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目的探讨应用小干扰 RNA(siRNA)敲除突变 K-ras 基因对肺癌细胞 H441体内外生长的抑制作用。方法构建真核表达载体 pSilencer3.1-K-ras~(V12),转染 H441细胞后应用半定量 RT-PCR 及 Western blotting 检测突变 K-ras 基因 mRNA 及蛋白质的表达变化,噻唑蓝法检测 H441细胞增殖速度的变化,流式细胞仪检测 H441细胞凋亡率的变化。裸鼠皮下移植 pSilencer3.1-K-ras~(V12)处理的H441细胞,观察其成瘤性的改变。结果测序证实 pSilencer3.1-K-ras~(V12)真核表达载体构建成功。RT-PCR 灰度比值结果示空载体组、阴性对照组、实验组 K-ras mRNA 相对表达量分别为:93.7%±2.2%、95.1%±2.5%、43.6%±3.1%,差异有统计学意义(F=78,P<0.01);Western blotting 结果示空载体组、阴性对照组、实验组 K-ras 蛋白相对表达量分别为:98.1%±2.4%、97.5%±2.0%、33.5%±3.7%,差异有统计学意义(F=93,P<0.01);经 pSilencer3.1-K-ras~(V12)作用的 H441细胞生长受到明显抑制(P<0.05),凋亡率较对照组明显升高(F=8.9,P<0.01),H441细胞在裸鼠体内生长受到明显抑制。结论靶向突变 K-ras 基因的 siRNA 可以抑制肺癌 H441细胞在体内外的生长速度,诱导细胞凋亡,为肺癌的基因治疗提供了新的思路和方法。
Objective To investigate the inhibitory effect of K-ras gene knockdown on the growth of lung cancer cell line H441 in vitro and in vivo. Methods The eukaryotic expression vector pSilencer3.1-K-ras ~ (V12) was constructed and transfected into H441 cells. Semi-quantitative RT-PCR and Western blotting were used to detect the mRNA and protein expression of K-ras mutant. H441 Cell proliferation rate changes, flow cytometry H441 cell apoptosis rate changes. H441 cells treated with pSilencer3.1-K-ras ~ (V12) were subcutaneously transplanted into nude mice, and the changes of tumorigenicity were observed. Results Sequencing confirmed that the eukaryotic expression vector pSilencer3.1-K-ras ~ (V12) was successfully constructed. RT-PCR results showed that the relative expression of K-ras mRNA in empty vector group, negative control group and experimental group were 93.7% ± 2.2%, 95.1% ± 2.5%, 43.6% ± 3.1% respectively, the difference was statistically significant Western blotting results showed that the relative expression of K-ras protein in empty vector group, negative control group and experimental group were 98.1% ± 2.4%, 97.5% ± 2.0% and 33.5% ± (F = 78, P <0.01) (P <0.01). The growth of H441 cells treated with pSilencer3.1-K-ras ~ (V12) was significantly inhibited (P <0.05), and the apoptosis rate was significantly higher than that of the control group (F = 8.9, P <0.01). The growth of H441 cells in nude mice was significantly inhibited. Conclusions siRNA targeting K-ras gene can inhibit the growth of H441 cells in vitro and in vivo and induce apoptosis of lung cancer cells, providing new ideas and methods for gene therapy of lung cancer.