本文選題：腎透明細(xì)胞癌 + microRNA��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:據(jù)《柳葉刀》報(bào)道,全世界每年超過(guò)350,000人診斷為腎細(xì)胞癌(renal cell cancer,RCC),約140,000人死于這種癌癥。腎透明細(xì)胞癌(clear cell renal cell cancer,ccRCC)是RCC常見病理類型,約占所有RCC患者的60%-80%,目前對(duì)腎透明細(xì)胞癌的主要治療方法為手術(shù)切除,其對(duì)于放、化療均不敏感,而且大約30%的患者確診時(shí)已發(fā)生遠(yuǎn)處轉(zhuǎn)移,這明顯限制了腎透明細(xì)胞癌患者的術(shù)后生存率。microRNA(miRNA)是一類高度保守的小非編碼RNA,通過(guò)與靶mRNA 3’端非翻譯區(qū)(3’-UTR)配對(duì),降解靶mRNA或抑制其翻譯,從而調(diào)節(jié)靶基因的表達(dá)。越來(lái)越多研究表明,mRNA 3’-UTR基因突變減弱了其與microRNA特異性結(jié)合,從而影響靶基因表達(dá),并參與多種腫瘤的發(fā)生發(fā)展。SET8是迄今發(fā)現(xiàn)的唯一可特異性單甲基化H4K20的賴氨酸甲基轉(zhuǎn)移酶,其在維持基因組穩(wěn)定性、調(diào)節(jié)基因表達(dá)、DNA損傷修復(fù)、調(diào)控細(xì)胞周期中發(fā)揮著重要作用。已有多個(gè)研究報(bào)道SET8 mRNA 3’-UTR區(qū)miR502結(jié)合位點(diǎn)單核苷酸多態(tài)性與肝細(xì)胞肝癌、乳腺癌等多種腫瘤的發(fā)生和/或預(yù)后明顯相關(guān)。同時(shí)有研究報(bào)道SET8蛋白在腫瘤組織的表達(dá)水平明顯高于鄰近非腫瘤組織,且SET8與腫瘤的預(yù)后存在緊密聯(lián)系。本研究旨在探討SET8 mRNA 3’-UTR區(qū)miR502結(jié)合位點(diǎn)單核苷酸多態(tài)性和SET8表達(dá)與腎透明細(xì)胞癌患者發(fā)病與預(yù)后的相關(guān)性,觀察敲低內(nèi)源性SET8表達(dá)對(duì)腎透明細(xì)胞癌786-O細(xì)胞生物學(xué)行為的影響。方法:1選取2006年~2010年在河北醫(yī)科大學(xué)第四醫(yī)院泌尿外科經(jīng)術(shù)后組織病理確診為原發(fā)性腎透明細(xì)胞癌患者110例。留取患者術(shù)前靜脈血標(biāo)本,準(zhǔn)確記錄所有患者的性別、年齡、臨床分期、腫瘤大小、轉(zhuǎn)移情況等臨床特征。通過(guò)門診復(fù)查、電話隨訪腎透明細(xì)胞癌患者術(shù)后的生存情況,隨訪期限為5年,截止時(shí)間為2015年12月。另收集2012年10月~2013年10月于河北醫(yī)科大學(xué)第四醫(yī)院體檢的130例健康人,詳細(xì)記錄體檢資料,并除外與腎透明細(xì)胞癌相關(guān)疾病,采集靜脈血標(biāo)本。2采用基因測(cè)序的方法檢測(cè)miR502結(jié)合位點(diǎn)基因型,分析其與腎透明細(xì)胞癌發(fā)病風(fēng)險(xiǎn)及預(yù)后的相關(guān)性;免疫組織化學(xué)法檢測(cè)腎透明細(xì)胞癌患者組織標(biāo)本SET8蛋白表達(dá)情況及其與ccRCC患者臨床病理特征及預(yù)后的關(guān)系。3體外培養(yǎng)腎透明細(xì)胞癌786-O細(xì)胞,將細(xì)胞分3組:(1)Control照組(未轉(zhuǎn)染),(2)NS-siRNA組(轉(zhuǎn)染陰性對(duì)照siRNA),(3)SET8-siRNA組(轉(zhuǎn)染SET8敲低質(zhì)粒)。MTT實(shí)驗(yàn)和克隆形成實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后786-O細(xì)胞增殖能力,劃痕實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后786-O細(xì)胞遷移能力,Transwell實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后786-O細(xì)胞侵襲能力。