贲门癌细胞凋亡相关长链非编码RNA的分析
发布时间：2018-08-11 09:35
【摘要】：目的:本研究旨在运用生物信息学方法在贲门癌组织中找到差异表达的基因,并构建基因间的共表达网络关系,在网络图中挑选相关基因并通过实验验证某些个基因与贲门癌细胞凋亡相关。方法:我们从河南科技大学第一附属医院及安阳肿瘤医院肿瘤外科获得15对贲门癌组织和癌旁组织,随后应用Human LncRNA Array v2.0芯片(8×60K,Arraystar)对癌组织和癌旁组织进行lncRNA(Long non-coding RNA)表达的全基因组分析,通过基因功能富集分析找到凋亡相关基因本体论(GO)条目,从中选取mRNA并验证,通过皮尔森系数与差异表达lncRNA构建共表达网络图,从中挑选lncRNA进行qRT-PCR验证,并分析mRNA与lncRNA之间的相关性,最后通过贲门癌细胞系的验证证实该基因的差异表达与细胞凋亡相关。结果:芯片结果显示,癌组织中有5139个lncRNAs差异表达。它们中有3026个上调的lncRNAs的差异表达倍数大于2,有2113个下调的lncRNAs差异表达倍数大于2。GO分析显示在“抗凋亡”的生物学过程的GO条目(GO term:0006916)中有42个蛋白编码基因显著富集。在mRNA-lncRNA共表达网络图中,共包含2个mRNA和50个lncRNA,二者的皮尔森相关系数大于等于0.97。随后,我们通过实时定量聚合酶链反应的方法,随机的挑选了1个下调和5个上调基因在另外29对贲门癌及贲门癌旁组织中进行验证,结果1个下调的lncRNA(RP11-98F14.10)表达与基因芯片结果一致,接下来对PCR验证过的2个mRNA与这个下调lncRNA进行相关性分析,结果显示PEA15与lncRNA(RP11-98F14.10)存在相关性。最后,我们构建重组过表达质粒,通过慢病毒来转染了1株贲门癌细胞系(SK-GT-2),然后利用流式细胞仪检测细胞凋亡状态,发现过表达该基因后增加了贲门癌细胞的凋亡。结论:贲门癌中存在大量差异表达的基因,mRNA(PEA15)与lncRNA(RP11-98F14.10)存在负性调控关系,且lncRNA(RP11-98F14.10)可能与贲门癌细胞凋亡相关,影响了贲门癌的发生发展。
[Abstract]:Objective: the purpose of this study was to find differentially expressed genes in gastric cardia cancer tissues by bioinformatics, and to construct the coexpression network of genes. The related genes were selected from the network diagram and some genes were confirmed to be associated with apoptosis of cardiac cancer cells. Methods: we obtained 15 pairs of cardiac cancer tissues and adjacent tissues from the first affiliated Hospital of Henan University of Science and Technology and Anyang Oncology Hospital. Then Human LncRNA Array v2.0 chip (8 脳 60 KG Arraystar) was used to analyze the whole genome of lncRNA (Long non-coding RNA expression in cancer tissues and adjacent tissues. The ontological (GO) entries of apoptosis-related genes were found by gene function enrichment analysis, and mRNA was selected and verified. The coexpression network diagram was constructed by Pearson coefficient and differential expression lncRNA, lncRNA was selected for qRT-PCR verification, and the correlation between mRNA and lncRNA was analyzed. Finally, the differential expression of this gene was confirmed to be related to apoptosis through the verification of cardiac cancer cell line. Results: the microarray showed that there were 5139 lncRNAs differentially expressed in cancer tissues. In 3026 of them, the differential expression multiple of up-regulated lncRNAs was more than 2, and 2113 down-regulated lncRNAs differential expression multiple was greater than that of 2.GO analysis showed that 42 protein coding genes were significantly enriched in go term:0006916 of "anti-apoptotic" biological process. In the mRNA-lncRNA coexpression network, 2 mRNA and 50 LNC RNAs were included, and their Pearson correlation coefficient was greater than 0.97. Subsequently, we randomly selected one down-regulated and five up-regulated genes by real-time quantitative polymerase chain reaction (RQPCR) to verify the expression of one down-regulated gene and five up-regulated genes in another 29 gastric cardia carcinomas and paracentric tissues. Results one down-regulated lncRNA (RP11-98F14.10) expression was consistent with the results of gene chip. Then the correlation analysis between two mRNA verified by PCR and the down-regulated lncRNA showed that PEA15 was correlated with lncRNA (RP11-98F14.10). Finally, we constructed a recombinant overexpression plasmid and transfected a cardia cancer cell line (SK-GT-2) by lentivirus, then detected the apoptosis by flow cytometry, and found that the expression of the gene increased the apoptosis of gastric cardia cancer cells. Conclusion: there is a negative regulatory relationship between PEA15 and lncRNA (RP11-98F14.10) in cardiac carcinoma, and lncRNA (RP11-98F14.10) may be associated with apoptosis of gastric cardia cancer cells, which may affect the occurrence and development of gastric cardia carcinoma.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
