pGL3-4×PRE-E4-luc报告基因质粒的构建及在p44信号通路中的应用鉴定

Construction of pGL3-4×PRE-E4-luc reporter gene vector and its application on p44 signaling pathway

p53基因是一种重要的抑癌基因,其产物能够与靶DNA特异性结合并激活转录参与诱导细胞凋亡,调控细胞周期,控制细胞的增殖与分化.p44蛋白主要定位于前列腺癌细胞的细胞质,促进癌细胞的增殖.通过shRNA沉默p44的表达后,LNCaP细胞的生长受抑制,p53靶基因TIGAR和GLIPR1的表达增加.为探寻p53是否介导p44对TIGAR和GLIPR1的作用,本研究构建了pGL3-4×PRE-E4-luc报告基因质粒,并将其分别转染经NT-shRNA慢病毒和p44-shRNA慢病毒感染的LNCaP细胞,比较两者荧光素酶活性.结果显示,重组质粒pGL3-4×PRE-E4-luc构建成功,为进一步研究p53在肿瘤发生发展过程中的作用通路提供了新的手段.但LNCaP细胞中p44并非通过p53作用于TIGAR和GLIPR1基因,具体机制仍有待进一步研究.

As an important tumor suppressor,p53 plays central roles in cell proliferation inhibition and cell cycle control through specific DNA binding and transcriptional activation.Located in the cytoplasm,p44 is required for proliferation of prostate epithelial cells.The shRNAmediated silencing of p44 expression strongly inhibited the proliferation of prostate cancer cells and activated the expression of TP53-induced glycolysis and apoptosis regulator（TIGAR）and Glioma pathogenesis-related protein 1（GLIPR1）,which were two target genes of p53.To explore whether p53 participates in mediating p44 pathway,the recombinant reporter plasmid pGL3-4×PRE-E4-luc was constructed and transfected into LNCaP cells infected by lentivirus containing NT-shRNA or p44-shRNA,respectively.Then,their luciferase activities were compared.The results showed that the recombinant reporter plasmid pGL3-4×PRE-E4-luc were successfully constructed,providing a novel method for study on the p53 pathway in cancer initiation and development.However,the luciferase activity and western blot results indicated that p44 regulated expression of TIGAR and GLIPR1 independent of the p53 signaling.The detail mechanism requires further investigation.