{"paper_id":"909ea01e-c64d-4803-a405-1d2aca2c3691","body_text":"Endometriosis (EMs) is a chronic and recurrent, but benign, disease in women of reproductive age, with a morbidity of approximately 10%. It is characterized by the presence of functional endometrial glands and stroma outside the uterine cavity[ 1 , 2 ]. Typical symptoms of EMs include cyclic pelvic pain, dysmenorrhea, dyspareunia, and infertility. Previous studies have reported that EMs patients have a high risk of developing gynecological tumors and autoimmune disorders[ 3 , 4 ]. Thus, EMs can cause severe psychological and physiological harm to those affected by it and imposes a substantial social burden[ 5 , 6 ].\nDespite its high prevalence and incapacitating symptoms, the etiology of EMs is not clear. Evidence suggests that it is a multifactorial disease. Retrograde menstruation, immune system disorders, and genetic and environmental factors have been proposed as susceptibility factors for EMs[ 7 - 9 ]. The susceptibility factor of retrograde menstruation proposed by Sampson is the most widely accepted[ 10 ]. However, almost all women of reproductive age exhibit some degree of retrograde menstruation, and only 10% to 15% suffer from EMs[ 11 , 12 ]. Recently, more evidence has emerged to support the theory that genetic changes in the eutopic endometrium may be the key molecular events in the pathogenesis of EMs[ 13 ].\nMicroRNAs (miRNAs) are short noncoding RNA molecules that regulate genetic expression post-transcriptionally and are implicated in several biological processes, such as cell proliferation, differentiation, and apoptosis[ 14 , 15 ]. Some miRNAs have been reported to be abnormally expressed in reproductive cancers[ 12 , 16 , 17 ], and miR-451 is of particular interest, as it acts as a tumor suppressor and is relevant to the poor prognosis of cancers. Aberrant miR-451 expression has been shown in eutopic and ectopic endometrial tissues; however, data regarding differences in miR-451 expression in the eutopic endometrium from healthy patients and those with EMs remain inconclusive[ 18 , 19 ].\nIn our study, we examined miR-451 expression in the eutopic endometrium of women with and without EMs and evaluated the role of miR-451 in cell proliferation. Finally, we predicted possible targets of miR-451 and the related signaling pathways.\n\nPathologic tissues were collected from patients with grade III cervical intraepithelial neoplasia, including 40 with EMs and 20 without. All 60 subjects underwent total hysterectomy at the Shengjing Hospital of China Medical University between 2009 and 2010. The EMs group included 2, 6, 20, and 12 cases at American Society for Reproductive Medicine (ASRM) stage I, II, III, and IV, respectively, of the disease. None of the patients had a history of endocrine, immune, or metabolic disorders and none had received any hormonal or antibiotic treatments within 3 mo prior to surgery.\nThe ectopic and eutopic endometrial tissues were digested overnight at 37 °C with Dispase IV for 70 min and Dispase II for 50 min (Sigma, United States). After filtration through 100 and 400 mesh nylon screens, the obtained primary cells were rinsed in PBS and then cultured for 24 h in DMEM/F12 medium supplemented with 15% fetal bovine serum and antibiotics at 37 °C in an atmosphere containing 5% CO 2 .\nTotal RNA was isolated from the EMs tissues using the TRIzol reagent. cDNA was synthesized from miR-451 using a TaqMan ®  miRNA Reverse Transcription Kit and used in a 1:5 dilution ratio for qRT-PCR, which was performed, using an miRNA Assays kit and Universal Master Mix following the kit protocols (Applied Biosystems). U6 was used as the endogenous control. Conditions of reverse transcription were as follows: 16 °C for 30 min, 42 °C for 30 min, 85 °C for 5 min, and then holding at 4 °C. Conditions for qRT-PCR were as follows: enzyme activation at 95 °C for 10 min followed by 40 cycles of denaturation for 15 s at 95 °C and annealing and extension for 60 s at 60 °C. All experimental samples were run in triplicate and each qRT-PCR reaction was repeated at least two times. MiR-451 expression levels were calculated and analyzed using the 2 −ΔΔCt  relative quantitation method.\nUsing Lipofectamine TM  2000 reagent, miR-451 mimics and miR-451 inhibitors were transfected into EMs cells and normal endometrial cells, respectively. The oligonucleotide sequence of the miR-451 mimic is 5′-AAA CCG UUA CCA UUA CUG AGUU-3′, and its NC sequence is 5′-UUC UCC GAA CGU GUC ACG UTT-3′. The oligonucleotide sequence of the miR-451 inhibitor is 5′-AAC UCA GUA AUG GUA ACG GUUU-3′, and the sequence of the scrambled siRNA is 5′-CAG UAC UUU UGU GUA GUA CAA-3’. In addition, cells transfected with or without the empty vector were used as the control groups. All cells were incubated at 37 °C in an atmosphere containing 5% CO 2  for 24 to 96 h post transfection.