miR502结合位点单核苷酸多态性和SET8表达与肾透明细胞癌患者预后关系的研究

发布时间：2018-05-24 09:15

  本文选题：肾透明细胞癌 + microRNA　； 参考：《河北医科大学》2017年硕士论文

【摘要】：目的:据《柳叶刀》报道,全世界每年超过350,000人诊断为肾细胞癌(renal cell cancer,RCC),约140,000人死于这种癌症。肾透明细胞癌(clear cell renal cell cancer,ccRCC)是RCC常见病理类型,约占所有RCC患者的60%-80%,目前对肾透明细胞癌的主要治疗方法为手术切除,其对于放、化疗均不敏感,而且大约30%的患者确诊时已发生远处转移,这明显限制了肾透明细胞癌患者的术后生存率。microRNA(miRNA)是一类高度保守的小非编码RNA,通过与靶mRNA 3’端非翻译区(3’-UTR)配对,降解靶mRNA或抑制其翻译,从而调节靶基因的表达。越来越多研究表明,mRNA 3’-UTR基因突变减弱了其与microRNA特异性结合,从而影响靶基因表达,并参与多种肿瘤的发生发展。SET8是迄今发现的唯一可特异性单甲基化H4K20的赖氨酸甲基转移酶,其在维持基因组稳定性、调节基因表达、DNA损伤修复、调控细胞周期中发挥着重要作用。已有多个研究报道SET8 mRNA 3’-UTR区miR502结合位点单核苷酸多态性与肝细胞肝癌、乳腺癌等多种肿瘤的发生和/或预后明显相关。同时有研究报道SET8蛋白在肿瘤组织的表达水平明显高于邻近非肿瘤组织,且SET8与肿瘤的预后存在紧密联系。本研究旨在探讨SET8 mRNA 3’-UTR区miR502结合位点单核苷酸多态性和SET8表达与肾透明细胞癌患者发病与预后的相关性,观察敲低内源性SET8表达对肾透明细胞癌786-O细胞生物学行为的影响。方法:1选取2006年~2010年在河北医科大学第四医院泌尿外科经术后组织病理确诊为原发性肾透明细胞癌患者110例。留取患者术前静脉血标本,准确记录所有患者的性别、年龄、临床分期、肿瘤大小、转移情况等临床特征。通过门诊复查、电话随访肾透明细胞癌患者术后的生存情况,随访期限为5年,截止时间为2015年12月。另收集2012年10月~2013年10月于河北医科大学第四医院体检的130例健康人,详细记录体检资料,并除外与肾透明细胞癌相关疾病,采集静脉血标本。2采用基因测序的方法检测miR502结合位点基因型,分析其与肾透明细胞癌发病风险及预后的相关性;免疫组织化学法检测肾透明细胞癌患者组织标本SET8蛋白表达情况及其与ccRCC患者临床病理特征及预后的关系。3体外培养肾透明细胞癌786-O细胞,将细胞分3组:(1)Control照组(未转染),(2)NS-siRNA组(转染阴性对照siRNA),(3)SET8-siRNA组(转染SET8敲低质粒)。MTT实验和克隆形成实验检测转染后786-O细胞增殖能力,划痕实验检测转染后786-O细胞迁移能力,Transwell实验检测转染后786-O细胞侵袭能力。应用qPCR和Western Blot技术检测增殖相关基因Survivin和Caspase3 mRNA和蛋白的表达,粘附分子E-cadherin mRNA和蛋白表达。结果:1 miR502结合位点单核甘酸多态性与肾透明细胞癌患者发病风险的相关性110例肾透明细胞癌患者和130例健康人之间,以CC基因型为对照,分别与CT、TT和CT+TT基因型进行比较,差异有统计学意义(P0.05),OR值分别为3.188(95%CI=1.344~7.557)、2.912(95%CI=1.319~6.427)、3.000(95%CI=1.393~6.463)。2 miR502结合位点单核甘酸多态性与肾透明细胞癌患者发病预后的相关性性别、确诊时年龄以及miR502结合位点基因型与术后5年生存率无明显相关性(P0.05);肿瘤分期、肿瘤直径以及是否存在淋巴结转移与术后5年生存率存在明显相关性(P0.05)。3 miR502结合位点基因型与SET8表达的相关性及SET8蛋白表达与肾透明细胞癌患者预后的相关性(1)与CT和TT基因型进行比较,基因型为CC的患者SET8蛋白表达明显下降,差异均具有统计学意义(P0.05)。(2)与SET8高表达患者相比,低表达患者5年生存率更高,差异具有统计学意义(P0.05)。(3)Cox比例风险回归模型显示SET8蛋白、肿瘤大小、肿瘤分期均为肾透明细胞癌患者预后的独立危险因素(P0.05)。4干扰SET8基因表达对肾透明细胞癌786-O细胞的影响(1)敲低SET8蛋白内源性表达后,786-O细胞增殖、侵袭和转移能力均受到抑制,凋亡增加。(2)q PCR及Western结果显示,敲低SET8蛋白内源性表达后,Survivin mRNA和蛋白表达明显降低(P0.05);而Caspase3、E-cadherin mRNA和蛋白表达明显升高(P0.05)。结论:1 SET8 miR502结合位点基因型CT、TT与肾透明细胞癌发病风险升高密切相关。2 SET8 miR502结合位点基因型CT、TT与肾透明细胞癌SET8蛋白表达水平升有关,且SET8是影响肾透明细胞癌患者术后5年预后的独立危险因素。3 SET8可能是通过调节Survivin、Caspase3和E-cadherin表达,调控肾透明细胞癌786-O细胞增殖、凋亡、侵袭和转移,进而影响肾透明细胞癌的发生发展。4分析miR502结合位点单核苷酸多态性和SET8表达有助于我们发现潜在的肾透明细胞癌患者。
[Abstract]:Objective: according to the lancet, more than 350000 people worldwide are diagnosed with renal cell cancer (RCC) every year in the world, and about 140000 people die of this kind of cancer. Renal clear cell carcinoma (clear cell renal cell cancer, ccRCC) is a common pathological type of RCC, accounting for all RCC patients. The main treatment for renal clear cell carcinoma is the main treatment of renal cell carcinoma. Surgical excision is insensitive to radiotherapy and chemotherapy, and about 30% of the patients have distant metastasis, which obviously restricts the postoperative survival rate of renal clear cell carcinoma patients.MicroRNA (miRNA) is a highly conservative small noncoding