沉默CCND1对肝细胞癌细胞5-氟尿嘧啶耐药性的影响及机制

Effect and mechanism of silencing CCND1 on 5-Fu resistance in hepatocellular carcinoma cells

目的探讨沉默细胞周期蛋白D1基因(CCND1)对肝细胞癌(HCC)细胞(SMMC-7721、HepG2细胞)5-氟尿嘧啶(5-Fu)耐药性的影响及机制。方法常规培养SMMC-7721、HepG2细胞,并随机分别分为HepG2+NC-siRNA组、HepG2+CCND1-siRNA组和SMMC-7721+NC-siRNA组、SMMC-7721+CCND1-siRNA组,分别用靶向CCND1的siRNA(CCND1-siRNA)、阴性对照siRNA(NC-siRNA)转染细胞。转染48 h后取各组细胞分别置于含10μg/mL 5-Fu的培养基中培养48 h。用荧光定量PCR法检测CCND1 mRNA表达,用CCK-8实验检测细胞活力,用流式细胞术检测细胞凋亡率,用Western blotting法检测CCND1、DNA修复相关蛋白[磷酸化组蛋白(γ-H2AX)、同源重组修复蛋白(RAD51)]表达。结果转染后,HepG2+CCND1-siRNA组、SMMC-7721+CCND1-siRNA组CCND1 mRNA、蛋白表达均低于其对照组(HepG2+NC-siRNA组、SMMC-7721+NC-siRNA组),比较差异均有统计学意义(P均<0.05)。5-Fu暴露下72 h,HepG2+CCND1-siRNA组、SMMC-7721+CCND1-siRNA组细胞活力(OD值)低于其对照组,凋亡率高于其对照组,γ-H2AX蛋白表达高于其对照组,RAD51蛋白表达低于其对照组,比较差异均有统计学意义(P均<0.05)。结论沉默CCND1可提高HCC对5-Fu的耐药性,该作用可能与调控DNA修复相关蛋白表达有关。

Objective To investigate the effect and mechanism of silencing the cyclin D1 gene(CCND1)on 5-fluorouracil(5-Fu)resistance in hepatocellular carcinoma(HCC)cell lines(SMMC-7721 and HepG2 cells).Methods SMMC-7721 and HepG2 cells were cultured and randomly divided into the HepG2+NC-siRNA group,HepG2+CCND1-siRNA group,SMMC-7721+NC-siRNA group,and SMMC-7721+CCND1-siRNA group,respectively.The cells were transfected with small interfering RNA targeting CCND1(CCND1-siRNA)or negative control siRNA(NC-siRNA).After 48 h of transfection,cells of each group were cultured in the medium containing 10μg/mL 5-Fu for 48 h.The level of CCND1 mRNA was detected by qRT-PCR.The cell viability was detected by CCK-8.The apoptosis rate was detected by flow cytometry.The expression levels of CCND1 and DNA repair-related proteins[phosphorylated histone(γ-H2AX)and RAD51]were detected by Western blotting.Results After transfection,the expression levels of CCND1 mRNA and protein in the HepG2+CCND1-siRNA group and SMMC-7721+CCND1-siRNA group were lower than those of the control groups(HepG2+NC-siRNA group and SMMC-7721+NC-siRNA group),and the differences were statistically significant(all P<0.05).After 72 h of exposure to 5-Fu,the cell viability(OD)of the HepG2+CCND1-siRNA group and SMMC-7721+CCND1-siRNA group was lower than that of the control groups,while the apoptosis rates were higher(all P<0.05).The expression levels ofγ-H2AX protein in the HepG2+CCND1-siRNA group and SMMC-7721+CCND1-siRNA group were higher than those of the control groups,while the expression levels of RAD51 protein were lower,with statistically significant differences(all P<0.05).Conclusion Silencing CCND1 can increase the 5-Fu sensitivity of HCC,which may be related to regulating the expression of DNA repair-related proteins.