Study The HuR Gene Silence On The Proliferation And Apotosis Of Endometrial Carcinoma Cell Lines Ishikawa In Vitro

ObjectiveEndometrial cancer is one of the highest incidence in the female malignant reproductive tract tumors.In recent years,duing to women's life expectancy, the elderly and obese women increased, the incidence of endometrial cancer increased year by year. For a long time,on the etiology of endometrial cancer research has been the focus. But so far, the exact occurrence and the mechanism of endometrial cancer remains unclear.HuR is a member of RNA-binding protein in the embryonic lethal abnormal vision (ELAV)family. Studies have shown that HuR can plays its role in mRNA post-transcriptional regulation by combining and stabling the fragments of A, U (AREs) in the 3' untranslated region (UTR). It is implemented to post-transcriptional gene regulation by changing the translation efficiency and mRNA stability.Encoding cytokines, lymphokine, and in a kind of early response genes (ERGS) mRNA in proto-oncogene which all contain AREs.Previous studies confirmed that HuR can be expressed in a variety of cell lines, such as:ovarian cancer, breast cancer, lung cancer, and thus played its important part in promoting tumor growth. In the current study,however, It is still unclear that wether the expression of HuR in endometrial carcinoma or not and what its accurate mechanism of tumorigenic action. Endometrial carcinoma is one kind of estrogen-dependent tumor. There are two types of estrogen receptor, named ER alpha and ER beta which belong to superfamily members of the ligand mediated nuclear receptor.ER expression can alter by regulating the transcription,post-transcription and epigenetic level. RNA bingding protein can bind with ER alpha mRNA and effectively control its protein expression. The 3 'UTR of ER is a period of 45 nucleotide, which contains 7 AREs, belonging to the classification of the I AREs, namely in the rich U area contains 1-3 AUUUA copy [4].But in the rencent study, the expression of HuR in endometrial carcinoma and the relationship between HuR and ER alpha is unclear.In our study, we used the cellular immune fluorescence to detect the alteration of HuR's expression in the endometrial cancer after transfecting HuR siRNA expression plasmid into Ishikawa cells.We investigated the change of the expression of HuR and ER-a protein by RNAi technology and studied preliminarily the role of HuR in the pathogenesis of endometrial cancer and explored its relationship with the occurrence and development of endometrial cancer.MethodsUsing the cellular immunity fluorescence method to measure the change of HuR expression in the cells after transfection; HuR siRNA expression plasmid transfected into Ishikawa cells with liposomes transfection method. Using RT-PCR and Western-blot observed the mRNA and protein expression of HuR in the Ishikawa cells after transfection 24h,,48h,72h. Then the flow cytometry and MTT were used separately to detect the Ishikawa cell cycle, apoptosis and proliferation in the different transfection hours.Results1.Building expression plasmid of HuR siRNAs sequence as HuR-siRNA1,HuR-siRNA2,HuR,-siRNA3,and using DNA sequencing technology to insure the resulting sequence is conform to the design. We transfected the expression plasmid of HuR siRNAs into Ishikawa cells respectively with lipofectamine2000.The rate of inhibition of HuR mRNA expression in Ishikawa cells respectively as follows:55.48%,82.81%, and 67.59%.We used the HuR-siRNA2 as the best sequence on following experiments.2.Immunofluorescence results showed that HuR expressed in nucleus and cytoplasm in Ishikawa cells, especially expressed in nucleus.We are also found that HuR fluorescence intensity decreased significantly after transfecting HuR-siRNA2 48h.3.By RT-PCR,We can observed that the brightness in beta actin are similar.However, the brightness of tansfecting HuR-siRNA2 expression plasmid group is weaker than blank control, Liposomes controls and negative control.The relative content of HuR mRNA of the blank control group,liposomes control group,HuR-siRNA-neg group and HuR-siRNA2 transfection group cells respectively was 0.6082±0.01562,0.5965±0.02080,0.5699±0.01390,0.2650±0.01458,0.11801±,0.0160,0.08135±0.05473. By comparing 24h,48h,72h groups with three control groups,we can conclude that the difference was statistically significant (P<0.00).The difference between the group of HuR-siRNA-neg group and the blank control group, it was statistically insignificant(P>0.05).4.By Western-Blot,HuR protein expression dramatically decreased between transfection group,especially at 72h. It was statistically insignificant(P>0.05). Between the blank group,liposomes control and HuR-siRNA-neg group to compare.. The relative content of HuR protein of the HuR-siRNA2 transfection group cells respectively was 0.5053±0.0233,0.2515±0.0155,0.1773±0.0104. By comparing 24h,48h,72h groups with three control groups,we can conclude that the difference was statistically significant (F=746.595,P<0.01). After transfection 72h,the rate of inhibition is 73.76%. By comparing 24h,48h,72h groups with three control groups,we can conclude that the difference of ER-a was statistically significant (P<0.00).It shows the positive correlation between HuR protein and ER alpha proteins expression in Ishikawa cells by Spearman rank analysis.5.By MTT method, After transfeting HuR-siRNA2 expression plasmid in Ishikawa cells,the rate of proliferation decreased,especially at 72h and the rate of inhibition was 34.26%. There was no significant difference (P> 0.05) between transfection 24h group and the blank by compared. We also concluded that there is a significant difference (P< 0.01) comparing transfection 48h,72h group.The rate of Ishikawa cells inhibition is 15.31%,26.99%,34.26%,19.58% respectively.6.By flow cytometry,comparing toHuR-siRNA-neg group,the rate of G1 phase in the cell cycle increasd in the group of HuR-siRNA, G2/M and S phase decreased.The ratio of cell apoptosis was 0.41%±0.49%,5.09%±1.58% respectively.ConclusionHuR has expression in the cytoplasm and cell nucleus in Ishikawa cells, but mainly in the cell nucleus. HuR siRNA expression plasmid has silent effect to HuR gene expression, but also inhibited Ishikawa endometrial cancer cells apoptosis, promoting it proliferation. HuR has important effect in the occurrence and development of tumor cells.