\nCellular proliferation analysis was performed using the Cell Counting Kit-8 (CCK-8) assay. After transfection with miR-451 mimics/inhibitors for 24h, 48h, 72h, and 96 h, 2 × 10 3  cells were added to 96-well plates and incubated overnight at 37 °C in an atmosphere containing 5% CO 2 . Then, 10 μL of CCK-8 was added to each well (Beyotime Biotechnology). The cells were incubated for another 4 h at 37 °C in an atmosphere containing 5% CO 2 , and then cell viability was determined by measuring the optical density at 450 nm.\nAnnexin V-FITC/PI double-staining assays were performed for analysis of apoptosis. Cells were collected and suspended in PBS 24 h after transfection. Cells were then stained in 500 μL of binding buffer with 5 μL of each of annexin V-FITC and PI (KeyGen Biotech), incubated in the dark at room temperature for 5-15 min, and subjected to flow cytometric analysis to assess cellular apoptosis within 1 h.\nUsing miRDB ( http://mirdb.org/miRDB/index.html ) and miRcode ( http://www.mircode.org/ ), we predicted the target genes of miR-451. Expression levels of the identified targeted genes were determined by analyzing the  GSE7846  gene profile from the GEO database (https:// www.ncbi.nlm.nih.gov/geo/ ). This dataset includes the expression data of endometrial cells derived from patients with EMs (ectopic group) and without EMs (normal group). We screened the differentially expressed genes with a  P -value < 0.01 and an adjusted  P -value < 0.01 between the EMs and control groups.\nData are expressed as the mean ± SEM. Statistical comparisons between groups were determined using the  t -test and  χ 2  test. Statistical significance was defined as  P  < 0.05. Analyses were performed using the R 3.4.2 and SPSS 22.0 software.\nThis study was approved by the China Medical University Research Ethics Committee according to the Helsinki Declaration, and written informed consent was obtained from each study participant.\n\nqRT-PCR was performed to quantitatively analyze the expression levels of miR-451 in eutopic tissues from the EMs and control groups. As shown in Figure  1A , we observed a significant reduction in miR-451 expression in the EMs group compared to the control group (EMs, 0.22 ± 0.06; control, 1.12 ± 0.11,  P  < 0.01). Consistent with the tissue results, miR-451 expression in cells was significantly lower in the EMs group than in the control group (Figure  1B ). The correlation of miR-451 expression levels with ASRM stage was then analyzed, as shown in Table  1 . No significant association was found between miR-451 expression and ASRM stage ( P  > 0.05).\nComparison of miR-451 expression in patients at different american society for reproductive medicine stages\nASRM: American Society for Reproductive Medicine.\nExpression of miR-451 in eutopic tissues and cell lines. A: MiR-451 expression in eutopic tissues from endometriosis (EMs) and control groups was quantified using quantitative real-time PCR. Data are expressed as 2 −ΔΔCt  (mean ± SE,  n  = 4).  b P  < 0.01  vs  control; B: Expression of miR-451 was compared between EMs and control cell lines. Data are expressed as 2 −ΔΔCt  (mean ± SEM,  n  = 4).  b P  < 0.01  vs  control.\nMiR-451 levels in eutopic cells transfected with miR-451 mimic were higher than those in the non-transfected control and scrambled mimic oligomer groups (miR-451 mimic, 1.33 ± 0.28; control, 0.25 ± 0.06; scrambled, 0.32 ± 0.09,  P  < 0.01) (Figure  2A ). CCK-8 assay results showed that transfection with miR-451 mimic suppressed the proliferation rate of EMs cells (Figure  2B ). To investigate whether the reduced cell proliferation resulted from apoptosis, we evaluated the effect of miR-451 mimic on cellular apoptosis using flow cytometry. MiR-451 mimic induced early apoptosis in a larger number of cells compared to scrambled oligonucleotides, and this difference was statistically significant ( P  < 0.01) (Figure  2C and 2D ). Thus, overexpression of miR-451 in EMs cells induces apoptosis and inhibits cell proliferation.\nTransfection with miR-451 mimic inhibits cell proliferation by inducing the apoptosis of eutopic cells in endometriosis. A: MiR-451 expression was significantly increased after transfection with miR-451 mimic. Data are expressed as 2 −ΔΔCt  (mean ± SEM,  n  = 4).  