RNAi特异性抑制FoxO3a对软脂酸诱导HepG2.2.15细胞凋亡的影响

RNA interference-mediated silencing of the FoxO3a gene inhibits palmitate-induced apoptosis in human hepatoma cell line HepG2.2.15

目的:观察FoxO3a基因干扰对软脂酸诱导HepG2.2.15细胞凋亡的影响.方法:HepG2.2.15细胞分五组:mock组(加脂质体)、FoxO3asiRNA组、FoxO3asiRNA+软脂酸组、阴性siRNA对照组、阴性siRNA+软脂酸组;Westernblot法检测细胞的FoxO3a蛋白表达水平.MTT法检测细胞存活率;AnnexinFITC/PI双染流式细胞术检测细胞凋亡率;检测细胞的caspase-3活性;RT-PCR检测细胞Bim、p27kipmRNA表达水平;荧光显微镜观察荧光蛋白所在位置.结果:FoxO3asiRNA+软脂酸组和FoxO3asiRNA组FoxO3a总蛋白明显减少(P<0.05),而其他各组基本相同.与阴性siRNA+软脂酸组相比,FoxO3asiRNA+软脂酸组的存活率增加,凋亡率、Caspase3活性下降,BimmRNA、p27kipmRNA表达减少(P<0.05);与FoxO3asiRNA组相比,FoxO3asiRNA+软脂酸组的存活率减少,凋亡率、caspase-3活性、BimmRNA、p27kipmRNA增加(P<0.05);阴性siRNA对照组、FoxO3asiRNA组、mock组的存活率、凋亡率、caspase-3活性、BimmRNA、p27kipmRNA差异无统计学学意义(P>0.05);FoxO3asiRNA组细胞质的绿色荧光比细胞核多;而FoxO3asiRNA+软脂酸正相反.结论:FoxO3a-siRNA单独不能诱导HepG2.2.15细胞凋亡,但抑制FoxO3a的表达后能通过降低Caspase3活性、抑制Bim、p27Kip的表达,从而减少软脂酸诱导的细胞凋亡.并且FoxO3a是通过去磷酸化(失活)即核移位调控这一过程.

AIM： To investigate the impact of small interfering RNA （siRNA）-mediated silencing of the FoxO3a gene on palmitate-induced apoptosis of HepG2.2.15 cells.METHODS： Cultured HepG2.2.15 cells were divided into five groups： mock group （cells cultured in DMEM medium containing LipofectamineTM2000）, FoxO3a siRNA group, FoxO3a siRNA＋palmitate group, negative siRNA group, and negative siRNA＋palmitate group. The protein expression of FoxO3a was detected by Western blot. Cell viability was measured by MTT assay. Apoptosis was evaluated by propidium iodide （PI） staining and flow cytometry. Caspase-3 activity was measured by colorimetric assay. The mRNA expression of Bim and p27kip was examined by reverse transcription （RT）-PCR. The location of fluorescent protein was examined by fluorescence microscopy.RESULTS： After transfection, the levels of to- tal FoxO3a protein decreased in the FoxO3a siRNA＋PA group and FoxO3a siRNA group, while the other groups showed no significant difference. The survival rate was higher, and the apoptosis rate, caspase-3 activity, and mRNA levels of Bim and p27kip were lower in the FoxO3a siRNA＋PA group than in the negative siRNA＋PA group, while the survival rate was lower, and the apoptosis rate, caspase3 activity, and mRNA levels of Bim and p27kip were higher in the FoxO3a siRNA＋PA group than in the FoxO3a siRNA group （all P 〈 0.05）. However, these parameters showed no significant changes among the negative siRNA group, FoxO3a siRNA group and mock group （all P 〉 0.05）. Stronger green fluorescence was noted in the cytoplasm than in the nucleus in the FoxO3a siRNA group, whereas the nucleus had stronger green fluorescence in the FoxO3a siRNA＋PA group.CONCLUSION： FoxO3a siRNA itself does not in- duce apoptosis of HepG2.2.15 cells. FoxO3a siRNA-mediated knockdown of the FoxO3a gene inhibits palmitate-induced apoptosis of HepG2.2.15 cells by decreasing caspase3 activity and down- regulating Bim and p27Kip expression.