MiR-320a And Mir-340-5p Inhibit The Invasion And Metastasis Of Endometrial Cancer By Targeting EIF4E

1.Objective.Endometrial cancer(EC)is the most common malignancy of the female genital tract which seriously threaten women's health.Metastasis is the main cause of EC treatment failure.Epithelial-mesenchymal transition(EMT)play an important role in tumor metastasis and is an integral step in embryonic development,but it also plays an important role in the development of tumor,especially in the progress of tumor invasion and metastasis.Through EMT,tumor cells lose some of the characteristics of epithelial cells and obtain some characteristics of mesenchymal cells,thus enabling tumor cells to acquire stronger ability of metastasis and invasion.Therefore,explore the therapeutic targets of EMT inhibition is a new direction of tumor metastasis research.Eukaryotic translation initiation factor 4E(eIF4E),one of the eukaryotic translation initiation complexes,is the most effective regulator of translation initiation.As an oncogene,high eIF4E expression was found in a variety of tumors,moreover,it even plays an important role in EMT and tumor invasion.MicroRNAs(miRNA)are small non-coding RNAs,act as oncogenes or tumor suppressor genes in tumorigenesis and development.MiRNA binds to the 3'-untranslated regions(3 'UTRs)of the target mRNAs,inhibit their translation or cause their degradation.miR-320a and miR-340-5p have been reported to play the role of inhibiting tumor growth,proliferation,invasion and apoptosis in a variety of tumors,such as liver cancer,non-small cell lung cancer,cervical cancer and breast cancer,but little is known about them in EC as yet.The purpose of this study was to examine the effect and mechanism of miR-320a and miR-340-5p in the metastasis of EC.In this study,Oncomine database data was collected to explore the relationship between eIF4E expression and progress and prognosis of EC.eIF4E,miR-320a and miR-340-5p expressions were detected in 8 cases of EC and adjacent normal tissues.The targeting relationship between eIF4E and miR-320a or miR-340-5p was determined.We also detected the effects of miR-320a and miR-340-5p on the proliferation,apoptosis,invasion and metastasis of EC cells.EMT of EC cells was induced by TGF-?1 to detect whether EMT was involved in the molecular mechanism of the effects of miR-320a and miR-340-5p on the invasion and metastasis of EC.?.MethodsA.Expression of eIF4E,miR-320a and miR-340-5p in human endometrial carcinoma tissues and cells1.Data collection and analysis of Oncomine database Kaplan-meier survival curve analysis was performed in the outcome of EC patients from Oncomine database,we also detected the correlation between eIF4E expression level and tumor grade.2.Validation of eIF4E expression in endometrial cancer and noncancerous tissues 8 pairs of fresh EC and adjacent normal tissues were collected,eIF4E expression was detected by western blotting.3.Detection of the expression of miR-320a and miR-340-5p in endometrial carcinoma and adjacent tissuesqRT-PCR were performed to detect the expressions of miR-320a and miR-340-5p in endometrial carcinoma and adjacent tissues.4.Expression of eIF4E in different EC cell lines We evaluated the expression of eIF4E in EC cell lines HEC-1A?RL95-2 and Ishikawa by western blotting.5.Expression of miR-320a and miR-340-5p in different EC cell lines Expression of miR-320a and miR-340-5p were evaluated in EC cell lines HEC-1A?RL95-2 and Ishikawa by qRT-PCR.B.Correlation between miR-320a/miR-340-5p and eIF4E1.Prediction by bioinformatics analysis Predicted target genes were examined for miR-320a and miR-340-5p by bioinformatics analysis.2.Construction of pcDNA-GFP-eIF4E-3 UTR vector Get the eIF4E-3'UTR sequence from NCBI and primers was designed and synthesized,then the sequence was amplified by PCR and cloned to the pcDNA-GFP after the restriction enzyme digestion3.Detection of the influence of miR-320a and miR-340-5p on eIF4E expression The pcDNA-GFP-eIF4E-3'UTR was co-transfected with miR-320a or miR-340-5p mimics into endometrial cancer cells,respectively.Then intensity of GFP fluorescence were observed under a fluorescence microscope and the rate of GFP positive cells were detected by flow cytometryC.Detection the effect of miR-320a and miR-340-5p on the biological character-ristics of EC cellsAfter the transfection of miR-320a,miR-340-5p,mu-320a and mu-340-5p into the cells of HEC-1A and RL95-2,respectively,MTT,Annexin V-FITC/PI,transwell cell invasion assay and cell scratch assay were performed to detect cell proliferation,apoptosis,invasion and migration.The molecular mechanism of miR-320a and miR-340-5p inhibiting the metastasis of EC cells1.Detection of the changes of MMP-3 and MMP-9 in HEC-1 A cells transfected with miR-320a and miR-340-5p by western blotting After the transfection of miR-320a or miR-340-5p into HEC-1 A cells,the expressions of MMP-3 and MMP-9 were detected by western blotting2.Detection the effects of miR-320a and miR-340-5p on TGF-?1-induced EMT Different concentrations of TGF-?1 were applied to HEC-1A cells,and western blotting was performed to detect the expression of eIF4E and p-eIF4E in TGF-?1-induced EC cells.The effect of miR-320a and miR-340-5p transfection on the expression of p-eIF4E induced by TGF-?1 was also detected.3.Detection of the effects of miR-320a and miR-340-5p on the EMT-related cell behavior induced by TGF-?1.The effect of miR-320a and miR-340-5p on the TGF-pl-induced mesenchymal morphology of EC cells was detected by light microscopes.Cell invasion assay detected the changes in the invasion ability of EC cells induced by TGF-?1 and the effects of miR-320a and miR-340-5p on these changes4.Detection the changes of EMT-related proteins.To further understand the molecular mechanism of its function,western blotting was used to detect the efrect of miR-320a or miR-340-5p on the expression of EMT-related molecule Snail,E-cadherin,?