microrna-21对膀胱癌t24细胞增殖的影响及对vegf和ap1蛋白的调节

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1、microRNA-21对膀胱癌T24细胞增殖的影响及对VEGF和AP1蛋白的调节作者：杨国胜刘百川尹智峰【摘要】【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

2、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。【关键词】microRNA-21;VEGF;AP1;T

3、24细胞;膀胱肿瘤　Abstract：【Objective】ToinvestigatetheroleofmiR-2115ontheexpressionofvascularendothelialgrowthfactor(VEGF)andactivatorprotein-1(AP1)proteinanditsmRNA，andtoexploretheeffectofmicroRNA-21inregulationoftheproliferationofT24bladdercancercells.【Methods】Usingthesyntheticdouble-strandbasepa

4、ir(bp)DNA，whichisbasedonthesequenceofthemiR-21，insertedtheDNAofmiR-21intothevectorofpSilencerTM3.1-H1neo，andclonedthemicroRNAhighlyexpressionplasmid.ThenthemiR-21plasmidweretransferredintoT24withLipofectamine2000，consequentlycelllinewiththemiR-21highlyexpressionwasestablishedbyscreeningwith

5、G418.ThelevelofVEGFandAP1mRNAandproteinweremeasuredbyusingRT-PCRandWesternblotting，respectively.Methylthiazolyltetrazolium(MTT)wasusedtoanalyzetheproliferationoftheT24.【Results】TheWesternblotresultsdisplayedthatmiR-21promotedtheexpressionoftheVEGFandAP1protein.RT-PCRalsoindicatedthatthemRNA

6、ofVEGFandAP1increased.Comparedwiththecontrolgroup，MTTmethoddemonstratedthatthegrowthrateofT24treatedwiththemiR-21highexpressionwasalsosignificantlyincreased.【Conclusion】miR-21canpromotetheproliferationofT24，suggestingthatitwasrelatedtothelowexpressionofVEGFandAP1.15　　]膀胱肿瘤是泌尿系统中最常见的肿瘤，在国内膀胱

7、肿瘤的发病率在男性泌尿生殖器肿瘤中占首位，其中男性发病率约为女性的3～4倍，年龄以50～70岁为多，严重危害人类的健康。microRNAs(miRNAs)是一类可调控基因表达的内源性非编码单链小分子RNA，长度约18～24个核苷酸，通过与靶mRNA特异的碱基配对，引起靶mRNA降解或者翻译抑制，产生转录后基因沉默。miRNAs的作用涉及多种生理和病理过程，已有大量实验证据表明，miRNAs在肿瘤[1-2]、心血管[3-4]、糖尿病[5]、胚胎发育[6-7]中异常表达。最新研究显示，microRNA与膀胱