miR-122調(diào)控網(wǎng)絡(luò)及其生物學(xué)功能
發(fā)布時(shí)間：2019-01-06 05:02
【摘要】：miR-122是肝臟特異性高表達(dá)的microRNA,但其表達(dá)調(diào)控網(wǎng)絡(luò)、生物學(xué)功能及其與疾病的關(guān)系尚不清楚。本研究中,我們通過螢光素酶報(bào)告系統(tǒng)、凝膠遷滯實(shí)驗(yàn)以及染色體免疫共沉淀等方法鑒定了miR-122的啟動(dòng)子區(qū)及其轉(zhuǎn)錄激活因子。運(yùn)用小鼠肝發(fā)育、人肝癌組織及細(xì)胞株、黃曲霉素誘導(dǎo)轉(zhuǎn)化的細(xì)胞株等模型,鑒定了miR-122調(diào)控回路及其在肝臟生理和病理過程中的作用。我們發(fā)現(xiàn):miR-122的轉(zhuǎn)錄起始位點(diǎn)位于miR-122前體上游的4811bp(chr18: 54264475)處,其啟動(dòng)子則位于miR-122前體(pre-miRNA)上游-5.3～-4.8 kb區(qū)域。進(jìn)一步研究揭示,在miR-122的啟動(dòng)子區(qū)存在著轉(zhuǎn)錄因子C/EBPα的多個(gè)結(jié)合序列,刪除這些位點(diǎn)能明顯降低miR-122的啟動(dòng)子活性;而且,C/EBPα能與miR-122啟動(dòng)子序列在體內(nèi)外直接結(jié)合;如果抑制C/EBPα的表達(dá),則可導(dǎo)致miR-122啟動(dòng)子活性及內(nèi)源miR-122表達(dá)水平的顯著下降。這些結(jié)果表明C/EBPα可直接激活miR-122的轉(zhuǎn)錄表達(dá)。我們的結(jié)果還顯示:miR-122通過直接抑制IGF-1R的表達(dá),使得GSK-3β保持活性狀態(tài);活化的GSK-3β不僅抑制細(xì)胞增殖,還能激活C/EBPα,進(jìn)一步促進(jìn)miR-122的轉(zhuǎn)錄,加強(qiáng)對IGF-1R表達(dá)的抑制作用。這些發(fā)現(xiàn)揭示了肝細(xì)胞內(nèi)一條新的GSK-3β-C/EBPα-miR-122-IGF-1R-GSK-3β調(diào)控回路。同時(shí),我們在肝癌組織中,檢測到高頻率的miR-122與C/EBPα表達(dá)下調(diào)以及IGF-1R異常增高,而且miR-122的低表達(dá)與肝癌患者生存期短密切相關(guān)。更重要的是,過表達(dá)HBx蛋白或黃曲霉素處理會(huì)導(dǎo)致GSK-3β和C/EBPα活性下降以及miR-122表達(dá)下調(diào),從而使IGF-1R水平上升;而回復(fù)miR-122的表達(dá)則能顯著抑制肝癌細(xì)胞株以及黃曲霉素轉(zhuǎn)化細(xì)胞的成瘤能力。這提示GSK-3β-C/EBPα-miR-122-IGF-1R調(diào)控回路的失調(diào)與肝癌發(fā)生發(fā)展密切相關(guān)。本論文的研究結(jié)果不僅加深了人們對miR-122調(diào)控網(wǎng)絡(luò)和生物學(xué)功能以及肝癌發(fā)病機(jī)制的認(rèn)識,而且為肝癌防治提供了新的分子靶點(diǎn)。
[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 偽 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 偽 could directly bind to the miR-122 promoter sequence in vitro and in vivo, and if the expression of C/EBP 偽 was inhibited, the activity of miR-122 promoter and the expression level of endogenous miR-122 decreased significantly. These results suggest that C/EBP 偽 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 偽, 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 偽-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 偽 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 偽 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 偽-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.
【學(xué)位授予單位】：中山大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363
本文編號：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 偽 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 偽 could directly bind to the miR-122 promoter sequence in vitro and in vivo, and if the expression of C/EBP 偽 was inhibited, the activity of miR-122 promoter and the expression level of endogenous miR-122 decreased significantly. These results suggest that C/EBP 偽 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 偽, 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 偽-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 偽 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 偽 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 偽-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.
【學(xué)位授予單位】：中山大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363
【引證文獻(xiàn)】
相關(guān)期刊論文 前2條
1 李紹祥;李浩然;張成平;曹建彪;;miR-122的研究進(jìn)展[J];昆明醫(yī)科大學(xué)學(xué)報(bào);2012年09期
2 馬義濤;李艷華;周輝云;王穎;徐寧迎;;閹割對金華豬肝臟miR-122和miR-378表達(dá)量和膻味性狀的影響[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2013年08期
，本文編號：2402378
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