-SMA in TGF-?1 induced EC cells.?.ResultsA.Expression of eIF4E,miR-320a and miR-340-5p in EC tissues and cells1.High eIF4E expression correlated with poor prognosis and high pathological grade of EC.Kaplan-meier survival curve analysis was performed in the outcome of EC patients from Oncomine database,the results showed that high eIF4E expression in EC patients was significantly correlated with poor prognosis(p<0.05).Further analysis of the correlation between eIF4E expression level and tumor grade showed that revealed a significant correlation between eIF4E expression level and the histological grade of EC(p<0.01).2.The expression of eIF4E in EC tissues was higher than that in adj acent normal tissues.The results of western blotting showed that eIF4E expression in EC was higher than that in adjacent tissues,and the difference was statistically significant(p<0.05).3.MIR-320a or miR-340-5p was downregulated in EC tissues.The results of qRT-PCR showed that the relative expression levels of the miR-320a or miR-340-5p were significantly decreased in EC compared with the adjacent normal tissues,the difference was statistically significant(P<0.01).4.eIF4E expression was higher in HEC-1A cells and RL95-2 cells than that in Ishikawa cells.Western blotting showed that the expression of eIF4E protein was markedly increased in HEC-IA and RL95-2 but was low in Ishikawa(P<0.01).5.The expression levels of miR-320a and miR-340-5p in HEC-1A cells and RL95-2 cells were lower than those in Ishikawa cells.qRT-PCR results showed that the expression levels of miR-320a and miR-340-5p in both HEC-1A and RL95-2 cells were lower than those in Ishikawa cells(p<0.05).B.eIF4E is a direct target of miR-320a and miR-340-5p,which inhibit the expression of eIf4E and p-eIf4E.1.Based on the results of online miRNA analysis software TargetScan,we confirmed that there are target site of miR-320a and miR-340-5p on the 3'-UTR of eIF4E.2.After pcDNA-GFP-eIF4E-UTR vector was cotransfected with miR-320a or miR-340-5p into EC cells for 48h,GFP fluorescence intensity and positiverate decreased significantly in both miR-320a and miR-340-5p treated cells compared with the control treatment.3.Western blotting analysis confirmed that miR-320a or miR-340-5p induced the greatest decline of not only eIF4E but also p-eIF4E expression.C.miR-320a and miR-340-5p inhibit the invasion and metastasis of EC cells,inhibit proliferation and promote apoptosis,and play different roles in different EC cell lines.1.miR-320a and miR-340-5p effectively inhibited the invasion and metastasis of HEC-IA cells.Transwell experiment showed that compared with the control group,both miR-320a and miR-340-5p could significantly inhibit the invasion ability of HEC-1 A cells,whereas had no significant effect on the invasion ability of RL95-2 cells.Cell scratch experiment also showed that both miR-320a and miR-340-5p could significantly inhibit the migration ability of HEC-1A cells.2.miR-320a and miR-340-5p inhibit cell proliferation and induce apoptosis of RL95-2 cells.MTT assay indicated that compared with the control group and the mutant group,both miR-320a and miR-340-5p could effectively inhibit the proliferation of RL95-2 cells but had no significant effect on the proliferation of HEC-1A cells.Annexin V-FITC/PI results showed the same trend,that is,miR-320a and miR-340-5p could effectively promote the apoptosis of RL95-2,but had no effect on HEC-1A.D.miR-320a and miR-340-5p inhibit EC cells metastasis are associated with the inhibition of MMP-3 and MMP-9 expression and the reversal of TGF-pl-induced EMT.1.The results of western blotting showed that compared with the control group,the expression levels of MMP-3 and MMP-9 were significantly down-regulated after transfection2.TGF-?1 of 10ng/mL upregulates the expression of p-eIF4E and is blocked by miR-320a or miR-340-5p.3.MIR-320a or mIR-340-5p reverses the biological outcome of EMT in EC cells induced by TGF-?1.Following 10ng/mL TGF-pl treatment for 48 h,HEC-1A cells shows fibroblast-like appearance,become long fusiform shape and dispersed in a loose funicular,but when TGF-?1 were applied together with miR-320a or miR-340-5p,the TGF-pl-induced EMT is blocked.Cell invasion assay showed that TGF-?1 enhanced the ability of cell invasion of EC cells,but miR-320a or miR-340-5p could partially reverse this phenomenon.4.miR-320a and miR-340-5p attenuates the change of EMT markers induced by TGF-?1 in EC cells.The results showed that the changes of Snail,E-Cadherin and a-SMA caused by TGF-?1 could be reversed by miR-320a or miR-340-5p.Finally,siRNA targeting eIF4E was applied for control,we get the same results as miR-320a and miR-340-5p treatment.Conclusion:1.eIF4E is involved in the development of endometrial cancer,and high eIF4E expression is associated with poor prognosis and high grade of EC.2.Loss of miR-320a or miR-340-5p expression might be closely associated with the genesis and development of EC.3.eIF4E is a direct target of miR-320a and miR-340-5p,which inhibit the expression of eIF4E and p-eIF4E4.miR-320a or miR-340-5p can effectively inhibit the proliferation,apoptosis,migrationand invasion of EC cells through targeted regulation of eIF4E and p-eIF4E,and show different roles in different cell lines5.miR-320a or miR-340-5p can effectively inhibit the invasion and metastasis of HEC-1A.The mechanism is associated with decreasing expression of MMP-3 and MMP-9 as well as reversing TGF-?-induced EMT through target p-eIF4E.