本文選題：LINC00460 + KLF2�。� 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景:惡性腫瘤的發(fā)生發(fā)展涉及基因組、表觀遺傳等方面的劇烈變化。新近研究表明長非編碼RNA(lncRNA)在調(diào)控腫瘤細(xì)胞增殖、凋亡以及侵襲轉(zhuǎn)移等生物學(xué)功能中發(fā)揮重要的作用。lncRNA的表達(dá)發(fā)生紊亂與包括結(jié)直腸癌在內(nèi)的多種惡性腫瘤密切相關(guān)。研究方法:(1)通過分析TCGA、GEO數(shù)據(jù)庫中結(jié)直腸癌-正常組織的lncRNA表達(dá)譜,篩選出差異表達(dá)lncRNA LINC00460,qRT-PCR在60對結(jié)直腸癌組織和癌旁組織、四種結(jié)直腸癌細(xì)胞株和正常結(jié)腸上皮細(xì)胞株中檢測LINC00460的表達(dá)水平。(2)采用敲低及過表達(dá)實驗在體外探究LINC00460對結(jié)直腸癌細(xì)胞增殖和凋亡功能的影響。(3)生物信息學(xué)分析預(yù)測LINC00460下游靶基因,并進(jìn)一步通過qRT-PCR、Western blotting、RIP、ChIP、雙熒光素酶報告基因、核質(zhì)分離以及拯救實驗加以驗證。研究結(jié)果:(1)生物信息學(xué)分析大樣本芯片數(shù)據(jù)及組織樣本中采用qRT-PCR驗證發(fā)現(xiàn),相比癌旁組織,結(jié)直腸癌組織中LINC00460的表達(dá)顯著增高;qRT-PCR檢測發(fā)現(xiàn)與正常結(jié)腸上皮細(xì)胞相比,結(jié)直腸癌細(xì)胞株中LINC00460的表達(dá)亦增高。(2)在結(jié)直腸癌細(xì)胞中敲低LINC00460能夠顯著抑制其增殖能力、誘導(dǎo)細(xì)胞凋亡,過表達(dá)LINC00460則促進(jìn)細(xì)胞增殖;裸鼠皮下移植瘤證實敲低LINC00460的表達(dá)抑制結(jié)直腸癌細(xì)胞瘤體形成能力。(3)生物信息學(xué)預(yù)測及敲低LINC00460后qRT-PCR驗證發(fā)現(xiàn)許多參與細(xì)胞增殖、凋亡相關(guān)基因表達(dá)發(fā)生了變化。其中抑癌基因KLF2表達(dá)上調(diào),癌基因CUL4A表達(dá)下調(diào)。(4)RIP實驗證實 LINC00460 能與 EZH2(enhancer of zeste homolog 2)蛋白以及 AG02(Argonaute2)蛋白結(jié)合,ChIP實驗檢測發(fā)現(xiàn)EZH2參與調(diào)控KLF2的轉(zhuǎn)錄表達(dá)。雙熒光素酶報告基因顯示LINC00460能通過競爭性吸附miR-149-5p間接促進(jìn)CUL4A蛋白翻譯進(jìn)程。研究結(jié)論:(1)LINC00460在結(jié)直腸癌組織中的相對表達(dá)量明顯高于癌旁組織,提示其高表達(dá)與結(jié)直腸癌的發(fā)生發(fā)展密切相關(guān)。(2)LINC00460能夠促進(jìn)結(jié)直腸癌細(xì)胞增殖、抑制細(xì)胞凋亡。(3)LINC00460 一方面通過綁定組蛋白EZH2,在轉(zhuǎn)錄水平抑制抑癌基因KLF2的表達(dá)。另一方面通過競爭性吸附miR-149-5p繼而間接促進(jìn)癌基因CUL4A轉(zhuǎn)錄后表達(dá)進(jìn)程。(4)LINC00460/EZH2/KLF2以及LIINC00460/miR-149-5p/CUL4A調(diào)控軸的激活在結(jié)直腸癌發(fā)生發(fā)展中起到重要作用。
[Abstract]:Background: the occurrence and development of malignant tumors involve dramatic changes in genome, epigenetics and so on. Recent studies have shown that long non-coding RNA-lncRNAs play an important role in regulating the biological functions of tumor cell proliferation, apoptosis, invasion and metastasis. The disorder of expression of LNNRNAs is closely related to various malignant tumors, including colorectal cancer. Methods by analyzing the lncRNA expression profiles of colorectal carcinom-normal tissues in TCGAGA-GEO database, we screened out the differential expression of lncRNA LINC00460qRT-PCR in 60 pairs of colorectal cancer tissues and adjacent tissues. Detection of LINC00460 expression level in four colorectal cancer cell lines and normal colonic epithelial cell lines.) bioinformatics analysis of the effects of LINC00460 on the proliferation and apoptosis of colorectal cancer cells in vitro using knockdown and overexpression experiments Predict the downstream target gene of LINC00460, The results were further verified by qRT-PCR Western blotting technique, double luciferase reporter gene, nuclear and cytoplasmic isolation and rescue experiments. Results: the bioinformatics analysis of large sample microarray data and qRT-PCR verification in tissue samples showed that the expression of LINC00460 in colorectal cancer tissues was significantly higher than that in adjacent tissues. The results of qRT-PCR showed that the expression of LINC00460 in colorectal cancer tissues was higher than that in normal colonic epithelial cells. The expression of LINC00460 in colorectal cancer cell line was also increased. 2) knockout of LINC00460 could significantly inhibit the proliferation and induce apoptosis of colorectal cancer cell line. Overexpression of LINC00460 could promote cell proliferation. In nude mice subcutaneously transplanted tumor confirmed that low expression of LINC00460 inhibited tumor formation ability of colorectal cancer cells. Bioinformatics prediction and qRT-PCR test showed that many genes involved in cell proliferation and apoptosis-related gene expression changed after knocking down LINC00460. The expression of tumor suppressor gene KLF2 was upregulated, and the down-regulation of oncogene CUL4A showed that LINC00460 could bind to EZH2(enhancer of zeste homolog 2 and AG02 Argonaute2 protein. EZH2 was found to be involved in the regulation of KLF2 transcription. Double luciferase reporter gene showed that LINC00460 could indirectly promote the process of CUL4A protein translation through competitive adsorption of miR-149-5p. Conclusion the relative expression of LINC00460 in colorectal cancer tissues is significantly higher than that in adjacent tissues, suggesting that the high expression level is closely related to the occurrence and development of colorectal cancer, and it can promote the proliferation of colorectal cancer cell line C00460. On the one hand, inhibiting the expression of tumor suppressor gene KLF2 at the transcriptional level by binding histone EZH2. On the other hand, through competitive adsorption of miR-149-5p, the process of CUL4A posttranscriptional expression of oncogene is indirectly promoted. The activation of EZH2 / KLF2 and LIINC00460/miR-149-5p/CUL4A regulatory axis plays an important role in the development of colorectal cancer.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34

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相關(guān)期刊論文 前2條

1 Farooq Rashid;Abdullah Shah;Ge Shan;;Long Non-coding RNAs in the Cytoplasm[J];Genomics,Proteomics & Bioinformatics;2016年02期

2 楊峰;易凡;曹慧青;梁子才;杜權(quán);;長鏈非編碼RNA研究進(jìn)展[J];遺傳;2014年05期

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本文編號：1800868