现代妇产科进展2016，Vol.25Issue(3)：161-165,5.DOI:10.13283/j.cnki.xdfckjz.2016.03.001
Dicer1在卵巢癌间质成纤维细胞中调控DNA损伤修复相关基因
Dicer1 regulation of DNA damage response related molecules in ovarian cancer associat-ed fibroblasts
摘要
Abstract
Objective:To explore the expression of Dicer1 in ovarian cancer CAFs (cancer-associated fibroblasts) and its the regulation of DNA damage response(DDR). Meth-ods:GSEA( gene set enrichment analysis) analysis was performed on a public dataset named GSE40595 including mRNA profiling data of normal and ovarian tumor stroma cells,to obtain the potential regulation of Dicer1 on DSB related pathway and relevent genes. MRC5 cells were prompted by transforming growth factor beta 1 ( TGFβ1 ) to differentiate into cancer-associated fibroblasts( CAFs ) , namely MRC5-CAFs. A Dicer1 specific siRNA was used to knock down Dicer1 expression in MRC5-CAFs,with NC-siRNA being a negative control. The expression of Dicer1 and obtained genes from GSEA analyisis,such as XRCC6、TP53BP1、H2AFX、BRCA1、ATR and RAD51,were evaluated by qRT-PCR 24h after siRNA transfection. Western blot assay was conducted to observe the alteration of the γ-H2AX and RAD51 proteins,the marker mole-cules of DDR 48h after siRNA transfection. Meanwhile,CCK8 assay was used to detect the cell viability at various time points in the presence of 20umol/L cisplatin. Furthermore,confocal mi-croscope was adopted to check the production of DSB biomarkerγ-H2 AX in both groups with or without cisplatin treatment. Results:The results of GSEA indicated the notablely positive corre-lation between Dicer1 and DSB pathway(NES=1. 7942109,FDRq=0. 0067545176) and DNA repair pathway(NES=2. 4433048,FDRq=0),with the screened out involved gene sets listed in the heatmap. qRT-PCR and western blot confirmed the remarkably reduced mRNA and protein levels of all tested markers in MRC5-CAFs after Dicer1 knocking down,which was in line with the results of GSEA. Confocal microscopic assay showed that γ-H2AX foci in the nuclei of the Dicer1-siRNA transfected cells was generously decreased in comparison with the control cells. Moreover Dicer1 silence increased the sensitivity to cisplatin in MRC5-CAFs(P<0. 001). Con-clusions:Dicer1 positively regulated DNA damage response related genes in ovarian cancer stro-mal fibroblasts,and silencing the expression of Dicer1 enforced the sensitivity to cisplatin in the fibroblasts.关键词
卵巢癌/成纤维细胞/Dicer1/顺铂/DNA损伤应答/DNA双键断裂损伤/同源重组修复Key words
Ovarian cancer/Fibroblasts/Dicer1/Cisplatin/DNA damage response/DNA double-strand breaks/Homologous recombination repair分类
医药卫生引用本文复制引用
靳平,杨宗元,徐森,张陶然,高庆蕾..Dicer1在卵巢癌间质成纤维细胞中调控DNA损伤修复相关基因[J].现代妇产科进展,2016,25(3):161-165,5.基金项目
国家自然科学基金资助项目(No:81372801) （No:81372801）
