microRNA-21对膀胱癌T24细胞增殖的影响及对VEGF和AP1蛋白的调节

Effect of Cell Proliferation and Regulation of VEGF and AP1 Protein via microRNA-21 in T24 Cell

【目的】研究microRNA-21(miR-21)对膀胱癌T24细胞系中血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)和活化因子蛋白-1(activatorprotein-1,AP1)及mRNA表达的调节作用,探讨miR-21对膀胱癌T24细胞增殖的影响。【方法】用人工合成的miR-21相应的双链互补DNA片段,插入pSilencerTM3.1-H1neo载体表达载体,经测序鉴定后作为microRNA的表达质粒;用脂质体将miR-21高表达质粒转染进T24膀胱癌细胞,经G418筛选获得阳性克隆。用RT-PCR技术和Westernblot方法分别检测该细胞系中VEGF和AP1的mRNA和蛋白表达情况。四甲基偶氮唑蓝(MTT)法检测膀胱癌T24细胞增殖的变化情况。【结果】Westernblot结果显示miR-21能够增加VEGF和AP1蛋白的表达,RT-PCR方法分析显示VEGF和AP1mRNA水平升高。MTT显示转染后细胞的生长速率比未转染的细胞明显加快。【结论】miR-21促进T24膀胱癌细胞增殖,提示可能与VEGF和AP1的低表达有关。

【Objective】To investigate the role of miR-21 on the expression of vascular endothelial growth factor（VEGF）and activator protein-1（AP1）protein and its mRNA,and to explore the effect of microRNA-21 in regulation of the proliferation of T24 bladder cancer cells.【Methods】Using the synthetic double-strand base pair（bp）DNA,which is based on the sequence of the miR-21,inserted the DNA of miR-21 into the vector of pSilencerTM 3.1-H1 neo,and cloned the microRNA highly expression plasmid.Then the miR-21 plasmid were transferred into T24 with Lipofectamine 2000,consequently cell line with the miR-21 highly expression was established by screening with G418.The level of VEGF and AP1 mRNA and protein were measured by using RT-PCR and Western blotting,respectively.Methylthiazolyltetrazolium（MTT）was used to analyze the proliferation of the T24.【Results】The Western blot results displayed that miR-21 promoted the expression of the VEGF and AP1 protein.RT-PCR also indicated that the mRNA of VEGF and AP1 increased.Compared with the control group,MTT method demonstrated that the growth rate of T24 treated with the miR-21 high expression was also significantly increased.【Conclusion】miR-21 can promote the proliferation of T24,suggesting that it was related to the low expression of VEGF and AP1.