长非编码RNA LINC00460调控结直肠癌细胞增殖和凋亡的机制探究

发布时间：2018-04-25 10:05

本文选题：LINC00460 + KLF2　；参考：《南京医科大学》2017年硕士论文

【摘要】：研究背景:恶性肿瘤的发生发展涉及基因组、表观遗传等方面的剧烈变化。新近研究表明长非编码RNA(lncRNA)在调控肿瘤细胞增殖、凋亡以及侵袭转移等生物学功能中发挥重要的作用。lncRNA的表达发生紊乱与包括结直肠癌在内的多种恶性肿瘤密切相关。研究方法:(1)通过分析TCGA、GEO数据库中结直肠癌-正常组织的lncRNA表达谱,筛选出差异表达lncRNA LINC00460,qRT-PCR在60对结直肠癌组织和癌旁组织、四种结直肠癌细胞株和正常结肠上皮细胞株中检测LINC00460的表达水平。(2)采用敲低及过表达实验在体外探究LINC00460对结直肠癌细胞增殖和凋亡功能的影响。(3)生物信息学分析预测LINC00460下游靶基因,并进一步通过qRT-PCR、Western blotting、RIP、ChIP、双荧光素酶报告基因、核质分离以及拯救实验加以验证。研究结果:(1)生物信息学分析大样本芯片数据及组织样本中采用qRT-PCR验证发现,相比癌旁组织,结直肠癌组织中LINC00460的表达显著增高;qRT-PCR检测发现与正常结肠上皮细胞相比,结直肠癌细胞株中LINC00460的表达亦增高。(2)在结直肠癌细胞中敲低LINC00460能够显著抑制其增殖能力、诱导细胞凋亡,过表达LINC00460则促进细胞增殖;裸鼠皮下移植瘤证实敲低LINC00460的表达抑制结直肠癌细胞瘤体形成能力。(3)生物信息学预测及敲低LINC00460后qRT-PCR验证发现许多参与细胞增殖、凋亡相关基因表达发生了变化。其中抑癌基因KLF2表达上调,癌基因CUL4A表达下调。(4)RIP实验证实 LINC00460 能与 EZH2(enhancer of zeste homolog 2)蛋白以及 AG02(Argonaute2)蛋白结合,ChIP实验检测发现EZH2参与调控KLF2的转录表达。双荧光素酶报告基因显示LINC00460能通过竞争性吸附miR-149-5p间接促进CUL4A蛋白翻译进程。研究结论:(1)LINC00460在结直肠癌组织中的相对表达量明显高于癌旁组织,提示其高表达与结直肠癌的发生发展密切相关。(2)LINC00460能够促进结直肠癌细胞增殖、抑制细胞凋亡。(3)LINC00460 一方面通过绑定组蛋白EZH2,在转录水平抑制抑癌基因KLF2的表达。另一方面通过竞争性吸附miR-149-5p继而间接促进癌基因CUL4A转录后表达进程。(4)LINC00460/EZH2/KLF2以及LIINC00460/miR-149-5p/CUL4A调控轴的激活在结直肠癌发生发展中起到重要作用。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

【参考文献】