| Objective: Endometriosis is a common inflammatory disease characterized by the presence of functional endometrial glands and stroma outside the uterine cavity.Typical clinical manifestations include chronic pelvic pain,dysmenorrhoea,dyspareunia,and infertility.Endometriosis affects up to 10% of women of reproductive age and up to 50% of women with infertility and/or pelvic pain.Despite being a benign disease,endometriosis shows malignant biological behaviors such as distant metastasis,adhesion,invasion,implantation,and recurrence.Transforming growth factor ?(TGF-?)is a multifunctional cytokine regulating cell differentiation,proliferation,angiogenesis,and immune responses.Studies have shown that the level of TGF-? elevates in peritoneal fluid and serum of endometriosis patients,indicating that TGF-? signaling pathways play important roles in pathogenesis of endometriosis.TGF-? initiates the downstream signaling pathways through transmembrane type I(TGF-?receptor 1,TGFBR1)and type II(TGF-?receptor 2,TGFBR2)receptors.When binding to TGF-?,TGFBR2 recruits and activates TGFBR1,which in turn phosphorylates its downstream key signaling molecules,SMAD2 and SMAD3.The activated SMADs then bind to SMAD4 and transfer to the nucleus to regulate subsequent gene expression.TGF-?/SMAD signaling pathway is involved in the pathogenesis of endometriosis by regulating a variety of pathophysiological processes,among which epithelial-mesenchymal transition(EMT)is widely studied.EMT is characterized by the loss of cell polarity and cell-cell contacts and gain of mesenchymal phenotypes,which in turn induces increased migration and invasiveness in epithelial cells,and exerts an important role in the pathogenetic process of endometriosis.Micro RNAs(mi RNAs or mi Rs)are a class of endogenous single strand non-coding small RNAs with 20-25 nucleotides in length,which bind to the 3' untranslated region(3'UTR)of target genes and negatively regulate their expression by inhibition of translation or degradation of m RNAs in a post-transcriptional pattern.micro RNA has been shown to be involved in a variety of cellular functions,including cell differentiation,proliferation,apoptosis,migration and invasion.Recently,many studies have reported significant dysregulation of micro RNAs in the circulation in endometriosis patients,suggesting micro RNAs may be potential therapeutic targets of endometriosis.mi R-96-5p is a member of the mi R-183-96-182 cluster,and has been reported to act as oncogene or tumor suppressor in different kinds of tumors,for example hepatocellular carcinoma,thyroid carcinoma,pancreatic cancer,colorectal cancer,renal cancer,cervical cancer,breast cancer and osteosarcoma.However,to the best of our knowledge,the specific role of mi R-96-5p in endometriosis has not been investigated so far.Methods: Real-time PCR was used to detect the expression of mi R-96-5p in eutopic and ectopic endometrial tissue of patients with endometriosis.Real-time PCR and western blot were used to detect the expression of TGFBR1 and SMAD3 in eutopic and ectopic endometrial tissue of patients with endometriosis.Endometrial cell lines Ishikawa and End1/E6E7 were transfected with mi R-96-5p mimics or inhibitor to overexpress or knockdown mi R-96-5p,and the transfection efficiency was validated by RT-q PCR.CCK-8 assay was adopted to detect the change of endometrial cell proliferation.Wound healing assay was used to detect the change in cell migration ability,and transwell assay was used to detect the change in cell invasion ability.Endometrial cell lines Ishikawa and End1/E6E7 were transfected with TGFBR1 small interfering RNA(si RNA)to knockdown the expression of TGFBR1,and the transfection efficiency was validated by RT-q PCR.CCK-8 assay was adopted to detect the change of endometrial cell proliferation.Wound healing assay was used to detect the change in cell migration ability,and transwell assay was used to detect the change in cell invasion ability.According to bioinformatics prediction,mi R-96-5p might bind the 3?UTR of TGFBR1.The HEK293 T cells were cotransfected with wild-type TGFBR1 3?UTR or mutant TGFBR1 3?UTR along with mi R-96-5p mimics or NC and dual-luciferase reporter assay