高通量筛选肿瘤转移相关基因的研究

High-throughput screening for tumor metastasis-associated human gene

目的利用高通量双萤光素酶报告基因系统筛选与肿瘤转移相关的人类功能基因。方法利用与肿瘤转移相关的重要因子——血管内皮生长因子(VEGF),将含有VEGF反应序列的萤光素酶报告基因(VEGF-LUC)质粒与409个待筛人类功能基因真核表达质粒共转染Hela细胞以筛选影响VEGF-LUC表达的人类功能基因,进一步通过Western blot在蛋白质水平验证初筛阳性的基因对细胞内VEGF蛋白表达量的影响。结果染色质重塑蛋白4A(CHMP4A)明显增强VEGF-LUC相对萤光素酶值(P<0.05),Western blot结果表明CHMP4A明显增强Hela细胞内VEGF的表达。结论初步筛选到一个可能通过影响VEGF活性增强肿瘤侵袭性的基因CHMP4A。

Objective To identify novel tumor metastasis-associated human gene by cell-based high-throughput dual- luciferase reporter system assay. Method We cotransfected plasmid encoding VEGF-responsive element-luciferase （VEGF-LUC） reporter gene with 409 human genes clones into Hela cell respectively, for high-throughput screening of tumor metastasis-associated gene, and then, we used Western blot to detect the VEGF protein expression level to further verify the VEGF-LUC activity screening. Results Only identified one gene （chromatin modifying protein 4A, CHMPgA） that could significantly up-regulate VEGF-LUC activity. The result of Western blot showed that CHMP4A can increase the congregation of VEGF protein. Conclusion CHMP4A is a potential gene that increases the stability and transcriptional activity of VEGF which related to tumor metastasis.