本文選題：肝癌　切入點：乙型肝炎病毒　出處：《安徽大學(xué)》2017年碩士論文

【摘要】：肝癌(HCC)是一種全球發(fā)病率較高的惡性腫瘤,全球每年的新發(fā)病例超過600000例。肝癌根據(jù)其疾病的惡性程度劃分,在所有的腫瘤中排名第五,致死率居全球第三。乙型肝炎病毒(HBV)感染是肝癌形成的重要誘因。隨著肝癌研究的深化以及現(xiàn)代技術(shù)的發(fā)展,發(fā)現(xiàn)肝癌在緩慢的形成的過程中,往往會造成許多分子和信號通路的改變。因此,從微觀分子的角度探究肝癌發(fā)生發(fā)展的機(jī)制,尤其是HBV感染導(dǎo)致的分子及信號通路的改變,并將特異的分子看作抗腫瘤研究新的靶點,可以為肝癌的臨床治療提供新的思路。長鏈非編碼RNA(LncRNA)是一類內(nèi)源性RNA,從細(xì)胞中轉(zhuǎn)錄出來的轉(zhuǎn)錄本長度大于200nt,在進(jìn)化上保守性較差,能夠在多個水平上調(diào)控基因的表達(dá),包括染色質(zhì)修飾、轉(zhuǎn)錄和轉(zhuǎn)錄后加工。近年研究表明,大量的LncRNAs具有復(fù)雜的生物學(xué)功能,而且參與到許多癌癥的形成過程。例如,結(jié)直腸癌組織與對應(yīng)的非癌組織相比,HOTAIR的表達(dá)水平有明顯上升,它可以通過直接與PRC2復(fù)合體的結(jié)合,影響依賴PRC2的染色質(zhì)修飾,進(jìn)而調(diào)節(jié)多種基因的表達(dá)。通過對比HBx轉(zhuǎn)基因小鼠以及對照組小鼠肝臟中l(wèi)ncRNA量的差異,研究者發(fā)現(xiàn)LncRNA Dreh表達(dá)量在轉(zhuǎn)基因小鼠中顯著下調(diào),并且這種下調(diào)與鼠齡和性別沒有相關(guān)性,由HBx抑制表達(dá)的LncRNAs Dreh能夠通過抑制中間纖維蛋白Vimentin的表達(dá)來調(diào)控細(xì)胞骨架的修飾狀況,從而使HCC的生長、轉(zhuǎn)移均處在一個抑制的狀態(tài)。本課題進(jìn)行了以下3部分的研究:(1)通過RNA-seq檢測肝癌細(xì)胞系HepG2及整合了 HBV基因組的肝癌細(xì)胞系4D14中差異表達(dá)的LncRNAs,進(jìn)而利用定量PCR確定了一條在4D14中顯著下調(diào)的長鏈非編碼RNA,命名為LncRNA-DR4作為本課題的研究對象。(2)構(gòu)建了 LncRNA-DR4過表達(dá)的肝癌細(xì)胞系,通過CCK8,細(xì)胞周期等實驗,發(fā)現(xiàn)LncRNA-DR4的過量表達(dá)能夠顯著抑制肝癌細(xì)胞系HepG2的細(xì)胞增殖,且這種抑制作用可能是HepG2細(xì)胞周期中S期的阻滯造成的。(3)利用定量PCR確定了 HBV特有基因HBx與LncRNA-DR4表達(dá)的負(fù)相關(guān)性,進(jìn)一步的熒光素酶活性實驗,發(fā)現(xiàn)HBx在轉(zhuǎn)錄水平上下調(diào)了LncRNA-DR4表達(dá),并且這種調(diào)控可能依賴p53蛋白�？傊�,我們鑒定了一條在HBV陽性細(xì)胞系中下調(diào)的長鏈非編碼RNA,命名為LncRNA-DR4。確定了 HBx蛋白對LncRNA-DR4的負(fù)調(diào)控作用,并且這種調(diào)控在轉(zhuǎn)錄水平上進(jìn)行,這種調(diào)控作用是依賴p53的。另外,對于LncRNA-DR4的生物學(xué)功能,我們發(fā)現(xiàn)過表達(dá)LncRNA-DR4能顯著抑制肝癌細(xì)胞的增殖,引起S期細(xì)胞阻滯。
[Abstract]:Hepatocellular carcinoma (HCC) is a malignant tumor with a high incidence worldwide, with more than 600000 new cases per year. Liver cancer ranks fifth among all tumors according to the degree of malignancy of the disease. Hepatitis B virus hepatitis B virus (HBV) infection is an important inducement for the formation of liver cancer. With the deepening of liver cancer research and the development of modern technology, it is found that liver cancer is slowly forming. Therefore, from the micromolecular perspective, to explore the mechanism of the development of liver cancer, especially the changes in the molecular and signal pathways caused by HBV infection, The specific molecules are regarded as new targets for antitumor research, which can provide a new idea for the clinical treatment of liver cancer. LncRNAs are a class of endogenous RNAs that transcribe from cells with a length larger than 200nt, which is less evolutionarily conservative. The ability to regulate gene expression at multiple levels, including chromatin modification, transcription and post-transcriptional