| Objective：Ovarian cancer is a kind of malignancy tumors which threats female’s life. It isone of the three most common malignant cancer of the female reproductive system.The study found that hypermehylated in cancer1(HIC1) occurs hypermethylatedwhich results expression silencing frequently, but the HIC1whether affect ovariancancer and its mechanism has no in-depth studies. This study investigated themolecular mechanisms of the HIC1and its regulation of HIC1/SIRT1pathway inovarian cancer.Methods：Ovarian cancer tissues:Using immunohistochemical method to detect DNMT1,DNMT3a, HIC1, SIRT1protein expression of26cases of ovarian cancer clinicaltissue samples and statistical analysing the relationship between the DNMTs andHIC1expression and the HIC1downstream regulation of SIRT1protein expression,and further using the Western Blot method to detect HIC1and SIRT1proteinexpression of ovarian cancer and normal ovarian tissues.Cell experiments: Culturing epithelial ovarian cancer cell line SKOV-3, a DNAmethylation inhibitor5-azacytidine as a tool for drug intervention, divided intocontrol group and administration groups. MTT assay was used to detect the effect oncell proliferation of the different time points and different concentrations of the drugs,and drew the cell growth curve; using Western Blot to test HIC1, SIRT1proteinexpression of cells before and after administration; inverted microscope observationof the morphological changes of the cells before and after administration; flowcytometry testing SKOV-3cell cycle arrest and apoptosis.Results:1.Immunohistochemistry results show that the negative rate of HIC1proteinexpression of the DNMTs positive samples is much higher than that found of theDNMTs negative samples in ovarian tissues. Statistical analysing show that DNMTs protein expression and HIC1protein expression was negatively correlated.2.Immunohistochemistry and Western Blot experiments observed HIC1proteinis downregulated in ovarian cancer, SIRT1protein expression is elevated comparedwith expression in normal ovarian tissues.It has significant difference (P <0.05).3.MTT results show that the5-azacytidine can inhibit the growth of SKOV-3cells in different degrees, and the inhibition depend on the time and concentration. Arelatively low concentration of5-azacytidine didn’t significantly inhibit theproliferation of SKOV-3cells,while an excessive concentration has strong inhibitionof tumor cells, but there are also significant cytotoxicity.4. Observe the cell morphology of SKOV-3cells treated with drugs72h, wefound that a small part of1μM drug treated cells from spindle to round nucleus, cellmembrane integrity, cell gap; cell morphology of a lot of cells with2.5μM drugtreatment tends to round, foaming, particles in the cytoplasm are more, adherentability decreased; with5μM drug treatment group cell size tends to be uniform, hascell debris, some cell necrosis, but the control group has strong adherent ability andcell proliferation rate was significantly faster than that of treatment groups.5.Flow cytometry showed that SKOV-3cells after treatment with5-azacytidine72h, the proportion of cells in G1phase increased, cells were blocked at the G1phase.And with the increase of5-azacytidine concentration, the cycle arrest trend was morepronounced. Different treated group compared with control group were statisticallysignificant difference (P <0.05),5μM treated compared with1μM treatment group hadsignificant difference (P <0.05).6. Flow cytometry showed that SKOV-3cells after treatment with5-azacytidine72h, the apoptosis percentage of cells gradually increased with the increase of theconcentration of5-azacytidine. Different treated group compared with control groupwere statistically significant difference (P <0.05),5μM treated compared with1μM,2.5μM treatment groups had significant difference (P <0.05).7.Western Blot detection HIC1ptotein expression of untreated SKOV-3cellsappeared weak, but after5-azacytidine treated, the expression enhanced. Withincreasing of drug concentration, the expression enhanced.Compared with control group it has significant difference (P <0.01). In addition, SIRTl protein expressiondecreased after treated5-azacytidine, and with the increase of the concentration of thedrug the decreased degrees up. Compared with control group it has significantdifference (P <0.01).Conclusions:1.Compared with normal ovarian tissues，DNMTs protein expression in ovariancancer tissues raised while HIC1protein expression decreased. We can tell DNMTsplay an important role in affecting HIC1protein expression.2.Compared with normal ovarian tissues, SIRT1protein expression in ovariancancer tissues increased.It provides the basis for further researching HIC1proteinexpression change in vitro.3.The methyltransferase inhibitor5-azacytidine can up-regulate HIC1proteinexpression and down-regulate SIRT1protein expression in ovarian cancer cell SKOV-3.It also can slow down the cell proliferation rate,make cell division stay in G1phaseand induce apoptosis.We can tell the regulation of HIC1’s DNA methylation canrestore HIC1protein expression and HIC1protein inhibit the growth of ovariancancer through HIC1/SIRT1pathway. |
