MiR-206 Inhibits Cell Proliferation,Migration,and Invasion By Targeting BAG3 In Human Cervical Cancer

IntroductionCervical cancer is one of the most common malignant tumors of the genital tract in women worldwide.According to statistics,there are about 500,000 new cases every year,and most of them occur in developing countries.China accounts for about one third of the world's total annual new cases.At present,cervical intraepithelial neoplasia(CIN)has been recognized as a precancerous lesion of cervical cancer,while persistent infection of high-risk human papillomavirus(HR-HPV)can induce about 1%of CIN patients to progress to cervical cancer.According to statistics,the occurrence period of cervical cancer from CIN to cervical cancer is about 8-10 years,which shows that the occurrence of cervical cancer involves a variety of factors.Therefore,this paper aims to elucidate the potential molecular mechanism of cervical cancer and explore the mechanism of abnormal expression of molecules in cervical cancer cells in the occurrence,development and lymphatic metastasis of cervical cancer.The research results will contribute to the development and application of targeted drugs for cervical cancer.MicroRNAs(microRNAs)have been reported as a non-coding RNA with a length of about 19-23 nucleotides.In recent decades,microRNAs have played a role in many malignant tumors as oncogenes or tumor suppressor genes.Generally speaking,microRNAs inhibit the transcription of tumor-related genes and down-regulate their expression by binding to the complementary sequence of the 3'-untranslated region(3'-UTR)of the corresponding RNA.It is noteworthy that miR-206 can regulate the expression of different target genes by targeting 3'-UTR of multiple genes,and play an important role in cell differentiation,proliferation and apoptosis.Previous studies have confirmed that Bcl2-associated athanogene 3(BAG3)is also involved in many biological processes of cervical cancer,including cell proliferation,migration and invasion.BAG3 possesses Bag region,which is one of the family proteins of molecular chaperone.Bag3 can bind with HSP22 to form a molecular chaperone complex.HSP22 is responsible for recognizing misfolded proteins,whereas BAG3 with proline domain attracts and activates macrophages through this domain,and further relies on macrophage autophagy to promote the degradation of misfolded proteins,thereby promoting the development of cancer cells.However,the role of the miR-206/BAG3 pathway in cervical cancer remains unknown.In this study,we will detect the expression of miR-206 and BAG3 in cervical squamous cell carcinoma tissues and cervical cancer cell lines SiHa and HeLa,and then explore the effects of miR-206 and BAG3 on the proliferation,migration and invasion of cervical cancer cells in vitro and in vivo.Finally,we will evaluate whether BAG3 is an effective target for miR-206.The purpose of this study was to investigate the expression of miR-206 in cervical cancer,it's possible molecular regulation mechanism and its relationship with the occurrence,development and metastasis of cervical cancer,and to provide new ideas for clinical treatment.Part I Expression and Significance of miRNA-206 and BAG3 in Cervical Squamous Cell CarcinomaObjective:To clarify the expression of miR-206,BAG3 and their proteins in cervical cancer tissues and adjacent tissues,and to analyze the relationship between miR-206,BAG3 and the occurrence and metastasis of cervical squamous cell carcinoma combined with clinicopathological data.Methods:Firstly,50 clinical samples of fresh cervical squamous cell carcinoma were collected,and total RNA and protein were extracted from cancer tissues and corresponding adjacent tissues;the expression of miR-206 and BAG3 was detected by RT-PCR and real-time fluorescence quantitative PCR;the expression of BAG3 protein was detected by Western blot;and the relationship between miR-206 and BAG3 was analyzed statistically to clarify the correlation between miR-206 and BAG3,and clinicopathological data of patients with cervical squamous cell carcinoma.The correlation between the expression of miR-206 and BAG3 and pelvic lymph node metastasis was analyzed.Result:1.Compared with the adjacent