. Comparing with the CEA (46.8%) and FOBT (43.5%) results, the sensitivity and AUC of miR -139-5p PMR was more in plasma, which shows potential as a diagnostic biomarker for the CRC, pending further validation. 5. Conclusion In this investigation, we assessed the methylation pattern of the miR -139-5p gene in liquid and soft samples of CRC patients compared to the healthful individuals for the first experience. We illustrated that miR-139-5p is significantly under expressed in the case group compared to the healthy group. Also, compared to the normal group, the rate of gene methylation in the case group was meaningfully greater, indicating the role of epigenetic factors, especially gene methylation, in the miR-139-5p down-regulation. The superiority of current survey was the concurrent assessment of miR-139-5p expression and RAP-1b protein and admeasuring the methylation mode of promoter region of the gene in liquid and soft tissue specimens, which prepared the feasibility of further comparison of the results. Finally, it could be supposed that hyper methylation inside the promoter area of the miR -139-5p gene is one of the critical epigenetic agents implicated in reducing miR-139-5p expression. Due to the occurrence of methylation alterations in primary steps of carcinogenesis, it can be proposed as a miRNA-based therapeutic target and an early detection biomarker for CRC diagnosis in plasma samples. However, further study on the more number of clinical samples is necessary to confirm it as a diagnostic biomarker in patients' plasma. Abbreviations CRC Colorectal Cancer miRNAs microRNAs RAP- 1b Ras-Related Protein RAP-1b PMR Percentage of Methylated Reference ROC analysis Receiver Operating Characteristics analysis AUC The Area Under the ROC curve CEA Carcino Embryonic Antigen MSP Methylation-Specific PCR Declarations Acknowledgments The current study, supported by Vice Chancellery for Research and Technology, Hamadan University of Medical Sciences (No. 9609286007). The authors would like to thank the staff of the Medical Genetics Laboratory, Reference Labora­tory, and Kermanshah University of Medical Sciences for their help during the current research. Ethical approval Informed consent was obtained from all participants. The Ethics Committee of Hamadan University of medical sciences approved this survey (IR.UMSHA.REC.1396.533). Funding : This study was funded by Vice Chancellery for Research and Technology, Hamadan University of Medical Sciences (No. 9609286007). Author contributions All the authors have played key roles in various sections of the present investigation, including study design, blood and tissue sampling, bioinformatics studies, carrying out experiments, data analysis, writing, reading and approving the final manuscript. Consent for publication Not applicable Availability of data and materials Data from this survey are accessible on a reasonable request by AM. Competing interests The authors declare no competitive interests. References Guren MG. The global challenge of colorectal cancer. Lancet Gastroenterol Hepatol. 2019;4(12):894–5. Yasudome R, Seki N, Asai S, Goto Y, Kita Y, Hozaka Y, et al. Molecular pathogenesis of colorectal cancer: Impact of oncogenic targets regulated by tumor suppressive miR-139-3p. Int J Mol Sci. 2022;23(19):11616. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. Cancer J Clin. 2015;65(2):87–108. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. Cancer J Clin. 2011;61(2):69–90. Ni Y, Xie G, Jia W. Metabonomics of Human Colorectal Cancer: New Approaches for Early Diagnosis and Biomarker Discovery. J Proteome Res. 2014;13(9):3857–70. Al-Sohaily S, Biankin A, Leong R, Kohonen‐Corish M, Warusavitarne J. Molecular pathways in colorectal cancer. J Gastroenterol Hepatol. 2012;27(9):1423–31. Hammond WA, Swaika A, Mody K. Pharmacologic resistance in colorectal cancer: a review. Therapeutic Adv Med Oncol. 2016;8(1):57–84. Xie N, Meng Q, Zhang Y, Luo Z, Xue F, Liu S, et al. MicroRNA–142–3p suppresses cell proliferation, invasion and epithelial–to–mesenchymal transition via RAC1–ERK1/2 signaling in colorectal cancer. Mol Med Rep. 2021;24(2):1–12. Sengupta D, Deb M, Kar S, Shilpi A, Parbin S, Mallick B, et al. Epigenetic MicroRNA Regulation of Multiple Chromatin Functions: A Perspective in Cancer. Epigenetic Diagnosis Therapy. 2015;1(2):81–90. Bardhan K, Liu K. Epigenetics and colorectal cancer pathogenesis. Cancers. 2013;5(2):676–713. Dwivedi RS, Herman JG, McCaffrey TA, Raj DS. Beyond genetics: epigenetic code in chronic kidney disease. Kidney Int. 2011;79(1):23–32. Herman JG. Hypermethylation pathways to colorectal cancer: implications for prevention and detection. Gastroenterol Clin. 2002;31(4):945–58. Jelski W, Mroczko B. Biochemical Markers of Colorectal Cancer - Present and Future. Cancer Manag Res. 2020;12:4789–97. Zhang P, Yin J, Yuan L, Wang Q, Du X, Dong R, et al. MicroRNA–139 suppresses hepatocellular carcinoma cell proliferation and migration by directly targeting Topoisomerase I. Oncol Lett. 2019;17(2):1903–13. Khalili N, Nouri-Vaskeh M, Segherlou ZH, Baghbanzadeh A, Halimi M, Rezaee H, et al. Diagnostic, prognostic, and therapeutic significance of miR-139-5p in cancers. Life Sci. 2020;256:117865. Bozgeyik İ. miRNAs, cancer, and unconventional miRNA functions. Bull Biotechnol. 2023;4(1):36–41. Shen K, Mao R, Ma L, Li Y, Qiu Y, Cui D, et al. Post-transcriptional regulation of the tumor suppressor miR‐139‐5p and a network of miR‐139‐5p‐mediated m RNA interactions in colorectal cancer. FEBS J. 2014;281(16):3609–24. Sun C, Sang M, Li S, Sun X, Yang C, Xi Y, et al. Hsa-miR-139-5p inhibits proliferation and causes apoptosis associated with down-regulation of c-Met. Oncotarget. 2015;6(37):39756. Finlin BS, Andres DA. Rem Is a New Member of the Rad- and Gem/Kir Ras-related GTP-binding Protein Family Repressed by Lipopolysaccharide Stimulation*. J Biol Chem. 1997;272(35):21982–8. Fischer TH, Gatling MN, McCormick F, Duffy CM. White 2nd G. Incorporation of Rap 1b into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium. J Biol Chem. 1994;269(25):17257–61. Shen K, Liang Q, Xu K, Cui D, Jiang L, Yin P, et al. MiR-139 inhibits invasion and metastasis of colorectal cancer by targeting the type I insulin-like growth factor receptor. Biochem Pharmacol. 2012;84(3):320–30. Asefi M, Saidijam M, Rezvani N, Soltanian AR, Khalilian AR, Mahdavinezhad A. A novel epigenetic biomarker, plasma miR-138-5p gene promoter-methylated DNA, for colorectal cancer diagnosis. Per Med. 2022;19(4):315–25. Lange CP, Campan M, Hinoue T, Schmitz RF, van der Meulen-de AE, Slingerland H, et al. Genome-scale discovery of DNA-methylation biomarkers for blood-based detection of colorectal cancer. PLoS ONE. 2012;7(11):e50266. Kim H-J, Yu M-H, Kim H-G, Byun J-H, Lee C. Noninvasive molecular biomarkers for the detection of colorectal cancer. BMB Rep. 2008;41(10):685–92. Eads CA, Lord RV, Wickramasinghe K, Long TI, Kurumboor SK, Bernstein L, et al. Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res. 2001;61(8):3410–8. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008;3(6):1101–8. Nahm FS. Receiver operating characteristic curve: overview and practical use for clinicians. Korean J Anesthesiol. 2022;75(1):25–36. Amirkhah R, Schmitz U, Linnebacher M, Wolkenhauer O, Farazmand A. MicroRNA–mRNA interactions in colorectal cancer and their role in tumor progression. Genes Chromosom Cancer. 2015;54(3):129–41. Huang LL, Huang LW, Wang L, Tong BD, Wei Q, Ding XS. Potential role of miR-139-5p in cancer diagnosis, prognosis and therapy. Oncol Lett. 2017;14(2):1215–22. Jiang L-h, Sun D-w, Li J, Tang J-h. MiR-139-5p: promising biomarker for cancer. Tumor Biology. 2015;36(3):1355–65. Breving K, Esquela-Kerscher A. The complexities of microRNA regulation: mirandering around the rules. Int J Biochem Cell Biol. 2010;42(8):1316–29. Ando T, Yoshida T, Enomoto S, Asada K, Tatematsu M, Ichinose M, et al. DNA methylation of microRNA genes in gastric mucosae of gastric cancer patients: its possible involvement in the formation of epigenetic field defect. Int J Cancer. 