發(fā)布時間：2018-08-11 09:35

【摘要】：目的:本研究旨在運(yùn)用生物信息學(xué)方法在賁門癌組織中找到差異表達(dá)的基因,并構(gòu)建基因間的共表達(dá)網(wǎng)絡(luò)關(guān)系,在網(wǎng)絡(luò)圖中挑選相關(guān)基因并通過實驗驗證某些個基因與賁門癌細(xì)胞凋亡相關(guān)。方法:我們從河南科技大學(xué)第一附屬醫(yī)院及安陽腫瘤醫(yī)院腫瘤外科獲得15對賁門癌組織和癌旁組織,隨后應(yīng)用Human LncRNA Array v2.0芯片(8×60K,Arraystar)對癌組織和癌旁組織進(jìn)行l(wèi)ncRNA(Long non-coding RNA)表達(dá)的全基因組分析,通過基因功能富集分析找到凋亡相關(guān)基因本體論(GO)條目,從中選取mRNA并驗證,通過皮爾森系數(shù)與差異表達(dá)lncRNA構(gòu)建共表達(dá)網(wǎng)絡(luò)圖,從中挑選lncRNA進(jìn)行qRT-PCR驗證,并分析mRNA與lncRNA之間的相關(guān)性,最后通過賁門癌細(xì)胞系的驗證證實該基因的差異表達(dá)與細(xì)胞凋亡相關(guān)。結(jié)果:芯片結(jié)果顯示,癌組織中有5139個lncRNAs差異表達(dá)。它們中有3026個上調(diào)的lncRNAs的差異表達(dá)倍數(shù)大于2,有2113個下調(diào)的lncRNAs差異表達(dá)倍數(shù)大于2。GO分析顯示在“抗凋亡”的生物學(xué)過程的GO條目(GO term:0006916)中有42個蛋白編碼基因顯著富集。在mRNA-lncRNA共表達(dá)網(wǎng)絡(luò)圖中,共包含2個mRNA和50個lncRNA,二者的皮爾森相關(guān)系數(shù)大于等于0.97。隨后,我們通過實時定量聚合酶鏈反應(yīng)的方法,隨機(jī)的挑選了1個下調(diào)和5個上調(diào)基因在另外29對賁門癌及賁門癌旁組織中進(jìn)行驗證,結(jié)果1個下調(diào)的lncRNA(RP11-98F14.10)表達(dá)與基因芯片結(jié)果一致,接下來對PCR驗證過的2個mRNA與這個下調(diào)lncRNA進(jìn)行相關(guān)性分析,結(jié)果顯示PEA15與lncRNA(RP11-98F14.10)存在相關(guān)性。最后,我們構(gòu)建重組過表達(dá)質(zhì)粒,通過慢病毒來轉(zhuǎn)染了1株賁門癌細(xì)胞系(SK-GT-2),然后利用流式細(xì)胞儀檢測細(xì)胞凋亡狀態(tài),發(fā)現(xiàn)過表達(dá)該基因后增加了賁門癌細(xì)胞的凋亡。結(jié)論:賁門癌中存在大量差異表達(dá)的基因,mRNA(PEA15)與lncRNA(RP11-98F14.10)存在負(fù)性調(diào)控關(guān)系,且lncRNA(RP11-98F14.10)可能與賁門癌細(xì)胞凋亡相關(guān),影響了賁門癌的發(fā)生發(fā)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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2

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