miR-122调控网络及其生物学功能
发布时间：2019-01-06 05:02
【摘要】：miR-122是肝脏特异性高表达的microRNA,但其表达调控网络、生物学功能及其与疾病的关系尚不清楚。本研究中,我们通过萤光素酶报告系统、凝胶迁滞实验以及染色体免疫共沉淀等方法鉴定了miR-122的启动子区及其转录激活因子。运用小鼠肝发育、人肝癌组织及细胞株、黄曲霉素诱导转化的细胞株等模型,鉴定了miR-122调控回路及其在肝脏生理和病理过程中的作用。我们发现:miR-122的转录起始位点位于miR-122前体上游的4811bp(chr18: 54264475)处,其启动子则位于miR-122前体(pre-miRNA)上游-5.3～-4.8 kb区域。进一步研究揭示,在miR-122的启动子区存在着转录因子C/EBPα的多个结合序列,删除这些位点能明显降低miR-122的启动子活性;而且,C/EBPα能与miR-122启动子序列在体内外直接结合;如果抑制C/EBPα的表达,则可导致miR-122启动子活性及内源miR-122表达水平的显著下降。这些结果表明C/EBPα可直接激活miR-122的转录表达。我们的结果还显示:miR-122通过直接抑制IGF-1R的表达,使得GSK-3β保持活性状态;活化的GSK-3β不仅抑制细胞增殖,还能激活C/EBPα,进一步促进miR-122的转录,加强对IGF-1R表达的抑制作用。这些发现揭示了肝细胞内一条新的GSK-3β-C/EBPα-miR-122-IGF-1R-GSK-3β调控回路。同时,我们在肝癌组织中,检测到高频率的miR-122与C/EBPα表达下调以及IGF-1R异常增高,而且miR-122的低表达与肝癌患者生存期短密切相关。更重要的是,过表达HBx蛋白或黄曲霉素处理会导致GSK-3β和C/EBPα活性下降以及miR-122表达下调,从而使IGF-1R水平上升;而回复miR-122的表达则能显著抑制肝癌细胞株以及黄曲霉素转化细胞的成瘤能力。这提示GSK-3β-C/EBPα-miR-122-IGF-1R调控回路的失调与肝癌发生发展密切相关。本论文的研究结果不仅加深了人们对miR-122调控网络和生物学功能以及肝癌发病机制的认识,而且为肝癌防治提供了新的分子靶点。
[Abstract]:MiR-122 is a liver specific high expression microRNA, but its expression regulatory network, biological function and its relationship with disease is unclear. In this study, we identified the promoter region of miR-122 and its transcriptional activator by luciferase reporting system, gel hysteresis assay and chromosome immunoprecipitation. Using mouse liver development, human liver cancer tissues and cell lines, and aflatoxin-induced transformation of cell lines, the regulatory loop of miR-122 and its role in the physiological and pathological process of liver were identified. We found that the transcriptional initiation site of miR-122 was located at 4811bp (chr18: 54264475) upstream of miR-122 precursor, and the promoter was located at -5.3 ~ 4.8 kb region upstream of miR-122 precursor (pre-miRNA). Further studies revealed that there were several binding sequences of transcription factor C/EBP 伪 in the promoter region of miR-122, and the deletion of these sites could significantly reduce the promoter activity of miR-122. Moreover, C/EBP 伪 could directly bind to the miR-122 promoter sequence in vitro and in vivo, and if the expression of C/EBP 伪 was inhibited, the activity of miR-122 promoter and the expression level of endogenous miR-122 decreased significantly. These results suggest that C/EBP 伪 can directly activate the transcriptional expression of miR-122. Our results also showed that miR-122 could keep GSK-3 尾 active by directly inhibiting the expression of IGF-1R. Activated GSK-3 尾 not only inhibited cell proliferation, but also activated C/EBP 伪, further promoted the transcription of miR-122 and enhanced the inhibition of IGF-1R expression. These findings reveal a new regulation circuit of GSK-3 尾-C/EBP 伪-miR-122-IGF-1R-GSK-3 尾 in hepatocytes. At the same time, we detected the down-regulated expression of miR-122 and C/EBP 伪 and the abnormal increase of IGF-1R in HCC tissues, and the low expression of miR-122 was closely related to the short survival time of HCC patients. More importantly, overexpression of HBx protein or aflatoxin treatment led to the decrease of GSK-3 尾 and C/EBP 伪 activity and the down-regulation of miR-122 expression, which led to the increase of IGF-1R level. The expression of miR-122 could significantly inhibit the tumorigenesis of hepatoma cell lines and aflatoxin transformed cells. This suggests that the maladjustment of GSK-3 尾-C/EBP 伪-miR-122-IGF-1R regulatory circuit is closely related to the occurrence and development of HCC. The results of this paper not only deepen the understanding of the regulatory network and biological function of miR-122 and the pathogenesis of HCC, but also provide a new molecular target for the prevention and treatment of HCC.