2009;124(10):2367–74. Guo H, Hu X, Ge S, Qian G, Zhang J. Regulation of RAP1B by miR-139 suppresses human colorectal carcinoma cell proliferation. Int J Biochem Cell Biol. 2012;44(9):1465–72. Stork PJ. Does Rap1 deserve a bad Rap? Trends Biochem Sci. 2003;28(5):267–75. Selvakumar SC, Preethi KA, Sekar D. MicroRNAs as important players in regulating cancer through PTEN/PI3K/AKT signalling pathways. Biochimica et Biophysica Acta (BBA) -. Reviews Cancer. 2023;1878(3):188904. Howell PM, Liu Z, Khong HT. Demethylating Agents in the Treatment of Cancer. Pharmaceuticals (Basel). 2010;3(7):2022–44. Lai X, Eberhardt M, Schmitz U, Vera J. Systems biology-based investigation of cooperating microRNAs as monotherapy or adjuvant therapy in cancer. Nucleic Acids Res. 2019;47(15):7753–66. Ibrahim AF, Weirauch U, Thomas M, Grünweller A, Hartmann RK, Aigner A. MicroRNA replacement therapy for miR-145 and miR-33a is efficacious in a model of colon carcinoma. Cancer Res. 2011;71(15):5214–24. Fan M, Ma X, Wang F, Zhou Z, Zhang J, Zhou D, et al. MicroRNA-30b-5p functions as a metastasis suppressor in colorectal cancer by targeting Rap1b. Cancer Lett. 2020;477:144–56. Tang Z, Peng H, Chen J, Liu Y, Yan S, Yu G, et al. Rap1b enhances the invasion and migration of hepatocellular carcinoma cells by up-regulating Twist 1. Exp Cell Res. 2018;367(1):56–64. Wang P, Gu J, Wang K, Shang J, Wang W. miR-206 inhibits thyroid cancer proliferation and invasion by targeting RAP1B. J Cell Biochem. 2019;120(11):18927–36. Zhang J, Li Z, Han J, Tian Z, Meng Q, Niu W. KLF7 enhances the invasion and migration of colorectal cancer cells via the miR-139-5p/TPD52 axis. Cancer Biol Ther. 2024;25(1):2385172. Deris Zayeri Z, Parsi A, Shahrabi S, Kargar M, Davari N, Saki N. Epigenetic and metabolic reprogramming in inflammatory bowel diseases: diagnostic and prognostic biomarkers in colorectal cancer. Cancer Cell Int. 2023;23(1):264. Kalimutho M, Di Cecilia S, Blanco GDV, Roviello F, Sileri P, Cretella M, et al. Epigenetically silenced miR-34b/c as a novel faecal-based screening marker for colorectal cancer. Br J Cancer. 2011;104(11):1770–8. Chen J, Chen Y, Chen Z. miR-125a/b Regulates the Activation of Cancer Stem Cells in Paclitaxel-resistant Colon Cancer. Cancer Invest. 2013;31(1):17–23. Jun HH, Kwack K, Lee KH, Kim JO, Park HS, Ryu CS, et al. Association between TP53 genetic polymorphisms and the methylation and expression of miR-34a, 34b/c in colorectal cancer tissues. Oncol Lett. 2019;17(5):4726–34. Wu XD, Song YC, Cao PL, Zhang H, Guo Q, Yan R, et al. Detection of miR-34a and miR-34b/c in stool sample as potential screening biomarkers for noninvasive diagnosis of colorectal cancer. Med Oncol. 2014;31(4):894. Piñero J, Rodriguez Fraga PS, Valls-Margarit J, Ronzano F, Accuosto P, Lambea Jane R, et al. Genomic and proteomic biomarker landscape in clinical trials. Comput Struct Biotechnol J. 2023;21:2110–8. Jelski W, Mroczko B, Szmitkowski M. The diagnostic value of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) measurement in the sera of colorectal cancer patients. Dig Dis Sci. 2010;55(10):2953–7. Additional Declarations No competing interests reported. Supplementary Files Supplementarytable1.docx Supplementarytable2.docx Supplementaryfigure1.docx Cite Share Download PDF Status: Published Journal Publication published 15 May, 2025 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 24 Apr, 2025 Reviews received at journal 24 Apr, 2025 Reviewers agreed at journal 24 Apr, 2025 Reviews received at journal 22 Apr, 2025 Reviewers agreed at journal 21 Apr, 2025 Reviewers agreed at journal 19 Apr, 2025 Reviewers agreed at journal 09 Apr, 2025 Reviewers invited by journal 07 Apr, 2025 Submission checks completed at journal 07 Apr, 2025 First submitted to journal 06 Apr, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-5527622","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":439337898,"identity":"5c260648-11b9-4981-8c6c-c20e4c26b38d","order_by":0,"name":"Masoud Asefi","email":"","orcid":"","institution":"Hamedan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Masoud","middleName":"","lastName":"Asefi","suffix":""},{"id":439337901,"identity":"01af92a2-d7a4-4b5c-81e4-05f682dd7f0c","order_by":1,"name":"Nayebali Rezvani","email":"","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nayebali","middleName":"","lastName":"Rezvani","suffix":""},{"id":439337903,"identity":"46478c58-b6e6-4c03-8dc1-62229e6cae89","order_by":2,"name":"Massoud Saidijam","email":"","orcid":"","institution":"Hamedan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Massoud","middleName":"","lastName":"Saidijam","suffix":""},{"id":439337904,"identity":"b21a2ce8-2b1d-4bfa-a8ec-e6ab771555c8","order_by":3,"name":"Ali Reza Soltanian","email":"","orcid":"","institution":"Hamedan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"Reza","lastName":"Soltanian","suffix":""},{"id":439337905,"identity":"9a8050ae-387d-48e7-a01a-1948e47b4c80","order_by":4,"name":"Ali Reza Khalilian","email":"","orcid":"","institution":"Hamedan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"Reza","lastName":"Khalilian","suffix":""},{"id":439337906,"identity":"c7129158-1584-43f8-b8d2-e0dbe60a9edf","order_by":5,"name":"Ali Mahdavinezhad","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDACdsYGBsYGZgYGZgbGB0A+Dx9BLcwILcwGIC1shLUAMVgLAwObBJgkpIO/mbl1w88d1vIGx3mPVX7NsZNhY2B++OgGHi0ShxnbbvaeSTfccJgv7bbstmSgw9iMjXPwWQPUcoO37TDjzGYes9uS25iBWnjYpPFpkQfZ8rftsD1IS7HktnrCWgyAWm4DbUnsZ+YxY/y47TBhLYYgLbJt6clALcbSjNuO87AxE/CL3PH2ZzfftlnbtvGfMfz4c1u1PT9788PHeL2PDJh5wCSxykGA8QcpqkfBKBgFo2DEAACDZ0QxouPXSAAAAABJRU5ErkJggg==","orcid":"","institution":"Hamedan University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Ali","middleName":"","lastName":"Mahdavinezhad","suffix":""}],"badges":[],"createdAt":"2024-11-26 12:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5527622/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5527622/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-025-14290-x","type":"published","date":"2025-05-15T15:57:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80278576,"identity":"49ed8c8c-3327-4f6c-91ed-6b86661d707b","added_by":"auto","created_at":"2025-04-10 05:28:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50825,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot of PMR level of the \u003cem\u003emiR\u003c/em\u003e-139-5p gene in clinical samples. The median PMR values for CRC patients were higher in plasma samples in comparison with those of control persons (12.4 vs. 0.66, p\u0026lt;0.0001) as well as in tissue specimens (0.78 vs. 0.1, p (\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5527622/v1/9b098cefbb0cdaafb69bf0ab.png"},{"id":80276351,"identity":"74f1f8c1-1770-4b19-9362-ebd4a176555d","added_by":"auto","created_at":"2025-04-10 05:04:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45676,"visible":true,"origin":"","legend":"\u003cp\u003eOverall 5.7 and 4.69 fold (2\u003csup\u003e−ΔΔCT\u003c/sup\u003e) lower expression of miR-139-5p in tissue and plasma specimens of CRC affected patients, respectively (Mean ±SEM, n=40, p\u0026lt;0.0001 and p\u0026lt;0.001, respectively.)\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5527622/v1/cbf503521ed799843592da91.png"},{"id":80278566,"identity":"b351ca6d-00b8-4f54-827f-3f96b064d8e3","added_by":"auto","created_at":"2025-04-10 05:28:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56082,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic value of studied plasma biomarkers using ROC curve analysis shows an excellent AUC, and higherspecificity and sensitivity for the plasma \u003cem\u003emiR\u003c/em\u003e-139-5p methylated biomarker which is able to detect 75%(sensitivity) of CRC patients and distinguishes 92.5% (specificity) of individuals without CRCa; PMR \u003cem\u003emiR\u003c/em\u003e-139-5p (AUC =0.958, the sensitivity and specificity were 75% and 92.5%, respectively ), b; miR-139-5p expression (AUC=0.767, the sensitivity and specificity were 70% and 65%, in the same order), c; RAP-1b (AUC=0.718, sensitivity=73.3% ,and specificity = 73.3%), d; CEA (AUC=0.708, sensitivity=46.8% ,and specificity =98% ).