长链非编码RNA-n346372在膀胱癌发生发展中的作用及相关基因研究

发布时间：2018-03-04 21:38

本文选题：膀胱癌　切入点：长链非编码RNA　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:1.基于全转录组高通量测序结果,筛选在膀胱癌组织与癌旁组织中显著差异表达的lncRNA分子;2.验证lncRNA-n346372在膀胱癌组织与癌旁组织,膀胱癌细胞系中的表达差异情况,以及与患者临床病理信息的相关性;3.探讨lncRNA-n346372在膀胱癌细胞增殖、迁移、侵袭、凋亡等生物学功能中的作用;4.进一步分析验证lncRNA-n346372与mRNA Cyclin B1的相关性,预测可能涉及的调控网络。方法:1.通过新一代测序技术,对10对膀胱癌组织/癌旁组织进行全转录组高通量测序,采用Wilcoxon rank sum test、DESeq2、EdgeR等统计学分析方法,以p0.05、fdr0.05、log2FoldChange2为筛选策略,对测序结果中差异表达的lncRNA进行再次筛选,为后续验证缩小范围;2.提取40对膀胱癌/癌旁组织,及5个不同恶性程度的膀胱癌细胞系中的RNA,反转录成cDNA,采用Real Time qPCR技术和FISH技术对上述筛选结果中lncRNA n346372的表达情况进行进一步组织和细胞水平的验证,同时对不同肿瘤级别中lncRNA n346372中的表达量进行t test比较分析,明确其相关性;3.分别构建lncRNA-n346372 shRNA T24慢病毒及过表达5637稳转细胞株,通过CCK-8,小室,流式等实验技术,观察不同处理组相对对照组细胞功能变化情况;4.在高通量测序发现的众多差异表达lncRNA及mRNA的基础上,通过生物信息学的相关性分析,预测可能与lncRNA-n346372相关的mRNA分子,进一步通过Real Time qPCR,免疫组化,Weston Blot等技术验证两者在组织和细胞表达水平的相关性,为后续功能相关性以及调控机制研究提供前提依据。结果:1.按照上述策略,共筛选出32条log2FoldChange2的lncRNAs,其中差异倍数3的lncRNAs共9条,lncRNA-n346372 T/N的差异倍数最高,为3.96倍;Tumor组的相对表达量平均值为4.29,Normal组为0.20;2.40对组织中有18对膀胱癌组织大于癌旁组织,肿瘤组的表达量显著高于对照组(P0.05),并在FISH实验中得以证实。在高级别肿瘤组织中的表达高于低级别肿瘤组织(P0.05)。在T24(高侵袭性)膀胱癌细胞株的表达量显著高于5637(低侵袭性)膀胱癌细胞系;3.成功构建干扰/过表达稳转膀胱癌细胞株,干扰T24膀胱癌细胞内的lncRNA-n346372表达后后,其增殖能力、迁移及侵袭能力受到抑制,而过表达后细胞增殖、迁移及侵袭能力显著增强。4.生物信息学相关性分析显示lncRNA-n346372与细胞周期蛋白Cyclin B1具有显著相关性(P0.05),相关系数r=0.76。并在组织核酸水平的验证中得以证实,而且免疫组化、Weston Blot实验发现Cyclin B1在膀胱癌组织中表达水平高于癌旁组织。干扰lncRNA-n346372的细胞株的Cyclin B1的表达量显著低于对照组。结论:1.LncRNA-n346372在膀胱癌组织的表达量显著高于癌旁组织,病理级别越高lncRNA-n346372的表达量越高,与恶性表型存在显著相关性。2.LncRNA-n346372参与膀胱癌细胞的增殖、迁移及侵袭等功能3.LncRNA-n346372与mRNA Cyclin B1在表达水平上存在相关性,lncRNA-n346372可能通过靶向调节mRNA Cyclin B1表达影响膀胱癌细胞的功能。
[Abstract]:Objective: 1. based on whole transcriptome high-throughput sequencing results, screening lncRNA expression significant difference in bladder tumor tissues and adjacent cancer in 2.; verification of lncRNA-n346372 in bladder cancer tissues and adjacent tissues. The differential expression of bladder cancer cell lines, and the correlation with clinical pathological information of lncRNA-n346372 in 3.; the proliferation of bladder cancer cell migration, invasion, apoptosis and other biological function; 4. further correlation analysis of lncRNA-n346372 and mRNA Cyclin B1 to verify the regulation of network prediction may be involved. Methods: 1. by a new generation of sequencing technology, whole transcriptome high-throughput sequencing of 10 bladder cancer / paracancerous tissues. The Wilcoxon rank sum test, DESeq2, EdgeR and other statistical analysis methods, P0.05, fdr0.05, log2FoldChange2 for the screening strategy, differential expression of sequencing results in lncRNA screening again, For the subsequent verification of the narrow scope; 2. from 40 for bladder cancer / tumor adjacent tissues, and 5 different malignant degree of bladder cancer cell lines RNA, reverse transcription to cDNA, further verify the tissue and cell level by using Real Time qPCR technology and FISH technology on the screening results of expression of lncRNA in n346372 at the same time, the