應(yīng)用qPCR和Western Blot技術(shù)檢測(cè)增殖相關(guān)基因Survivin和Caspase3 mRNA和蛋白的表達(dá),粘附分子E-cadherin mRNA和蛋白表達(dá)。結(jié)果:1 miR502結(jié)合位點(diǎn)單核甘酸多態(tài)性與腎透明細(xì)胞癌患者發(fā)病風(fēng)險(xiǎn)的相關(guān)性110例腎透明細(xì)胞癌患者和130例健康人之間,以CC基因型為對(duì)照,分別與CT、TT和CT+TT基因型進(jìn)行比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05),OR值分別為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結(jié)合位點(diǎn)單核甘酸多態(tài)性與腎透明細(xì)胞癌患者發(fā)病預(yù)后的相關(guān)性性別、確診時(shí)年齡以及miR502結(jié)合位點(diǎn)基因型與術(shù)后5年生存率無(wú)明顯相關(guān)性(P0.05);腫瘤分期、腫瘤直徑以及是否存在淋巴結(jié)轉(zhuǎn)移與術(shù)后5年生存率存在明顯相關(guān)性(P0.05)。3 miR502結(jié)合位點(diǎn)基因型與SET8表達(dá)的相關(guān)性及SET8蛋白表達(dá)與腎透明細(xì)胞癌患者預(yù)后的相關(guān)性(1)與CT和TT基因型進(jìn)行比較,基因型為CC的患者SET8蛋白表達(dá)明顯下降,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。(2)與SET8高表達(dá)患者相比,低表達(dá)患者5年生存率更高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)Cox比例風(fēng)險(xiǎn)回歸模型顯示SET8蛋白、腫瘤大小、腫瘤分期均為腎透明細(xì)胞癌患者預(yù)后的獨(dú)立危險(xiǎn)因素(P0.05)。4干擾SET8基因表達(dá)對(duì)腎透明細(xì)胞癌786-O細(xì)胞的影響(1)敲低SET8蛋白內(nèi)源性表達(dá)后,786-O細(xì)胞增殖、侵襲和轉(zhuǎn)移能力均受到抑制,凋亡增加。(2)q PCR及Western結(jié)果顯示,敲低SET8蛋白內(nèi)源性表達(dá)后,Survivin mRNA和蛋白表達(dá)明顯降低(P0.05);而Caspase3、E-cadherin mRNA和蛋白表達(dá)明顯升高(P0.05)。結(jié)論:1 SET8 miR502結(jié)合位點(diǎn)基因型CT、TT與腎透明細(xì)胞癌發(fā)病風(fēng)險(xiǎn)升高密切相關(guān)。2 SET8 miR502結(jié)合位點(diǎn)基因型CT、TT與腎透明細(xì)胞癌SET8蛋白表達(dá)水平升有關(guān),且SET8是影響腎透明細(xì)胞癌患者術(shù)后5年預(yù)后的獨(dú)立危險(xiǎn)因素。3 SET8可能是通過(guò)調(diào)節(jié)Survivin、Caspase3和E-cadherin表達(dá),調(diào)控腎透明細(xì)胞癌786-O細(xì)胞增殖、凋亡、侵襲和轉(zhuǎn)移,進(jìn)而影響腎透明細(xì)胞癌的發(fā)生發(fā)展。4分析miR502結(jié)合位點(diǎn)單核苷酸多態(tài)性和SET8表達(dá)有助于我們發(fā)現(xiàn)潛在的腎透明細(xì)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.11
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本文編號(hào)：1928507