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5527622/v1/fc8359ff34a58a0ee8cd568c.png"},{"id":83067906,"identity":"7ff3514e-5afc-4094-9e93-e59c86bdc0c8","added_by":"auto","created_at":"2025-05-19 16:07:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1145796,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5527622/v1/0962da93-3e18-4b84-bf33-dc2ccbd21bca.pdf"},{"id":80278600,"identity":"5dde5f22-9644-48fa-96b0-ad6d115c7506","added_by":"auto","created_at":"2025-04-10 05:29:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":24266,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5527622/v1/5d2bd0efbcc958d2d75a0c58.docx"},{"id":80278565,"identity":"6bf29c9d-3a4b-40f9-afe4-916a615852d5","added_by":"auto","created_at":"2025-04-10 05:28:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22392,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5527622/v1/3b62182af35e8e96d977e261.docx"},{"id":80276357,"identity":"67cf44c7-c1ab-4449-aba4-8cf3e536db47","added_by":"auto","created_at":"2025-04-10 05:04:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":255697,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5527622/v1/146e53910acddc800ba24274.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of Epigenetic Silencing of the miR-139-5p gene in the pathogenesis of colorectal cancer and its diagnostic biomarker capability in plasma samples","fulltext":[{"header":"1. Background","content":"\u003cp\u003eThe second and third most commonly diagnosed malignancy is attributed to colorectal cancer (CRC) in women and men, respectively, and the second leading cause of cancer death globally (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). There is a noticeable difference in CRC incidence and mortality (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). It is frequently diagnosed at advanced phases due to the limitations of current clinical screening methods (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Relative CRC risk is specified by genetic potency and environmental influences, with age being the most imperative risk factor for sporadic CRC. Transformation of normal colonic mucosa into invasive cancer occurs through the accumulation of genetic and epigenetic changes (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Despite the identifying of several molecular pathways intervened in the development of CRC, the detailed molecular pathogenesis of this cancer has not yet been clarified (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). MicroRNAs (miRNAs) with their target genes organize a very complex network in cells, and it is expected that abnormal expression of miRNAs interrupts this RNA system. Many surveys have revealed that deregulation of miRNAs act as tumor suppressors or oncogenes in CRC cells by targeting neoplasm-related genes (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). New advances in molecular biology have demonstrated the role of miRNAs in various biological and pathological functions including, down-regulation of tumor suppressor miRNAs in many cancers, particularly CRC (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Epigenetic variations, in particular DNA methylation and histone modifications, perform major pathophysiological role in the beginning and progression of CRC (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Covalent bonding of a methyl group to the 5-carbon status of the cytosine is widespread throughout the genome; however, when added to CpG in the promoter region, it generally results in lower expression of methylated gene. The mechanism of gene silencing by DNA methylation may be related to a barrier of the transcriptional machinery or modification in chromatin configuration or recruitment of repressors (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Owing to the truth that methylation in a specific region of the genome occurs in the primary steps of tumorigenesis (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and significantly, most cancer deaths are due to metastatic spread of tumor cells not due to the primary tumor (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), early diagnosis increases the chance of recovery and prevention of subsequent complications. \u003cem\u003eMiR\u003c/em\u003e-139, located on 11q13.4 and is down-regulated in several cancers, exposes antioncogenic and anti-metastatic activity in human tumors(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). MiR-139-5p, encoded within the second intron of the phosphodiesterase 2A gene (16), was recognized as, a tumor-suppressor miRNA, and is down regulated in a diversity of cancers, such as, gastric cancer and CRC(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Ras family of low molecular weight GTP-binding proteins have been occupied in a numerous cellular processes, comprising differentiation and cell growth, intracellular vesicular transferring, nucleocytoplasmic transport, and cytoskeletal restructuring (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Ras-Related Protein Rap-1b ( RAP- 1b ) is a low molecular mass GTP-binding protein (22-kDa) which is both a substrate for cAMP-dependent protein kinase and a member of the Ras superfamily (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In previous studies, RAP-1b has been identified as one of the straight target of miR-139-5p (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). We aim to determine and evaluate the expression of miR-139-5p, RAP-1b protein, and the methylation condition of the \u003cem\u003emiR\u003c/em\u003e-139-5p promoter region in plasma samples of CRC affected individuals, healthy controls and in tumor tissue and healthy near specimens. Furthermore, we examined the diagnostic value of these biomarkers and their relationship with clinical and pathological features.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Tissue and plasma samples\u003c/h2\u003e \u003cp\u003e A total of 80 plasma samples from CRC patients and healthy matched individuals, as well as 40 paired tumor and adjacent healthy tissues were provided in the present survey. The inclusion and exclusion criteria of clinical samples have been reported in our previous study (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Written informed was signed by all participants. This survey authorized by the Ethics Committee of Hamadan University of medical sciences (IR.UMSHA.REC.1396.533).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Methylation Analysis using MethyLight method\u003c/h2\u003e \u003cp\u003eThis section of the experiments has explained in detail in the previous article (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and it is briefly as follows: The promoter area of the \u003cem\u003emiR\u003c/em\u003e-139-5p gene was identified using the miR Start and Ensemble Databases. After that, the most specific primers and probes for fully methylated bisulfiteconverted DNA (one set for \u003cem\u003emiR\u003c/em\u003e-139-5p gene and one for ALU-C4)were designed for a GC-rich area inside the promoter region using Beacon designer software (version 8.13) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The probes and the primers were synthesized by the German company Metabion.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe primers and probes specifications.