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目的探讨抑制JTV1基因表达对大黄素引起的人白血病K562细胞系凋亡的影响及其机制。方法将pGene-Sil-1-JTV1-1.1 siRNA重组质粒、pGeneSil-1-N.1阴性对照质粒及pGeneSil-1空载体分别转染人K562细胞,通过集落形成试验检测细胞的增殖能力;各组转染K562细胞经80μmol/L大黄素处理后,流式细胞术分析细胞周期和细胞凋亡率,RT-PCR法检测细胞中凋亡相关基因Bcl-2、Bax、C-myc转录水平的变化,Western blot法检测Bcl-2、Bax、C-myc翻译水平的变化。结果抑制JTV1基因的表达后,K562细胞的增殖能力明显提高;抑制JTV1基因表达可使经80μmol/L大黄素处理的K562细胞G1期细胞比例下降,并减轻大黄素引起的细胞凋亡,同时,细胞Bax基因mRNA的转录水平和蛋白表达水平均明显下降,而Bcl-2基因和C-myc基因mRNA的转录水平和蛋白表达水平则明显上调。结论抑制JTV1基因表达可能通过上调Bcl-2和C-myc基因的表达,下调Bax基因的表达,促进K562细胞的增殖,并阻止细胞凋亡。
Objective To investigate the effect of inhibiting JTV1 gene expression on emodin-induced apoptosis of human leukemia K562 cell line and its mechanism. Methods The pGene-Sil-1-JTV1-1.1 siRNA recombinant plasmid, pGeneSil-1-N.1 negative control plasmid and pGeneSil-1 empty vector were respectively transfected into human K562 cells and the proliferation ability of the cells was detected by colony formation assay. Transfection of K562 cells with 80μmol / L emodin treatment, cell cycle and apoptosis rate was analyzed by flow cytometry, and the changes of apoptosis-related genes Bcl-2, Bax and C-myc transcription were detected by RT-PCR Western blot was used to detect the changes of Bcl-2, Bax and C-myc levels. Results Inhibition of JTV1 gene expression significantly enhanced the proliferation of K562 cells. Inhibition of JTV1 gene expression decreased the percentage of G1 phase cells in K562 cells treated with 80μmol / L emodin and reduced the apoptosis induced by emodin. At the same time, The mRNA and protein expression levels of Bax gene were significantly decreased, while the transcription and protein levels of Bcl-2 gene and C-myc mRNA were significantly up-regulated. Conclusion Inhibition of JTV1 gene expression may promote the proliferation of K562 cells and prevent apoptosis by up-regulating the expression of Bcl-2 and C-myc genes, down-regulating the expression of Bax gene.