RNA结合蛋白QKI-5在肾癌发生发展中的功能研究

发布时间：2018-05-05 08:57

本文选题：RNA结合蛋白QKI + RASA1　；参考：《新疆医科大学》2016年博士论文

【摘要】：目的:1)探讨RNA结合蛋白QKI-5在肾透明细胞癌中的生物学功能;2)探索引发QKI-5发挥生物学功能的潜在分子机制及其相关信号通路。方法:1)q PCR和western blot检测QKI-5在肾透明细胞癌组织和配对的癌旁非肿瘤组织中的表达差异,并利用TCGA数据库中肾透明细胞癌患者临床资料分析QKI-5表达水平的高低和肾透明细胞癌患者的临床特征及预后的关系;2)应用以慢病毒为载体的过表达技术观察过表达QKI-5,体内及体外实验观察对肾癌细胞增殖作用的影响,以及对细胞周期的影响;3)在上述功能变化的基础上,进一步探索RNA结合蛋白QKI-5发挥上述作用的可能分子机制。结果:1)QKI-5的mRNA表达水平在肾透明细胞癌组织中较其对应的癌旁组织中明显下调,同时在肾癌细胞系中的表达也较永生化肾小管正常上皮细胞的表达水平降低;TCGA数据库资料分析显示QKI-5低表达的患者的T分期更晚,病理分级更高,QKI-5低表达的患者生存率明显差于QKI-5高表达的患者。2)过表达QKI-5能够抑制肾癌细胞株786-0和A-498细胞体外的增殖能力、克隆形成能力以及在裸鼠体内的生长能力;流式细胞术结果显示,过表达QKI-5使786-0和A-498细胞产生G1期阻滞,而这种阻滞与周期相关蛋白cyclin D1表达降低及p21,p27表达增高有关;3)RT-PCR和Western Blot结果显示,过表达QKI-5可以增加RASA1 mRNA稳定性的表达,促进RASA1的表达,提示QKI-5对RASA1是存在正性调节的,并且在组织标本中验证了QKI-5的表达与RASA1的表达呈正相关。进一步的细胞增殖实验验证QKI-5通过直接稳定RASA1的mRNA表达,从而抑制RAS/MAPK/ERK信号通路发挥其抑癌基因的功能。同时采用RNA免疫共沉淀实验确认了QKI-5可通过结合于RASA1的mRNA的3’UTR对其进行正性调控。结论:通过本课题的研究,我们发现QKI-5在肾透明细胞癌肿瘤组织中表达下调,并且QKI-5的表达水平随肿瘤体积的增大而降低,随肿瘤病理分级的增加而降低,与患者的生存期呈正相关。体外实验及体内实验发现QKI-5在肾透明细胞癌中发挥着抑癌基因的功能,可以使细胞周期阻滞在G1期,进一步的细胞实验初步阐明了QKI-5通过对RASA1的转录后调控抑制RAS/MAPK/ERK信号通路发挥其抑癌基因的功能,本研究结果加深了我们对QKI-5在肾透明细胞癌中的生物学功能的认识,而且很可能为日后肾透明细胞癌的治疗提供一个新的治疗靶点。
[Abstract]:Objective: to investigate the biological function of RNA binding protein QKI-5 in renal clear cell carcinoma (RCC). Methods PCR and western blot were used to detect the expression of QKI-5 in renal clear cell carcinoma (RCC) and matched non-cancerous tissues. The clinical data of patients with renal clear cell carcinoma in TCGA database were used to analyze the relationship between the level of QKI-5 expression and the clinical characteristics and prognosis of patients with renal clear cell carcinoma. The effect of QKI-5 on the proliferation of renal cell carcinoma cells was observed in vivo and in vitro. On the basis of the above functional changes, the possible molecular mechanism of RNA binding protein QKI-5 was further explored. Results the expression level of mRNA in the cell carcinoma of renal clear cell was significantly lower than that in the adjacent tissues of renal clear cell carcinoma, and the expression of QKI-5 was significantly decreased in the renal clear cell carcinoma tissues than in the corresponding adjacent tissues. At the same time, the expression of TCGA in renal carcinoma cell line was lower than that in immortalized renal tubular normal epithelial cells. The analysis of TCGA database showed that the T stage of patients with low expression of QKI-5 was later than that of immortalized renal tubular normal epithelial cells. The survival rate of patients with lower expression of QKI-5 was significantly lower than that of patients with high expression of QKI-5. The overexpression of QKI-5 could inhibit the proliferation, clone formation and growth in nude mice of 786-0 and A-498 cell lines in vitro. Flow cytometry showed that overexpression of QKI-5 resulted in G1 phase arrest in 786-0 and A-498 cells, which was related to the decrease of cyclin D1 expression and the increase of p21 p27 expression. Overexpression of QKI-5 can increase the expression of RASA1 mRNA stability and promote the expression of RASA1, suggesting that QKI-5 has positive regulation on RASA1, and the expression of QKI-5 is positively correlated with the expression of RASA1 in tissue samples. Further cell proliferation experiments demonstrated that QKI-5 inhibits the function of tumor suppressor gene by directly stabilizing the mRNA expression of RASA1 and thus inhibiting the RAS/MAPK/ERK signaling pathway. At the same time, RNA immunoprecipitation assay was used to confirm that QKI-5 can be positively regulated by the 3'UTR of mRNA bound to RASA1. Conclusion: through this study, we found that the expression of QKI-5 was down-regulated in renal clear cell carcinoma, and the expression of QKI-5 decreased with the increase of tumor volume and pathological grade. There was a positive correlation with the survival time of the patients. In vitro and in vivo experiments, it was found that QKI-5 plays a role as a tumor suppressor gene in renal clear cell carcinoma and can block cell cycle in G1 phase. Further cell experiments have preliminarily clarified the role of QKI-5 in tumor suppressor genes through posttranscriptional regulation of RASA1 and inhibition of RAS/MAPK/ERK signaling pathway. The results of this study have deepened our understanding of the biological function of QKI-5 in renal clear cell carcinoma. It is also likely to provide a new therapeutic target for the future treatment of renal clear cell carcinoma.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.11

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