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimers and probes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTm\u003csup\u003eo\u003c/sup\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLength\u003c/p\u003e \u003cp\u003ebp\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALU-C4 Forward primer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'GGTTAGGTATAGTGGTTTATATTTGTAATTTTAGTA-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALU-C4 Reverse primer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'ATTAACTAAACTAATCTTAAACTCCTAACCTCA-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALU-C4 Probe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CGCTAACCGAACGCAACGAC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiR-139 Reverse Primer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TTTCGTTTGAGGAATTGGATAATAGCG-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiR-139 Forward Primer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-6-FAM-ACCGACGAACGAACGAACGACTACGAAC-BHQ-1-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiR-139 Probe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-6-FAM-CCTAAACCACAAACAACCACGAACCTCGCC-BHQ-1-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNext, DNA of all specimens extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Germany, Cat.No:51104) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) followed by bisulfite modification using an EpiTect Bisulfite kit (Qiagen.Cat.No.59104) in duplicate. Sodium bisulfite has no impression on methylated cytosine, but it causes deamination of non-methylated cytosine and modifies it to uracil. This difference is the basis for distinguishing methylated from non-methylated DNA (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFinally, the qPCR reactions were done in 96well plates on an ABI 7500 RealTime PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in duplicate, using the EpiTect\u0026reg; MethyLight PCR\u0026thinsp;+\u0026thinsp;ROX Vial kit (Qiagen GmbH). ALU-C4, a consensus DNA sequence, was applied as the housekeeping gene for normalization.\u003c/p\u003e \u003cp\u003eThe percentage of methylated reference (PMR), the degree of methylation of every sample relatively to the entirely methylated control, of the samples, calculated using saleable methylated human bisulfite converted DNA (EpiTect PCR Control DNA; Qiagen GmbH), as an entirely methylated control.\u003c/p\u003e \u003cp\u003ePMR, at a defined locus, was assessed using the subsequent equation : [(miR/ALU-C4)\u003csup\u003esample\u003c/sup\u003e / (miR/ALU-C4)\u003csup\u003epositive control\u003c/sup\u003e] x 100 (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. MiR-139-5p expression and measurement of RAP-1b Protein in Clinical Samples\u003c/h2\u003e \u003cp\u003eA favorgen miRNA (Favorgen, Taiwan, Cat.No:FAMIK002) and NucleoSpin\u0026reg; miRNA Plasma (Macherey-Nagel, Germany) extraction kits were applied to extract miRNAs from tissue and plasma samples, respectively. The extraction of miRNA from approximately 10 mg of tissue sample consists of four following steps: 1- Cell lysis 2- Binding of DNA to the silica column membrane, 3- Column washing, and 4- Dilution of DNA in buffer which in the first step ,tissue sample was ground into smaller pieces using a scalpel blade. Then the sample was transferred to a 1.5 ml microtube and the next steps were performed according to the kit instructions. The quality and quantity of RNA was evaluated using the nanodrop (ND-1000). BON miR Kit (Bon yakhteh, Inc) was applied to synthesis cDNA. Finally, qPCR was carried out to assess the expression of miR-139-5p supported by the BON miR SYBR\u0026reg;green PCR Kit (Bon yakhteh, Inc) in accordance with the company\u0026rsquo;s instructions. U6 as a housekeeping gene used to normalize data and the expression level was computed by the 2\u003csup\u003e(\u0026minus;ΔΔCT )\u003c/sup\u003e manner (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). ELISA kit (Cat.no:E2153Hu) was applied to measure RAP-1b Protein in an ELISA reader (Rayto 2100C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe Chi-square test was used to analyze descriptive data. The normality of data distribution evaluated by Kolmogorov-Smirnov test. Therefore, non-parametric Mann-Whitney, Kruskal-Wallis tests, Anova, and independent t-tests were used depending on the results of normality test. Using SPSS 16 software, P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as the level of statistical meaningful. The specificity and sensitivity of studied biomarkers evaluated by Receiver Operating Characteristics (ROC) analysis using PMR, ΔCT, and RAP-1b quantities. In general, the area under the ROC curve (AUC)\u0026thinsp;\u0026ge;\u0026thinsp;0.8 is assumed acceptable and AUC\u0026thinsp;\u0026ge;\u0026thinsp;0.9 is interpreted excellent (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eForty tissue samples from CRC affected patients and 40 from marginal normal tissue (22 females and 18 males) with a mean age of (57.7\u0026thinsp;\u0026plusmn;\u0026thinsp;17.2) and the same number of plasma samples from case group and healthy individuals ( 22 females and 18 males) with a matched ages as a control participants were included in the study.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Hyper methylation of \u003cem\u003emiR\u003c/em\u003e-139-5p in CRC without any clinical and pathological association was observed\u003c/h2\u003e \u003cp\u003eMedian PMR values of 0.78 (95% CI, 0.12\u0026ndash;6.58) and 0.1 (95% CI, 0.01\u0026ndash;0.29) were obtained in tumor and adjacent normal tissues, at the same order. The uppermost PMR value got from marginal normal tissues was assumed as a threshold level; specimen with a PMR\u0026thinsp;\u0026gt;\u0026thinsp;5.1 were translated positively for methylation status (36/40, (90%)). As demonstrated in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the PMR level was remarkably larger in the cancer tissues compared with that of the normal tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eThe median PMR levels of 12.4 (95% CI, 3.23\u0026ndash;32.25) and 0.66 (95%CI, 0.51-1.0) in plasma samples from the CRC and the normal groups, were obtained, sequentially. Also, the maximum PMR value from the healthy plasma specimens was assumed as the threshold of methylation condition; thus, all samples with a PMR\u0026thinsp;\u0026gt;\u0026thinsp;4.8 were supposed positively methylated (29/40, (72.5%)). The PMR values for CRC patients were noticeably greater in plasma samples in comparison with those of control subjects (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResults from PMR values showed no statistically meaningful association between the tissue and plasma specimens and clinicopathological characteristics of CRC patients (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: Box plot of PMR level of the \u003cem\u003emiR\u003c/em\u003e-139-5p gene in clinical samples. The median PMR values for CRC patients were higher in plasma samples in comparison with those of control persons (12.4 vs. 0.66, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) as well as in tissue specimens (0.78 vs. 0.1, p (\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelationship between \u003cem\u003emiR\u003c/em\u003e-139-5p methylation and clinicopathological specifications in clinical samples of case group.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTissue PMR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlasma PMR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinicopathological characteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of patients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.19\u0026thinsp;\u0026plusmn;\u0026thinsp;7.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.353\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17.22\u0026thinsp;\u0026plusmn;\u0026thinsp;17.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.338\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMucinous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.89\u0026thinsp;\u0026plusmn;\u0026thinsp;19.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e21.14\u0026thinsp;\u0026plusmn;\u0026thinsp;18.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistological grading\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.96\u0026thinsp;\u0026plusmn;\u0026thinsp;10.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.490\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e21.74\u0026thinsp;\u0026plusmn;\u0026thinsp;19.