RNA, which is paired with the target mRNA 3 'untranslated region (3' -UTR) to degrade target mRNA or inhibit its turn over. To regulate the expression of target genes, more and more studies have shown that the mRNA 3 '-UTR gene mutation weakens its specific binding to microRNA, thus affects the expression of target genes and participates in the development of a variety of tumors, and.SET8 is the only specific monomethylation H4K20 of the lysine methyltransferase that has been found so far, which maintains the stability of the genome. Qualitative, regulatory gene expression, DNA damage repair and regulation of cell cycle play an important role. Many studies have reported that the single nucleotide polymorphisms of the miR502 binding site at the SET8 mRNA 3 '-UTR region are closely related to the occurrence and / or prognosis of a variety of tumors such as hepatocellular carcinoma, breast cancer and other tumors. The level of SET8 was significantly higher than that of adjacent non tumor tissues, and there was a close relationship with the prognosis of the tumor. The aim of this study was to explore the correlation between the single nucleotide polymorphisms of the miR502 binding site at the SET8 mRNA 3 '-UTR region and the expression of SET8 in the pathogenesis and prognosis of renal clear cell carcinoma, and to observe the SET8 expression of knockdown endogenous SET8 to the 786-O cells of renal clear cell carcinoma. The effects of biological behavior. Methods: 1 to select 110 cases of primary renal clear cell carcinoma in the Department of Urology, Fourth Hospital of Hebei Medical University, 2006 ~2010, which were diagnosed as primary renal clear cell carcinoma after operation. The preoperative venous blood samples were collected to accurately record the clinical features of all patients' sex, age, bed stage, tumor size, metastasis and so on. The survival of renal clear cell carcinoma patients was followed up by telephone follow-up. The duration of follow-up was 5 years, the deadline was December 2015. In addition, 130 healthy people in the fourth hospital of Hebei Medical University in October October 2012 were collected, and the physical examination data were recorded in detail, with the exception of renal clear cell carcinoma related diseases and collecting veins. Blood specimen.2 detected the genotype of miR502 binding site by gene sequencing, analyzed the correlation with the risk and prognosis of renal clear cell carcinoma, and the relationship between the expression of SET8 protein in the tissue specimens of renal clear cell carcinoma and its relationship with the clinical pathophysiology and prognosis of ccRCC patients.3 in vitro culture of renal permeability The cell carcinoma 786-O cells were divided into 3 groups: (1) Control group (untransfected), (2) NS-siRNA group (transfected negative control siRNA), (3) SET8-siRNA group (transfected SET8 knockout plasmid).MTT experiment and clone formation test to detect the proliferation ability of 786-O cells after transfection, and the scratch