microRNA抑制鼻咽癌VEGF基因表达的实验研究

Inhibition of the expression of VEGF gene in nasopharyngeal carcinoma cells by microRNA

目的:观察人工构建的microRNA对鼻咽癌CNE-2细胞VEGF基因表达的调控效应,探讨microR-NA在鼻咽癌基因治疗中的应用前景。方法:构建靶向VEGF基因的microRNA表达质粒,脂质体法转染鼻咽癌CNE-2细胞,RT-PCR检测转染细胞中VEGF基因的mRNA水平,Western Blotting法观察microRNA对VEGF蛋白表达水平的调控效应,CCK-8比色法检测靶向VEGF的人工构建microRNA对鼻咽癌细胞生长的抑制作用,皮下注射稳定转染microRNA质粒的CNE-2细胞,构建裸鼠移植瘤模型,观察肿瘤生长情况。结果:在mi-croRNA表达质粒转染CNE-2细胞后,VEGF的mRNA表达水平下调,蛋白表达水平降低,但是光镜和荧光显微镜下的初步观察显示CNE-2细胞生长未发生变化,以CCK-8比色法检测的细胞增殖抑制率也无明显改变,而在初步的裸鼠移植瘤实验中,发现调控VEGF表达的microRNA重组质粒能有效抑制肿瘤生长。结论:人工构建表达microRNA的质粒,可在鼻咽癌CNE-2细胞中有效下调VEGF基因的表达水平,抑制裸鼠移植瘤的生长,为鼻咽癌基因治疗研究奠定了一定的基础。

Objective：To explore the regulative effect of expression of VEGF gene in nasopharyngeal carcinoma,and to discuss the future application of microRNA in the gene therapy for nasopharyngeal carcinoma.Method：We constructed the recombination miRNA plasmid vectors which target VEGF gene and plasmids were transfected into CNE-2 cells by using Lipofectamine 2000 Reagent. The VEGF mRNA and VEGF protein were detected by reverse transcriptase polymerase chain reaction（RT-PCR） and Western blotting respectively. WST-8 assay was used to determine the inhibitory effect of microRNA on cell growth. Stable cell lines and wild type CNE-2 cell line were inoculated to subcutis of nude miceto establish animal models. The tumor growth and volume were observed.Result：After the transfection of CNE-2 cells ,the expressions of VEGF mRNA and VEGF protein were down-regulated at different degree. Whereas,CNE-2 cell growth showed no change by observation of fluorescence microscopy,and cell proliferation was not inhibited in WST-8 assay. However,in vivo,growth of xenograft was inhibited in preliminary experiments of nude mice.Conclusion：By miRNA plasmid constructed artificially,miRNA can effectively interfere nasopharyngeal carcinoma cells by down-regulating the expressions of VEGF gene,therefore can inhibit the growth of tumor xenografted in vivo. Future application of microRNA in the gene therapy of nasopharyngeal carcinoma might be expected.