HBV誘導(dǎo)表達(dá)的novel-miR-10對肝癌細(xì)胞惡性行為的作用與機(jī)制研究
發(fā)布時(shí)間：2018-07-22 20:58
【摘要】：【目的】肝細(xì)胞癌(hepatocellular carcinoma,HCC)是一種常見的高度惡性腫瘤。由于缺乏早期診斷方法及有效地治療策略,預(yù)后極差。HBV感染是肝細(xì)胞癌發(fā)生的重要危險(xiǎn)因素之一,其導(dǎo)致慢性肝炎肝硬化直至最終發(fā)展為肝癌是個(gè)漫長的過程并受多種因素調(diào)控,但其具體機(jī)制尚不明確。miRNAs(microRNAs)是一類約22nt大小的非編碼RNA分子,在轉(zhuǎn)錄后水平調(diào)控基因的表達(dá)。研究表明HBV感染可導(dǎo)致肝細(xì)胞內(nèi)多種mi RNAs表達(dá)水平的變化。miRNAs可通過調(diào)控多個(gè)癌基因和抑癌基因的表達(dá)影響肝細(xì)胞的生物學(xué)功能,包括細(xì)胞活性,增殖能力,遷移和侵襲能力等等,進(jìn)而影響肝癌的發(fā)生發(fā)展。由此我們猜想,HBV感染導(dǎo)致肝癌發(fā)生發(fā)展可能是通過調(diào)控部分mi RNAs的表達(dá)實(shí)現(xiàn)的。本課題著重研究HBV感染導(dǎo)致的mi RNAs表達(dá)變化及其這些變化對肝癌的發(fā)生發(fā)展產(chǎn)生的作用。提出了HBV促進(jìn)肝癌發(fā)生的新機(jī)制�！痉椒ā渴紫壤蒙疃葴y序技術(shù)分析HBV陽性肝癌組織和HBV陰性肝癌組織中miRNAs的表達(dá)譜并選擇了差異性表達(dá)較明顯的新miRNA—novel-miR-10作為研究對象;隨后用qRT-PCR技術(shù)驗(yàn)證了HBV對novel-miR-10表達(dá)的調(diào)控作用;構(gòu)建novel-miR-10的啟動子報(bào)告質(zhì)粒并通過雙熒光素酶報(bào)告系統(tǒng)驗(yàn)證HBV以及HBx對novel-miR-10啟動子的調(diào)控,同時(shí)驗(yàn)證了轉(zhuǎn)錄因子SP1和CREB1對novel-miR-10啟動子活性的影響;接下來通過MTT實(shí)驗(yàn)、平板克隆形成實(shí)驗(yàn)、Transwell遷移和侵襲實(shí)驗(yàn)進(jìn)一步確定novel-miR-10在肝癌細(xì)胞中的生物學(xué)功能;通過FACS技術(shù)和western blot實(shí)驗(yàn)驗(yàn)證了novel-miR-10對肝癌細(xì)胞凋亡和EMT過程的影響;繼而通過生物信息學(xué)方法預(yù)測novel-miR-10的靶基因并通過EGFP熒光報(bào)告系統(tǒng)結(jié)合qRT-PCR和western blot實(shí)驗(yàn)進(jìn)行驗(yàn)證;然后干擾靶基因的表達(dá)并通過MTT實(shí)驗(yàn)、平板克隆形成實(shí)驗(yàn)、Transwell遷移和侵襲實(shí)驗(yàn)來確定靶基因在肝癌中的生物學(xué)功能并通過FACS技術(shù)和western blot實(shí)驗(yàn)驗(yàn)證了靶基因?qū)Ω伟┘?xì)胞凋亡和EMT過程的影響;最后通過一系列挽救實(shí)驗(yàn)證明novel-miR-10對肝癌的生物學(xué)作用是通過對靶基因的調(diào)控實(shí)現(xiàn)的�！窘Y(jié)果】深度測序結(jié)果顯示在HBV陽性肝癌組織中存在多個(gè)差異性表達(dá)的miRNAs,其中新發(fā)現(xiàn)的miRNA-novel-miR-10在HBV陽性肝癌組織中表達(dá)量高于HBV陰性肝癌組織;表達(dá)HBV1.3copy可促進(jìn)肝癌細(xì)胞中novel-miR-10的表達(dá);表達(dá)HBV1.3copy和HBx可增強(qiáng)novel-miR-10啟動子的活性;過表達(dá)轉(zhuǎn)錄因子SP1抑制,CREB1促進(jìn)novel-miR-10啟動子的活性,并且敲降CREB1后HBV1.3copy和HBx對novel-miR-10啟動子的促進(jìn)作用被阻斷;肝癌表型實(shí)驗(yàn)證實(shí)novel-miR-10可促進(jìn)肝癌細(xì)胞的細(xì)胞活性、增殖能力、遷移和侵襲能力;生物信息學(xué)預(yù)測TNFRSF19和RAB43是novel-miR-10的兩個(gè)直接靶基因,EGFP熒光報(bào)告載體驗(yàn)證novel-miR-10直接靶定TNFRSF19和RAB43的3’UTR區(qū)域并下調(diào)其表達(dá);qRT-PCR和western blot實(shí)驗(yàn)證實(shí)novel-miR-10抑制內(nèi)源性TNFRSF19和RAB43的表達(dá);敲降TNFRSF19和RAB43這兩個(gè)靶基因的表達(dá)促進(jìn)肝癌細(xì)胞的細(xì)胞活性、增殖能力、遷移和侵襲能力;挽救實(shí)驗(yàn)證明敲降TNFRSF19和RAB43能逆轉(zhuǎn)由降低novel-miR-10表達(dá)引起的肝癌細(xì)胞生物學(xué)功能的抑制作用�！窘Y(jié)論】HBV可通過HBx與CREB1增強(qiáng)novel-miR-10啟動子的活性,從而促進(jìn)其表達(dá)。novel-miR-10促進(jìn)肝癌細(xì)胞的細(xì)胞活性、增殖能力、遷移和侵襲能力,起到癌基因的作用,并且其癌基因作用是或至少部分是通過靶定并下調(diào)其靶基因TNFRSF19和RAB43的表達(dá)來實(shí)現(xiàn)的�？傊�,這些結(jié)果證明了HBV可通過調(diào)節(jié)miRNA表達(dá)促進(jìn)肝癌發(fā)生,并闡明了其具體機(jī)制。
[Abstract]:[Objective] hepatocellular carcinoma (HCC) is a common high malignant tumor. Due to the lack of early diagnosis and effective treatment strategy, the poor prognosis of.HBV is one of the most important risk factors for the occurrence of hepatocellular carcinoma. It is a long process to lead to the hardened liver of chronic hepatitis until it is eventually developed into liver cancer. It is regulated by a variety of factors, but its specific mechanism is not yet clear that.MiRNAs (microRNAs) is a class of about 22nt size of non coded RNA molecules that regulate the expression of genes at the post transcriptional level. The study shows that HBV infection can lead to a variety of MI RNAs expression levels in the liver cells..miRNAs