【作者】 李雷；

【导师】 陈小楠；

【作者基本信息】 中国医科大学 ， 外科学， 2023， 硕士

【摘要】 目的:在肾脏肿瘤中,最常见的肾恶性肿瘤是肾细胞癌(renal cell carcinoma,RCC),导致每年超过14,000人死亡。其中,最常见并且恶性程度最高和预后最差亚型是肾透明细胞癌(clear cell renal cell carcinoma,cc RCC),其大约占据肾癌70%的病例。虽然关于cc RCC的治疗取得了长足的进步。然而,总生存期(overall survive,OS)仍需改善。本文旨在探究NSUN5(NOP2/Sun RNA methyltransferase 5,NSUN5)在肾透明细胞癌细胞系和人肾小管上皮细胞(HK-2)中的表达水平,以及其与患者预后和临床病理特征的关系;探讨NSUN5对肾透明细胞癌增殖、迁移、侵袭和凋亡等的影响。方法:从TCGA数据库下载肾透明细胞癌患者的信息矩阵,在矩阵中筛选出m~5C(5-Methylcytosine,m~5C)甲基化基因并分析m~5C甲基化基因在肾透明细胞癌中的表达。进一步探究NSUN5与肾透明细胞癌患者预后的关系。同时收集15对肾透明细胞癌患者的组织标本,免疫组化实验检测NSUN5在癌组织和癌旁正常组织中的表达水平,生物信息分析学进一步分析NSUN5与肾透明细胞癌患者临床病理特征的关系。在786-O及769-P细胞系中敲低NSUN5表达,利用CCK-8实验检测NSUN5对细胞增殖的影响;NSUN5对细胞迁移能力的影响通过划痕愈合实验检测;NSUN5对细胞侵袭能力的影响通过Transwell实验检测;流式细胞术探究NSUN5对细胞凋亡的影响;Western blot法检测NSUN5和p53通路等蛋白的水平。裸鼠细胞成瘤实验为了评价NSUN5在体内的致瘤作用。结果:生物信息分析学及实验结果证明NSUN5在cc RCC中表达上调。通过TCGA转录组数据分析,在cc RCC患者中NSUN5的高表达与预后不良相关,并与肿瘤高级别分期和晚期淋巴转移呈正相关。并且GO与GSVA分析表明NSUN5在肾透明细胞癌中与肿瘤免疫微环境相关。实验敲低NSUN5后,786-O和769-P细胞的增殖能力下降,迁移速度减慢,侵袭能力降低,凋亡细胞百分比增加。在敲低NSUN5的cc RCC细胞中BAX,caspase8、caspase9和p53的蛋白表达水平显著增加,而Bcl2、CCND1、CCND3和MMP9的蛋白表达显著降低。此外,在成瘤模型中NSUN5的下调导致裸鼠体内肿瘤体积减小和重量减轻。结论:NSUN5在cc RCC组织中表达上调,敲低NSUN5可能通过激活p53信号通路抑制cc RCC细胞的侵袭、增殖与迁移能力,同时促进其凋亡。更多还原

【Abstract】 Background:Among renal tumors,the most common renal malignant tumor is renal cell carcinoma(RCC),which causes more than 14000 deaths every year.Among them,the most common subtype with the worst malignancy and prognosis is clear cell renal cell carcinoma(cc RCC),which accounts for about 70%of cases of renal cancer.Although great progress has been made in the treatment of cc RCC.However,the overall survival(OS)rate still needs to be improved.The aim of this study was to explore the expression level of NSUN5(NOP2/Sun RNA methyltransferase 5,NSUN5)in cc RCC cell lines and human renal tubular epithelial cells(HK-2),and its relationship with prognosis and clinicopathological characteristics of patients;To explore the effect of NSUN5 on proliferation,migration,invasion and apoptosis of clear cell renal cell carcinoma.Methods:Download the matrix of cc RCC patients from TCGA databases,screen out m~5C(5-Methylcytosine,m~5C)methylation genes in the matrix and analyze the expression of m~5C methylation genes in clear cell renal cell carcinoma.Further explore the relationship between NSUN5 and prognosis of cc RCC.At the same time,the tissue samples of 15patients with cc RCC were collected,and the expression level of NSUN5 in tumor tissue and normal tissues adjacent to the tumor was detected by immunohistochemistry,and bioinformatics further analyze the relationship between the expression of NSUN5 and the clinicopathological characteristics of patients with cc RCC.Knocking down the expression of NSUN5 in 786-O and 769-P cell lines,and detecting the effect of NSUN5 on cell proliferation by CCK-8 experiment;The effect of NSUN5 on cell migration was detected by wound healing assays;Transwell invasion assay was used to detect the effect of NSUN5 on cell invasion;The effect of NSUN5 on apoptosis was investigated by cell apoptosis assay;Western blot was used to detect the level of NSUN5,p53 pathway-associated proteins.The tumorigenic experiment of nude mice was conducted to evaluate the tumorigenic effect of NSUN5 in vivo.Results:Bioinformatics and experimental results proved that NSUN5 is overexpressed in cc RCC.The high expression of NSUN5 in patients with cc RCC is associated with poor prognosis,high-level tumor stage and terminal lymph node metastasis by the analysis of TCGA transcriptome data.The GO and GSVA analysis showed that NSUN5 was associated with tumor immune microenvironment in cc RCC.After knocking out NSUN5,the proliferation ability of 786-O and 769-P cells decreased,the migration speed slowed down,the invasion ability decreased,and the percentage of apoptotic cells increased.The protein expression levels of BAX,caspase8,caspase9 and p53 in NSUN5 knockout cells increased significantly,while the protein expression levels of Bcl2,CCND1,CCND3 and MMP9decreased significantly.In addition,the down-regulation of NSUN5 in the tumorigenic model led to the reduction of tumor volume and weight in nude mice.Conclusion:NSUN5 is highly expressed in cc RCC tissue and may inhibit the invasive,proliferative and migratory abilities of cc RCC cells by activating the p53 signaling pathway,while promoting their apoptosis.更多还原