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.583\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.98\u0026thinsp;\u0026plusmn;\u0026thinsp;15.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e15.15\u0026thinsp;\u0026plusmn;\u0026thinsp;13.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e21.91\u0026thinsp;\u0026plusmn;\u0026thinsp;25.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor location\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.41\u0026thinsp;\u0026plusmn;\u0026thinsp;17.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.732\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e13.03\u0026thinsp;\u0026plusmn;\u0026thinsp;14.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.168\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProximal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.71\u0026thinsp;\u0026plusmn;\u0026thinsp;9.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20.83\u0026thinsp;\u0026plusmn;\u0026thinsp;18.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;50 mm\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.89\u0026thinsp;\u0026plusmn;\u0026thinsp;14.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.390\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e19.70\u0026thinsp;\u0026plusmn;\u0026thinsp;19.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.452\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\ge\\:50\\:\\varvec{m}\\varvec{m}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.14\u0026thinsp;\u0026plusmn;\u0026thinsp;9.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e16.75\u0026thinsp;\u0026plusmn;\u0026thinsp;15.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNM Stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.422\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e22.55\u0026thinsp;\u0026plusmn;\u0026thinsp;16.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.348\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;9.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14.47\u0026thinsp;\u0026plusmn;\u0026thinsp;16.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.91\u0026thinsp;\u0026plusmn;\u0026thinsp;21.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20.49\u0026thinsp;\u0026plusmn;\u0026thinsp;20.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node involvement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.91\u0026thinsp;\u0026plusmn;\u0026thinsp;21.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.209\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20.49\u0026thinsp;\u0026plusmn;\u0026thinsp;20.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.209\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;8.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17.75\u0026thinsp;\u0026plusmn;\u0026thinsp;16.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eStandard deviation, a: Mann-Whitney U two-tailed tests; b: ANOVA; c: Kruskal-Wallis *\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2. Down-regulation of miR-139-5p and greater quantity of RAP-1b protein in both plasma and tissue samples without any association with clinicopathological parameters\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe expression of miR-139-5p in tumor tissue (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and plasma of patients (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) had a significant decrease, in comparison with the healthy participants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere was no remarkable association between down regulation of miR-139-5p and any clinical and pathological characteristics (supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: Overall 5.7 and 4.69 fold (2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e) lower expression of miR-139-5p in tissue and plasma specimens of CRC affected patients, respectively (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;40, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively.)\u003c/p\u003e \u003cp\u003eThe concentration of RAP-1b, a valid target of miR-139-5p, was higher in CRC patients than in control groups. (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and supplementary Fig.\u0026nbsp;1). Also, the results revealed that there is no statistical meaningful association amongst RAP-1b concentration and clinicopathological characteristics (supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe greater quantity of RAP-1b protein in clinical samples of CRC patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003ePercentiles\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNo. of patients\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMinimum\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eMaximum\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e25th\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e50th\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e75th\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTissue RAP-1b(ng/l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e64.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlasma RAP-1b(ng/l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e93.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3. The results of diagnostic value of studied biomarkers using ROC curve analysis\u003c/h2\u003e \u003cp\u003eA high level of carcinoembryonic antigen (CEA) in plasma specimens of CRC subjects was observed in our previous work (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). As observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the specificity and sensitivity of the \u003cem\u003emiR\u003c/em\u003e-139-5p methylated biomarker in plasma is higher than CEA and FOBT tests.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMiRNAs have essential effects on gene expression and signaling pathways so that deregulated miRNAs could be related to different diseases, including CRC (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In previous studies, miR-139-5p, as a tumor suppressor, has been identified as an influential factor in cancer diagnosis, prognosis, and treatment in various types of human cancers (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Many molecular mechanisms lead to miRNA deregulation (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), including transcriptional tumor suppressor miRNAs silencing by special DNA methylation in cancer cells (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Haiyan Guo et al. demonstrated that miR-139 decreases proliferation in CRC through targeting RAP-1b directly (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The current research examined methylation pattern of \u003cem\u003emiR\u003c/em\u003e-139-5p, its expression and target (RAP-1b) in both cancer and healthy groups to acquire more vision about the miR-139-5p function in CRC disease. This study showed that the methylation level of the promoter area of the \u003cem\u003emiR\u003c/em\u003e-139-5p gene was significantly higher in the plasma and tissue specimens of CRC patients. On the other hand, the level of miR-139-5p significantly decreased in the tumoral group compared with the healthy group in both tissue and plasma samples.