can regulate the expression of multiple oncogenes and tumor suppressor genes. The biological functions of hepatocytes, including cell activity, proliferation, migration and invasion, etc., and thus affect the development and development of liver cancer. Therefore, we suspect that HBV infection may lead to the development of liver cancer by regulating the expression of partial mi RNAs. This topic focuses on the changes in the expression of MI RNAs caused by HBV infection and these The effect of change on the development and development of liver cancer was proposed. A new mechanism for HBV to promote the occurrence of liver cancer was proposed. [method] first, the expression profiles of miRNAs in HBV positive liver cancer tissues and HBV negative liver cancer tissues were analyzed by deep sequencing technology, and the new miRNA novel-miR-10 was selected as the research object, and then qRT-PC was used as the research object. The R technique verified the regulation of HBV on the expression of novel-miR-10, constructed the promoter report plasmid of novel-miR-10 and verified the regulation of HBV and HBx on novel-miR-10 promoter through the double luciferase reporter system, and verified the effect of the transcription factor SP1 and CREB1 on novel-miR-10 promoter activity. Transwell migration and invasion experiments further determine the biological function of novel-miR-10 in hepatoma cells; the effects of novel-miR-10 on the apoptosis and EMT process of hepatoma cells are verified by FACS and Western blot experiments. Then, the target gene of novel-miR-10 is pretested by bioinformatics and the EGFP fluorescence report is reported. The system was verified by qRT-PCR and Western blot experiments, and then the target gene was interfered with the expression of the target gene and through the MTT experiment, the flat clone formation experiment, the Transwell migration and invasion experiment to determine the biological function of the target gene in the liver cancer. The target gene was tested for the apoptosis and EMT process of the liver cancer cells by the FACS technique and the Western blot test. Finally, through a series of rescue experiments, the biological effect of novel-miR-10 on liver cancer was achieved through the regulation of the target gene. [results] the results of deep sequencing showed that there were multiple differentially expressed miRNAs in the HBV positive liver cancer tissues, and the newly discovered miRNA-novel-miR-10 was expressed in the HBV positive liver cancer tissues. Higher than HBV negative liver cancer tissue; expression of HBV1.3copy can promote the expression of novel-miR-10 in hepatoma cells; expression of HBV1.3copy and HBx can enhance the activity of novel-miR-10 promoter; over expression of transcription factor SP1 inhibits, CREB1 promotes the activity of novel-miR-10 promoter, and HBV1.3copy and HBx to promoter promoter after knocking down CREB1 Novel-miR-10 can promote the cell activity, proliferation, migration and invasion of hepatoma cells; bioinformatics predicts that TNFRSF19 and RAB43 are the two direct target genes of novel-miR-10, and EGFP fluorescent reporter vectors verify that novel-miR-10 directly targets the 3 'UTR region of TNFRSF19 and RAB43 and down its table. QRT-PCR and Western blot experiments confirmed that novel-miR-10 inhibited the expression