processing. Recent studies have shown that a large number of LncRNAs have complex biological functions and are involved in the formation of many cancers. The expression level of HOTAIR in colorectal cancer tissues was significantly higher than that in the corresponding non-cancerous tissues. It could affect the chromatin modification dependent on PRC2 by directly binding with PRC2 complex. By comparing the amount of lncRNA in the liver of HBx transgenic mice and control mice, the researchers found that the expression of LncRNA Dreh was significantly down-regulated in transgenic mice. And this down-regulation is not related to age and sex of mice. LncRNAs Dreh, which is inhibited by HBx, can regulate the modification of cytoskeleton by inhibiting the expression of intermediate fibrin Vimentin, thus making HCC grow. In this study, we studied the following three parts: 1) Detection of differential expression of LncRNAsin hepatocellular carcinoma cell line HepG2 and 4D14 cell line with HBV genome by RNA-seq, and then use quantitative PCR to confirm the expression of LncRNAs. A liver cancer cell line with overexpression of LncRNA-DR4 was constructed by targeting a long chain noncoding RNAs that were significantly down-regulated in 4D14 and named LncRNA-DR4 as the research object of this study. By CCK8 and cell cycle experiments, it was found that overexpression of LncRNA-DR4 could significantly inhibit the proliferation of hepatoma cell line HepG2. The inhibitory effect may be caused by the arrest of S phase in HepG2 cell cycle. (3) quantitative PCR was used to determine the negative correlation between HBx and LncRNA-DR4 expression of HBV specific gene, and further luciferase activity experiment was carried out. We found that HBx down-regulated the expression of LncRNA-DR4 at the transcriptional level, and this regulation may depend on p53 protein. In conclusion, we identified a long chain noncoding RNAs down-regulated in HBV positive cell lines, named LncRNA-DR4. The negative regulation of HBx protein on LncRNA-DR4 was determined. In addition, we have found that overexpression of LncRNA-DR4 can significantly inhibit the proliferation of hepatoma cells and induce S phase cell arrest for the biological function of LncRNA-DR4.
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 Catherine MN Croagh;John S Lubel;;Natural history of chronic hepatitis B:Phases in a complex relationship[J];World Journal of Gastroenterology;2014年30期

2 Michael C Kew;;Hepatocellular carcinoma in African Blacks: Recent progress in etiology and pathogenesis[J];World Journal of Hepatology;2010年02期

3 王金瑩,王立新,林三仁;抗氧化劑對黃曲霉毒素染毒的轉(zhuǎn)基因小鼠肝腫瘤的防護(hù)[J];中華預(yù)防醫(yī)學(xué)雜志;1999年02期

，

本文編號：1690831