Effect Of 5-Aza-dC On Cell Proliferation In Endometrial Cancer Cell Lines And IGFBP7 Gene Expression

Backgrounds and objectivesEndometrial cancer is one of the most common malignant tumors of the female reproductive system, the incidence rate is only second to cervical cancer, and increased year by year. In China, endometrial cancer is the ninth common incident cancer in women, and about 62000 patients were diagnosed with endometrial cancer in 2012. But its pathogenesis is still not clear. Insulin like growth factor binding protein 7 is a secreted protein with the molecular weight of 30 kda, the studies have found that in prostate cancer, malignant melanoma, colorectal cancer, thyroid cancer and other solid tumors its expression is decreased or deletion and the abnormal expression is associated with promoter hypermethylation and application of DNA methyltransferase inhibitor 5-Aza-2 deoxycytidine can upregulate the expression of IGFBP7.Early research showed that the expression of IGFBP7 in endometrial cancer cell lines was decreased or absent. up-regulating the expression of IGFBP7 by Lipofectamine can decrease cell proliferation rate and increase apoptosis in endometrial cancer cell lines; in addition, added human recombinant IGFBP7 to endometrial cancer cell culture medium can inhibit the proliferation of endometrial cancer cells through the regulation of the ERK signaling pathway. The application of 5-aza-2 deoxycytidine to treat the endometrial cancer HEC-1A and Ishikawa cells is to observed the proliferation of endometrial cancer cells and effect of IGFBP7 expression and explore the therapeutic possibility of endometrial carcinoma. Methods:(1)adding 5-Aza-dC to the culture medium of endometrial cancer HEC-1A and Ishikawa cells,so that the final concentration was 2.5μmol/L, 5μmol/L, 10μmol/L, 20μmol/L, and treated for 24, 48, 72 h, respectively. Then Counting Kit-8 Cell(CCK-8) method was used to detect the effect of 5-Aza-dC on the proliferation activity of HEC-1A and Ishikawa cells at different concentrations and different time, and calculated the inhibitory rate on the tumor cells.(2)The expression of IGFBP7 mRNA in HEC-1A and Ishikawa cells was detected by real-time fluorescence quantitative PCR(qRT-PCR) technology after the treatment of 5μmol/L 5-Aza-dC for 72 hours.(3)The expression of IGFBP7 protein in HEC-1A and Ishikawa cells was detected by Western blot after the treatment of 5μmol/L 5-Aza-dC for 72 hours.(4)All dates were analysised by SPSS17.0 statistical software, and the results in the form of sx ±. and quantitative datas conformed to the normal distribution were analysised by one-way ANOVA(homogeneity of variance),LSD-t was used in comparison between groups. Statistical significance level was 0.05.Results: 1.CCK-8 method was used to detect the effects of different concentrations of 5-Aza-dC on the proliferative activity of HEC-1A and Ishikawa cells in endometrial carcinomaThe results showed that 2.5μmol/L, 5μmol/L, 10μmol/L, 20μmol/L5-Aza-dC could inhibit the proliferation of HEC-1A and Ishikawa cells at 24 h 、 48 h 、72h,respectiavely. At the same concentration, with the extension of time, cell proliferation was decreased significantly, while the inhibition rate increased(P<0.05); at the same time, with the increase of drug concentration, the rate of cell proliferation decreased, the inhibition rate increased, has obvious dose dependent manner, the difference is statistically significant(P < 0.05). 2.Effect of 5-Aza-dC on the expression of IGFBP7 mRNA in endometrial carcinoma HEC-1A and Ishikawa cell linesThe results showed that IGFBP7 mRNA relative expression levels in HEC-1A and Ishikawa cells after treatment for 72 hours with 5μmol/L 5-Aza-dC were significantly increased compared with before treatment, the difference has statistical significance(P < 0.05). 3.Effect of 5-Aza-dC on the expression of IGFBP7 protein in endometrial carcinoma HEC-1A and Ishikawa cell linesThe results showed that IGFBP7 protein relative expression levels in HEC-1A and Ishikawa cells after treatment for 72 hours with 5μmol/L 5-Aza-dC were significantly increased compared with before treatment, the difference has statistical significance(P < 0.05). Conclusions:1. 5-Aza-dC shows a proliferation inhibition effect on endometrial cancer HEC-1A and Ishikawa cells;2. 5-Aza-dC can elevate the expression of IGFBP7 in vitro endometrial cancer cell lines; methylation mechanism involved in IGFBP7 expression and regulation, but the promoter methylation status requires further analysis.