应用短发夹状RNA研究HBV X基因表达对三氧化二砷诱导HepG2细胞凋亡特性的影响

Study on relationship between HBV X gene expression level and arsenic trioxide with apoptosis of hepatocellular carcinoma cell by shRNA

目的:应用shRNA(短发夹状RNA)介导的RNA干扰研究HBV X基因表达对三氧化二砷(As2O3)诱导HepG2细胞凋亡特性的影响。方法:用噻唑蓝(MTT)还原法分析经2.0μmol/L As2O3处理的HepG2细胞和表达HBVX基因的HepG2-X细胞的生长抑制率。以未处理细胞为对照,流式细胞术分析细胞凋亡比和细胞周期,采用Caspase3/cpp32活性比色试剂盒测定As2O3处理后细胞裂解物中Caspase3的活性。应用脂质体介导HBX序列特异性shRNA表达载体pXi-1、pXi-2和序列无关对照pXi-3转染HepG2-X,再次分析细胞凋亡比、细胞周期和Caspase3活性的变化。结果:MTT法表明2.0μmol/L As2O3处理36小时具有理想的细胞生长抑制率。As2O3作用后细胞凋亡比明显增高(P<0.05),G2/M期细胞增多(P<0.05)。不同HBV X基因表达水平的细胞As2O3诱导的细胞凋亡比有统计学差异(P<0.05),HepG2-X与HepG2比较,其As2O3诱导的细胞凋亡比下降,且细胞裂解物的Caspase3活性下降(P<0.05),但应用序列特异性shRNA抑制HBV X基因表达后凋亡比和Caspase3活性可再次增高,而作为对照的序列无关的shRNA则无此效应,但这种效应与细胞周期无关。结论:As2O3可诱导HepG2细胞凋亡。表达HBVX基因后As2O3诱导的HepG2细胞凋亡比和Caspas3活性下降,特异性shRNA抑制HBV X基因表达后则无此效应,但这种效应与细胞周期无关。

To study the effects of HBV X gene expression and arsenic trioxide （ As2O3 ） on cell apoptosis in HepG2 cells by small hairpin RNA （shRNA） -mediated RNA interference. Methods： Cell line HepG2 and cells with stable expression of HBV X gene, HepG2-X, were treated with 2μmol/L As2O3 , the growth inhibition rates were detected by Methyhhiazolyldiphenyl-tetrazolium bromide （MTT） assay. With untreated corresponding cells as controls, the cell cycle and apoptosis ratio were detected by flow cytometry. Caspase-3 activity of cells treated with As2O3 was measured by Caspase-3/cpp32 colorimetric assay kit. HBV X gene sequence-specific shRNA expression vector, pXi-1, pXi-2, and sequence-unrelated control pXi-3 were transfected into HepG2-X by plasmid. The cell cycle, apoptosis ratio and Caspase-3 activity were re-observed. Results： MTT assay revealed that the growth inhibition rate was acceptable when HepG2 and HepG2-X cells were treated with 2μmol/L As2O3 for 36 hours. After As2O3 treatment, the cell apoptosis ratio were significantly elevated （P 〈 O. O5 ） , and Cells trapped in G2/M phase were markedly increased （ P 〈 O. O5 ） . As2O3-induced apoptosis displayed significant difference among various groups with different HBV X gene expression （P 〈 O. O5 ） . As compared with HepG2, apoptosis induced by As2 O3 and Caspase 3 activity in HepG2-X were significantly down-regulated. The down-regulations were removed after HBV X gene expression was re- pressed by sequence-specific shRNAs （ P 〈 O. O5 ） . In contrast, sequece-unrelated control shRNA did not produce these effects, and these effects did not have relation with cell cycle. Conclusion. As2O3 could induce apoptosis in HepG2cells. Apoptosis induced by As2O3 and Caspase 3 activity were downregulated in HepG2 with HBV X gene expression. The downregulations could be removed after HBV X gene expression was repressed by sequence-specific shRNA, and this effect did not correlate with cell cycle.