葛根素联合γ突触核蛋白-微小RNA对肺癌H1299细胞增殖凋亡的影响

Effect of puerarin combined with SNCG-siRNA on proliferation and apoptosis of lung cancer H1299 cells and its mechanism

目的探讨葛根素联合γ突触核蛋白-微小RNA(SNCG-siRNA)对肺癌H1299细胞增殖、凋亡的影响及其机制。方法以0、20、40、80、160和320μg/mL葛根素作用于体外培养的H1299细胞24 h后,采用MTT法检测细胞增殖并计算出葛根素的IC50。在H1299细胞中转染SNCG干扰序列SNCG-siRNA及其阴性对照NC-siRNA后,采用逆转录-聚合酶链反应(RT-PCR)和蛋白质免疫印迹(Western blot)检测转染效果。以150μg/mL葛根素处理转染SNCG-siRNA或NC-siRNA的H1299细胞24 h后,采用MTT法、克隆形成实验和流式细胞仪分别检测细胞的增殖、克隆形成和凋亡能力,Western blot检测细胞中肿瘤增殖抗原(Ki67)、细胞周期蛋白D1(Cyclin D1)、B淋巴细胞瘤-2(Bcl-2)蛋白和Bcl-2相关X蛋白(Bax)的表达情况。结果与0μg/mL处理组相比,20、40、80、160和320μg/mL葛根素处理后H1299细胞存活率均明显降低(P<0.05),IC50为150.40μg/mL。与NCsiRNA组相比,SNCG-siRNA组细胞中SNCG mRNA和蛋白的表达水平均明显降低(P<0.05)。与NC-siRNA组相比,SNCG-siRNA、葛根素+NC-siRNA和葛根素+SNCG-siRNA处理组细胞的存活率、克隆形成率和细胞中Ki67、CyclinD1、Bcl-2蛋白的表达水平均明显降低,而细胞凋亡率和细胞中Bax蛋白的表达水平均明显升高(P<0.05),且葛根素+SNCG-siRNA处理对H1299细胞的作用强度明显大于葛根素+NC-siRNA或者SNCG-siRNA组。结论葛根素素和SNCG-siRNA联合可协同抑制H1299细胞增殖并促进细胞凋亡,其作用机制可能与共同下调Ki67、CyclinD1、Bcl-2和上调Bax蛋白的表达有关。

Objective To investigate the effect of puerarin combined with SNCG-siRNA on proliferation and apoptosis of lung cancer H1299 cells and its mechanism.Methods Puerarin(0,20,40,80,160 and 320μg/mL)was used to treat H1299 cells in vitro for24 hours.MTT method was used to detect cell proliferation and calculate the IC50 of puerarin.After transfection of SNCG interference sequence SNCG-siRNA and its negative control NC-siRNA into H1299 cells,the transfection effect was detected by RT-PCR and Western blot.The proliferation,clone formation and apoptosis of H1299 cells transfected with SNCG-siRNA or NC-siRNA were detected by MTT,clone formation test and flow cytometry after treatment with 150μg/mL puerarin for 24 hours.The expressions of Ki67,Cyclin D1,Bax and Bcl-2 protein were detected by Western blot.Results cells stimulated by 20,40,80,160 and 320μg/mL puerarin was significantly decreased(P<0.05),and the IC50 was 150.40μg/mL.Compared with NC-siRNA group,the expression levels of SNCG mRNA and protein in SNCG-siRNA group were significantly lower(P<0.05).Compared with NC-siRNA group,the cell survival rate,cloning formation rate and the expression level of Ki67,Cyclin D1 and Bcl-2 protein were reduced significantly in SNCG-siRNA,puerarin+NC-siRNA and puerarin+SNCG-siRNA treatment groups,while the apoptotic rate and the expression level of Bax protein in cells were significantly increased(P<0.05),and the effect of puerarin+SNCG-siRNA treatment on H1299 cells was stronger than that of puerarin+NC-siRNA or SNCG-siRNA.Conclusion SNCG-siRNA can synergistically inhibit the proliferation and promote apoptosis of H1299 cells.The mechanism may be related to the co-down-regulation of Ki67,Cyclin D1,Bcl-2 and up-regulation of Bax protein expression.