test was used to detect the migration ability of 786-O cells after transfection, Transwell test was used to detect transfection The invasiveness of post 786-O cells. QPCR and Western Blot techniques were used to detect the expression of Survivin and Caspase3 mRNA and protein of proliferation related genes, E-cadherin mRNA and protein expression of adhesion molecules. Results: the correlation between the 1 miR502 binding site mononuclear glycyrrhizic acid polymorphism and the risk of renal clear cell carcinoma in 110 cases of renal clear cell carcinoma Compared with CT, TT and CT+TT genotypes, the differences were statistically significant (P0.05), and OR values were 3.188 (95%CI=1.344~7.557), 2.912 (95%CI=1.319~6.427) and 3 (95%CI=1.393~6.463).2 miR502 junction site mononuclear glycyrrhizic acid polymorphism and the prognosis of renal clear cell carcinoma in 130 healthy people. Correlation sex, diagnosis age and miR502 binding site genotype had no significant correlation with the 5 year survival rate after operation (P0.05); tumor staging, tumor diameter, and lymph node metastasis were associated with 5 year survival rates (P0.05), the correlation between the.3 miR502 binding site genotype and the expression of SET8 and the expression of SET8 protein The correlation of prognosis in patients with renal clear cell carcinoma (1) was compared with CT and TT genotypes. The expression of SET8 protein in patients with CC was significantly decreased, and the difference was statistically significant (P0.05). (2) the 5 year survival rate of the patients with low expression of SET8 was higher and the difference was statistically significant (P0.05). (3) a Cox proportional risk regression model. SET8 protein, tumor size and tumor staging are independent risk factors for the prognosis of renal clear cell carcinoma (P0.05).4 interfering SET8 gene expression on 786-O cells of renal clear cell carcinoma (1) after knocking low SET8 protein endogenous expression, 786-O cell proliferation, invasion and transfer ability are inhibited and apoptosis increases. (2) Q PCR and Western junction The results showed that after the endogenous expression of low SET8 protein, the expression of Survivin mRNA and protein decreased significantly (P0.05), while Caspase3, E-cadherin mRNA and protein expression increased significantly (P0.05). Conclusion: 1 SET8 miR502 binding site genotype CT. The level of SET8 protein expression in clear cell carcinoma is related, and SET8 is an independent risk factor affecting the 5 year prognosis of renal clear cell carcinoma..3 SET8 may be regulated by Survivin, Caspase3 and E-cadherin expression to regulate the proliferation, apoptosis, invasion and metastasis of renal clear cell carcinoma 786-O cells, and then affect the development and development of renal clear cell carcinoma. .4 analysis of miR502 binding sites single nucleotide polymorphisms and SET8 expression can help us find potential renal clear cell carcinoma patients.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11
，

本文编号：1928507