Effect Of IEX-1Gene Transfection On Proliferation And Sensitivity To Cisplatin In Human Ovarian Carcinoma Cell Line

Background and ObjectiveOvarian cancer is the most common cause of mortality in gynecologic cancers, although5-year survival has improved. Owing to non-specific clinical symptoms, short duration and early invasion, most patients have distant spread of this disease (stage Ⅲ-Ⅳ) at the time of diagnosis. Current treatment is mainly surgery in combination with chemotherapy, radiation therapy, and biological treatment. Now one hot research point is to look for an effective gene target to suppress the progression of human ovarian cancer. The successful application of gene therapy in tumor field provides new ideas for the treatment of ovarian cancer. Immediate early response gene X-1(IEX-1) as one member of the immediate early gene family, is located at human chromosome6p21.3and adjacent to the major histocompatibility complex locus. Recent studies have shown that IEX-1expression and transcription are down-regulated or absent in many tumor tissues and cells. IEX-1over-expression can regulate tumor cell growth and reverse malignancy, which implys IEX-1has close relation to cancer occurrence and development. The latest research shows that IEX-1expression decreased with the development of ovarian tumor from benign to malignance. Apoptosis in ovarian cancer tissues had positive correlation with IEX-1 expression. The low expression of IEX-1might play an important role in biological behavior of ovarian cancer.In our research, a recombinant plasmid pEGFP-N1-IEX-1was transfected to SKOV3cells with IEX-1low expression by lipofectamine, to observe the effect of IEX-1on cell proliferation, cell cycle and sensitivity to cisplatin of human ovarian cancer cell line SKOV3respectively, and to further investigate the effect of IEX-1on ovarian cancer development and the significance as ovarian cancer gene therapy target, and to provide valuable basis for the clinical therapy of ovarian cancer.Methods1. The recombined plasmid pEGFP-N1-IEX-1was purified and verified by PCR. Ovarian cancer cell line SKOV3was transfected with recombinant plasmid pEGFP-N1-IEX-1and empty plasmid pEGFP-Nl by lipofectamine. Three groups were divided:SKOV3group, SKOV3-EGFP group and SKOV3-IEX-1group. The expressions of IEX-1mRNA and protein were examined by RT-PCR and immunocytochemistry respectively, and transfection efficiency was observed by fluorescence inverted microscope.2. The cell cycle was detected by flow cytometry, to analyze the effect of IEX-1gene transfection on cell cycle process in human ovarian carcinoma cell line.3. The cell viability was determined by MTT assay, to analyze the effects of IEX-1gene transfection on proliferation and sensitivity to cisplatin of ovarian cancer cell line SKOV3.4. Statistical software SPSS17.0was used to analyze the data results. One-way analysis of variance and bonferroni test were used to measure mean differences between three groups. Bilateral a=0.05was considered as test standards.Results1. The amplification length of recombinant plasmid pEGFP-N1-IEX-1by PCR method was about412bp; there were low expressions of IEX-1mRNA and protein in SKOV3cells. IEX-1mRNA and protein were detected in SKOV3cells after the IEX-1transfection.2. After24hours transfection, a large number of green fluorescent particles were observed in the cells transfected pEGFP-Nl-IEX-1and pEGFP-Nl under fluorescence inverted microscope.3. Flow cytometry results testing cell cycle showed that:the proportion of Go/G1phase in SKOV3-IEX-1, SKOV3-EGFP and SKOV3groups was (62.93±1.27)%,(75.16±1.32)%,(75.34±0.43)%respectively; and it was significantly less than those of SKOV3-EGFP and SKOV3groups(F=378.76, P<0.000); the proportion of S phase in three groups was (32.30±1.20)%,(16.89±0.29)%,(17.98±1.05)%respectively; the proportion of G2/M phase in three groups was (4.41±0.97)%,(7.17±0.18)%,(6.82±0.27)%;Compared with SKOV3-EGFP and SKOV3groups, cells of proliferative phase(S and G2/M) in SKOV3-IEX-1group increased significantly;The difference had statistically significancy.4. The MTT results showed that after transfection of recombinant plasmid pEGFP-Nl-IEX-1, cell proliferation rate was much faster than the control cells and cells transfected with empty vector (P<0.05). The difference between SKOV3group and SKOV3-pEGFP-N1group was not statistically significant(P>0.05).5. The cells with transfected pEGFP-N1-IEX-1had enhanced sensitivity to cisplatin, compared the normal group and the empty vector control group. The difference was significant (P<0.05), while there was no significant difference between SKOV3group and SKOV3-pEGFP group (P>0.05).Conclusions1. IEX-1can promote the proliferation ability of ovarian cancer cells in vitro partly through the G1/S check point.2. IEX-1can enhance the sensitivity of ovarian cancer cells to cisplatin in vitro.3. In ovarian cancer cell line SKOV3, the over-expression of IEX-1by regulating the cell cycle can promote cell proliferation in good growth conditions, and promote cell apoptosis in adverse conditions.