磷脂酰肌醇蛋白多糖3基因沉默联合三氟拉嗪对肝癌细胞增殖和凋亡的影响

Effects of phosphatidylinositol proteoglycan 3 gene silencing combined with trifluoperazine on proliferation and apoptosis of liver cancer cells

目的研究磷脂酰肌醇蛋白多糖3(GPC3)基因沉默联合三氟拉嗪(TFP)对肝癌细胞增殖和凋亡的影响。方法将肝癌细胞HepG2分为对照组、TFP组、TFP+si-NC组和TFP+si-GPC3组。TFP组用20μmol·L^(-1)TFP处理,TFP+si-NC组转染si-NC且用20μmol·L^(-1)TFP处理,TFP+si-GPC3组转染si-GPC3且用20μmol·L^(-1)TFP处理,对照组不转染且不用TFP处理。用细胞计数试剂盒-8(CCK-8)法检测不同浓度TFP对HepG2细胞生长的抑制作用,计算药物的半抑制浓度(IC50)并选择最适药物浓度。用流式细胞仪检测各组细胞凋亡情况;用蛋白质印迹(Western blot)法检测细胞凋亡相关蛋白裂解的胱天蛋白酶3(Cl-caspase-3)、B细胞淋巴瘤-2相关X蛋白(Bax)的表达;用实时荧光定量聚合酶链反应(qRT-PCR)和Western blot检测GPC3基因和蛋白的表达;用CCK-8法检测各组细胞活力。结果对照组、TFP组、TFP+si-NC组和TFP+si-GPC3组的GPC3蛋白表达水平分别为0.90±0.06,0.57±0.07,0.57±0.08和0.27±0.06,细胞活力分别为(100.00±0.00)%,(54.47±5.95)%,(52.24±4.10)%和(22.70±2.86)%,细胞凋亡率分别为(2.84±0.61)%,(22.37±1.36)%,(20.82±0.81)%和(31.72±1.92)%,Cl-caspase-3蛋白表达水平分别为0.37±0.05,0.81±0.06,0.91±0.03和1.42±0.14,Bax蛋白表达水平分别为0.29±0.01,0.75±0.09,0.79±0.12和1.21±0.06。上述指标,TFP组与对照组相比,TFP+si-GPC3组与TFP组相比,差异均有统计学意义(均P<0.05)。结论肝癌细胞增殖、凋亡与GPC3基因沉默联合TFP干预有显著相关性。

Objective To analyze the effect of phosphatidylinositol proteoglycan 3(GPC3)gene silting combined with trifluoperazine(TFP)on the proliferation and apoptosis of liver cancer cells.Methods HepG2 cells were divided into control group,TFP group,TFP+si-NC group and TFP+si-GPC3 group.TFP group was treated with 20μmol·L^(-1) TFP,TFP+si-NC group transfected si-NC and treated with 20μmol·L^(-1) TFP,TFP+si-GPC3 group transfected si-GPC3 and treated with 20μmol·L^(-1) TFP,control group was neither transfected nor treated with TFP.Cell counting kit-8(CCK-8)method was used to detect the inhibition of different concentrations of TFP on the growth of HepG2 cells.The semi-inhibitory concentration(IC50)of TFP was calculated and the optimal concentration was selected.The apoptosis of hepatoma cells were analyzed by flow cytometry.Western blot was used to detect the expression of apoptosis-related proteins Cl-caspase-3,B-cell lymphoma-2-associated X(Bax).Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting were used to detect the expression of GPC3 mRNA and GPC3 protein.The cell viability of HepG2 cells was detected by CCK-8 assay.Results The protein expression levels of GPC3 protein in control group,TFP group,TFP+si-NC group and TFP+si-GPC3 group were 0.90±0.06,0.57±0.07,0.57±0.08 and 0.27±0.06,respectively;the cell viability were(100.00±0.00)%,(54.47±5.95)%,(52.24±4.10)%,(22.70±2.86)%,respectively;the apoptosis rates were(2.84±0.61)%,(22.37±1.36)%,(20.82±0.81)%,(31.72±1.92)%,respectively;Cl-caspase-3 protein expression levels were 0.37±0.05,0.81±0.06,0.91±0.03,1.42±0.14,respectively;Bax protein expression levels were 0.29±0.01,0.75±0.09,0.79±0.12,1.21±0.06,respectively.Compared between control group and TFP group,between TFP group and TFP+si-GPC3 group,the differences were statistically significant(all P<0.05).Conclusion The proliferation and apoptosis of hepatoma cells were significantly correlated with GPC3 gene silencing combined with TFP intervention.