\u003c/p\u003e \u003cp\u003eAlso, a significant rise of RAP-1b protein was observed in mentioned clinical specimens of the patients group compared to those in the healthy participants (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, negative regulation of RAP-1b by underexpressed miR-138-5p occurs through epigenetic changes and RAP-1b is able to induce MAPK-dependent signaling cascade and cause cell growth and proliferation of CRC cells (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The potential of miRNAs as a therapeutic target is being considered (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) to control this cascade such as miRNA mimics or demethylating agents like 5-azacytidine which can reactivate miRNA expression (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). For example, systemic or local application of intact miRNA molecules, miR-145, into mouse xenograft colon carcinoma reduces tumor growth, increases apoptosis, and simultaneously suppresses the c-Myc and ERK5 oncogenes (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn line with our study, several studies had reported that RAP-1b is overexpressed in various cancers, such as thyroid cancer, hepatocellular carcinoma, and CRC, which in turn leading to proliferation and invasion of tumoral cells (\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAgree with our study, it was revealed that miR-139 is under expressed in CRC tissues. The outcomes verified that miR-139-5p reduces proliferation, invasion and migration by straightly targeting RAP-1b and tumor protein D52, manifesting miR-139 as a putative tumor suppressor miRNA in patients with CRC(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Also, miR-139-5p might be suggested as a biomarker to predict the transformation of inflammatory bowel disease to CRC (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, we realized that methylation and expression levels of miR-139-5p as well as its target, RAP-1b protein, are not correlated with clinicopathological parameters, including histological grade, tumor stage, tumor size, lymph node metastasis, tumor type, age, gender, and distant metastasis, because on one hand, all eligible untreated patients, pathologically confirmed CRC, irrespective of sex, age, tumor type and location, histological grading, TNM staging, etc. were included in the study and the control group was matched with the patient group by age and sex (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) ; therefore, the number of patients was divided between the mentioned subgroups, including different grades and different stages, etc. ,and on the other hand we were not able to increase the number of samples in the study subgroups because of limitation of financial resources. In our opinion, it is better to calculate the sample size for subgroups to assess the relationship between the level of gene expression as well as gene methylation with clinical and pathological features of patients. Another suggestion in future surveys is that considering the larger sample size to provide a more appropriate distribution of cases between subgroups.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, although the methylation of \u003cem\u003emiR\u003c/em\u003e-139-5p in CRC disease has not been yet evaluated, a decrease in methylation-based expression has been reported for other miRNAs such as miR-148a, is hypermethylated in tissue of 65% of CRC patients with no meaningful relationship with clinicopathological specifications (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) ; and other researches have illustrated that DNA methylation has an essential impression on deregulation of some miRNAs, consist of miR-125 and 34 in CRC, and accelerating the tumor development (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe use of diagnostic tests to determine the presence or absence of a suspected disease is critical in clinical situations. The ideal test is defined by a sensitivity and specificity of 100% which is rare in the clinical setting, because sensitivity and specificity decrease as each of them increases. Also, an ideal test has a ROC curve with AUC\u0026thinsp;=\u0026thinsp;1.0, but in general, AUC\u0026thinsp;\u0026ge;\u0026thinsp;0.8 is assumed suitable and AUC\u0026thinsp;\u0026ge;\u0026thinsp;0.9 is interpreted excellent (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Graphical plot of diagnostic classification, ROC curve, presented a great AUC of 0.767, 0.958, and 0.718 of miR-139-5p, PMR value, and RAP-1b protein for plasma specimens, in the same order (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggest the ability of these biomarkers to recognize CRC disease from healthful persons. To our knowledge, there are no surveys have previously assessed the diagnostic value and methylation pattern of \u003cem\u003emiR\u003c/em\u003e-139-5p and its expression in plasma specimens. Therefore, our results compared to other miRNA\u003csup\u003e,\u003c/sup\u003es methylation modes and other classes of molecular biomarkers.\u003c/p\u003e \u003cp\u003eUsing conventional methylation-specific PCR (MSP), the sensitivity of 76.8% and a specificity of 93.6% for stool \u003cem\u003emiR\u003c/em\u003e-34a methylation test were reported (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), although compared to quantitative MSP, the mentioned technique is not quantitative and has lower accuracy for detection of CRC. Also, compared to the sensitivity (55%) and the specificity (82.5%) of plasma level of methylated \u003cem\u003emiR\u003c/em\u003e-138-5p (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), the present study has higher diagnostic value with an excellent AUC.\u003c/p\u003e \u003cp\u003eThere are different classes of molecular biomarkers by source or by type including traditional or new kind. Measurement of gene expression at mRNA level and DNA methylation in plasma belongs to genomic new biomarkers while protein detection is traditional (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Jelski Wojciech et al. reported diagnostic value of aldehyde dehydrogenase isoenzyme I (ADH I) in the serum of patients with CRC with the sensitivity and specificity of 76% and 82%, respectively (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Thus, compared to traditional type of biomarkers, the diagnostic value of \u003cem\u003emiR\u003c/em\u003e-139-5p promoter methylated marker is more favorable (the sensitivity was 75%, and specificity was 92.5% with AUC\u0026thinsp;=\u0026thinsp;0.958).\u003c/p\u003e \u003cp\u003eBecause methylation changes befalls in the primitive stages of tumor formation (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), perhaps it is possible to get an admissible clinical sensitivity by evaluating this methylation marker in the biological samples including, plasma and serum. Comparing with the CEA (46.8%) and FOBT (43.5%) results, the sensitivity and AUC of \u003cem\u003emiR\u003c/em\u003e-139-5p PMR was more in plasma, which shows potential as a diagnostic biomarker for the CRC, pending further validation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this investigation, we assessed the methylation pattern of the \u003cem\u003emiR\u003c/em\u003e-139-5p gene in liquid and soft samples of CRC patients compared to the healthful individuals for the first experience. We illustrated that miR-139-5p is significantly under expressed in the case group compared to the healthy group. Also, compared to the normal group, the rate of gene methylation in the case group was meaningfully greater, indicating the role of epigenetic factors, especially gene methylation, in the miR-139-5p down-regulation. The superiority of current survey was the concurrent assessment of miR-139-5p expression and RAP-1b protein and admeasuring the methylation mode of promoter region of the gene in liquid and soft tissue specimens, which prepared the feasibility of further comparison of the results. Finally, it could be supposed that hyper methylation inside the promoter area of the \u003cem\u003emiR\u003c/em\u003e-139-5p gene is one of the critical epigenetic agents implicated in reducing miR-139-5p expression. Due to the occurrence of methylation alterations in primary steps of carcinogenesis, it can be proposed as a miRNA-based therapeutic target and an early detection biomarker for CRC diagnosis in plasma samples. However, further study on the more number of clinical samples is necessary to confirm it as a diagnostic biomarker in patients' plasma.