本文编号：2176609
[Abstract]:Objective: the purpose of this study was to find differentially expressed genes in gastric cardia cancer tissues by bioinformatics, and to construct the coexpression network of genes. The related genes were selected from the network diagram and some genes were confirmed to be associated with apoptosis of cardiac cancer cells. Methods: we obtained 15 pairs of cardiac cancer tissues and adjacent tissues from the first affiliated Hospital of Henan University of Science and Technology and Anyang Oncology Hospital. Then Human LncRNA Array v2.0 chip (8 脳 60 KG Arraystar) was used to analyze the whole genome of lncRNA (Long non-coding RNA expression in cancer tissues and adjacent tissues. The ontological (GO) entries of apoptosis-related genes were found by gene function enrichment analysis, and mRNA was selected and verified. The coexpression network diagram was constructed by Pearson coefficient and differential expression lncRNA, lncRNA was selected for qRT-PCR verification, and the correlation between mRNA and lncRNA was analyzed. Finally, the differential expression of this gene was confirmed to be related to apoptosis through the verification of cardiac cancer cell line. Results: the microarray showed that there were 5139 lncRNAs differentially expressed in cancer tissues. In 3026 of them, the differential expression multiple of up-regulated lncRNAs was more than 2, and 2113 down-regulated lncRNAs differential expression multiple was greater than that of 2.GO analysis showed that 42 protein coding genes were significantly enriched in go term:0006916 of "anti-apoptotic" biological process. In the mRNA-lncRNA coexpression network, 2 mRNA and 50 LNC RNAs were included, and their Pearson correlation coefficient was greater than 0.97. Subsequently, we randomly selected one down-regulated and five up-regulated genes by real-time quantitative polymerase chain reaction (RQPCR) to verify the expression of one down-regulated gene and five up-regulated genes in another 29 gastric cardia carcinomas and paracentric tissues. Results one down-regulated lncRNA (RP11-98F14.10) expression was consistent with the results of gene chip. Then the correlation analysis between two mRNA verified by PCR and the down-regulated lncRNA showed that PEA15 was correlated with lncRNA (RP11-98F14.10). Finally, we constructed a recombinant overexpression plasmid and transfected a cardia cancer cell line (SK-GT-2) by lentivirus, then detected the apoptosis by flow cytometry, and found that the expression of the gene increased the apoptosis of gastric cardia cancer cells. Conclusion: there is a negative regulatory relationship between PEA15 and lncRNA (RP11-98F14.10) in cardiac carcinoma, and lncRNA (RP11-98F14.10) may be associated with apoptosis of gastric cardia cancer cells, which may affect the occurrence and development of gastric cardia carcinoma.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
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