tissues,the expression of miR-206 in cervical squamous cell carcinoma was significantly down-regulated,and the expression of BAG3 and its protein in cervical squamous cell carcinoma was significantly up-regulated(P<0.01).2.Compared with cervical squamous cell carcinomas without pelvic lymph node metastasis,the expression of miR-206 in cervical squamous cell carcinomas with metastasis was significantly down-regulated;however,the expression of BAG3 and its protein was significantly up-regulated in cervical squamous cell carcinomas with metastasis(P<0.01).3.In cervical squamous cell carcinoma,the expression of miR-206 was negatively correlated with FIGO stage and pelvic lymph node metastasis(P<0.05),but not with age and tumor diameter(P>0.05).In addition,the expression levels of BAG3 gene and protein were positively correlated with clinical stage and lymph node metastasis(P<0.05),but not with age and tumor diameter(P>0.05).4.Linear regression and correlation analysis showed that the expression of miR-206 was negatively correlated with BAG3 gene or protein(R2 = 0.841,P<0.001;R2 = 0.766,P<0.001).Conclusion:1.The down-regulated expression of miR-206 and up-regulated expression of BAG3 and BAG3 protein in cervical squamous cell carcinoma suggest that miR-206 may act as an anti-oncogene and BAG3 may act as a carcinogenic gene in cervical cancer.2.miR-206 and BAG3 are negatively correlated and significantly correlated with FIGO staging and lymph node metastasis,suggesting that they may be involved in the invasion and metastasis of cervical squamous cell carcinoma.Part II:The effect of miR-206 on proliferation,migration and invasion of cervical cancer cells by targeting BAG3 expressionObjective:1.In view of the negative correlation between the expression of miR-206 and BAG3 in cervical cancer tissues,this part will study the effects of miR-206 and BAG3 on the proliferation,migration and invasion of cervical cancer cells in vitro and their target relationship.2.To explore the effect of miR-206 on the growth and proliferation of cervical cancer cells through animal experiments,and explore the related mechanisms,in order to clarify the mechanism of miR-206 in the process of cervical cancer deterioration,and lay the foundation for finding new molecular markers and drug targets.Method:1.SiHa and HeLa cells were cultured in vitro.The expression of miR-206 and BAG3 was detected by RT-PCR and real-time fluorescence quantitative PCR.The expression of BAG3 protein was detected by Western Blot.2.SiHa and HeLa cells were transfected with mimics or control RNA s.The proliferation of cervical cancer cells was analyzed by CCK-8 method.3.The effects of miR-206 on cell migration and invasion were identified by wound healing and Transwell method of Matrigel.4.Bioinformatics and double luciferase reporter gene analysis of the target relationship between miR-206 and BAG3;5.To construct a lentivirus stably overexpressed or silenced by BAG3 siRNA(si-BAG3)and infect SiHa and HeLa cells.To analyze whether interfering with the expression of BAG3 can reverse the biological effects of miR-206 on cancer cells by co-transfecting mimic or mir-NC.6.SiHa cells were randomly divided into four groups:miR-206 + si-BAG3 transfection group;miR-206 + Si-Control transfection group;Mi-NC + si-BAG3 transfection group;Mi-NC + Si-Control transfection group.The expression of miR-206 and BAG3 was detected by fluorescence quantitative PCR.Cells of each group were inoculated subcutaneously into nude mice to establish cervical cancer xenograft model in nude mice and observe the tumorigenesis of nude mice.The size of the tumor was measured by vernier caliper,the volume and weight of the transplanted tumor were calculated and the volume of the tumor was plotted.The expression of BAG3 protein was detected by Western blot.Results:1.The expression of miR-206 and BAG3 in cervical cancer cellsCompared with normal cells,the expression of miR-206 in SiHa and HeLa cells decreased significantly(P<0.01).On the other hand,the expression of BAG3 in SiHa and HeLa cells was significantly higher than that in normal cells(P<0.01).2.miR-206 inhibits the proliferation of cervical cancer cells CCK-8 assay showed that the over-expression of miR-206 