靶向突变K-ras基因的小干扰RNA体内外抑制肺癌细胞生长的研究

Study of growth inhibition of lung cancer cells by siRNA targeting mutant K-ras gene in vitro and in vivo

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 study the inhibitory effect of mutant K-ras gene depletion by small interfering RNA on the growth of lung cancer cell line-H441 cells in vitro and in vivo. Methods One pair of 63 bp reverse repeated sequence targeting mutant K-rasv12 mRNA spaced by 9 bp nucleotide were synthesized and inserted into plasmid pSilencer3.1 eukaryotic expression vector. After transient and stable transfection into H441 cells, the mutant K-ras mRNA and protein level were measured by RT-PCR and Western blotting, and the H441 cells proliferation was measured by MTT method, and the apoptosis rate was detected by flow-cytometry. H441 cells treated with pSilencer3. 1-K-rasv12 were transplanted subcutaneously in nude mice and their tumorigenesis ability was observed. Results The recombinant plasmid pSilencer3. 1- K-rasv12 was successfully constructed by sequencing. The introduction of pSilencer3.1-K-rasv12 was showed to efficiently and specifically inhibit the expression of K-rasv12 gene according to the results of RT-PCR and Western blotting （ P 〈 0, 01, as compared with controls）. The inhibitory effect on cell proliferation was confirmed by MTI＂ test （P 〈0. 05, as compared with controls）. Apoptosis rate of H441 cells treated with pSilencer3.1-K-rasv12 was significantly higher than that of the control cells （ P 〈 0. 01 ）. The test in vivo showed that downregulation of K-rasv12 expression in H441 cells apparently affected their ability to form tumors in nude mice. Conehlsions siRNA targeting mutant K-ras mRNA can specifically suppress the expression of mutant K-ras gene in H441 cells, and therefore has a substantially inhibitory effect on cell proliferation in vitro and in vivo, it provides a new method and material to the gene therapy of lung cancer.