Suppression Of Dicer1 Influenced Ovarian Cancer-associated Fibroblasts Sensitivity To Cisplatin

?Objective?To explore the expression of Dicer1 in ovarian CAFs(cancer-associated fibroblasts)and its regulation on cellular activation and subsequent effects on cisplatin sensitivity.?Methods?Intergrated analysis of GSE40595(m RNA profiling data of normal and ovarian tumor stroma cells included)was performed to detect Dicer1 expression in different tissues,followed by q RT-PCR in primary fibroblasts to verify the results.GSEA(gene set enrichment analysis)was performed on GSE40595 to obtain the potential regulation of Dicer1 on stromal chemoresistance related pathways and relevant genes.MRC5 cells were transformed to CAFs by transforming growth factor beta 1(TGF?1),namely MRC5-CAFs.A Dicer1 specific si RNA was used to silence Dicer1 expression in MRC5-CAFs,with a NC-si RNA being the negative control.The expression of Dicer1 and included genes from GSEA,such as XRCC6?TP53BP1?BRCA1?BRCA2?ATR and RAD51 were evaluated by q RT-PCR 24 hours after si RNA transfection.Then,western blot assay was conducted to observe the alteration of the marker molecules of DDR-?-H2 AX and RAD51 48 hours after si RNA transfection.Meanwhile,CCK8 assay was used to detect the cell viability at various time points in the presence of 5 umol/L cisplatin.Furthermore,confocal microscope was adopted to check the production of DSB biomarker ?-H2 AX in both groups with or without cisplatin treatment.Finally,co-culture models and murine xenograft models were adopted to study the potential infuluence of Dicer1 inhibition on the pro-carcinogenic effect of MRC5-CAFs in both untreated and cispatin-treated groups.?Results?Compared with the normal stroma,Dicer1 was found overexpressed in the ovarian cancer stroma and was found exclusively expressed in stromal CAFs.GSEA indicated a notable positive correlation between Dicer1 and DSB repair pathway(NES=1.7942109,FDRq=0.0067545176)and DNA repair pathway(NES=2.4433048,FDRq=0),with the enrolled gene sets listed in the heatmap.q RT-PCR and western blot assays confirmed the remarkably reduced levels of DNA damage response in MRC5-CAFs after Dicer1 knocking down,which was in line with the results of GSEA.Confocal microscopic assay showed that ?-H2 AX foci in the nuclei of the Dicer1-si RNA transfected cells was generously increased in comparison with the control cells.Moreover,Dicer1-silence increased the sensitivity to cisplatin in MRC5-CAFs(P<0.001)in vitro and subsequently undermined their tumor-promoting ability.?Conclusions?Dicer1 was highly expressed in CAFs and positively regulated DNA damage response related genes in ovarian CAFs.Inhibition of Dicer1 expression enhanced ovarian CAFs sensitivity to cisplatin and finally hamper their tumorpromoting effects in OC.