RNAi下调STAT3基因表达促进Bel-7402肝癌细胞凋亡

RNAi-induced down-regulation of STAT3 expression promotes the apoptosis of Bel-7402 Hepatocarcinoma

目的探讨JAK／STAT3信号转导途径对肝癌细胞凋亡的影响。方法以BEL-7402肝癌细胞为模型，采用RNAi技术，将STAT3-siRNA转染BEL-7402肝癌细胞，沉默STAT3基因表达，另设细胞对照组，阴性质粒组（pGCsi．U6／neoRFPscramble-siRNA载体转染BEL-7402）。分别通过流式细胞术检测细胞凋亡比率的变化，JC-1荧光染色观察线粒体膜电势AW。变化，并利用WesternBlot方法检测Caspase3蛋白质水平的变化。结果STAT3-siRNA组凋亡率为（38．82±0．88）％，明显高于其他组[对照组（9．22±0．38）％，阴性质粒组（16．47±1．04）％，P〈0．05]，并引起细胞线粒体膜电势明显降低[（59．06±1．89）％vs（91．33±1．78）％和（89．90±1．92）％，P〈0．05]，活性Caspase3蛋白在STAT3-siRNA组表达明显高于其他组（0．48±0．05VS0．22±0．04和0．26±0．06，P〈0．05）。结论RNAi沉默STAT3基因，抑制JAK／STAT3信号转导途径，促进BEL-7402肝癌细胞凋亡。

Objective To explore influence of JAK/STAT3 signaling pathway on the apoptosis .of HCC cells. Methods DNA-vector-based RNAi approach silence was used to down-regnlate STAT3 expres- sion in Bel-7402 cells. According to the STAT3 cDNA sequence in the GeneBank database, the plasmid pGCsi. U6/neoRFP -STAT3 that was designed for expression of STAT3 siRNA was constructed and synthe- sized, and then transfected into the Bel-7402 cells with lipofectamine 2000. The apoptotic rate was meas- ured with flow cytometry （FCM） and annexinV/PI apoptosis detection kit staining. The mitochondrial mem- brane potential （B^m） was visualized by the JC-1 fluorescence staining and the inverted fluorescence mi- croscope. Moreover, the expression of caspase-3 protein was analyzed by Western blotting. Results The apoptotie ratio of STAT3-siRNA group was （ 38. 82 ± 0. 88 ） % , which was significantly higher than that in other group [ control group（9. 22 ± 0. 38 ） %, scramble-siRNA group（ 16.47± 1. 04 ） %, P 〈 0.05 ]. The mitochondrial membrane potential of STAT3-siRNA group observed by the JC-1 fluorescence staining was decreased significantly[ （91.33±1.78 ） % ] and [ （ 89.90 ± 1.92 ） % vs （ 59.06 ±1.89 ） %, P 〈 0. 05 ]. The Western blot results showed that the protein expression of active caspase-3 in STAT3-siRNA group was significantly higher than other groups （ 0. 48± 0. 05 vs 0. 22 ±0. 04 and 0. 26 ±0. 06, P 〈 0. 05 ）. Conclu- sions STAT3 gene silencing significantly improves the apoptotic effect in the Bel-7402 cells.