Study On The Expression Of MiR-16 In Epithelial Ovarian Cancer And The Effect And Mechanism Of Inhibiting Invasion And Metastasis Of Ovarian Cancer Cells

Ovarian cancer?OC?is the fifth leading cause of cancer death in women.It is the most fatal gynecological malignant tumor in the world and a serious threat to the health of women^[1,2].Epithelial ovarian cancer?EOC?accounts for90%of ovarian cancer.Due to the lack of early and effective screening methods,most patients are diagnosed at an advanced stage and the survival rate of patients with epithelial ovarian cancer is very poor.Based on SEER data from 2006 to 2012,the 5-year survival rate of early epithelial ovarian cancer was about 92%,while that of advanced patients was less than 30%.And it is particularly unfortunate that more than 70%of the patients of ovarian cancer are diagnosed at advanced stages and with local or distal metastasis^[3,4].In order to improve the survival rate of patients with ovarian cancer,it is urgently necessary to find the early detection and screening methods and explore a more effective treatment in the advanced stage of disease.Despite the efforts of scientists over the past few decades to find new serum biomarkers and treatment strategies?such as gene therapy?,a trend survey of5-year survival rates in women with ovarian cancer showed that the survival rate of women with ovarian cancer has improved only slightly in the last 30years^[5].In addition,the highly heterogeneous nature of epithelial ovarian cancer complicates the diagnosis and treatment of ovarian cancer,histologically defined subsets such as serous,mucinous,endometrial and transparent cell carcinoma and all have different clinical manifestations and potential molecular genetic events^[6].However,it is not clear which genetic factors determine the differentiation of ovarian epithelial cells.Therefore,in order to improve the diagnosis and treatment of ovarian cancer,we must improve the understanding of the molecular mechanism of ovarian cancer.So far,scientists have discovered that a variety of micro RNA?miRNA?molecules are involved in regulating the genesis and development of ovarian cancer^[1,7].miRNA is a single-stranded endogenous RNA with a length of about20-22 nucleotides.Its main role is to participate in the formation of RNA silencing complex and act as a post-transcriptional regulator of gene expression^[8-10].The mechanism of miRNA involved in post transcriptional regulation is usually due to its interaction with target mRNA's 3'untranslated region?UTR?,which specifically inhibits target mRNA expression through sequence complementation^[11,12].Existing studies have shown that miRNA plays an important role in many basic life activities,such as differentiation,cell growth,stress response and cell death,which involve a wide range of human diseases,including malignant tumors^[13-16].miRNAs also act as oncogenes and tumor-suppressor genes.With the deepening of the research,the key role of miRNAs in regulating human epithelial ovarian cancer has been increasingly understood.For example,miR-193a plays a role in tumor inhibition by inhibiting DNA synthesis in human epithelial ovarian cancer^[17].miR-212 inhibits the proliferation of epithelial ovarian cancer cells by targeting heparin and epidermal growth factor^[18].In addition,upregulation of expression in advanced ovarian epithelial carcinoma may promote epithelial stromal transformation and development of ovarian cancer^[19].In 2002,scientists discovered that miR-16 had a role similar to anti-oncogene in chronic lymphoblastic leukemia^[20].In subsequent studies,miR-16 has been found to be associated with human glioma^[21],colorectal cancer^[22],bladder cancer^[23],liver cancer^[24],prostate cancer^[25]and different types of lymphoma or leukemia^[26-27].Increasing the expression of miR-16 in tumor cells can enhance cell apoptosis and inhibit the cell cycle of cancer cells.There have been studies aimed at enhancing chemosensitivity of tumors by up-regulating the expression of miR-16.In addition,miR-16 has also been reported to regulate the angiogenesis^[28]and the immune response^[29].Researches are under way on targeted drugs for