Her-2/neu小分子干扰RNA对肺腺癌细胞周期和凋亡机制的影响

Effects of Her-2/neu siRNA-mediated gene silencing on cell cycle and apoptosis of lung adenocarcinoma cells

目的研究人类表皮生长因子受体2(Her2/neu)小分子干扰RNA(siRNA)对Her2/neu过表达的肺腺癌细胞的细胞周期和凋亡机制的影响。方法用化学合成的靶向Her2/neusiRNA转染肺腺癌calu3细胞,转染前后通过逆转录聚合酶链反应(RTPCR)测定细胞中Her2/neu、细胞周期蛋白(Cyclin)D1mRNA水平;流式细胞术检测Her2/neu蛋白水平和细胞周期的变化;AnnexinⅤ异硫氢酸荧光素(FITC)试剂盒检测肺癌细胞的凋亡情况;荧光分光光度法测定Caspase3活性;酶联免疫吸附试验(ELISA)技术检测培养液上清中血管内皮生长因子(VEGF)水平。结果Her2/neusiRNA能在mRNA和蛋白水平下调肺癌细胞株calu3中Her2/neu基因的表达。calu3细胞转染Her2/neusiRNA48h后处于G0/G1期的细胞增多,同时S期的细胞比例减少,与未转染对照组、空载体组和非特异性siRNA组比较差异有统计学意义(F=6.1,P<0.01)。转染Her2/neusiRNA后CyclinD1mRNA水平下降,同时培养上清液中的VEGF含量为176pg/ml±6pg/ml(F=24.7,P<0.01),Caspase3活性为135%±4%(F=8.9,P<0.01),细胞凋亡率为25.1%±1.2%(F=10.3,P<0.01)。结论化学合成的靶向Her2/neusiRNA能够下调Her2/neu基因表达,继而降低CyclinD1和VEGF水平,激活Caspase3途径,从而阻滞细胞周期于G0/G1期和诱导凋亡。

Objective To investigate the effect of synthesized Her-2/neu specific siRNA on the cell cycle and apoptosis of Her-2/neu upregulating human lung adenocarcinoma cells.Methods Human lung cancer cells of the line calu-3 were cultured and divided into 4 groups: untreated control group, blank vector group transfected with blank vector, non-specific siRNA group transfected with unrelated siRNA, and Her2/neu siRNA group transfected with Her2/neu siRNA. RT-PCR was used to examine the Her-2/neu and cyclin D_1 mRNA expression. Flow cytometry was used to examine the Her-2/neu protein expression and cell cycle. The apoptosis rate was analyzed by using annexin V-FITC kit. The vascular endothelial growth factor (VEGF) level in the culture supernatant was detected by ELISA.Results Twenty-four hours after transfection, the expressions of Her-2/neu mRNA and cyclin D1 mRNA in the Her2/neu siRNA group decreased remarkably, both significantly lower than those in the other 3 groups. Forty-eight hours after transfection, the expression rate of Her-2/neu protein was 25.0%±1.6% in the calu-3 cells transfected with Her-2/neu siRNA, significantly lower than in the control group, blank vector group, and non-specific siRNA group (98.2%±2.2%, 95.7%±2.0%, and 94.8%±1.6% respectively, all P<0.01); the proportion of the cells in G_0/G_1 stage increased and those in the S stage decreased in the celu-3 cells transfected with Her-2/neu siRNA, and the proportions of the cells in G_0/G_1 stage and Stage did not significantly change (F=6.1, P<0.01); the apoptotic rate of the Her2/neu siRNA group was 25.1%±1.2%, significantly higher than those of the other 3 groups (4.8%±0.5%, 8.6%±0.9%, and 10.3%±0.3% respectively, all P<0.01); the caspase-3 activity ratio of the Her2/neu siRNA group was 134.6%±4.5%, significantly higher than those in the blank vector group and non-specific siRNA group (105.0%±2.5% and 112.0%±2.8% respectively, both P<0.01), and the VEGF level in the supernatant of the Her2/neu siRNA group was 176 pg/ml±6 pg/ml, significantly lower than those of the control group, blank vector group, and non-specific siRNA group (476 pg/ml±13 pg/ml , 426 pg/ml± 9 pg/ml , and 406 pg/ml± 9 pg/ml respectively, all P<0.01). Conclusion Chemically synthesized Specific Her-2/neu targeting siRNA effectively inhibits Her-2/neu expression and leads to the decline of cyclin D_1 and VEGF levels and activation of caspase-3 pathway thus arresting the cell cycle at G_0/G_1 stage and enhancing cell apoptosis.