小干扰RNA干扰高迁移率族蛋白1对HepG2细胞增殖和凋亡的影响

Effect of HMGB1-siRNA on proliferation and apoptosis of HepG2 cells

目的 探讨小干扰RNA（siRNA）抑制高迁移率族蛋白1（HMGB1）基因对人肝癌细胞株HepG2细胞增殖和凋亡的影响. 方法设计并化学合成针对HMGB1的3对siRNA分子序列（siRNAH1、siRNH2、siRNAH3）,同时设未转染对照组（Lipo组）及转染阴性siRNA组（阴性siRNA对照组）.脂质体法瞬时转染HepG2细胞,通过PCR法和Western blot检测细胞中HMGB1基因在mRNA水平和蛋白质水平的变化,利用四甲基偶氮唑盐法检测转染HMGB1-siRNA的HepG2细胞增殖情况,TdT酶介导的缺口末端标记法检测细胞凋亡情况.两组数据间比较用t检验,组内数据比较用方差分析,细胞增殖数据采用可重复双因素方差分析.结果 3对特异性HMGB1-siRNA转染后,HMGB1 mRNA相对表达量分别为1.147±0.024、1.014±0.042和0.435±0.055,较Lipo组的1.411±0.065明显减少（t值分别为7.187、9.018和20.689,P值均＜0.01）;HMGB1蛋白相对表达量分别为0.369±0.035、0.340±0.028和0.097±0.020,较Lipo组的0.553±0.051明显减少（t值分别为6.678、7.919和17.287,P值均＜0.01）,其中以siRNAH3抑制效果最好,抑制率可达到80%.转染siRNAH3后HepG2细胞的增殖能力受到明显抑制,与Lipo组相比,72、96、120h时F值分别为34.651、540.550、3778.197,P值均＜0.01,HepG2细胞凋亡指数明显增高（F=130.696,P＜0.01）.结论 靶向HMGB1基因的siRNA分子片段可以有效地抑制HepG2细胞生长,促进HepG2细胞凋亡,其作用与下调HMGB1的表达有关,HMGB1可能是肝癌治疗的一个潜在靶点.

Objective To investigate the effect of decreased expression of high mobility group Box-1 on the proliferation and apoptosis of HepG2 cells. Methods Three specific siRNAs of HMGB 1 were designed and synthesized, and were transiently transfected into HepG2 cells by LipofectamineTM 2000. The HMGB1 expression in HepG2 cells was detected by RT-PCR and Western blotting respectively. The proliferation activity in vitro was assessed by MTT assay. In situ apoptosis was evaluated by terminal deoxynucleotidyl transferase-deoxyuridine triphosphate nick end labeling （TUNEL） assay. Results All of these specific HMGB1-siRNAs （1, 2, 3） efficiently and specifically inhibited the expression of the HMGB 1 gene, and the levels of HMGB 1 mRNA were 1.147 ± 0.024, 1.014 ± 0.042, 0.435 ± 0.055, respectively, in HMGB 1-siRNAs transfection group, which were significantly lower than that in LipofectamineTM2000 alone group （1.411 ± 0.065,P 〈 0.01）; Correspondingly, all of these specific HMGB 1-siRNAs （1, 2, 3） could efficiently and specifically inhibit the expression of the HMGB1 protein, and the levels of HMGB1 protein were 0.369 ± 0.035, 0.340 ± 0.028, 0.097 ± 0.020, respectively, in HMGB1-siRNAs transfection group, which were significantly lower than that in LipofectamineTM2000 alone group （0.553 ± 0.051, P 〈 0.01）. Of the 3 specific HMGB 1-siRNAs,HMGB 1-siRNA-3 （siRNAH3） had the highest inhibition rate （80%）. The proliferation of HepG2 cells was markedly inhibited by siRNAH3 transfection. Compared to mock-transfection, siRNAH3 transfection dramatically suppressed the proliferation of HepG2 cells （P 〈 0.01）. Moreover, siRNAH3 can induce apoptosis （P 〈 0.01）. Conclusion siRNA targeting HMGB 1 mRNA can specifically reduce HMGB 1 gene and protein expression. siRNAH3 can effectively suppress the proliferation and induce apoptosis of HepG2 cells.