Cytoplasmic MTDH associates with mRNAs. Homo sapiens

To examine whether MTDH is a novel RNA binding protein and regulates either metabolism or translation of various mRNAs, we performed a RNA-binding protein immunoprecipitation (RIP) with MTDH and IgG antibodies, and the resulting immunoprecipitated RNA was subjected to a microarray to identify transcripts associating with MTDH. In addition we tested the effect of PI3K inhibition using BEZ235 (a dual PI3K/mTOR inhibitor) on the association of MTDH with target mRNAs. Overall design: The microarray was performed on three biological triplicates as well as three experimental triplicates of immunoprecipitated MTDH and IgG in Hec50co endometrial cancer cells. Magna RIP (RNA-binding protein immunoprecipitation, Millipore) Kit and microarray were used to immunoprecipitate MTDH associated RNAs and to identify mRNAs that associate with MTDH in the absence or presence of 100nM BEZ235 (LC Labs). Fold changes represent immunoprecipitated MTDH RNA compared to IgG immunoprecipitated RNA. Representative mRNAs associated with MTDH and the effect of BEZ235 on the association of MTDH with mRNAs are shown.

为探究MTDH是否为新型RNA结合蛋白，及其是否调控多种mRNA的代谢或翻译过程，我们使用MTDH抗体与免疫球蛋白G（IgG）抗体开展了RNA结合蛋白免疫沉淀（RNA-binding protein immunoprecipitation, RIP）实验，并将免疫沉淀获得的RNA进行芯片检测，以鉴定与MTDH结合的转录本。此外，我们采用BEZ235——一种双靶点磷脂酰肌醇3-激酶/哺乳动物雷帕霉素靶蛋白（PI3K/mTOR）抑制剂——处理细胞，探究PI3K/mTOR通路抑制对MTDH与靶mRNA结合的影响。
实验整体设计：本芯片实验以Hec50co子宫内膜癌细胞为模型，针对MTDH免疫沉淀组与IgG免疫沉淀组，分别设置3次生物学重复与3次实验重复。我们使用密理博（Millipore）公司的Magna RIP（RNA结合蛋白免疫沉淀）试剂盒与芯片技术，分别在100nM BEZ235（LC Labs）存在或缺失的条件下，免疫沉淀与MTDH结合的RNA，并鉴定与之结合的mRNA。富集倍数指MTDH免疫沉淀组RNA与IgG免疫沉淀组RNA的信号比值。本研究展示了与MTDH结合的代表性mRNA，以及BEZ235对MTDH与mRNA结合的调控作用。