of endogenous TNFRSF19 and RAB43, and the expression of two target genes that knocked down TNFRSF19 and RAB43 promoted the cell activity, proliferation, migration and invasion ability of liver cancer cells; the rescue experiment showed that the knockdown TNFRSF19 and RAB43 can reverse the liver caused by decreasing novel-miR-10 expression. [Conclusion] HBV can enhance the activity of novel-miR-10 promoter through HBx and CREB1, thus promoting the expression of.Novel-miR-10 to promote the cell activity, proliferation, migration and invasion of hepatoma cells, and play the role of the oncogene, and its oncogene effect is or at least partly through the target. The expression of the target gene TNFRSF19 and RAB43 was downregulated. In conclusion, these results showed that HBV could promote the development of liver cancer by regulating the expression of miRNA and elucidate its specific mechanism.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.7
本文編號：2138557
[Abstract]:[Objective] hepatocellular carcinoma (HCC) is a common high malignant tumor. Due to the lack of early diagnosis and effective treatment strategy, the poor prognosis of.HBV is one of the most important risk factors for the occurrence of hepatocellular carcinoma. It is a long process to lead to the hardened liver of chronic hepatitis until it is eventually developed into liver cancer. It is regulated by a variety of factors, but its specific mechanism is not yet clear that.MiRNAs (microRNAs) is a class of about 22nt size of non coded RNA molecules that regulate the expression of genes at the post transcriptional level. The study shows that HBV infection can lead to a variety of MI RNAs expression levels in the liver cells..miRNAs can regulate the expression of multiple oncogenes and tumor suppressor genes. The biological functions of hepatocytes, including cell activity, proliferation, migration and invasion, etc., and thus affect the development and development of liver cancer. Therefore, we suspect that HBV infection may lead to the development of liver cancer by regulating the expression of partial mi RNAs. This topic focuses on the changes in the expression of MI RNAs caused by HBV infection and these The effect of change on the development and development of liver cancer was proposed. A new mechanism for HBV to promote the occurrence of liver cancer was proposed. [method] first, the expression profiles of miRNAs in HBV positive liver cancer tissues and HBV negative liver cancer tissues were analyzed by deep sequencing technology, and the new miRNA novel-miR-10 was selected as the research object, and then qRT-PC was used as the research object. The R technique verified the regulation of HBV on the expression of novel-miR-10, constructed the promoter report plasmid of novel-miR-10 and verified the regulation of HBV and HBx on novel-miR-10 promoter through the double luciferase reporter system, and verified the effect of the transcription factor SP1 and CREB1 on novel-miR-10 promoter activity. Transwell migration and invasion experiments further determine the biological function of novel-miR-10 in hepatoma cells; the effects of novel-miR-10 on the apoptosis and EMT process of hepatoma cells are verified by FACS and Western blot experiments. Then, the target gene of novel-miR-10 is pretested by bioinformatics and the EGFP fluorescence