Effects Of RNAi On HER-2/neu Gene Of Ishikawa Endometrial Cancer Cells And The Effect On Their Sensitivity To Progesterone

IntroductionEndometrial Carcinoma(EMC) is one of the most common seen malignant tumors of female genital tract.Recent years,its incidence rate has the great tendency to increase.Especially in some European countries,it has come to the first place in the malignant tumors of female genital tract.The degree of the expression of HER-2 defers in different types of endometrial carcinoma and it demonstrates that HER-2 functions in several types of endometrial carcinoma.Recent research has found that the expression degree of HER-2 in endometrial carcinoma correlates with the poor prognosis of the patients.RNA interference is a new gene blockage technique which develops recent years. It's a kind of sequence-specific gene silencing tool which functions through 21-23nt double strand RNA(dsRNA).Now,it has become one of the most athletic hot pots in molecular biologic research.Compared with antisense RNA,RNAi has higher achievement ratio and can suppress the expression of target gene to a lower level hence achieve the effect of gene knock-out.As a specific gene knock-out tool which has high performance,RNAi not only can be used to investigate functional genome but also provide a broad prospect for gene therapy.ObjectiveTo investigate the effects of HER-2/neu gene silencing on Ishikawa endometrial cancer cells and the change of their sensitivity to progesterone.MethodsDesign and synthesize two shRNAs based on the sequence of HER-2/neu mRNA. They were separately subcloned into the plasmid of pSilencer4.1 containing CMV promoter.The pSilencer vectors of the RNA interference specific to HER-2/neu gene were constructed.The pSilencer vectors were transfected into Ishikawa endometrial cancer cells with Lipofectamine^TM2000 in vitro.Then the effects of RNAi on the expression of p185 protein were detected by Western Blot after 24,48 and 72 hours and the apoptotic rate was assayed by Annexinâ…¤in vitro.Flow cytometry was used to examine cell cycle after 72h of transfection.Twelve hours after transfection,culture the cells for 48h with progesterone in different concentration.Then,the cellular growth activities were assayed by MTT in different groups.ResultsDNA sequencing of the plasmids verified the successful construction of the HER-2/neu shRNA vectors.Compared with the control group,expressions of protein 185 were remarkably down-regulated in the RNAi groups and the apoptotic rate increased.The proportion of the cells in G₀/G₁ stage increased and those in the S stage decreased in the Ishikawa cells transfected with HER-2/neu shRNA.Their sensitivity to progesterone also increased.ConclusionWe successfully construct the recombinant plasmids of shRNAs specific to HER-2/neu gene and transfect them into the Ishikawa endometrial cancer cells in vitro. The RNAi inhibited the expression of protein 185 and subsequently suppressed the proliferation of Ishikawa cells,increased their apoptotic rate and arrested the cell cycle at G₀/G₁ stage.RNAi also increased the cells' sensitivity to progesterone.