【作者】 徐鑫；

【导师】 黄百渠；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2008， 博士

【摘要】 人PTPRO是受体型酪氨酸磷酸酯酶家族的一员,其功能主要涉及肾脏肾小球细胞的结构和功能,神经组织中轴突的生长和导向,以及肿瘤抑制。与其功能相对应,PTPRO主要表达于不同发育时期的肾脏组织和神经组织,而在很多肿瘤细胞中则通过DNA甲基化沉默。例如,在肾脏中,PTPRO表达于发育早期的肾小球细胞顶部和发育成熟的肾小球足细胞顶部;在脑中,PTPRO最高表达出现于妊娠中后期,这一时期也是脑部轴突发生的主要时期;由高甲基化介导的PTPRO的沉默常见于不同的肿瘤和肿瘤细胞系中。尽管存在表达差异,但是我们对PTPRO表达的调控机制了解很少。我们详细地研究了PTPRO表达调控的机制。首先,我们发现PTPRO是一个新的E2F1的靶基因。过表达E2F1能上调PTPRO的mRNA水平并且激活PTPRO启动子荧光素酶报告基因活性;PTPRO启动子上存在两个E2F1的结合位点;EMSA和ChIP实验表明E2F1能够结合于PTPRO的启动子。其次,我们发现一个小的microRNA簇—miR-17-92也能够调控PTRPO的表达。生物信息学分析发现PTPRO mRNA的3’UTR区存在miR-17-92的结合位点;报告基因的实验结果表明这些位点对于PTPRO mRNA的稳定性是重要的,miR-17-92能下调PTPRO的mRNA和蛋白水平。最后,我们发现E2F1和miR-17-92在细胞周期进程中协同调控PTPRO的表达。PTPRO的mRNA在细胞周期的S期上调,这与E2F1上调于G1/S期,以及miR-17-5p(miR-17-92家族的一个成员)上调于后S期的结果相一致。我们还分别检测了PTPRO启动子和3’UTR荧光素酶报告基因的活性,发现启动子的活性高峰出现在S早期,而3’UTR的活性则于S后期下降,这与E2F1和miR-17-92的表达是一致的。总之,我们发现PTPRO能够被E2F1和miR17-92协同调控。更多还原

【Abstract】 Human PTPRO is a member of the receptor-type protein tyrosine phosphatases (RPTPs) family, which is involved in podocyte function, axon pathfinding and tumor suppression. The expression of PTPRO varies under different conditions. For example, in kidney, PTPRO is expressed on the apical surface of the podocyte early in development and is present on the apical surface of podocyte foot processes in the mature phenotype; in brain, maximal expression of PTPRO is coincident with mid to late gestation and axonogenesis; in tumors, DNA hypermethylation-mediated silencing of PTPRO was reported in primary human tumors and cancer cell lines. However, we know little so far about the regulation mechanisms for PTPRO expression.This thesis describes the detailed study on the regulatory mechanisms of PTPRO expression. First, we discovered that PTPRO was a target of E2F1. E2F1 could up-regulate PTPRO mRNA level and activated PTPRO promoter. We also found that there existed two functional E2F1 binding sties in PTPRO promoter. By using EMSA and ChIP, we showed that E2F1 bound to PTPRO promoter in vitro and in vivo. Second, we showed that microRNA cluster miR-17-92, another target of E2F1, participated in PTPRO regulation. Bioinformatic analysis unraveled the existence of miR17-92 binding site in the 3’UTR of PTPRO mRNA, and luciferase reporter assay demonstrated the importance of miR-17-92 binding site in PTPRO post-transcriptional regulation. In addition, miR-17-92 could down-regulate PTPRO mRNA and protein level. Finally, we found that E2F1 and miR-17-92 were simultaneously involved in PTPRO regulation in cell cycle progression. We discovered that the promoter reporters inserted with PTPRO 3’UTR responded more weakly to E2F1 activation, implying that E2F1 first up-regulated PTPRO mRNA and then down-regulated PTPRO protein expression through up-regulating miR-17-92 transcription. Moreover, E2F1 overexpession indeed upregulated miR-17-5p, a member of miR-17-92 cluster. We then monitored PTPRO mRNA expression in synchronized HeLa cells, and found that PTPRO transcription was up-regulated at the S phase. Next, we examined the expression profiling of E2F1 and two miR-17-92 members, i.e., miR-17-5p and miR-20a, in synchronized HeLa cells. We found that both E2F1 mRNA and protein were up-regulated in G1 phase (2hr after released from double-thymidine arrest) whereas the expression of miR-17-5p was high in late S phase (6hr after released from double-thymidine arrest). These results demonstrated a positive correlation between E2F1 and PTPRO, and a negative correlation between miR-17-92 cluster, especially miR-17-5p and PTPRO. In addition, these results were coincided with our in vitro data that the PTPRO promoter activity was high in early S phase while the PTPRO 3’UTR reporter activity was low in late S phase in synchronized HeLa cells. Altogether, this study provides evidence that PTPRO gene is co-regulated by both E2F1 and miR-17-92.更多还原