Sox4表达抑制对宣威地区女性肺癌细胞凋亡影响及其机制的探讨

Sox4 targeted inhibition effects on lung cancer cell apoptosis of Xuanwei female patients and its mechanism research

目的:研究靶向抑制sox4基因的表达对宣威地区女性肺癌细胞凋亡的影响,并初步探讨其作用机制。方法:构建靶向抑制转录因子sox4的shRNA重组载体pGFP-V-RS-sox4shRNA并转染宣威地区女性肺癌细胞XWLC-05,以转染pGFP-V-RS-scramshRNA的XWLC-05细胞及亲本XWLC-05细胞作为对照,研究sox4表达抑制对Caspase-3表达、细胞形态、细胞周期和凋亡率等凋亡指标的影响。使用Caspase-3抑制剂z-VAD-FMK处理转染细胞,检测上述细胞凋亡指标。结果:成功构建了能高效抑制sox4基因表达的pGFP-V-RS-A-sox4shRNA重组载体;转染后48及72h,实验组细胞sox4mRNA表达水平分别为0.09±0.018及0.44±0.06,比转染后24h降低了88%和40%,与同一时段中的阴性对照及空白对照组相比表达水平明显降低(P<0.05),其中以转染后48h的抑制最为明显,而Caspase-3mRNA表达与阴性对照及空白对照组相比均明显升高(P<0.05),以48h升高最明显;空白对照组与阴性对照组间比较,转染48及72h后sox4mRNA(P=0.071;P=0.063)和Caspase-3mRNA(P=0.103;P=0.229)的表达水平差异无统计学意义;抑制sox4基因的表达后细胞出现典型的凋亡形态学改变;流式细胞术(FCM)检测显示,转染干扰质粒后48h,细胞出现明显的亚二倍体峰,实验组细胞的凋亡率平均为(34.8±3.37)%,显著高于阴性对照组(0.45±0.05)%及空白对照组(0.44±0.06)%,P均为0.000。经z-VAD-FMK阻断Caspase-3酶活性后,实验组细胞中活化的Caspase-3(13.6±1.76)%显著低于阴性对照组(50.5±6.15)%,差异有统计学意义,P=0.000;实验组细胞的凋亡率(10.8±1.05)%也明显低于阴性对照组细胞(38.3±3.16)%,P=0.000。结论:抑制sox4的表达可促进宣威地区女性肺癌细胞凋亡;转录因子sox4可能通过抑制Caspase-3依赖的凋亡途径而促进宣威地区女性肺癌的发生发展。

OBJECtIVE：To study the effects of targeted inhibiting sox4 gene expression on promoting apoptosis of Xuanwei female lung cancer cells and its possible mechanism in vitro. METHODS： Recombinant plasmid pGFP-V-RS-sox4shRNA was constructed and transfected into Xuanwei female lung cancer cell line XWLC-05 to inhibit the expression of transcription factor sox4 taking parental XWLC-05 cells and the cells transfected by hlasmid pGFP-V-RS-scramshRNA as control. Then cell apoptosis was identified by analyzing expression of Caspase-3, cell cycle,morphology and apoptosis rate of transfected cells treated with or without Caspase-3 inhibitor z-VAD-FMK. RESULTS： Recombinant plasmid pGFP-V-RS-A-sox4shRNA was successfully constructed and efficiently transfected into XWLC-05 cells. The sox4 mRNA expression in sox4 inhibited cells was 0.09±0. 018 and 0.44±0.06 at 48 h and 72 h after transfection,decreased 88% and 40% when compared with the expression at 24 h,and their expression was significantly lower than that in the negative control and blank control in the same period （P〈0.05）. The inhibition effect was most obvious at 48 h after transfection. The Caspase-3 mRNA and protein were significantly up-regulated 48 h and 72 h after transfection when compared with non-transfected cells （P〈0.05）. There was no significant difference in the sox4 mRNA expression level between blank and negative control at 48 h and 72 h（P = 0.071 ; P= 0. 063） after plasmid transfection, also of Caspase-3 mRNA expression （ P = 0. 103 ; P = 0. 229 ）.The typical apoptosis morphological changes were observed in sox4-inhibited cells by microscope. There was obvious sub- G1 peak in sox4-inhibited cells and their average apoptosis rate（34.8 ±3.37）% was significantly higher than that in the negative control（0.45± 0.05） % and blank control（0.44 ±0.06） % （ both P were 0. 000） 48 h after transfection. After treating with z-VAD-FMK,the activated Caspase-3 of sox4 inhibited cells （13.6：1： 1.76）~ was significantly lower than that in the negative control （50.5 ±6.15）% （P： 0. 000） and their apoptosis rate （10.8±1.05）% also lower than that in the negative control （38.3 ± 3.16） % （P： 0. 000）. CONCLUSIONS： Inhibition of sox4 expression can promote the apoptosis of XWLC-05 cells;Transcription factor sox4 may play an important role in occurrence and progress of Xuanwei female lung cancer through inhibiting the Caspase-3 dependent apoptosis pathway.