BCLB经AMPK-mTOR途径增强饥饿诱导的肝癌细胞凋亡及自噬

发布时间：2018-05-03 03:05

本文选题：BCLB + 肿瘤抑癌基因　；参考：《浙江大学》2017年博士论文

【摘要】：原发性肝癌是世界上发病率最高的癌症之一,预后极差,也是世界上第三位肿瘤性致死原因。近几年研究发现,肿瘤抑制基因(tumor suppressor genes,TSGs)的表观遗传学调控变化,尤其是DNA甲基化改变,在原发性肝癌发生及进展中起到关键作用。研究目的:通过高通量表观遗传学筛查,我们发现BCLB基因启动子在原发性肝癌中高度甲基化。因此,我们进一步探索该基因的功能,研究其在原发性肝癌发生和发展中起的作用及其机制。研究方法:通过甲基化特异性PCR(methylation-specific PCR,MSP)和重亚硫酸氢盐全基因组测序(bisulfite genome sequencing,BGS)检测BCLB基因启动子的甲基化水平;通过逆转录cDNA PCR(reverse transcription PCR,RT-PCR)和免疫组化(immunohistochemistry,IHC)检测肝癌组织和"癌旁正常"组织中BCLB的表达情况;通过转染pCMV6.BCLB质粒,筛选出稳定表达BCLB蛋白的肝癌细胞系,观察BCLB基因对肝癌细胞的迁移、凋亡、自噬等行为的影响,并通过在裸鼠皮下注射转染肝癌细胞,进一步观察BCLB基因对肝癌发生及生长的影响。研究结果:BCLB基因在肝细胞性肝癌细胞系中以及原发性肝癌组织中表达水平下调甚至沉默,而这些表达降低或沉默的肿瘤细胞和肿瘤组织中,BCLB基因启动子均被检测到高度甲基化。105肝癌标本中,BCLB基因在47例(44.8%)癌组织中阳性表达;而在配对的癌旁正常组织中,有96例(91.4%)呈阳性表达,二者有显著性差异(p=0.04)。BCLB蛋白的表达与乙肝病毒感染(p=0.036)、甲胎蛋白水平(p=0.048)、肿瘤大小(p=0.006)以及肿瘤TNM分期(p=0.022)显著相关。癌组织中,BCLB阳性表达患者术后总体5年生存率以及无瘤生存率均显著高于阴性表达患者(p=0.032,p=0.027)。肝癌细胞株转染BCLB后,通过体内外试验发现,BCLB可显著抑制肿瘤生长。进一步研究发现,BCLB基因可通过AMPK-mTOR信号通路,促进饥饿诱导的肝细胞性肝癌的凋亡及自噬的发生。研究结论:BCLB是一个肝癌抑癌基因,BCLB蛋白表达能显著抑制肿瘤细胞的增殖及迁移侵袭,并能增强肝癌细胞对饥饿的敏感性从而通过MAPK-mTOR信号通路促进肿瘤细胞的凋亡和自噬。BCLB甲基化在肝癌中是一个经常发生的、肿瘤特异性的分子事件,甲基化的BCLB具有作为一个肿瘤标志物用于肝癌的诊断和预后评估的潜能。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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