significantly decreased the proliferation of SiHa and HeLa cells compared with the control group(P<0.01).At the same time,immunoblotting assay was used to detect cell proliferation-related proteins,including EGFR,Bcl-2 and Bax proteins.The results showed that the expression of EGFR and Bcl-2 in SiHa and HeLa cells was significantly decreased by the mimic of miR-206 compared with the control group(P<0.01).On the contrary,the expression of Bax protein in SiHa and HeLa groups transfected with miR-206 mimics was significantly higher than that in control group(P<0.01).3.miR-206 inhibits migration and invasion of cervical cancer cellsThe analysis of wound healing experiment showed that the overexpression of miR-206 significantly inhibited the migration of SiHa and HeLa cells(P<0.01).In addition,Transwell assay showed that the overexpression of miR-206 significantly inhibited the invasive ability of SiHa and HeLa cells compared with the control group(P<0.01).Western blot showed that the expression of MMP2 and MMP9 in SiHa and HeLa cells was significantly decreased by the mimic of miR-206(P<0.01).4.BAG 3's 3'-UTR is the direct target of miR-206The results of double luciferase reporter gene analysis showed that the fluorescence intensity of wild-type BAG3-3'UTR-wt cells was significantly decreased by miR-206 mimics in a dose-dependent manner(P<0.01),while the fluorescence intensity of BAG3-3'UTR-mut cells was not changed by miR-206 mimics(P>0.05).Western blot analysis showed that BAG3 protein expression was significantly decreased in SiHa and Hela cell lines co-transfected with miR-206 and 3'-UTR-wt,but not in SiHa and Hela cell lines co-transfected with miR-206 and 3'-UTR-mut(P>0.05).5.Overexpression of BAG3 partially reversed the inhibitory effect of miR-206.Overexpression of BAG3 effectively increased the expression of BAG3 in SiHa and HeLa cells.CCK-8 analysis showed that over-expression of BAG3 promoted the proliferation of SiHa and HeLa cells and partially reversed the inhibition of cell proliferation by miR-206(P<0.01).The wound healing and Transwell assay showed that BAG3 overexpression also partially reversed migration and invasion of SiHa and HeLa cells inhibited by mimics of miR-206(P<0.01).6.Reducing the expression of BAG3 partially promotes the inhibitory effect of miR-206BAG3 siRNAs significantly inhibited the expression of BAG3 protein.CCK-8 analysis showed that compared with the other groups,the mimic of miR-206 combined with BAG3 siRNA inhibited the proliferation of SiHa and HeLa cells more effectively(P<0.01).The results of wound healing and Transwell experiment showed that the co-transfection of miR-206 with BAG3 siRNA effectively inhibited the migration and invasion of SiHa and HeLa cells(P<0.01)compared with blank control,mimic alone or BAG3 siRNA alone treatment group.7.miR-206 inhibits the growth of transplanted tumors in nude miceNude mice with mimics of miR-206 or si-BAG3 in each group survived for 14 days.Western blot results showed that mimic or si-BAG3 significantly inhibited BAG3 expression compared with mir-NC or silent control group(P<0.01).In addition,the expression of BAG3 protein was inhibited by both miR-206 mimics and si-BAG3(P<0.01).Statistical analysis of tumor volume showed that the tumor volume in the mimic or si-BAG3 transfection group was significantly smaller than that in the control group(P<0.01).After 14 days,the weight of transplanted tumors in each group was significantly lower than that in the control group(P<0.01).It is noteworthy that compared with the other three groups,the co-transfection group of miR-206 and si-BAG3 showed the lowest tumor weight and volume(P<0.01).Conclusion:1.miR-206 may play a role as an anti-oncogene in cervical cancer,which can inhibit the proliferation,migration,invasion and transplantation of cervical cancer cells.BAG3 may play a role as a oncogene in cervical cancer,which can promote the growth,proliferation,migration and invasion of cervical cancer cells.2.miR-206 affects the proliferation,migration,invasion and transplantation of cervical cancer cells by targeting BAG3 expression.The up-regulation of miR-206 or the silence of BAG3 can inhibit the growth of transplanted tumors separately or jointly.