HMGB1对宫颈癌HeLa细胞凋亡的影响及其作用机制

目的 观察HMGB1对人宫颈癌HeLa细胞生长及凋亡的影响并研究其可能的作用机制.方法 构建HMGB1高表达质粒和RNA干扰质粒,将HMGB1高表达质粒与RNA干扰质粒分别转染到HeLa细胞中,采用MTT实验、PI单染流式细胞术和Annexin V-PI双标法流式细胞术检测HeLa细胞的增殖活性、细胞周期和凋亡情况.采用RT-PCR和western blot法检测HMGB1基因沉默对Caspase-3、bcl-2 和细胞色素C表达的影响.结果 HMGB1高表达载体(pEGFP-N1-HMGB1)与阴性对照质粒分别转染到HeLa细胞,RT-PCR和Western blot结果显示HMGB1重组质粒转染HeLa后可明显增加HMGB1的mRNA 及蛋白表达(P＜0.05).HMGB1干扰病毒载体(HMGB1 SiRNA)与空白病毒载体分别转染到HeLa细胞中,RT-PCR和Western blot结果显示HMGB1 SiRNA转染HeLa后可明显抑制HMGB1的mRNA及蛋白表达(P＜0.05).MTT法检测HMGB1高表达及基因沉默后HeLa的细胞生长曲线,结果显示HMGB1高表达可明显提高HeLa的细胞生长速度(P＜0.05),HMGB1沉默后HeLa的细胞生长明显受到抑制(P＜0.05).PI 染色流式细胞仪结果显示:HMGB1过表达后,细胞正常增殖周期被加速(P＜0.05);HMGB1沉默后,细胞增殖周期被抑制(P＜0.05).Annexin V-PI双标法流式细胞仪结果显示,HMGB1过表达后,HeLa细胞凋亡率明显下降(P＜0.05);HMGB1沉默后,HeLa细胞凋亡率明显上升(P＜0.05).RT-PCR方法检测不同细胞组Caspase-3和bcl-2 mRNA的表达,结果显示:HMGB1基因沉默后,Caspase-3 mRNA的表达明显增加(P＜0.05),bcl-2 mRNA的表达明显减少(P＜0.05).Western blot结果显示:HMGB1 基因沉默后,细胞色素C和Caspase-3蛋白表达水平增加,bcl-2蛋白表达水平减少(P＜0.05).结论 HMGB1可提高HeLa细胞生长速度、增殖周期,抑制HeLa细胞凋亡.HMGB1基因沉默可抑制Hela细胞增殖,促进其凋亡.影响Caspase-3—细胞色素C途径及调节bcl-2的表达可能是其主要作用机制.

Effect of HMGB1-mediated apoptosis on HeLa cells and its possible mechanism

Objective To study the effect of HMGBl-mediated apoptosis on HeLa cells and its possible mechanism.Methods The high expression of HMGB1 plasmid and SiRNA plasmid were constructed and transfected into human cervical cancer cell line Hela.RT-PCR and Western blot were performed to detect HMGB1 mRNA and protein expressions.The cell proliferation was tested by MTT method.Flow cytometry was performed to analyze the cell cycle of PI-stained Hela cells and the apoptotic rate of annexin V/PI-stained cells.The expressions of Caspase-3,bcl-2 and cytochrome C were tested in HMGB1 gene silencing cells by RTPCR and Western blot.Results pEGFP-N1-HMGB1 transfected Hela significantly increased the expressions of HMGB1 mRNA and protein (P＜0.05).HMGB1 SiRNA lentivirus vector significantly inhibited the expressions of HMGB1 mRNA and protein (P＜0.05).MTT assay demonstrated that HMGB1 overexpression significantly enhanced the cell proliferation and HMGB1 gene silencing significantly reduced the cell proliferation (P＜0.05).PI-staining test showed that HMGB1 overexpression significantly reduced cell G1/G2 phase and HMGB1 gene silencing significantly increased cell G1/G2 phase (P＜0.05).The results of annexin V-PI double staining flow cytometry showed that the apoptotic rate decreased significantly in the HMGB1 overexpression group (P＜0.05)and increased in the HMGB1 gene silencing group (P＜0.05).RT-PCR showed that the expression of caspase-3 mRNA increased significantly and the expression of bcl-2 mRNA reduced significantly in the HMGB1 gene silencing group (P＜0.05).Western blot analysis showed that Cytochrome C and Caspase-3 protein expression level increased and bcl-2 protein expression decreased in the HMGB1 gene silencing group (P＜0.05).Conclusion HMGB1 can significantly increase proliferation and cell cycle and inhibit apoptosis in human cervical cell line Hela.HMGB1 gene silencing could inhibit the proliferation and induce the apoptosis of Hela.The Caspase-3/Cytochrome C pathway and regulation of bcl-2 expression may be involved in its mechanism.