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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 effects of Her2 / neu siRNA on the cell cycle and apoptosis of lung cancer cells overexpressing Her2 / neu. Methods Her2 / neusiRNA was transfected into human lung adenocarcinoma calu3 cells by chemosynthesis. Her2 / neu and Cyclin D1 mRNA levels were determined by reverse transcriptase polymerase chain reaction (RTPCR) before and after transfection. Flow cytometry The levels of Her2 / neu protein and cell cycle were detected by enzyme linked immunosorbent assay (ELISA). The apoptosis of lung cancer cells was detected by Annexin V FITC kit. The activity of Caspase3 was detected by fluorescence spectrophotometry. The enzyme linked immunosorbent assay The level of vascular endothelial growth factor (VEGF) in culture supernatant was detected. Results Her2 / neusiRNA down-regulated the expression of Her2 / neu gene in lung cancer cell line calu3 at mRNA and protein levels. After 48h of Her2 / neusiRNA transfection, the number of cells in G0 / G1 phase of calu3 cells was increased and the proportion of cells in S phase decreased. Compared with untransfected control group, empty vector group and nonspecific siRNA group, there was significant difference (F = 6.1 , P <0.01). The level of CyclinD1 mRNA decreased after transfected with Her2 / neusiRNA, and the VEGF content in culture supernatant was 176 pg / ml ± 6 pg / ml (F = 24.7, P <0.01) and Caspase3 activity was 135% ± 4% P <0.01). The apoptosis rate was 25.1% ± 1.2% (F = 10.3, P <0.01). Conclusion Chemically synthesized Her2 / neusiRNA can down-regulate the expression of Her2 / neu gene and subsequently down-regulate the expression of CyclinD1 and VEGF and activate the Caspase3 pathway, thereby blocking the cell cycle in G0 / G1 phase and inducing apoptosis.