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eColorectal Cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emiRNAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicroRNAs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRAP- 1b\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRas-Related Protein RAP-1b\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePercentage of Methylated Reference\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROC analysis\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReceiver Operating Characteristics analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe Area Under the ROC curve\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarcino Embryonic Antigen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMethylation-Specific PCR\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study, supported by Vice Chancellery for Research\u003cbr\u003e\u0026nbsp;and Technology, Hamadan University of Medical Sciences (No. 9609286007). \u0026nbsp;The authors would like to thank the staff of the Medical Genetics Laboratory, Reference Labora\u0026shy;tory, and Kermanshah University of Medical Sciences for their help during the current research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Informed consent was obtained from all participants. The Ethics Committee of Hamadan University of medical sciences approved this survey (IR.UMSHA.REC.1396.533).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This study was funded by Vice Chancellery for Research and Technology, Hamadan University of Medical Sciences (No. 9609286007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have played key roles in various sections of the present investigation, including study design, blood and tissue sampling, bioinformatics studies, carrying out experiments, data analysis, writing, reading and approving the final manuscript.\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\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData from this survey are accessible on a reasonable request by AM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competitive interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGuren MG. The global challenge of colorectal cancer. Lancet Gastroenterol Hepatol. 2019;4(12):894\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYasudome R, Seki N, Asai S, Goto Y, Kita Y, Hozaka Y, et al. Molecular pathogenesis of colorectal cancer: Impact of oncogenic targets regulated by tumor suppressive miR-139-3p. Int J Mol Sci. 2022;23(19):11616.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTorre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. Cancer J Clin. 2015;65(2):87\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. Cancer J Clin. 2011;61(2):69\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi Y, Xie G, Jia W. Metabonomics of Human Colorectal Cancer: New Approaches for Early Diagnosis and Biomarker Discovery. J Proteome Res. 2014;13(9):3857\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Sohaily S, Biankin A, Leong R, Kohonen‐Corish M, Warusavitarne J. Molecular pathways in colorectal cancer. J Gastroenterol Hepatol. 2012;27(9):1423\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHammond WA, Swaika A, Mody K. Pharmacologic resistance in colorectal cancer: a review. Therapeutic Adv Med Oncol. 2016;8(1):57\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie N, Meng Q, Zhang Y, Luo Z, Xue F, Liu S, et al. MicroRNA\u0026ndash;142\u0026ndash;3p suppresses cell proliferation, invasion and epithelial\u0026ndash;to\u0026ndash;mesenchymal transition via RAC1\u0026ndash;ERK1/2 signaling in colorectal cancer. Mol Med Rep. 2021;24(2):1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSengupta D, Deb M, Kar S, Shilpi A, Parbin S, Mallick B, et al. Epigenetic MicroRNA Regulation of Multiple Chromatin Functions: A Perspective in Cancer. Epigenetic Diagnosis Therapy. 2015;1(2):81\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBardhan K, Liu K. Epigenetics and colorectal cancer pathogenesis. Cancers. 2013;5(2):676\u0026ndash;713.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDwivedi RS, Herman JG, McCaffrey TA, Raj DS. Beyond genetics: epigenetic code in chronic kidney disease. Kidney Int. 2011;79(1):23\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerman JG. Hypermethylation pathways to colorectal cancer: implications for prevention and detection. Gastroenterol Clin. 2002;31(4):945\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJelski W, Mroczko B. Biochemical Markers of Colorectal Cancer - Present and Future. Cancer Manag Res. 2020;12:4789\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang P, Yin J, Yuan L, Wang Q, Du X, Dong R, et al. MicroRNA\u0026ndash;139 suppresses hepatocellular carcinoma cell proliferation and migration by directly targeting Topoisomerase I. Oncol Lett. 2019;17(2):1903\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalili N, Nouri-Vaskeh M, Segherlou ZH, Baghbanzadeh A, Halimi M, Rezaee H, et al. Diagnostic, prognostic, and therapeutic significance of miR-139-5p in cancers. Life Sci. 2020;256:117865.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBozgeyik İ. miRNAs, cancer, and unconventional miRNA functions. Bull Biotechnol. 2023;4(1):36\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen K, Mao R, Ma L, Li Y, Qiu Y, Cui D, et al. Post-transcriptional regulation of the tumor suppressor miR‐139‐5p and a network of miR‐139‐5p‐mediated m RNA interactions in colorectal cancer. FEBS J. 2014;281(16):3609\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun C, Sang M, Li S, Sun X, Yang C, Xi Y, et al. Hsa-miR-139-5p inhibits proliferation and causes apoptosis associated with down-regulation of c-Met. Oncotarget. 2015;6(37):39756.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFinlin BS, Andres DA. Rem Is a New Member of the Rad- and Gem/Kir Ras-related GTP-binding Protein Family Repressed by Lipopolysaccharide Stimulation*. J Biol Chem. 1997;272(35):21982\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFischer TH, Gatling MN, McCormick F, Duffy CM. White 2nd G. Incorporation of Rap 1b into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium. J Biol Chem. 1994;269(25):17257\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen K, Liang Q, Xu K, Cui D, Jiang L, Yin P, et al. MiR-139 inhibits invasion and metastasis of colorectal cancer by targeting the type I insulin-like growth factor receptor. Biochem Pharmacol. 2012;84(3):320\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsefi M, Saidijam M, Rezvani N, Soltanian AR, Khalilian AR, Mahdavinezhad A. A novel epigenetic biomarker, plasma miR-138-5p gene promoter-methylated DNA, for colorectal cancer diagnosis. Per Med. 2022;19(4):315\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLange CP, Campan M, Hinoue T, Schmitz RF, van der Meulen-de AE, Slingerland H, et al. Genome-scale discovery of DNA-methylation biomarkers for blood-based detection of colorectal cancer. PLoS ONE. 2012;7(11):e50266.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim H-J, Yu M-H, Kim H-G, Byun J-H, Lee C. Noninvasive molecular biomarkers for the detection of colorectal cancer. BMB Rep. 2008;41(10):685\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEads CA, Lord RV, Wickramasinghe K, Long TI, Kurumboor SK, Bernstein L, et al. Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res. 2001;61(8):3410\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008;3(6):1101\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNahm FS. Receiver operating characteristic curve: overview and practical use for clinicians. Korean J Anesthesiol. 2022;75(1):25\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmirkhah R, Schmitz U, Linnebacher M, Wolkenhauer O, Farazmand A. MicroRNA\u0026ndash;mRNA interactions in colorectal cancer and their role in tumor progression. Genes Chromosom Cancer. 2015;54(3):129\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang LL, Huang LW, Wang L, Tong BD, Wei Q, Ding XS. Potential role of miR-139-5p in cancer diagnosis, prognosis and therapy. Oncol Lett. 2017;14(2):1215\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang L-h, Sun D-w, Li J, Tang J-h. MiR-139-5p: promising biomarker for cancer. Tumor Biology. 