b P  < 0.01  vs  control and scrambled; B: Cell Counting Kit-8 assays revealed a lower proliferation rate of cells transfected with miR-451 mimic compared to cells in the control and scrambled groups ( a P  < 0.05); C and D: Flow cytometric analysis of apoptosis in cells transfected with scrambled siRNA and miR-451 mimic, respectively. Cells are divided into four sections: Q1: Annexin V-FITC- PI+ represents mechanical error; Q2: Annexin V-FITC+ PI+ represents late apoptotic or necrotic cells; Q3: Annexin V-FITC- PI- represents non-apoptotic cells; Q4: Annexin V-FITC+ PI- represents early apoptotic cells.\nAs shown in Figure  3A , miR-451 expression was significantly attenuated in the siRNA-transfected group compared to the non-transfected and scrambled mimic oligomer groups (miR-451 siRNA, 0.41 ± 0.14; control, 1.23 ± 0.08; scrambled, 1.06 ± 0.06,  P  < 0.01). Additionally, the proliferation ability of miR-451 siRNA-transfected cells was greater than that of the other two groups (Figure  3B ). We used flow cytometric assay to evaluate the effect of miR-451 siRNA transfection on apoptosis. Our results showed that the proportion of early apoptotic cells was significantly lower in the miR-451 siRNA group compared to that in the scrambled group ( P  < 0.01) (Figure  3C and 3D ). These results indicate that, in eutopic cells, miR-451 reduces apoptosis and increases cell proliferation.\nTransfection with miR-451 siRNA decreases apoptosis and promotes cell proliferation in control eutopic cells. A: MiR-451 expression was significantly inhibited after transfection with miR-451 siRNA. Data are expressed as 2 −ΔΔCt  (mean ± SEM,  n  = 4).  b P  < 0.01  vs  control and scrambled; B: Cell Counting Kit-8 assays revealed that the proliferation rate of cells transfected with miR-451 siRNA was higher than those of cells in the control and scrambled groups ( a P  < 0.05); C and D: Flow cytometric analysis of apoptosis in cells transfected with scrambled siRNA and miR-451, respectively.\nUsing the miRDB and miRcode miRNA target prediction databases, we identified a total of 12 genes targeted by miR-451, namely,  OSR1, MEX3C, CUX2, ZNF644, TBC1D9B, DCAF5, CDKN2B, TTN, YWHAZ, CDKN2D, EIF2AK3 , and  TBX1  (Figure  4A ). As shown in Figure  4B , among the targeted genes, the expression levels of  OSR1 ,  YWHAZ ,  TTN , and  CDKN2D  were significantly different between the two groups according to  GSE7846  ( P  < 0.05, adj.  P  < 0.05). The logFC values of  OSR1 ,  YWHAZ ,  TTN , and  CDKN2D  were 0.76, 0.43, 0.33, and 0.63, respectively. Furthermore, according to the pathway analysis data in the Kyoto Encyclopedia of Genes and Genomes,  YWHAZ  and  CDKN2D  may have important roles in the cell cycle in EMs (Figure  4C ).\nResults of bioinformatics analysis of miR-451 target genes according to the  GSE7846  dataset. A: Potential target genes of miR-451 predicted based on miRDB and miRcode databases; B: Heatmap of expression levels of  OSR1 ,  YWHAZ ,  TTN , and  CDKN2D ; C: Role of  YWHAZ  and  CDKN2D  in the cell cycle according to Kyoto Encyclopedia of Genes and Genomes pathway analysis.\n\nIn this study, qRT-PCR analysis of eutopic endometrial tissues and cells showed that miR-451 was significantly downregulated in patients with EMs compared to normal controls ( P  = 0.011). Although we did not observe a significant association between miR-451 expression and the ASRM stage of EMs, ectopic overexpression of miR-451 in eutopic cells in EMs was shown to be associated with reduced cell proliferation and increased apoptosis. Conversely, siRNA-mediated knockdown of miR-451 promoted the proliferation and reduced the apoptosis of eutopic cells.\nThe “eutopic endometrium determinism” theory suggests that the occurrence of EMs is mainly dependent on the characteristics of eutopic endometrial lesions, and retrograde menstruation may act as a precipitating factor. Thus, genetic dysregulation in the endometrium is crucial in the pathogenesis of EMs. Identifying differentially expressed genes between patients with and without EMs would serve as a minimally invasive method to diagnose EMs and evaluate the risk of recurrence. For example, Mahdian et al[ 20 ] reported that  MIF ,  CD74 , and  COX-2  are essential in inflammation and endometrium reconstruction during the menstrual cycle, and increased expression of these genes is a molecular biomarker for the development and pathophysiology of EMs. In addition, Sapkota et al[ 21 ] also identified five novel loci ( CCDC170 ,  FN1 ,  SYNE1 ,  ESR1 , and  FSHB ) and nineteen independent single nucleotide polymorphisms that are significantly associated with the risk of EMs.