was performed to explore whether mi R-96-5p directly targets TGFBR1.RT-q PCR and western blot were used to detect the expression of TGFBR1 in Ishikawa and End1 / E6E7 endometrial cells following overexpression of mi R-96-5p.Ishikawa and End1/E6E7 cells were the End1/E6E7 and Ishikawa cells were treated with TGF-?1 of different concentration(0ng/ml,0.5ng/ml,1ng/ml,2.5ng/ml,5ng/ml,10ng/ml),RT-q PCR and western blot were used to detect the expression of TGFBR1,TGFBR2 and SMAD3.Western blot was used to detect the expression of E-cadherin,N-cadherin and vimentin.mi R-96-5p and negative control(NC)were transfected in Ishikawa and end1 / E6E7 cell lines treated with the optimal concentration of TGF-?1.Western blot was used to verify the changes in expression levels of TGFBR1,TGFBR2,SMAD3,E-cadherin,N-cadherin and vimentin.CCK-8 assay was used to detect the proliferation ability of both cell lines overexpressing mi R-96-5p after TGF-?1 treatment.Wound healing assay was used to detect cell migration ablity,and transwell assay was used to detect cell invasion ablity.The effect of mi R-96-5p on biological behaviors of endometrial cells through targeting TGFBR1 and TGF-?/SMAD signaling pathway was explored.Results: The expression of mi R-96-5p was significantly decreased in ectopic endometrial tissue compared with eutopic endometrial tissue of endometriosis patients.The expression of TGFBR1 and SMAD3 was increased in ectopic endometrium samples compared with eutopic endometrial samples.The expression of mi R-96-5p was negatively correlated with the expression of TGFBR1.CCK-8 assay indicated that overexpression of mi R-96-5p significantly suppressed cell proliferation in Ishikawa and End1/E6E7 cells.Wound healing assay and transwell assay indicated that overexpression of mi R-96-5p inhibited cell migration and invasion in Ishikawa and End1/E6E7 cell lines.The effect of knockdown of mi R-96-5p on cell biological behaviors was opposite to that of overexpressing mi R-96-5p.Additionally,CCK-8 assay indicated that knockdown of TGFBR1 significantly suppressed cell proliferation in Ishikawa and End1/E6E7 cells.Wound healing assay and transwell assay indicated that knockdown of TGFBR1 inhibited cell migration and invasion in Ishikawa and End1/E6E7 cell lines.According to bioinformatics prediction,mi R-96-5p might bind the 3?-UTR of TGFBR1,and dual-luciferase reporter assay confirmed that TGFBR1 is a direct target of mi R-96-5p.In addition,the m RNA and protein expression of TGFBR1 was significantly reduced following mi R-96-5p mimics transfection compared with NC transfection group.PCR and western-blot indicated that TGF-?1 activated TGF-?/SMAD signaling pathway and increased the expression of TGFBR1,TGFBR2 and SMAD3 in Ishikawa and End1/E6E7 cells.Furthermore,the expression of E-cadherin was downregulated,while the expression of N-cadherin and vimentin was upregulated,indicating that TGF-?1 could induce EMT in endometrial cell lines.Western blot showed that after TGF-?1 stimulation,mi R-96-5p overexpression suppressed the expression of TGFBR1 and SMAD3,however,the expression of TGFBR2 remained unchanged,indicating that mi R-96-5p reversed the TGF-?1 activation of TGF-?/SMAD signaling pathway,probably by targeting TGFBR1.Additionally,mi R-96-5p partially reversed the EMT induced by TGF-?1,which is characterized by elevated expression of E-cad and decreased expression of N-cad and vimentin.CCK-8 assay showed that TGF-?1 could promote cell proliferation,however,the promotive effect was reduced by mi R-96-5p overexpression.Moreover,transwell showed that mi R-96-5p could attenuate the promoting effect of TGF-?1 on cell migration.As expected,wound assay results also showed that mi R-96-5p could inhibit the migration ability of endometrial cells induced by TGF-?1.Conclusion:1.mi R-96-5p was downregulated in ectopic endometrial tissue of endometriosis patients.TGFBR1 was upregulated in ectopic endometrial tissue of endometriosis patients.2.TGFBR1 is a direct target of mi R-96-5p.3.mi R-96-5p inhibited the biological behaviors of endometrial cells and reversed EMT in endometrial cells through negative regulation of TGFBR1. |
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