本文選題：BCLB + 腫瘤抑癌基因　； 參考：《浙江大學(xué)》2017年博士論文

【摘要】：原發(fā)性肝癌是世界上發(fā)病率最高的癌癥之一,預(yù)后極差,也是世界上第三位腫瘤性致死原因。近幾年研究發(fā)現(xiàn),腫瘤抑制基因(tumor suppressor genes,TSGs)的表觀遺傳學(xué)調(diào)控變化,尤其是DNA甲基化改變,在原發(fā)性肝癌發(fā)生及進(jìn)展中起到關(guān)鍵作用。研究目的:通過高通量表觀遺傳學(xué)篩查,我們發(fā)現(xiàn)BCLB基因啟動(dòng)子在原發(fā)性肝癌中高度甲基化。因此,我們進(jìn)一步探索該基因的功能,研究其在原發(fā)性肝癌發(fā)生和發(fā)展中起的作用及其機(jī)制。研究方法:通過甲基化特異性PCR(methylation-specific PCR,MSP)和重亞硫酸氫鹽全基因組測(cè)序(bisulfite genome sequencing,BGS)檢測(cè)BCLB基因啟動(dòng)子的甲基化水平;通過逆轉(zhuǎn)錄cDNA PCR(reverse transcription PCR,RT-PCR)和免疫組化(immunohistochemistry,IHC)檢測(cè)肝癌組織和"癌旁正常"組織中BCLB的表達(dá)情況;通過轉(zhuǎn)染pCMV6.BCLB質(zhì)粒,篩選出穩(wěn)定表達(dá)BCLB蛋白的肝癌細(xì)胞系,觀察BCLB基因?qū)Ω伟┘?xì)胞的遷移、凋亡、自噬等行為的影響,并通過在裸鼠皮下注射轉(zhuǎn)染肝癌細(xì)胞,進(jìn)一步觀察BCLB基因?qū)Ω伟┌l(fā)生及生長(zhǎng)的影響。研究結(jié)果:BCLB基因在肝細(xì)胞性肝癌細(xì)胞系中以及原發(fā)性肝癌組織中表達(dá)水平下調(diào)甚至沉默,而這些表達(dá)降低或沉默的腫瘤細(xì)胞和腫瘤組織中,BCLB基因啟動(dòng)子均被檢測(cè)到高度甲基化。105肝癌標(biāo)本中,BCLB基因在47例(44.8%)癌組織中陽性表達(dá);而在配對(duì)的癌旁正常組織中,有96例(91.4%)呈陽性表達(dá),二者有顯著性差異(p=0.04)。BCLB蛋白的表達(dá)與乙肝病毒感染(p=0.036)、甲胎蛋白水平(p=0.048)、腫瘤大小(p=0.006)以及腫瘤TNM分期(p=0.022)顯著相關(guān)。癌組織中,BCLB陽性表達(dá)患者術(shù)后總體5年生存率以及無瘤生存率均顯著高于陰性表達(dá)患者(p=0.032,p=0.027)。肝癌細(xì)胞株轉(zhuǎn)染BCLB后,通過體內(nèi)外試驗(yàn)發(fā)現(xiàn),BCLB可顯著抑制腫瘤生長(zhǎng)。進(jìn)一步研究發(fā)現(xiàn),BCLB基因可通過AMPK-mTOR信號(hào)通路,促進(jìn)饑餓誘導(dǎo)的肝細(xì)胞性肝癌的凋亡及自噬的發(fā)生。研究結(jié)論:BCLB是一個(gè)肝癌抑癌基因,BCLB蛋白表達(dá)能顯著抑制腫瘤細(xì)胞的增殖及遷移侵襲,并能增強(qiáng)肝癌細(xì)胞對(duì)饑餓的敏感性從而通過MAPK-mTOR信號(hào)通路促進(jìn)腫瘤細(xì)胞的凋亡和自噬。BCLB甲基化在肝癌中是一個(gè)經(jīng)常發(fā)生的、腫瘤特異性的分子事件,甲基化的BCLB具有作為一個(gè)腫瘤標(biāo)志物用于肝癌的診斷和預(yù)后評(píng)估的潛能。
[Abstract]:Primary liver cancer (HCC) is one of the most common cancers in the world with poor prognosis and the third leading cause of cancer death in the world. In recent years, it has been found that the epigenetic regulation of tumor suppressor gene (suppressor), especially the change of DNA methylation, plays a key role in the development and progression of primary liver cancer. Objective: through high throughput epigenetic screening, we found that BCLB gene promoter is highly methylated in primary liver cancer. Therefore, we further explore the function of the gene and its role in the occurrence and development of primary liver cancer and its mechanism. Methods: methylation level of BCLB gene promoter was detected by methylation specific PCR(methylation-specific PCR mSPs and bisulfite genome sequencing. The expression of BCLB was detected by reverse transcriptase cDNA PCR(reverse transcription polymerase chain reaction (cDNA PCR(reverse transcription) and immunohistochemical staining (IHC) in hepatocellular carcinoma tissues and "adjacent normal" tissues, and the stable expression of BCLB protein in HCC cell lines was screened by transfection of pCMV6.BCLB plasmid, and the migration of BCLB gene to HCC cells was observed. The effects of apoptosis and autophagy on the occurrence and growth of hepatocellular carcinoma were further observed by subcutaneously transfection of BCLB gene into hepatoma cells in nude mice. Results the expression of BCLB gene was down-regulated or even silenced in hepatocellular carcinoma cell lines and primary liver cancer tissues. However, the expression of BCLB gene was detected in 47 cases of cancer tissues with hypermethylated. 105 HCC samples, while in matched normal tissues, the positive expression of BCLB gene was found in the tumor cells and tumor tissues with decreased or silenced expression, while in the normal tissues adjacent to the tumor, the positive expression of the BCLB gene was found in 47 cases of cancer tissues with hypermethylated. 105 HCC samples. There were significant differences between the positive expression of p0. 04%. BCLB protein and hepatitis B virus infection (p0. 036, P 0. 048), tumor size (p0. 006) and tumor TNM stage (p0. 022). The overall 5-year survival rate and tumor-free survival rate of patients with positive expression of BCLB were significantly higher than those of patients with negative expression of BCLB. After transfection of BCLB into HCC cell line, it was found in vitro and in vivo that BCLB could significantly inhibit tumor growth. It was further found that the BCLB gene could promote the apoptosis and autophagy of liver cancer induced by starvation through AMPK-mTOR signaling pathway. Conclusion the expression of BCLB protein, a tumor suppressor gene, can significantly inhibit the proliferation and invasion of tumor cells. It is a frequent, tumor-specific molecular event that it can enhance the sensitivity of hepatoma cells to starvation and promote apoptosis and autophagy of tumor cells through MAPK-mTOR signaling pathway, and the methylation of autophagy .BCLB is a frequently occurring, tumor-specific molecular event in hepatocellular carcinoma. Methylated BCLB has the potential to be used as a tumor marker for diagnosis and prognosis evaluation of liver cancer.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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相關(guān)期刊論文 前3條

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2 Mitsuro Kanda;Hiroyuki Sugimoto;Yasuhiro Kodera;;Genetic and epigenetic aspects of initiation and progression of hepatocellular carcinoma[J];World Journal of Gastroenterology;2015年37期

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本文編號(hào)：1836683