本文選題：膀胱癌　切入點：長鏈非編碼RNA　出處：《第二軍醫(yī)大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:1.基于全轉錄組高通量測序結果,篩選在膀胱癌組織與癌旁組織中顯著差異表達的lncRNA分子;2.驗證lncRNA-n346372在膀胱癌組織與癌旁組織,膀胱癌細胞系中的表達差異情況,以及與患者臨床病理信息的相關性;3.探討lncRNA-n346372在膀胱癌細胞增殖、遷移、侵襲、凋亡等生物學功能中的作用;4.進一步分析驗證lncRNA-n346372與mRNA Cyclin B1的相關性,預測可能涉及的調控網(wǎng)絡。方法:1.通過新一代測序技術,對10對膀胱癌組織/癌旁組織進行全轉錄組高通量測序,采用Wilcoxon rank sum test、DESeq2、EdgeR等統(tǒng)計學分析方法,以p0.05、fdr0.05、log2FoldChange2為篩選策略,對測序結果中差異表達的lncRNA進行再次篩選,為后續(xù)驗證縮小范圍;2.提取40對膀胱癌/癌旁組織,及5個不同惡性程度的膀胱癌細胞系中的RNA,反轉錄成cDNA,采用Real Time qPCR技術和FISH技術對上述篩選結果中l(wèi)ncRNA n346372的表達情況進行進一步組織和細胞水平的驗證,同時對不同腫瘤級別中l(wèi)ncRNA n346372中的表達量進行t test比較分析,明確其相關性;3.分別構建lncRNA-n346372 shRNA T24慢病毒及過表達5637穩(wěn)轉細胞株,通過CCK-8,小室,流式等實驗技術,觀察不同處理組相對對照組細胞功能變化情況;4.在高通量測序發(fā)現(xiàn)的眾多差異表達lncRNA及mRNA的基礎上,通過生物信息學的相關性分析,預測可能與lncRNA-n346372相關的mRNA分子,進一步通過Real Time qPCR,免疫組化,Weston Blot等技術驗證兩者在組織和細胞表達水平的相關性,為后續(xù)功能相關性以及調控機制研究提供前提依據(jù)。結果:1.按照上述策略,共篩選出32條log2FoldChange2的lncRNAs,其中差異倍數(shù)3的lncRNAs共9條,lncRNA-n346372 T/N的差異倍數(shù)最高,為3.96倍;Tumor組的相對表達量平均值為4.29,Normal組為0.20;2.40對組織中有18對膀胱癌組織大于癌旁組織,腫瘤組的表達量顯著高于對照組(P0.05),并在FISH實驗中得以證實。在高級別腫瘤組織中的表達高于低級別腫瘤組織(P0.05)。在T24(高侵襲性)膀胱癌細胞株的表達量顯著高于5637(低侵襲性)膀胱癌細胞系;3.成功構建干擾/過表達穩(wěn)轉膀胱癌細胞株,干擾T24膀胱癌細胞內的lncRNA-n346372表達后后,其增殖能力、遷移及侵襲能力受到抑制,而過表達后細胞增殖、遷移及侵襲能力顯著增強。4.生物信息學相關性分析顯示lncRNA-n346372與細胞周期蛋白Cyclin B1具有顯著相關性(P0.05),相關系數(shù)r=0.76。并在組織核酸水平的驗證中得以證實,而且免疫組化、Weston Blot實驗發(fā)現(xiàn)Cyclin B1在膀胱癌組織中表達水平高于癌旁組織。干擾lncRNA-n346372的細胞株的Cyclin B1的表達量顯著低于對照組。結論:1.LncRNA-n346372在膀胱癌組織的表達量顯著高于癌旁組織,病理級別越高lncRNA-n346372的表達量越高,與惡性表型存在顯著相關性。2.LncRNA-n346372參與膀胱癌細胞的增殖、遷移及侵襲等功能3.LncRNA-n346372與mRNA Cyclin B1在表達水平上存在相關性,lncRNA-n346372可能通過靶向調節(jié)mRNA Cyclin B1表達影響膀胱癌細胞的功能。
[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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.14

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本文編號：1567452