Studies On The Mechanism Of MiR-200c Inhibiting Epithelial-mesenchymal Transformation By Targeting BMI-1 Gene In Endometrial Cancer Cells

BackgroundEndometrial carcinoma is one of the most common gynecological malignancies.In 2012,there were about 320,000 new cases worldwide.The incidence and mortality of endometrial carcinoma are increasing yearly.In the United States,there were 54,870 women were diagnosed with endometrial carcinoma in 2015,and 10,170 of them died from this malignant tumor.In 2017,there were 61380 cases of new cases of endometrial carcinoma,which is the fourth in all new cases of cancer among women,and 10,920 of them died.The prognosis of endometrial carcinoma depends on many factors,including tumor staging and classification.Due to tumor recurrence and metastasis,as well as the insensitivity of some patients to hormone therapy,radiotherapy and chemotherapy,the median survival time of endometrial carcinoma was shortened.Radical surgery is an effective treatment for early endometrial carcinoma,while,after the operation,there are still metastatic tumor in 40%of cases.In order to improve the prognosis of patients,it is found that the molecular and signaling pathways of tumor metastasis may provide new biological therapeutic targets for diagnosis and treatment.It is well known that epithelial mesenchymal transformation(EMT)is associated with the progression and metastasis of cancer.The functional characteristics of common EMT in endometrial carcinoma including the loss of E-cadherin expression,and the increase of interstitial phenotype and downstream transcription factors have been confirmed.Recent studies have shown that EMT regulation of endometrial carcinoma is related to the expression of PI3K activity,estrogen signal and microRNA(miRNA).EMT has also been proved to be dependent on the expression of several common differentiation markers,including the snail/slug,Twist,ZEB 1/2,and AKT/PI3K,PI3K/AKT/mTOR pathway.These molecular markers are also an important cell cycle regulatory factor for EMT,cell proliferation and cancer progression.However,the further research is needed to understand the complex relationship between these factors.MicroRNAs(miRNAs)are a group of small endogenous non-coding RNAs.They affect a variety of biological processes by regulating transcriptional inhibition gene expression or by binding to the 3' terminal translation region(UTR)of a specific target gene.Recently,miRNAs have received more and more global attention due to its known transcriptional silencing gene and its ability to regulate multiple signaling pathways.Recently,due to miRNAs have its abilities of transcribing silencing gene and regulating multiple signaling pathways,miRNAs have received more and more global attention.Studies have shown that the expression of abnormal miRNA can promote EMT,anti-apoptosis and angiogenesis,so miRNA is associated with tumor occurrence,tumor metastasis and disease progression.It was found that the abnormal expression of miRNA was involved in the occurrence and development of endometrial carcinoma.Some studies have found that miRNA regulates the EMT of endometrial carcinoma,in which the most important miRNA regulating EMT is microRNA-200 family,but the specific regulation mechanism is not clear.Studies found that AKT/phosphorylation AKT(p-akt)was involved in the regulation of microRNA-200c(miR-200c)expression,but the specific mechanism was not clear.AKT/phosphorylation AKT(p-akt)was involved in the regulation of miR-200c expression,but the specific mechanism was not clear.BMI-1 gene is a polypectin(PcG)protein,which is an important transcription inhibitor.It regulates the changes of chromatin structure and regulates gene activity.Many PcG proteins have been altered in human cancers,including endometrial carcinoma.Studies have shown that some miRNAs inhibit EMT in cancer cells by targeting the BMI-1 gene.The expression of BMI-1 genen in endometrial carcinoma is still controversial.Currently,there is no study on the target effect of miR-200c on BMI-1 in endometrial carcer.BMI-1 gene not only serves as a gene for predicting tumor prognosis,but also provides a new direction for targeted therapy for malignant tumors.In this study,we discussed the expression of miR-200c and BMI-1 gene in endometrial adenocarcinoma and analyzed the relationship between BMI-1 gene and the clinicopathologic features of endometrial adenocarcinoma,and the correlation between miR-200c and BMI-1 gene.Furthermore we studied the effect of miR-200c on the invasion and metastasis in endometrial cancer cells in vitro and confirmed the targeting effect of miR-200c on BMI-1 gene.Furthermore,we aim to find the mechanism of miR-200c inhibiting the epithelial-mesenchymal transformation of endometrial cancer cells by targeting BMI-1 gene through p-AKT pathway.Part I:The expression