report is reported. The system was verified by qRT-PCR and Western blot experiments, and then the target gene was interfered with the expression of the target gene and through the MTT experiment, the flat clone formation experiment, the Transwell migration and invasion experiment to determine the biological function of the target gene in the liver cancer. The target gene was tested for the apoptosis and EMT process of the liver cancer cells by the FACS technique and the Western blot test. Finally, through a series of rescue experiments, the biological effect of novel-miR-10 on liver cancer was achieved through the regulation of the target gene. [results] the results of deep sequencing showed that there were multiple differentially expressed miRNAs in the HBV positive liver cancer tissues, and the newly discovered miRNA-novel-miR-10 was expressed in the HBV positive liver cancer tissues. Higher than HBV negative liver cancer tissue; expression of HBV1.3copy can promote the expression of novel-miR-10 in hepatoma cells; expression of HBV1.3copy and HBx can enhance the activity of novel-miR-10 promoter; over expression of transcription factor SP1 inhibits, CREB1 promotes the activity of novel-miR-10 promoter, and HBV1.3copy and HBx to promoter promoter after knocking down CREB1 Novel-miR-10 can promote the cell activity, proliferation, migration and invasion of hepatoma cells; bioinformatics predicts that TNFRSF19 and RAB43 are the two direct target genes of novel-miR-10, and EGFP fluorescent reporter vectors verify that novel-miR-10 directly targets the 3 'UTR region of TNFRSF19 and RAB43 and down its table. QRT-PCR and Western blot experiments confirmed that novel-miR-10 inhibited the expression of endogenous TNFRSF19 and RAB43, and the expression of two target genes that knocked down TNFRSF19 and RAB43 promoted the cell activity, proliferation, migration and invasion ability of liver cancer cells; the rescue experiment showed that the knockdown TNFRSF19 and RAB43 can reverse the liver caused by decreasing novel-miR-10 expression. [Conclusion] HBV can enhance the activity of novel-miR-10 promoter through HBx and CREB1, thus promoting the expression of.Novel-miR-10 to promote the cell activity, proliferation, migration and invasion of hepatoma cells, and play the role of the oncogene, and its oncogene effect is or at least partly through the target. The expression of the target gene TNFRSF19 and RAB43 was downregulated. In conclusion, these results showed that HBV could promote the development of liver cancer by regulating the expression of miRNA and elucidate its specific mechanism.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.7
【共引文獻(xiàn)】
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1 陳建宏;劉妍;李進(jìn);徐東平;;隱匿性乙型肝炎病毒感染研究進(jìn)展[J];實(shí)用肝臟病雜志;2015年02期
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1 張冬青;肝內(nèi)乙型肝炎病毒DNA甲基化在核苷（酸）類似物停藥復(fù)發(fā)中的作用研究[D];福建醫(yī)科大學(xué);2013年
2 王曉英;慢乙肝患者HBVcccDNA與血清HBVDNA及其標(biāo)志物相關(guān)性研究[D];寧夏醫(yī)科大學(xué);2013年
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6 倪東京;組蛋白甲基化修飾在HBV誘導(dǎo)肝細(xì)胞基因表達(dá)改變中的作用機(jī)制研究[D];第三軍醫(yī)大學(xué);2014年
7 王甜;HBV cccDNA的檢測方法及其在慢性乙型肝炎臨床應(yīng)用中的研究進(jìn)展[D];重慶醫(yī)科大學(xué);2014年
8 王瑩;青少年人群隱匿性乙型肝炎病毒感染及分子進(jìn)化特征分析[D];新疆醫(yī)科大學(xué);2014年
，本文編號：2138557
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