【学位授予单位】：中山大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
本文编号：2402378
[Abstract]:MiR-122 is a liver specific high expression microRNA, but its expression regulatory network, biological function and its relationship with disease is unclear. In this study, we identified the promoter region of miR-122 and its transcriptional activator by luciferase reporting system, gel hysteresis assay and chromosome immunoprecipitation. Using mouse liver development, human liver cancer tissues and cell lines, and aflatoxin-induced transformation of cell lines, the regulatory loop of miR-122 and its role in the physiological and pathological process of liver were identified. We found that the transcriptional initiation site of miR-122 was located at 4811bp (chr18: 54264475) upstream of miR-122 precursor, and the promoter was located at -5.3 ~ 4.8 kb region upstream of miR-122 precursor (pre-miRNA). Further studies revealed that there were several binding sequences of transcription factor C/EBP 伪 in the promoter region of miR-122, and the deletion of these sites could significantly reduce the promoter activity of miR-122. Moreover, C/EBP 伪 could directly bind to the miR-122 promoter sequence in vitro and in vivo, and if the expression of C/EBP 伪 was inhibited, the activity of miR-122 promoter and the expression level of endogenous miR-122 decreased significantly. These results suggest that C/EBP 伪 can directly activate the transcriptional expression of miR-122. Our results also showed that miR-122 could keep GSK-3 尾 active by directly inhibiting the expression of IGF-1R. Activated GSK-3 尾 not only inhibited cell proliferation, but also activated C/EBP 伪, further promoted the transcription of miR-122 and enhanced the inhibition of IGF-1R expression. These findings reveal a new regulation circuit of GSK-3 尾-C/EBP 伪-miR-122-IGF-1R-GSK-3 尾 in hepatocytes. At the same time, we detected the down-regulated expression of miR-122 and C/EBP 伪 and the abnormal increase of IGF-1R in HCC tissues, and the low expression of miR-122 was closely related to the short survival time of HCC patients. More importantly, overexpression of HBx protein or aflatoxin treatment led to the decrease of GSK-3 尾 and C/EBP 伪 activity and the down-regulation of miR-122 expression, which led to the increase of IGF-1R level. The expression of miR-122 could significantly inhibit the tumorigenesis of hepatoma cell lines and aflatoxin transformed cells. This suggests that the maladjustment of GSK-3 尾-C/EBP 伪-miR-122-IGF-1R regulatory circuit is closely related to the occurrence and development of HCC. The results of this paper not only deepen the understanding of the regulatory network and biological function of miR-122 and the pathogenesis of HCC, but also provide a new molecular target for the prevention and treatment of HCC.
【学位授予单位】：中山大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
【引证文献】
相关期刊论文 前2条
1 李绍祥;李浩然;张成平;曹建彪;;miR-122的研究进展[J];昆明医科大学学报;2012年09期
2 马义涛;李艳华;周辉云;王颖;徐宁迎;;阉割对金华猪肝脏miR-122和miR-378表达量和膻味性状的影响[J];农业生物技术学报;2013年08期
，本文编号：2402378
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