Dicer1在卵巢癌间质成纤维细胞中调控DNA损伤修复相关基因

目的:检测Dicer1在卵巢癌间质肿瘤相关成纤维细胞中的表达情况,探讨其对DNA双键断裂损伤(DSB)修复相关基因的影响。方法:应用基因集合富集分析(GSEA)方法分析卵巢癌间质和正常卵巢间质基因表达公共数据库GSE40595,探索Dicer1对DSB修复相关通路及关键基因的调控作用,并筛选出相关差异基因。应用重组人转化生长因子β1(h TGFβ1)细胞因子诱导成纤维细胞系MRC5细胞活化成为肿瘤相关成纤维细胞(CAFs),即MRC5-CAFs。以靶向Dicer1基因的siRNA转染MRC5-CAFs,下调其Dicer1表达水平。qRT-PCR、Western blot法分别检测Dicer1及分析所得相关基因,如XRCC6、TP53BP1、H2AFX、BRCA1、ATR、RAD51 mRNA水平及Dicer1和DSB损伤修复关键基因γ-H2AX(磷酸化H2AX)、RAD51蛋白水平。荧光共聚焦检测细胞核蛋白γ-H2AX表达;采用CCK8试验检测细胞活性。结果:GSEA分析结果显示,Dicer1表达水平与DSB(NES=1.7942109,FDRq=0.0067545176)及DNA损伤修复(NES=2.4433048,FDRq=0)显著正相关,进一步通过相关分析筛选出在上述通路中与Dicer1表达正相关的显著基因集。沉默MRC5-CAFs细胞中Dicer1表达后,DSB相关基因,如XRCC6、TP53BP1、H2AFX及损伤修复相关基因BRCA1、ATR、RAD51均较对照组显著下降,与GSEA分析结果一致。荧光共聚焦显微镜检测到沉默Dicer1 MRC5-CAFs细胞内聚集较少的DSB。CCK8试验结果显示,MRC5-CAFs细胞在20μmol/L顺铂作用下,Dicer1-si组不同时间梯度的细胞活性率均低于NC-si组。结论:Dicer1在卵巢癌间质成纤维细胞中正向调控DNA损伤修复相关基因,抑制Dicer1表达后其对顺铂敏感性显著增加。

Dicer1 regulation of DNA damage response related molecules in ovarian cancer associat-ed fibroblasts

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.