miR-16.Previous studies have shown that miR-16 is associated with the proliferation of OC cells.It remains unclear,however,whether the invasion and metastasis of OC is affected by miR-16.The purpose of our study was to investigate the expression and role of miR-16 in epithelial ovarian cancer and its relationship with invasion and metastasis of ovarian cancer cells and its related mechanisms.Part One Expression of miR-16 in epithelial ovarian cancer and its correlation with different clinicopathological featuresObjective:To detect the expression of miR-16 in tumor tissues of patients with epithelial ovarian cancer and to explore the correlation between miR-16 and epithelial ovarian cancer.Methods:Tumor tissue specimens from 35 cases of epithelial ovarian cancer were selected as the study group,25 cases of epithelial benign ovarian tumor and 25 cases of normal ovarian tissue as control groups.The expression level of miR-16 in three groups was detected by qRT-PCR to understand the expression characteristics of miR-16 in epithelial ovarian cancer.In addition,we also tested the expression of miR-16 in groups with different clinicopathological features to understood whether the expression of miR-16was correlated with the clinicopathological characteristics of epithelial ovarian cancer.Results:1.Clinicopathological characteristics of the EOC tumor tissue specimensThis study included 35 patients with EOC,13 patients under 50 years of age??50 years?and 22 patients older than 50 years old?>50 years old?.There were 3 cases of stage ?,5 cases of stage ?,25 cases of stage ? and 2 cases of stage ?.The majority of patients?about 77.14%?were FIGO ?-? stage.There were 23 cases of serous ovarian cancer,including 17 cases of high grade serous ovarian cancer?48.57%of the EOC patients?and 6 cases of low grade serous ovarian cancer?17.14%?.There were 6 cases of mucinous ovarian carcinoma?17.14%?,4 cases of endometrioid ovarian carcinoma?11.43%?and2 cases of clear cell carcinoma?5.72%?.Serous ovarian cancer is the most common EOC in histopathology of our study,accounting for about 65.71%.2.The expression of miR-16 was down-regulated in epithelial ovarian cancer tissuesThe expression of miR-16 in epithelial ovarian carcinoma?0.29�0.13?was significantly lower than that in epithelial benign ovarian tumor?1.07�0.16?and normal ovary?1.04�0.28??P<0.01?.And there was no significant difference in the expression of miR-16 between epithelial benign ovarian tumor group and normal ovarian group?P>0.05?.3.Expression of miR-16 in epithelial ovarian carcinoma with different clinicopathological featuresThe expression of miR-16 in epithelial ovarian carcinoma was not correlated with age and histopathologic type and there was no significant difference?P>0.05?,but was correlated with clinicopathological stage of EOC and the type of dualistic model in ovarian carcinogenesis.The expression level of miR-16 in stage ??? patients was?0.26�0.11?,which was lower than that in patients with stage ????0.41�0.16?.The expression level of miR-16 in patients with type ? EOC was?0.23�0.12?,which was lower than that in type ? patients?0.36�0.18?.And the differences were all statistically significant?P<0.05?.Summary:1.The expression of miR-16 in epithelial ovarian carcinoma is down-regulated,and miR-16 may be a tumor suppressor factor for EOC.2.The expression of miR-16 is related not only to the clinical stage of ovarian cancer,but also to the type of dualistic model in ovarian carcinogenesis.And the expression of miR-16 is not related to age of the patients and the histopathologic type.Part Two Expression of miR-16 in SKOV3 cells and its effect on proliferation,migration and invasion of ovarian cancer cellsObjective:In this part,the expression of miR-16 in human ovarian serous papillary cystadenocarcinoma cell line SKOV3 and ovarian epithelial cells were detected.And then in vitro functional experiments,we transfected the synthesized miR-16 mimic into SKOV3 cells,making it overexpression in cells,and detected the effect of miR-16 on the proliferation,migration and invasion of SKOV3 cells.Through that we could further verify the effect of miR-16 as the tumor suppressor factors of ovarian