Gao Yu-En, Wang Yuan, Chen Fu-Quan, Feng Jin-Yan, Yang Guang, Feng Guo-Xing, Yang Zhe, Ye Li-Hong, Zhang Xiao-Dong
State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071, China.
State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071, China.
Acta Pharmacol Sin. 2016 Jul;37(7):898-907. doi: 10.1038/aps.2016.18. Epub 2016 May 2.
Increasing evidence shows that mRNAs exert regulatory function along with coding proteins. Recently we report that a hairpin within YAP mRNA 3'UTR can modulate the Hippo signaling pathway. PTEN is a tumor suppressor, and is mutated in human cancers. In this study we examined whether PTEN mRNA 3'UTR contained a hairpin structure that could regulate gene regulation at the post-transcriptional level.
The secondary structure of PTEN mRNA 3'UTR was analyzed using RNAdraw and RNAstructure. Function of hairpin structure derived from the PTEN mRNA 3'UTR was examined using luciferase reporter assay, RT-PCR and Western blotting. RNA-immunoprecipitation (RIP) assay was used to analyze the interaction between PTEN mRNA and microprocessor Drosha and DGCR8. Endogenous siRNA (esiRNA) derived from PTEN mRNA 3'UTR was identified by RT-PCR and rt-PCR, and its target genes were predicted using RNAhybrid.
A bioinformatics analysis revealed that PTEN mRNA contained a hairpin structure (termed PTEN-sh) within 3'UTR, which markedly increased the reporter activities of AP-1 and NF-κB in 293T cells. Moreover, treatment with PTEN-sh (1 and 2 μg) dose-dependently inhibited the expression of PTEN in human liver L-O2 cells. RIP assay demonstrated that the microprocessor Drosha and DGCR8 was bound to PTEN-sh in L-O2 cells, leading to the cleavage of PTEN-sh from PTEN mRNA 3'UTR. In addition, microprocessor Dicer was involved in the processing of PTEN-sh. Interestingly, esiRNA (termed PTEN-sh-3p21) cleaved from PTEN-sh was identified in 293T cells and human liver tissues, which was found to target the mRNA 3'UTRs of protein phosphatase PPP2CA and PTEN in L-O2 cells. Treatment of L-O2 or Chang liver cells with PTEN-sh-3p21 (50, 100 nmol/L) promoted the cell proliferation in dose- and time-dependent manners.
The endogenous siRNA (PTEN-sh-3p21) cleaved from PTEN-sh within PTEN mRNA 3'UTR modulates PPP2CA and PTEN at the post-transcriptional level in liver cells.
越来越多的证据表明，mRNA在编码蛋白质的同时还发挥调节功能。最近我们报道，YAP mRNA 3'UTR内的一个发夹结构可以调节Hippo信号通路。PTEN是一种肿瘤抑制因子，在人类癌症中发生突变。在本研究中，我们检测了PTEN mRNA 3'UTR是否含有一个能在转录后水平调节基因调控的发夹结构。
使用RNAdraw和RNAstructure分析PTEN mRNA 3'UTR的二级结构。使用荧光素酶报告基因检测、RT-PCR和蛋白质印迹法检测源自PTEN mRNA 3'UTR的发夹结构的功能。采用RNA免疫沉淀（RIP）检测分析PTEN mRNA与微处理器Drosha和DGCR8之间的相互作用。通过RT-PCR和rt-PCR鉴定源自PTEN mRNA 3'UTR的内源性siRNA（esiRNA），并使用RNAhybrid预测其靶基因。
生物信息学分析显示，PTEN mRNA在3'UTR内含有一个发夹结构（称为PTEN-sh），该结构显著增加了293T细胞中AP-1和NF-κB的报告基因活性。此外，用PTEN-sh（1和2μg）处理可剂量依赖性地抑制人肝L-O2细胞中PTEN的表达。RIP检测表明，微处理器Drosha和DGCR8在L-O2细胞中与PTEN-sh结合，导致PTEN-sh从PTEN mRNA 3'UTR上切割下来。此外，微处理器Dicer参与了PTEN-sh的加工过程。有趣的是，在293T细胞和人肝组织中鉴定出从PTEN-sh切割而来的esiRNA（称为PTEN-sh-3p21），发现其在L-O2细胞中靶向蛋白磷酸酶PPP2CA和PTEN的mRNA 3'UTR。用PTEN-sh-3p21（50、100 nmol/L）处理L-O2或Chang肝细胞，以剂量和时间依赖性方式促进细胞增殖。
从PTEN mRNA 3'UTR内的PTEN-sh切割而来的内源性siRNA（PTEN-sh-3p21）在肝细胞的转录后水平调节PPP2CA和PTEN。