expression of lncRNA in different tumor grade in n346372 were t test comparative analysis, identify the correlation; 3. lncRNA-n346372 shRNA T24 were used to construct lentiviral expression and 5637 stable cell lines by CCK-8, cell, flow cytometry experiments, observation of different treatment group relative to the control group, the changes of cell function; the difference in the expression of a large number of 4. high-throughput sequencing found lncRNA and mRNA based on the correlation of bioinformatics analysis, predict the possible mRNA molecules associated with lncRNA-n346372, Real Time qPC further through R, immunohistochemistry, expression of Weston Blot in both tissue and cell technology to verify the relationship between the level of providing the basis for follow-up, functional dependency and the regulation mechanism study. Results: 1. in accordance with the above strategies, 32 were selected to log2FoldChange2 lncRNAs, the number of times the difference of 3 lncRNAs 9, lncRNA-n346372 T/N difference the highest multiples of 3.96 times; and the relative expression of Tumor group's average was 4.29, 0.20 in Normal group; 2.40 of 18 in bladder cancer tissues than adjacent tissues, the expression of tumor group was significantly higher than the control group (P0.05), and confirmed in the experiment. The expression of FISH in high grade tumors in higher than low grade tumors (P0.05). In T24 (high invasive) expression in bladder cancer cell lines was significantly higher than 5637 (low invasive bladder cancer cell line; 3.) the successful construction of interference / overexpression of stably transfected bladder cancer cell line, T2 interference The expression of 4 bladder cancer cells after lncRNA-n346372, the proliferation, migration and invasion ability was inhibited, and the expression of cell proliferation, migration and invasion ability was significantly enhanced.4. bioinformatics analysis showed that lncRNA-n346372 correlation with cell cycle protein Cyclin B1 has a significant correlation, correlation coefficient (P0.05) and confirmed in r=0.76. verify that the organization of nucleic acid, and immunohistochemistry, Weston Blot found that the expression of Cyclin B1 in bladder cancer tissues was higher than adjacent tissues. The expression of lncRNA-n346372 interference cell line Cyclin B1 was significantly lower than the control group. Conclusion: the expression of 1.LncRNA-n346372 in bladder cancer was significantly higher than that in the adjacent tissues, the expression of the higher the pathological grade of lncRNA-n346372 is high, and there was a significant correlation between the malignant phenotype of.2.LncRNA-n346372 in bladder cancer cell proliferation, migration and invasion There is a correlation between 3.LncRNA-n346372 and mRNA Cyclin B1 expression level. LncRNA-n346372 may affect the function of bladder cancer cells by targeting mRNA Cyclin B1 expression.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14

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