and correlation of miR-200c and BMI-1 gene in endometrial adenocarcinomaPurpose1.To detect the expression of miR-200c and BMI-1 gene in endometrial adenocarcinoma and normal endometrial tissues.And aim to find their correlation.2.To study the relationship between BMI-1 gene and the clinical characteristics of endometrial adenocarcinoma.MethodsIn this study,qRT-PCR was used to detect the relative expression of miR-200c and BMI-1 gene in 60 cases of endometrial adenocarcinoma and 40 normal endometrial tissues respectively.Immunohistochemical method was used to detect the expression of BMI-1 gene in 60 cases of endometrial adenocarcinoma and 40 normal endometrial tissues.T-test and Spearman statistical method were used to analyze the expression of miR-200c and BMI-1 gene in endometrial adenocarcinoma and normal endometrial tissues and to analyze the correlation between them in endometrial adenocarcinoma.The relationship between BMI-1 gene expression and clinicopathological features of endometrial adenocarcinoma was analyzed by using ?2/Fisher's test.Results1.The expression of miR-200c in endometrial adenocarcinoma and normal endometrial tissuesqRT-PCR method was used to detect the relative expression of miR-200c in 60 example endometrial adenocarcinoma and 40 cases of normal endometrium tissue.Result showed that the expression level of miR-200c in endometrial adenocarcinoma tissue was decreased,comparing with the expression level in normal endometrium tissue(P<0.001).2.The expression of BMI-1 gene in endometrial adenocarcinoma and normal endometrial tissuesqRT-PCR method was used to detect the relative expression of BMI-1 gene in 60 example endometrial adenocarcinoma and 40 cases of normal endometrium tissue.Result showed that the expression level of BMI-1 gene in endometrial adenocarcinoma tissue was increased,comparing with the expression level in normal endometrium tissue(P<0.001).3.The relationship between the expression of BMI-1 gene in endometrial adenocarcinoma and the clinicopathological features of endometrial adenocarcinomaAccording to the immunohistochemical results,60 cases of endometrial adenocarcinoma patients were divided into two groups.One is BMI-1 gene low expression group(-/+)and the other is BMI-1 gene high expression group(++/+++).?2/Fisher' s test showed that BMI-1 gene was high expression in 44 cases(73.33%)of 60 cases of endometrial adenocarcinoma and was high expression in 2 cases(5.00%)of 40 cases of normal endometrial tissues.The high expression of BMI-1 gene was significantly associated with the patients's age(P=0.0102),high FIGO staging(P=0.0274),muscular invasion(P=0.0274),and lymphatic metastasis(P=0.0495).No relationship was found between BMI-1 gene and the differentiation of endometrial adenocarcinoma(P=0.088).4.The correlation between miR-200c and BMI-1 gene in endometrial adenocarcinomaThe result of Spearman statistics showed there was negative correlation between miR-200c and BMI-1 gnen in endometrial adenocarcinoma(r=-0.4051,P=0.0013).Conclusions1.The degradation miR-200c and the up-regulation of BMI-1 gene may be cause the development of endometrial adenocarcinoma.2.BMI-1 gene is involved in the development of endometrial adenocarcinoma and promotes the invasion and metastasis of endometrial cancer.3.The negative correlation between miR-200c and BMI-1 gene in endometrial adenocarcinoma provides the basis for further study on the targeting effect of miR-200c on BMI-1 gene.Part ?:The mechanism of miR-200c inhibiting epithelial-mesenchymal transition by targeting the BMI-1 gene in endometrial carcinoma cellsPurpose1.To detect the expression of miR-200c in Ishikawa(high differentiation)and JEC(middle differentiation)cells.2.To detect the transfection efficiency after Ishikawa and JEC cells were respectively transfected with miR-200c mimics to up-regulate the expression of miR-200c and transfected with miR-200c inhibitor to down-regulate miR-200c expression.3.To detect the proliferation,migration and invasion of Ishikawa and JEC cells after the cells were respectively transfected with miR-200c mimics and miR-200c inhibitor.4.To detect the change of miR-200c on the EMT and the effect on BMI-1 and p-AKT after Ishikawa and JEC cells were respectively transfected with miR-200c mimics and miR-200c inhibitor.5.To find the mechanism of miR-200c inhibiting the EMT by targeting BMI-1 gene in Ishikawa and JEC cells,and verified whether p-AKT is involved.Methods1.qRT-PCR method was used to detect the relative expression of miR-200c in Ishikawa and JEC cells.According to the instructions of Lipofectamine 2000,Transient transfection was performed on miR-200c mimics and miR-200c inhibitor.The transfection efficiency of Ishikawa and JEC cells was detected by qRT-PCR.2.MTT was used to detect cell proliferation after the cells were transfected miR-200c mimics and