cancer.Methods:The expression of miR-16 in ovarian cancer SKOV3 cells was detected by qRT-PCR,and the correlation between miR-16 and epithelial ovarian cancer was discussed.The synthetic miR-16 mimic was transfected into SKOV3 cells to make it overexpression in cells,and real-time quantitative PCR was used to identify the transfection effect.The effects of overexpression of miR-16 on the proliferation,migration and invasion of SKOV3 cells were detected by CCK-8 proliferation assay,scratch test and transwell invasion experiment separately.Results:1.The expression of miR-16 in SKOV3 cells?0.49�0.03?was significantly lower than that in ovarian epithelial cells?1.16�0.06??P<0.01?.2.The relative expression levels of miR-16 in SKOV3 cells after 24hours of transfection of miR-16 mimics?experimental group?and negative controls?control group?were?1.92�0.12?and?1.00�0.11?respectively.After48 hours of transfection,the relative expression levels of miR-16 in SKOV3cells of the two groups were?1.96�0.22?and?1.00�0.21?respectively.After72 hours,the relative expression levels of miR-16 in the two groups were?1.83�0.09?and?1.00�0.04?respectively.The differences were all statistically significant?P<0.01?.3.CCK-8 proliferation test showed that the OD values of the experimental group and the control group were?0.60�0.02?and?0.67�0.03?respectively after 24 hours of transfection,with no significant difference?P>0.05?.After 48 hours of transfection,the OD values of the two groups were?1.05�0.10?and?1.35�0.08?respectively?P<0.05?.And those after 72hours of transfection were?1.55�0.07?and?2.12�0.14?respectively?P<0.01?.The differences were all significent.Overexpression of miR-16 inhibited the proliferation of SKOV3 cells.And after 3 days of transfection,the overexpression of miR-16 significantly inhibited the proliferation of SKOV3cells.Compared with the control group,the growth inhibition rates of 24h,48h,72h after transfection in the miR-16 overexpression group were 13%,29%,37%respectively.4.The scratch test showed that overexpression of miR-16 significantly inhibited the confluence of scratches in SKOV3 cells.The relative wound area of miR-16 overexpression group were?0.94�0.05?,?0.69�0.03?,?0.43�0.01?at0h,12h,24h after wound scratch respectively,and those were?1.04�0.06?,?0.49�0.02?,?0.21�0.01?in negative control group respectively.There was no significant difference in the relative wound area between the two groups at 0 h after wound scratch?P>0.05?.The relative wound areas of the miR-16overexpression group were all significantly higher than those of the corresponding negative control group at 12h and 24h after wound scratch.There were significant differences between the two groups?P<0.05?.The results showed that overexpression of miR-16 significantly inhibited the migration of SKOV3 cells.5.Transwell invasion experiments showed that,the average number of invasive cells per visual field in the miR-16 overexpression group was?70�10?after 24 hours of transfection,which was significantly lower than that in the NC group?100�20?/visual field?P<0.01?.Overexpression of miR-16significantly inhibited the invasion of SKOV3 cells.Summary:1.Compared with normal ovarian epithelial cells,the expression level of miR-16 in SKOV3 cells was down-regulated.2.Overexpression of miR-16 in SKOV3 cells may slow down cell proliferation,the ability of invasion and metastasis.3.It was further proved that miR-16 may play a role as a tumor suppressor in the development of epithelial ovarian cancer.Part Three Study of the mechanism of miR-16 inhibiting invasion and metastasis of ovarian cancer cellsObjective:To investigate the regulation and signal pathway involved in the process of miR-16 affecting the invasion and metastasis of SKOV3 cells.Methods:Western blot assay was used to detect the associated protein of possible regulatory methods and signal pathways involved in the process of miR-16 inhibiting the invasion and metastasis of SKOV3 cells.We detected the expression of MMP2 and MMP9 in SKOV3 cells after overexpression of miR-16.Besides,we also detected the expression of E-cadherin,Slug,Snail,Vimentin and twist which associated with EMT and the expression of GSK3?,Wnt3a and?