發(fā)布時(shí)間：2018-06-03 05:16

  本文選題：長(zhǎng)非編碼RNA + DUXAP8��； 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[研究背景]胃癌是我國(guó)最常見的惡性消化道腫瘤之一,其發(fā)病率和死亡率分別占我國(guó)惡性腫瘤的第2位和第3位。胃癌發(fā)生發(fā)展的生理學(xué)過程受多種因素調(diào)控,近年來最新研究發(fā)現(xiàn),假基因(pseudogenes)廣泛參與了細(xì)胞的生物學(xué)過程,假基因是一類與編碼基因非常相似,但無法表達(dá)具有功能蛋白質(zhì)的細(xì)胞內(nèi)非編碼基因。部分假基因可以轉(zhuǎn)錄非編碼RNA,這些長(zhǎng)度超過200nt的非編碼RNA為長(zhǎng)非編碼 RNA(long noncoding RNAs,lncRNAs)的一類。LncRNAs 在腫瘤細(xì)胞中的異常表達(dá)與腫瘤的發(fā)生發(fā)展有著密切的聯(lián)系,充當(dāng)原癌基因或抑癌基因的角色。DUXAP8為假基因來源的lncRNA,目前DUXAP8在胃癌細(xì)胞中的生物學(xué)功能及潛在機(jī)制尚不明確。[研究目的]探討DUXAP8在胃癌中的表達(dá)及其生物學(xué)功能,并研究其調(diào)控機(jī)制。[研究方法](1)通過GEO數(shù)據(jù)庫中胃癌病例-對(duì)照芯片數(shù)據(jù)分析DUXAP8在胃癌中的表達(dá)量;利用熒光實(shí)時(shí)定量PCR(qRT-PCR)驗(yàn)證結(jié)果,并分析臨床病理資料。(2)MTT、克隆形成實(shí)驗(yàn)、Edu實(shí)驗(yàn)檢測(cè)DUXAP8對(duì)胃癌細(xì)胞增殖能力的影響;流式細(xì)胞術(shù)檢測(cè)DUXAP8對(duì)細(xì)胞周期及凋亡的影響;Transwell實(shí)驗(yàn)檢測(cè)DUXAP8對(duì)胃癌細(xì)胞侵襲轉(zhuǎn)移能力的影響。(3)高通量測(cè)序檢測(cè)DUXAP8的下游靶基因,并利用qRT-PCR法驗(yàn)證結(jié)果。(4)RNA免疫共沉淀(RIP)實(shí)驗(yàn)及染色質(zhì)免疫共沉淀(ChIP)實(shí)驗(yàn)研究DUXAP8下游靶基因與PRC2的分子機(jī)制。(5)構(gòu)建靶基因過表達(dá)質(zhì)粒,探討靶基因功能并進(jìn)行拯救實(shí)驗(yàn)。(6)構(gòu)建裸鼠移植瘤模型,探討DUXAP8對(duì)胃癌細(xì)胞體內(nèi)成瘤能力的影響。[研究結(jié)果](1)通過分析GEO數(shù)據(jù)庫中兩份胃癌病例-對(duì)照芯片數(shù)據(jù)發(fā)現(xiàn),DUXAP8在胃癌組織中顯著性高表達(dá);同時(shí)利用qRT-PCR法對(duì)結(jié)果進(jìn)行驗(yàn)證。(2)通過分析胃癌患者的臨床病理資料,發(fā)現(xiàn)DUXAP8在TNM分級(jí)較高、已發(fā)生淋巴結(jié)轉(zhuǎn)移的胃癌樣本中呈顯著性高表達(dá),并且與患者的預(yù)后密切相關(guān)。(3)細(xì)胞功能學(xué)實(shí)驗(yàn)結(jié)果表明,在胃癌細(xì)胞系中敲低DUXAP8的表達(dá)可以抑制胃癌細(xì)胞增殖和轉(zhuǎn)移能力,促進(jìn)胃癌細(xì)胞凋亡。(4)轉(zhuǎn)錄組高通量測(cè)序技術(shù)檢測(cè)敲低DUXAP8后轉(zhuǎn)錄組的變化。結(jié)果顯示:敲低DUXAP8后有133條mRNA的表達(dá)水平發(fā)生上調(diào),315條mRNA下調(diào)。結(jié)合測(cè)序及qRT-PCR法結(jié)果最終確定PLEKHO1為DUXAP8的下游靶基因。(5)RIP和ChIP實(shí)驗(yàn)證實(shí),DUXAP8可以與多梳抑制復(fù)合物2(PRC2)結(jié)合進(jìn)而調(diào)控下游靶基因PLEKHO1。(6)構(gòu)建PLEKHO1過表達(dá)質(zhì)粒,通過功能學(xué)實(shí)驗(yàn)發(fā)現(xiàn)PLEKHO1在胃癌中行使抑癌基因的作用;拯救實(shí)驗(yàn)結(jié)果提示,過表達(dá)PLEKHO1可以拯救DUXAP8對(duì)胃癌細(xì)胞的促進(jìn)增殖作用。(7)通過構(gòu)建裸鼠移植瘤模型證實(shí)干擾DUXAP8的表達(dá)可以抑制胃癌細(xì)胞的腫瘤形成能力。[研究結(jié)論]本項(xiàng)研究首次闡明DUXAP8通過與PRC2結(jié)合表觀沉默PLEKHO1的表達(dá),從而促進(jìn)胃癌細(xì)胞的增殖與轉(zhuǎn)移。該研究提示DUXAP8在胃癌細(xì)胞中發(fā)揮原癌基因的作用,可作為胃癌患者長(zhǎng)期生存率的獨(dú)立預(yù)測(cè)靶標(biāo),參與評(píng)判胃癌的診斷及預(yù)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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本文編號(hào)：1971607