miR-200c inhibitor for 24h,48h,72h and 96h.3.Transwell chamber was used to detect the effects on migration and invasion of Ishikawa and JEC cells after transfected miR-200c mimics and miR-200c inhibitor.4.Western-blotting method was used to detect the expression of EMT epithelial phenotype E-cadherin,interstitial phenotype N-cadherin,downstream transcription factor Slug and the changes of BMI-1,AKT and p-AKT after the cells were transfected with miR-200c mimics and miR-200c inhibitor for 48h.5.The cells were transfected with siRNA BMI-1 to interfere the expression of BMI-1 after transfected with miR-200c inhibitor for 24h.And Western-blotting method was used to detect the expression of EMT epithelial phenotype E-cadherin,interstitial phenotype N-cadherin,downstream transcription factor Slug and the changes of BMI-1,AKT and p-AKT.Results1.The compare of the expression of miR-200c in Ishikawa and JECcells qRT-PCR method was used to detect the relative expression of miR-200c in Ishikawa and JEC cells.There was no significant difference in the expression of miR-200c in the two cells(P>0.05).2.The results of transfection efficiency and the effect on cell proliferation ability after Ishikawa and JEC were respectively transfected with miR-200c mimics and miR-200c inhibitorThe transfection of miR-200c mimics activated the expression of miR-200c in Ishikawa and JEC significantly(P<0.001).However,the transfection of miR-200c inhibitor inhibited miR-200c expression significantly(P<0.001).After transfection with miR-200c mimics and miR-200c inhibitor for 24h,48h,72h and 96h,MTT method was used to detect cells growth and proliferation curve.The results showed that the growth and proliferation was decreased after transfection with miR-200c mimics.In the 24-48h trasnfestion,it was the most effective in inhibiting the proliferation cells.The growth and proliferation was increased after transfection with miR-200c inhibitor.In the 24-48h trasnfestion,it was the most effective in promoting the proliferation cells.3.The effects of miR-200c on the invasion and migration of Ishikawa and JEC cellsAfter the Ishikawa and JEC cells were transfected with miR-200c mimics,miR-200c inhibitor and NC respectively for 24 hours,the cells' invasion and migration ability were detected by transwell chamber experiment.Transwell chamber experimental results showed Ishikawa and JEC cells' invasion and migration ability significantly lower than the control group after transfetion with miR-200c mimics(P<0.001),in contrast,the invasion and migration ability was obviously higher than that of control group after transfection with miR-200c inhibitor(P<0.001).4.The effects of miR-200c on the EMT of Ishikawa and JEC cells,which is the change of the epithelial phenotype of E-cadherin,the interstitial phenotype of N-cadherin and the transcription factor Slug,as well as the effect on BMI-1 and p-AKTAfter transfection with miR-200c mimics,miR-200c inhibitor,NC and inhibitor NC for 48 hours,the cell protein was extracted and western blotting was used to detecte the expression of epithelial phenotype of E-cadherin,interstitial phenotype of N-cadherin,the transcription factor Slug,as well as BMI-1,AKT and p-AKT in Ishikawa and JEC cells.The Western blotting results showed that,when the cells were transfected with miR-200c mimics,the expression of the mesenchymal marker N-cadherin and the downstream EMT transcription factor,Slug,were decreased,while the expression of the epithelial marker E-cadherin was increased relative to the control cells(P<0.001).Also,the results showed that the expression of p-AKT and BMI-1 were lower in both cell lines compared with the control after transfection with miR-200c mimics(P<0.001).However,all the results were reversed in the two cell lines after transfection with miR-200c inhibitor.4.The mechanism of miR-200c inhibiting EMT by targeting BMI-1 in Ishikawa and JEC cellsAfter tansfection of Ishikawa and JEC cells with miR-200c inhibitor for 24 hours,both cell lines were transfected with siRNA BMI-1 again.The results of Western blotting showed that the expression of BMI-1 was reduced(P<0.001).The expression of EMT-associated proteins,by Western blotting showed that the expression of N-cadherin,Slug and p-AKT were decreased,while the expression of E-cadherin was increased compared with the BMI-1 siRNA NC group.In this way,this experiment reversed the result of transfection of miR-200c inhibitor only.Conclusions1.miR-200c may be a therapeutic target which inhibits the invasion and metastasis of endometrial carcinoma2.The loss of miR-200c expression and the degradation of miR-200c in endometrial adenocarcinoma may promote the progression and metastasis of endometrial cancer3.P-AKT was involved in the inhibitory effect of miR-200c inhibiting EMT by targeting BMI-1 gene in endometrial cancer cells...