-catenin in Wnt/?-catenin signaling pathway in these cells.Results:1.The relative expression levels of MMP2 in miR-16 overexpression group and negative control group were 0.80�0.08)and?1.02�0.12?respectively?P<0.05?,and the relative expression levels of MMP9 were?0.45�0.05?and?1.01�0.18?in the two groups respectively?P<0.01?.The differences were all statistically significant.Compared with negative control group,the expression levels of MMP2 and MMP9 in SKOV3 cells transfected with miR-16 mimic were significantly down-regulated.2.The relative expression levels of E-cadherin in the miR-16overexpression group and the negative control group were?1.63�0.03?and?0.98�0.02?respectively.The relative expression levels of Slug in the two groups were?0.69�0.10?and?1.06�0.06?respectively.The relative expression levels of Snail in the two groups respectively were?0.63�0.08?and?1.05�0.05?.The relative expression levels of Vimentin in two groups were?0.38�0.01?and?1.01�0.15?and those of Twist were?0.68�0.16?and?1.01�0.15?respectively.The expression of E-cadherin which as a cell adhesion molecule in SKOV3 cells of miR-16 overexpression group was higher than that in control group,while the expression of Slug,Sail,Vimentin and Twist which as mesenchymal cells markers was lower than that in control group.The differences were all statistically significant?P<0.05?.3.The relative expression levels of GSK3?in the miR-16 overexpression group and the negative control group were?1.85�0.10?and?0.99�0.01?respectively,the Wnt3a relative expression levels in the two groups were?0.45�0.04?and?1.00�0.06?,and those of GSK3?in the two groups were?0.27�0.01?and?1.00�0.11?respectively.The expression of GSK3?in miR-16overexpression group was higher than that in control group,while the expression of Wnt3a and?-catenin was lower than that in control group.The differences were all statistically significant?P<0.05?.Summary:1.Overexpression of miR-16 may inhibit the migration and invasion of SKOV3 cells by inhibiting the expression of MMP2 and MMP9.2.miR-16 may upregulate the expression of E-cadherin,and downregulate the expression of some mesenchymal markers including slug,snail,vimentin and twist,inhibit the EMT process,thus inhibit the invasion and metastasis of ovarian cancer cells.3.miR-16 overexpression in SKOV3 resulted in a significant increase in GSK3?expression as well as decrease expressions of Wnt3a and?-catenin.And then Wnt/?-catenin signaling pathway in SKOV3 cells was affected,which may inhibit the invasion and metastasis of ovarian cancer cells.4.It was further confirmed that miR-16 may be a tumor suppressor factor for epithelial ovarian cancer,which may play an important role in carcinogenesis and progression,and be closely related to the invasion and metastasis of ovarian cancer cells.Conclusions:1.miR-16 may be a tumor suppressor to EOC,and its expression levels were significantly down-regulated in EOC tumor tissue and ovarian cancer cell line SKOV3.2.The expression levels of miR-16 were correlated with the operative pathological stage and EOC dualism,but not with age and histopathologic type.3.Overexpression of miR-16 in SKOV3 cells slowed down cell proliferation and reduced cell migration and invasion.4.Overexpression of miR-16 may inhibit the migration and invasion of SKOV3 cells by inhibiting the expression of MMP2 and MMP9.5.miR-16 may upregulate the expression of epithelial intercellular adhesion molecule E-cadherin,and downregulate the expression of some mesenchymal markers including slug,snail,vimentin and twist,inhibit the EMT process,thus inhibit the invasion and metastasis of ovarian cancer cells.6.miR-16 overexpression in SKOV3 resulted in a significant increase in GSK3?expression as well as decrease expressions of Wnt3a and?-catenin.And then Wnt/?-catenin signaling pathway in SKOV3 cells was affected,which may inhibit the invasion and metastasis of ovarian cancer cells.7.miR-16 was closely related to invasion and metastasis of ovarian cancer cells.Overexpression of miR-16 in SKOV3 cells may decrease the ability of cell invasion and metastasis.It was further demonstrated that miR-16 may be a tumor suppressor factor for ovarian cancer.