假基因来源的长非编码RNA DUXAP8表观沉默PLEKH01促进胃癌细胞增殖和转移的机制研究

发布时间：2018-06-03 05:16

本文选题：长非编码RNA + DUXAP8　；参考：《南京医科大学》2017年硕士论文

【摘要】：[研究背景]胃癌是我国最常见的恶性消化道肿瘤之一,其发病率和死亡率分别占我国恶性肿瘤的第2位和第3位。胃癌发生发展的生理学过程受多种因素调控,近年来最新研究发现,假基因(pseudogenes)广泛参与了细胞的生物学过程,假基因是一类与编码基因非常相似,但无法表达具有功能蛋白质的细胞内非编码基因。部分假基因可以转录非编码RNA,这些长度超过200nt的非编码RNA为长非编码 RNA(long noncoding RNAs,lncRNAs)的一类。LncRNAs 在肿瘤细胞中的异常表达与肿瘤的发生发展有着密切的联系,充当原癌基因或抑癌基因的角色。DUXAP8为假基因来源的lncRNA,目前DUXAP8在胃癌细胞中的生物学功能及潜在机制尚不明确。[研究目的]探讨DUXAP8在胃癌中的表达及其生物学功能,并研究其调控机制。[研究方法](1)通过GEO数据库中胃癌病例-对照芯片数据分析DUXAP8在胃癌中的表达量;利用荧光实时定量PCR(qRT-PCR)验证结果,并分析临床病理资料。(2)MTT、克隆形成实验、Edu实验检测DUXAP8对胃癌细胞增殖能力的影响;流式细胞术检测DUXAP8对细胞周期及凋亡的影响;Transwell实验检测DUXAP8对胃癌细胞侵袭转移能力的影响。(3)高通量测序检测DUXAP8的下游靶基因,并利用qRT-PCR法验证结果。(4)RNA免疫共沉淀(RIP)实验及染色质免疫共沉淀(ChIP)实验研究DUXAP8下游靶基因与PRC2的分子机制。(5)构建靶基因过表达质粒,探讨靶基因功能并进行拯救实验。(6)构建裸鼠移植瘤模型,探讨DUXAP8对胃癌细胞体内成瘤能力的影响。[研究结果](1)通过分析GEO数据库中两份胃癌病例-对照芯片数据发现,DUXAP8在胃癌组织中显著性高表达;同时利用qRT-PCR法对结果进行验证。(2)通过分析胃癌患者的临床病理资料,发现DUXAP8在TNM分级较高、已发生淋巴结转移的胃癌样本中呈显著性高表达,并且与患者的预后密切相关。(3)细胞功能学实验结果表明,在胃癌细胞系中敲低DUXAP8的表达可以抑制胃癌细胞增殖和转移能力,促进胃癌细胞凋亡。(4)转录组高通量测序技术检测敲低DUXAP8后转录组的变化。结果显示:敲低DUXAP8后有133条mRNA的表达水平发生上调,315条mRNA下调。结合测序及qRT-PCR法结果最终确定PLEKHO1为DUXAP8的下游靶基因。(5)RIP和ChIP实验证实,DUXAP8可以与多梳抑制复合物2(PRC2)结合进而调控下游靶基因PLEKHO1。(6)构建PLEKHO1过表达质粒,通过功能学实验发现PLEKHO1在胃癌中行使抑癌基因的作用;拯救实验结果提示,过表达PLEKHO1可以拯救DUXAP8对胃癌细胞的促进增殖作用。(7)通过构建裸鼠移植瘤模型证实干扰DUXAP8的表达可以抑制胃癌细胞的肿瘤形成能力。[研究结论]本项研究首次阐明DUXAP8通过与PRC2结合表观沉默PLEKHO1的表达,从而促进胃癌细胞的增殖与转移。该研究提示DUXAP8在胃癌细胞中发挥原癌基因的作用,可作为胃癌患者长期生存率的独立预测靶标,参与评判胃癌的诊断及预后。
[Abstract]:Background gastric cancer is one of the most common malignant digestive tract tumors in China. The physiological process of carcinogenesis and development of gastric cancer is regulated by many factors. In recent years, recent studies have found that pseudogenes are widely involved in the biological process of cells, and pseudogenes are very similar to coding genes. However, it is impossible to express non-coding genes with functional proteins. Some pseudogenic RNAs can transcribe non-coding RNAs. The abnormal expression of LncRNAs in tumor cells is closely related to the occurrence and development of tumor. The role of proto-oncogene or tumor suppressor gene. DUXAP8 is a pseudogene-derived LNC RNA. The biological function and potential mechanism of DUXAP8 in gastric cancer cells are unclear. Objective: to investigate the expression and biological function of DUXAP8 in gastric cancer and its regulatory mechanism. [methods] the expression of DUXAP8 in gastric cancer was analyzed by using the case control chip data in GEO database, and the results were verified by real-time fluorescence quantitative PCR qRT-PCR. The clinicopathological data were analyzed. The effect of DUXAP8 on the proliferation of gastric cancer cells was detected by clone formation assay and Edu assay. The effect of DUXAP8 on cell cycle and apoptosis was detected by flow cytometry. Transwell assay was used to detect the effect of DUXAP8 on the invasion and metastasis of gastric cancer cells. High throughput sequencing was used to detect the downstream target genes of DUXAP8. The results of qRT-PCR assay and chromatin immunoprecipitation assay were used to study the molecular mechanism of DUXAP8 downstream target gene and PRC2 to construct the target gene overexpression plasmid. Objective: to investigate the function of target gene and rescue experiment. 6) to establish a nude mouse model of transplanted tumor and to explore the effect of DUXAP8 on the tumorigenic ability of gastric cancer cells in vivo. [results] 1) by analyzing two gastric cancer case-control microarray data in GEO database, we found that DUXAP8 was significantly overexpressed in gastric cancer tissues, and the results were verified by qRT-PCR. 2) by analyzing the clinicopathological data of gastric cancer patients, we found that DUXAP8 was highly expressed in gastric cancer tissues. It was found that DUXAP8 was highly expressed in gastric cancer samples with high TNM grade and lymph node metastasis, and was closely related to the prognosis of the patients. The expression of knockout DUXAP8 in gastric cancer cell lines inhibited the proliferation and metastasis of gastric cancer cells and promoted the apoptosis of gastric cancer cells. The results showed that 133 mRNA were up-regulated and 315 mRNA down-regulated after knockout. The results of sequencing and qRT-PCR confirmed that PLEKHO1 was the downstream target gene of DUXAP8. The results of ChIP and DUXAP8 confirmed that DUXAP8 could be combined with the multicomb inhibitory complex 2HPRC2 to regulate the downstream target gene PLEKHO1. 6) to construct the PLEKHO1 overexpression plasmid. Functional experiments showed that PLEKHO1 acted as a tumor suppressor gene in gastric cancer. Overexpression of PLEKHO1 can save the proliferation of gastric cancer cells by DUXAP8.) through the establishment of xenograft tumor model in nude mice, it is proved that interfering with the expression of DUXAP8 can inhibit the tumor formation ability of gastric cancer cells. [conclusion] this study demonstrated for the first time that DUXAP8 could promote the proliferation and metastasis of gastric cancer cells by inhibiting the expression of PLEKHO1 in combination with PRC2. This study suggests that DUXAP8 plays a role as a proto-oncogene in gastric cancer cells and can be used as an independent predictor of long-term survival rate in patients with gastric cancer and participate in the evaluation of the diagnosis and prognosis of gastric 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】：R735.2

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