FXR上调miR-122的分子机制及在抗HCC中的作用研究

发布时间：2017-12-26 17:29

本文关键词：FXR上调miR-122的分子机制及在抗HCC中的作用研究　出处：《第三军医大学》2017年博士论文　论文类型：学位论文

【摘要】：肝细胞癌(hepatocellular carcinoma,HCC)是全球范围内最普遍的恶性肿瘤之一。其诱发因素有多个,包括病毒感染、原癌基因激活、抑癌基因表达异常等。目前关于肝细胞癌的发病机制有待深入研究,而进一步阐明与肝细胞癌发病过程中密切相关的基因及其相互作用无疑将有助于加深对HCC发病机制的理解以及发现靶向治疗HCC的新靶点。近年来,与HCC密切相关的法尼醇X受体(farnesoid X receptor,FXR)和microRNA日益受到重视。FXR属于核受体超家族成员,主要在肝脏、小肠、肾脏和肾上腺中表达,密切参与胆汁酸代谢、糖脂代谢,进而调节肝脏再生、肝纤维化等。大量的研究发现,FXR与HCC的发生发展密切相关。FXR作为一种多功能的转录因子,可调节多种肿瘤相关靶基因的表达和功能,进而发挥重要的抗HCC作用,但FXR的抗HCC机制尚不十分清楚,有待进一步的深入研究。mi R-122是一种在肝脏中特异性高表达的microRNA。在正常生理状态下,miR-122可以调控肝脏的细胞发育和细胞代谢,诱导细胞分化,并参与肝细胞应激应答等多种生命活动;而在病理状态下,mi R-122与HCC密切相关。研究发现,在HCC发生、发展过程中,mi R-122发挥着类似抑癌基因的作用,其主要通过抑制肿瘤相关基因的表达而发挥其抗肿瘤的功能。鉴于miR-122在抗HCC方面的重要作用,那么设法上调miR-122的表达,无疑将有助于肝癌的防治。目前的研究多集中在miR-122通过调节靶基因发挥抗肿瘤作用的机制方面,而关于miR-122自身的表达调控少有报道,最近的研究发现,一些转录因子(HNF4α、C/EBPα)可以促进miR-122的表达,但关于核受体FXR是否上调miR-122的表达,并通过此通路发挥抗HCC作用,目前未见报道。因此,结合相关文献和我们初步的研究结果,本课题率先鉴定了miR-122是FXR的一个新靶基因,揭示FXR促进mi R-122表达的具体分子机制;进而阐明“FXR-miR-122通路”在抗HCC作用中的新机制,为进一步深入而全面地揭示FXR的抗肝细胞癌机制以及miR-122自身的表达调控积累新的理论资料,为探索以“FXR-miR-122通路”为靶标的抗肿瘤治疗提供新的科学依据。目的:本研究从体内外探讨FXR上调miR-122的分子机制及“FXR-miR-122通路在抗HCC中的重要作用方法:1.收集20例hcc病人的肿瘤组织及癌旁组织,和7种hcc细胞系,经real-timepcr和westernblot实验,检测fxr与mir-122的表达,并通过相关性统计,分析fxr与mir-122表达的相关性。2.不同浓度fxr激动剂处理hcc细胞,以及采用rna干扰(rnainterference,rnai),通过real-timepcr和westernblot实验检测mir-122以及mir-122靶基因igf-1r和cycling1的表达,研究fxr对mir-122表达的影响。3.应用生物信息学分析,预测人mir-122的5'调控区fxr的可能结合位点,构建含可能结合位点报告基因质粒,转染细胞经luc报告基因检测实验,初步确定fxr可能结合位点的所在区域。进一步采用凝胶迁移率实验(emsa)和染色质免疫沉淀实验(chip),探讨fxr调节mir-122表达的具体分子机制。4.hcc细胞转染antagomir-122抑制mir-122功能后,采用cck-8法检测细胞的增殖情况。5.hcc细胞接种裸鼠成瘤后,以fxr的配体灌胃处理小鼠,一定时间后,在肝癌荷瘤鼠上观察肿瘤的生长情况,然后处死小鼠,切取肿瘤组织,real-timepcr和westernblot实验检测mir-122,igf-1r和cycling1的表达情况。免疫组化测定增殖标志物ki67的表达,在体内探讨“fxr-mir-122通路”的抗hcc作用。结果:1.fxr和mir-122在20例hcc病人肿瘤组织中的表达均显著低于癌旁组织,且相关性分析显示,fxr与mir-122的表达呈显著正相关;同样,相对于正常人肝细胞系l02,7种hcc细胞系中fxr和mir-122的表达也呈显著正相关。2.fxr激动剂处理hcc细胞活化fxr后,可显著增加mir-122的表达,并减弱其靶基因和igf-1r和cycling1的表达,同时,hcc细胞转染fxrsirna抑制fxr后,显著抑制了fxr激动剂诱导mir-122的表达。3.通过报告基因检测、emsa和chip实验证实了,fxr可通过直接结合于mir-122启动子区-338到-325的dr2反应元件,增强mir-122的启动子活性,进而促进mir-122的表达。4.细胞增殖检测实验显示,抑制mir-122后可显著减弱fxr对hcc细胞增殖的抑制作用。5.肝癌荷瘤鼠模型上,fxr可显著诱导mir-122的表达,并抑制其靶基igf-1r和cycling1的表达,进而抑制肿瘤生长。结论:FXR通过直接结合于miR-122启动子区的DR2(-338到-325)反应元件,促进mi R-122的表达,进而抑制肿瘤的生长,提示FXR/miR-122通路可能是防治HCC的新靶点。
[Abstract]:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. There are many inducing factors, including viral infection, activation of proto oncogene and abnormal expression of tumor suppressor genes. At present, the pathogenesis of hepatocellular carcinoma needs to be further studied. Further elucidation of the genes and their interactions closely related to the pathogenesis of hepatocellular carcinoma will undoubtedly help to further understand the pathogenesis of HCC and find new targets for targeted therapy of HCC. In recent years, farnesoid X receptor is closely related with HCC (farnesoid X receptor, FXR and microRNA) has been paid more and more attention. FXR is a member of the nuclear receptor superfamily. It is mainly expressed in liver, small intestine, kidney and adrenal gland, and closely participates in bile acid metabolism, glucose and lipid metabolism, and then regulates liver regeneration, liver fibrosis and so on. A large number of studies have found that FXR is closely related to the development of HCC. As a multifunctional transcription factor, FXR can regulate the expression and function of multiple tumor related target genes, and play an important role in anti HCC. However, the mechanism of FXR against HCC is not yet clear, and needs further study. Mi R-122 is a highly expressed microRNA in the liver. Under normal physiological condition, miR-122 can regulate liver cell development and cell metabolism, induce cell differentiation and participate in various kinds of life activities such as liver cell stress response, while in pathological state, MI R-122 is closely related to HCC. It is found that in the process of HCC occurrence and development, MI R-122 acts as a tumor suppressor gene, and its anti-tumor function is mainly achieved by inhibiting the expression of tumor related genes. In view of the important role of miR-122 in anti HCC, it will undoubtedly contribute to the prevention and treatment of liver cancer by trying to increase the expression of miR-122. The present study focused on miR-122 regulated target genes through the mechanism of the antitumor effect, and regulate