2015;36(3):1355\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBreving K, Esquela-Kerscher A. The complexities of microRNA regulation: mirandering around the rules. Int J Biochem Cell Biol. 2010;42(8):1316\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndo T, Yoshida T, Enomoto S, Asada K, Tatematsu M, Ichinose M, et al. DNA methylation of microRNA genes in gastric mucosae of gastric cancer patients: its possible involvement in the formation of epigenetic field defect. Int J Cancer. 2009;124(10):2367\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo H, Hu X, Ge S, Qian G, Zhang J. Regulation of RAP1B by miR-139 suppresses human colorectal carcinoma cell proliferation. Int J Biochem Cell Biol. 2012;44(9):1465\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStork PJ. Does Rap1 deserve a bad Rap? Trends Biochem Sci. 2003;28(5):267\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelvakumar SC, Preethi KA, Sekar D. MicroRNAs as important players in regulating cancer through PTEN/PI3K/AKT signalling pathways. Biochimica et Biophysica Acta (BBA) -. Reviews Cancer. 2023;1878(3):188904.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHowell PM, Liu Z, Khong HT. Demethylating Agents in the Treatment of Cancer. Pharmaceuticals (Basel). 2010;3(7):2022\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLai X, Eberhardt M, Schmitz U, Vera J. Systems biology-based investigation of cooperating microRNAs as monotherapy or adjuvant therapy in cancer. Nucleic Acids Res. 2019;47(15):7753\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIbrahim AF, Weirauch U, Thomas M, Gr\u0026uuml;nweller A, Hartmann RK, Aigner A. MicroRNA replacement therapy for miR-145 and miR-33a is efficacious in a model of colon carcinoma. Cancer Res. 2011;71(15):5214\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan M, Ma X, Wang F, Zhou Z, Zhang J, Zhou D, et al. MicroRNA-30b-5p functions as a metastasis suppressor in colorectal cancer by targeting Rap1b. Cancer Lett. 2020;477:144\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang Z, Peng H, Chen J, Liu Y, Yan S, Yu G, et al. Rap1b enhances the invasion and migration of hepatocellular carcinoma cells by up-regulating Twist 1. Exp Cell Res. 2018;367(1):56\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang P, Gu J, Wang K, Shang J, Wang W. miR-206 inhibits thyroid cancer proliferation and invasion by targeting RAP1B. J Cell Biochem. 2019;120(11):18927\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Li Z, Han J, Tian Z, Meng Q, Niu W. KLF7 enhances the invasion and migration of colorectal cancer cells via the miR-139-5p/TPD52 axis. Cancer Biol Ther. 2024;25(1):2385172.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeris Zayeri Z, Parsi A, Shahrabi S, Kargar M, Davari N, Saki N. Epigenetic and metabolic reprogramming in inflammatory bowel diseases: diagnostic and prognostic biomarkers in colorectal cancer. Cancer Cell Int. 2023;23(1):264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalimutho M, Di Cecilia S, Blanco GDV, Roviello F, Sileri P, Cretella M, et al. Epigenetically silenced miR-34b/c as a novel faecal-based screening marker for colorectal cancer. Br J Cancer. 2011;104(11):1770\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Chen Y, Chen Z. miR-125a/b Regulates the Activation of Cancer Stem Cells in Paclitaxel-resistant Colon Cancer. Cancer Invest. 2013;31(1):17\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJun HH, Kwack K, Lee KH, Kim JO, Park HS, Ryu CS, et al. Association between TP53 genetic polymorphisms and the methylation and expression of miR-34a, 34b/c in colorectal cancer tissues. Oncol Lett. 2019;17(5):4726\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu XD, Song YC, Cao PL, Zhang H, Guo Q, Yan R, et al. Detection of miR-34a and miR-34b/c in stool sample as potential screening biomarkers for noninvasive diagnosis of colorectal cancer. Med Oncol. 2014;31(4):894.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePi\u0026ntilde;ero J, Rodriguez Fraga PS, Valls-Margarit J, Ronzano F, Accuosto P, Lambea Jane R, et al. Genomic and proteomic biomarker landscape in clinical trials. Comput Struct Biotechnol J. 2023;21:2110\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJelski W, Mroczko B, Szmitkowski M. The diagnostic value of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) measurement in the sera of colorectal cancer patients. Dig Dis Sci. 2010;55(10):2953\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biomarkers, Colorectal Neoplasms, DNA Methylation and human MIRN139","lastPublishedDoi":"10.21203/rs.3.rs-5527622/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5527622/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: The pathogenesis of CRC requires primary genetic and epigenetic mechanisms including, methylation of CpG islands of the genes. In the current study, micro RNA-139-5p (\u003cem\u003emiR\u003c/em\u003e-139-5p) promoter methylated DNA was evaluated in tumor tissue and plasma samples from CRC affected patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003e\u003cem\u003eMiR\u003c/em\u003e-139-5p promoter methylation was investigated in 80 samples of tumoral tissue and healthy marginal tissue and the same number of plasma samples, using the MethyLight method. The miR-139-5p expression was assessed using the qPCR method. BT (Bioassay Technology) Elisa kit was applied to measure RAP-1b as a target gene of miR-139-5p.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Median PMR values of 12.4 (95% CI, 3.23-32.25) and 0.66 (95%CI, 0.51-1.0) were obtained from plasma samples of CRC patients and controls, sequentially. In plasma samples, the sensitivity and specificity of \u003cem\u003emiR\u003c/em\u003e-139-5p promoter methylated marker were 75% and 92.5%, in the same order (AUC =0.958).\u003c/p\u003e\n\u003cp\u003eLower expression of miR-139-5p in plasma and tumor tissue of patients (P\u0026lt; 0.001) was shown. Also, a significant rise of RAP-1b protein concentration was observed in both mentioned specimens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Hyper-methylation of \u003cem\u003emiR\u003c/em\u003e-139-5p could be suggested as high accuracy diagnostic biomarker for the detection of CRC in plasma samples.\u003c/p\u003e","manuscriptTitle":"Evaluation of Epigenetic Silencing of the miR-139-5p gene in the pathogenesis of colorectal cancer and its diagnostic biomarker capability in plasma samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 05:04:46","doi":"10.21203/rs.3.rs-5527622/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-24T11:25:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-24T11:09:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309915146932970686705619371326803577914","date":"2025-04-24T09:49:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-22T13:40:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18845800086936999441168253671547495134","date":"2025-04-21T07:12:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230831179765515070544762017136866888099","date":"2025-04-19T06:15:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221675101604117662149086538936672454719","date":"2025-04-09T07:13:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-07T06:59:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-07T05:05:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-04-06T09:35:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"388bf406-3a2d-4a41-a6ab-091ce06bc117","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:03:34+00:00","versionOfRecord":{"articleIdentity":"rs-5527622","link":"https://doi.org/10.1186/s12885-025-14290-x","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2025-05-15 15:57:26","publishedOnDateReadable":"May 15th, 2025"},"versionCreatedAt":"2025-04-10 05:04:46","video":"","vorDoi":"10.1186/s12885-025-14290-x","vorDoiUrl":"https://doi.org/10.1186/s12885-025-14290-x","workflowStages":[]},"version":"v1","identity":"rs-5527622","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5527622","identity":"rs-5527622","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}