\nFurthermore, miRNAs regulate the expression of target genes and key cellular processes in EMs. In 2009, Burney et al[ 14 ] reported the downregulation of the miR-9 and miR-34 miRNA families in eutopic cells in the setting of EMs, and this downregulation is closely related to progesterone resistance in early secretory endometrium. Laudanski et al[ 22 ] showed that miR-483-5p and miR-629-3p are downregulated in EMs, and this is associated with inflammation. Moreover, miR-21 was shown to be significantly upregulated in severe EMs (stage III/IV) compared to mild EMs (stage I/II)[ 23 ]. Notably, miR-451 has been established as a tumor-suppressor gene in gastric, colorectal, bladder, and non-small cell lung carcinomas[ 24 ], and it was also shown to be downregulated in ovarian cancer compared to its concurrent EMs[ 25 ]. In addition, Nothnick et al[ 26 ] showed that deficiency of miR-451 regulates fibrinogen alpha chain and reduces endometrial implantation in a mouse model. Similarly, we found that miR-451 was downregulated in the eutopic endometrium in EMs compared to normal controls in studies involving both tissues and cells.\nUsing miRNA target-predicting databases, we identified 12 potential target genes of miR-451 and analyzed their expression levels according to the  GSE7846  dataset. Finally, a total of four genes,  YWHAZ ,  OSR1 ,  TTN , and  CDKN2D , were selected for further analysis. Among these target genes of miR-451,  YWHAZ  has previously been shown to be overexpressed in tissues in EMs[ 19 , 27 ]. Joshi et al[ 19 ] reported that miR-451 regulates  YWHAZ  expression and promotes proliferation of eutopic cells in baboons with EMs[ 19 ]. However, the roles of  OSR1 ,  TTN , and  CDKN2D  in EMs have not been reported until now. Published reports suggest that  OSR1  inhibits proliferation and induces cellular apoptosis by acting on the WNK and NF-κB pathways, and  OSR1  is dramatically downregulated in several carcinomas[ 28 - 30 ]. In addition,  CDKN2D  has been shown to be involved in carcinogenesis and has been identified in gynecological cancers. This gene may be regulated by miR-451 in esophageal carcinoma cell lines[ 31 ]. Thus, our study provides several novel therapeutic targets for EMs.\nNotably, most studies on EMs have only focused on identifying differences between ectopic lesions and eutopic endometrium. For example, Graham et al[ 18 ] reported that miR-451 is overexpressed in ectopic lesions compared to eutopic lesions and reduces cell survival by regulating  MIF . In this study, we found significant differences in miR-451 expression in the eutopic endometrium of patients with and without EMs, which effectively supports the “eutopic endometrium determinism” theory. Furthermore, we identified four potential target genes of miR-451 by bioinformatics analysis and analyzed their downstream pathways.\nOur study has two limitations. First, the number of included patients was relatively small. Second, the  in silico -predicted targets of miR-451 need to be validated through experiments, such as 3′-UTR luciferase reporter assays. However, we believe that our results indicate a novel role of miR-451 in EMs and support several potential biomarkers in the form of miR-451 targets that may be used for future clinical diagnosis and therapy of this disease.\nIn conclusion, miR-451 is a novel biomarker for EMs and is downregulated in the eutopic endometrium.  YWHAZ ,  OSR1 ,  TTN , and  CDKN2D  are potential target genes of miR-451 and may have important roles in the pathogenesis of EMs.\n\nDespite the high prevalence of endometriosis (EMs), its etiology is unclear.\nMiR-451 acts as a tumor suppressor and is relevant to the poor prognosis of cancers.\nTo evaluate the expression levels and role of miR-451 in the eutopic endometrium and predict possible targets of miR-451 and related signaling pathways.\nQuantitative real-time PCR was used to evaluate miR-451 expression. Cell Counting Kit-8 and flow cytometric assays were performed to determine cell proliferation and survival rates.\nMiR-451 was downregulated in the eutopic endometrium and related with EMs cell proliferation and apoptosis.  YWHAZ ,  OSR1 ,  TTN , and  CDKN2D  were identified as potential target genes of miR-451.\nReduced miR-451 expression in the eutopic endometrium contributes to the pathogenesis of EMs by promoting cell proliferation and reducing apoptosis.\nMiR-451 is a novel biomarker for EMs.  YWHAZ ,  OSR1 ,  TTN , and  CDKN2D  are potential target genes of miR-451 and may have key roles in this disease.","source_license":"CC0","license_restricted":false}