the expression of miR-122 on its own is rarely reported, a recent study found that some transcription factors (HNF4 and C/EBP alpha) could promote the expression of miR-122, but the expression of nuclear receptor FXR is up-regulated the expression of miR-122, and play the role of anti HCC through this pathway has not been reported. Therefore, combined with relevant literature and our preliminary research results, this study first identified miR-122 as a new target gene of FXR, to reveal the molecular mechanism of MI R-122 FXR to promote specific expression; and then clarify the "FXR-miR-122 pathway" new mechanism in anti HCC effects, for further in-depth and comprehensive reveal the regulation of the expression of anti liver cell carcinoma mechanism of FXR and miR-122's own accumulation theory for exploring new data, provide new scientific basis for anti-tumor therapeutic targets in the "FXR-miR-122 pathway". Objective: the purpose of this study was to explore the molecular mechanism of FXR upregulation of miR-122 and FXR-miR-122 pathway plays an important role in the anti HCC method from the in vivo tumor tissue and cancer: 1. of 20 cases were collected with HCC tissues and 7 HCC cell lines by real-timepcr and Westernblot experiments, the expression of FXR and miR-122, and through the correlation statistical analysis of correlation between the expression of FXR and mir-122. 2. different concentrations of FXR agonists were used to treat HCC cells, and RNA interference (rnainterference, RNAi) was used to detect miR-122 and miR-122 target gene IGF-1R and cycling1 expression by real-timepcr and Westernblot experiments. 3. bioinformatics analysis was used to predict the possible binding sites of FXR in the 5'regulatory region of human miR-122, and construct a reporter plasmid containing the possible binding sites. The transfected cells were detected by Luc reporter gene assay, and the location of the possible binding sites of FXR was preliminarily determined. The gel mobility test (EMSA) and chromatin immunoprecipitation assay (chip) were used to explore the specific molecular mechanism of FXR to regulate the expression of miR-122. After transfection of 4.hcc cells with antagomir-122 to inhibit miR-122 function, the proliferation of cells was detected by CCK-8. 5.hcc cells were inoculated in nude mice after intragastric administration with FXR ligand treated mice, after a certain period of time, to observe the growth of tumor in tumor bearing mice, then the mice were killed, cut the tumor tissue, real-timepcr and Westernblot test miR-122, IGF-1R and cycling1 respectively. The expression of the proliferation marker Ki67 was determined by immunohistochemistry, and the anti HCC effect of the "fxr-mir-122 pathway" was discussed in the body. Results: the expression of 1.fxr and miR-122 in 20 cases of HCC patients were significantly lower than that in tumor tissue and paracancerous tissue, and the correlation analysis showed that there was a positive correlation between FXR and miR-122; also, compared with the positive correlation of normal human liver cell line l02,7 FXR and miR-122 HCC cell lines also. 2.fxr agonist treated HCC cells activated FXR, significantly increased the expression of miR-122, and reduced the expression of target genes and IGF-1R and cycling1. Meanwhile, HCC cells transfected fxrsirna inhibited FXR expression, and significantly inhibited the expression of miR-122 induced by FXR agonist. 3., by means of reporter gene detection, EMSA and chip experiments, it is confirmed that FXR can enhance the activity of miR-122 promoter and directly promote the expression of miR-122 through directly combining DR2 reaction element from miR-122 promoter -338 to -325. 4. cell proliferation assay showed that inhibition of miR-122 could significantly reduce the inhibitory effect of FXR on the proliferation of HCC cells. 5. FXR could significantly induce the expression of miR-122 and inhibit the expression of IGF-1R and cycling1, and then inhibit the growth of tumor. Conclusion: FXR can directly promote the expression of MI R-122 by inhibiting the expression of MI R-122 and directly inhibiting the growth of tumor by combining DR2 (-338 to -325) in miR-122 promoter region, suggesting that FXR/miR-122 pathway may be a new target for the prevention and treatment of HCC.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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