| Objective:Endometrial Cancer is one of the most common malignant tumor in the female reproductive.The incidence of endometrial cancer is upswing in these years,and recent clinical studies show that the age incidice is younger and younger.miRNAs play a vital role in the occurrence and development of endometrial carcinoma.Ubiquitin-specific protease 15(USP15)is a kind of Deubiquitinating enzyme(DUB),which can affect the development of tumor by deconjugating ubiquitin from targeted proteins.Our research is to investigate the relationship between endometrial and miR-195 or USP15,and we also analysed the function of miR-195 or USP15 in the occurrence and development.Methods1.Expression of miR-195 in endometrial carcinoma tissue,para-carcinoma tissue and normal endometrium by RT-PCR.2.Expression of USP15 in endometrial carcinoma tissue,para-carcinoma tissue and normal endometrium by RT-PCR.3.Expression and location of USP15 in endometrial carcinoma tissue,para-carcinoma tissue and normal endometrium by Immunohistochemistry.4.Cultivate endometrial carcinoma cell,then we transfected cells by miR-195 specific mimics antisense oligonucleotides and miR-195 specific inhibitor antisense oligonucleotides according to the instructions of LipofectamineTM 3000 reagent.Then we use RT-PCR to detect transient transfection of miR-195.5.The cellular growth activity was detected by MTT assay.6.The effect of cell migration was asstayed by Transwell.7.The effect of cell migration was assayed by wound healing in vitro.8.The changes of USP15 mRNA in endometrial carcinoma cell after transient transfetion of miR-195 were detected by RT-PCR.9.Statistical methods:SPSS 19.0 statistical software was used for statistical analysis,and the data were recorded in mean ± standard deviation(SD).Two groups comparison examined by t test,other compares among experimental groups were analyze by single factor analysis of variance,p<0.05 was considered statistically significant.,all datas were the results of 3 repeat experiments.Results:1.The expression of miR-195 in endometrial tissue:The results indicated that average endometrial carcinoma of miR-195 expression was significantly lower than the mean expression of miR-195 in para-carcinoma tissue(p<0.05),and it also lower than the mean expression of miR-195 in normal endometrium(p<0.05).2.The expression of USP15 in endometrial tissue:The results showed that mean endometrial carcinoma of USP15 expression was significantly higher than the average expression of USP15 in para-carcinoma tissue(p<0.05),and it also higher than the mean expression of USP15 in normal endometrium(p<0.05).3.The IOD/Area in in endometrial tissueThe results indicated that average IOD/Area Of USP15 in endometrial carcinoma was significantly higher than the mean IOD/Area Of USP15 in para-carcinoma tissue(p<0.05),and it also higher than the IOD/Area Of USP15 in normal endometrium(p<0.05).4.Transfection efficiency of miR-195 transient transfectionThe expression of miR-195 after transfection of mimics was increased compared with negative control group,and the expression of miR-195 after transfection of inhibitor was reduced compared with negative control group5.The MTT experimentsThe results showed that after transfection miR-195 mimics,the endometrial cancer cell growth(p<0.05)slowed;after transfection miR-195 inhibitor,the endometrial cancer cell growth rate accelerated(p<0.05).6.Transwell assay.After transfection of miR-195 mimics,the number of migratory HEC-1-B cells were fewer than negative control group(p<0.05).The number of migratory HEC-1-B cells transfected with miR-195 inhibitors were more than miR-195 inhibitor control(p<0.05)7.Woud healing experimentsAfter transfection 24h in human endometrial cancer cell line HEC-1-B,the mobility between the four groups were 6.93±3.18%?39.28±1.655%,43.30±0.64%;31.19±0.01%(p<0.05).8.The changes of USP 15 mRNA after transfection of miR-195After transfection of miR-195 mimics in HEC-1-B,the expression of USP15 mRNA were reduced by 87.20%(p<0.05);after transfection of miR-195 inhibitor,the expression of USP15 mRNA were increased by 24.14 times(p<0.05).Conclusion1.miR-195 was down-regulated in endometrial carcinoma compared with para-carcinoma tissue or normal endometrium.2.miR-195 plays a vital role in the occurrence and progression of endometrial carcinoma,through inhibiting the proliferation and migration of endometrial cancer cell.3.USP15 was up-regulated in endometrial carcinoma compared with para-carcinoma tissue or normal endometrium,it located in the cytoplasm of endometrial glandular cell4.In endometrial cancer,miR-195 may suppress USP15 transcription by targeting it,and suppress the occurance and development of endometrial carcinoma. |
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