本文關(guān)鍵詞：FXR上調(diào)miR-122的分子機(jī)制及在抗HCC中的作用研究　出處：《第三軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 微小RNA-122 法尼醇X受體 肝細(xì)胞癌 細(xì)胞生長 基因調(diào)控

【摘要】：肝細(xì)胞癌(hepatocellular carcinoma,HCC)是全球范圍內(nèi)最普遍的惡性腫瘤之一。其誘發(fā)因素有多個(gè),包括病毒感染、原癌基因激活、抑癌基因表達(dá)異常等。目前關(guān)于肝細(xì)胞癌的發(fā)病機(jī)制有待深入研究,而進(jìn)一步闡明與肝細(xì)胞癌發(fā)病過程中密切相關(guān)的基因及其相互作用無疑將有助于加深對(duì)HCC發(fā)病機(jī)制的理解以及發(fā)現(xiàn)靶向治療HCC的新靶點(diǎn)。近年來,與HCC密切相關(guān)的法尼醇X受體(farnesoid X receptor,FXR)和microRNA日益受到重視。FXR屬于核受體超家族成員,主要在肝臟、小腸、腎臟和腎上腺中表達(dá),密切參與膽汁酸代謝、糖脂代謝,進(jìn)而調(diào)節(jié)肝臟再生、肝纖維化等。大量的研究發(fā)現(xiàn),FXR與HCC的發(fā)生發(fā)展密切相關(guān)。FXR作為一種多功能的轉(zhuǎn)錄因子,可調(diào)節(jié)多種腫瘤相關(guān)靶基因的表達(dá)和功能,進(jìn)而發(fā)揮重要的抗HCC作用,但FXR的抗HCC機(jī)制尚不十分清楚,有待進(jìn)一步的深入研究。mi R-122是一種在肝臟中特異性高表達(dá)的microRNA。在正常生理狀態(tài)下,miR-122可以調(diào)控肝臟的細(xì)胞發(fā)育和細(xì)胞代謝,誘導(dǎo)細(xì)胞分化,并參與肝細(xì)胞應(yīng)激應(yīng)答等多種生命活動(dòng);而在病理狀態(tài)下,mi R-122與HCC密切相關(guān)。研究發(fā)現(xiàn),在HCC發(fā)生、發(fā)展過程中,mi R-122發(fā)揮著類似抑癌基因的作用,其主要通過抑制腫瘤相關(guān)基因的表達(dá)而發(fā)揮其抗腫瘤的功能。鑒于miR-122在抗HCC方面的重要作用,那么設(shè)法上調(diào)miR-122的表達(dá),無疑將有助于肝癌的防治。目前的研究多集中在miR-122通過調(diào)節(jié)靶基因發(fā)揮抗腫瘤作用的機(jī)制方面,而關(guān)于miR-122自身的表達(dá)調(diào)控少有報(bào)道,最近的研究發(fā)現(xiàn),一些轉(zhuǎn)錄因子(HNF4α、C/EBPα)可以促進(jìn)miR-122的表達(dá),但關(guān)于核受體FXR是否上調(diào)miR-122的表達(dá),并通過此通路發(fā)揮抗HCC作用,目前未見報(bào)道。因此,結(jié)合相關(guān)文獻(xiàn)和我們初步的研究結(jié)果,本課題率先鑒定了miR-122是FXR的一個(gè)新靶基因,揭示FXR促進(jìn)mi R-122表達(dá)的具體分子機(jī)制;進(jìn)而闡明“FXR-miR-122通路”在抗HCC作用中的新機(jī)制,為進(jìn)一步深入而全面地揭示FXR的抗肝細(xì)胞癌機(jī)制以及miR-122自身的表達(dá)調(diào)控積累新的理論資料,為探索以“FXR-miR-122通路”為靶標(biāo)的抗腫瘤治療提供新的科學(xué)依據(jù)。目的:本研究從體內(nèi)外探討FXR上調(diào)miR-122的分子機(jī)制及“FXR-miR-122通路在抗HCC中的重要作用方法:1.收集20例hcc病人的腫瘤組織及癌旁組織,和7種hcc細(xì)胞系,經(jīng)real-timepcr和westernblot實(shí)驗(yàn),檢測fxr與mir-122的表達(dá),并通過相關(guān)性統(tǒng)計(jì),分析fxr與mir-122表達(dá)的相關(guān)性。2.不同濃度fxr激動(dòng)劑處理hcc細(xì)胞,以及采用rna干擾(rnainterference,rnai),通過real-timepcr和westernblot實(shí)驗(yàn)檢測mir-122以及mir-122靶基因igf-1r和cycling1的表達(dá),研究fxr對(duì)mir-122表達(dá)的影響。3.應(yīng)用生物信息學(xué)分析,預(yù)測人mir-122的5'調(diào)控區(qū)fxr的可能結(jié)合位點(diǎn),構(gòu)建含可能結(jié)合位點(diǎn)報(bào)告基因質(zhì)粒,轉(zhuǎn)染細(xì)胞經(jīng)luc報(bào)告基因檢測實(shí)驗(yàn),初步確定fxr可能結(jié)合位點(diǎn)的所在區(qū)域。進(jìn)一步采用凝膠遷移率實(shí)驗(yàn)(emsa)和染色質(zhì)免疫沉淀實(shí)驗(yàn)(chip),探討fxr調(diào)節(jié)mir-122表達(dá)的具體分子機(jī)制。4.hcc細(xì)胞轉(zhuǎn)染antagomir-122抑制mir-122功能后,采用cck-8法檢測細(xì)胞的增殖情況。5.hcc細(xì)胞接種裸鼠成瘤后,以fxr的配體灌胃處理小鼠,一定時(shí)間后,在肝癌荷瘤鼠上觀察腫瘤的生長情況,然后處死小鼠,切取腫瘤組織,real-timepcr和westernblot實(shí)驗(yàn)檢測mir-122,igf-1r和cycling1的表達(dá)情況。免疫組化測定增殖標(biāo)志物ki67的表達(dá),在體內(nèi)探討“fxr-mir-122通路”的抗hcc作用。結(jié)果:1.fxr和mir-122在20例hcc病人腫瘤組織中的表達(dá)均顯著低于癌旁組織,且相關(guān)性分析顯示,fxr與mir-122的表達(dá)呈顯著正相關(guān);同樣,相對(duì)于正常人肝細(xì)胞系l02,7種hcc細(xì)胞系中fxr和mir-122的表達(dá)也呈顯著正相關(guān)。2.fxr激動(dòng)劑處理hcc細(xì)胞活化fxr后,可顯著增加mir-122的表達(dá),并減弱其靶基因和igf-1r和cycling1的表達(dá),同時(shí),hcc細(xì)胞轉(zhuǎn)染fxrsirna抑制fxr后,顯著抑制了fxr激動(dòng)劑誘導(dǎo)mir-122的表達(dá)。3.通過報(bào)告基因檢測、emsa和chip實(shí)驗(yàn)證實(shí)了,fxr可通過直接結(jié)合于mir-122啟動(dòng)子區(qū)-338到-325的dr2反應(yīng)元件,增強(qiáng)mir-122的啟動(dòng)子活性,進(jìn)而促進(jìn)mir-122的表達(dá)。4.細(xì)胞增殖檢測實(shí)驗(yàn)顯示,抑制mir-122后可顯著減弱fxr對(duì)hcc細(xì)胞增殖的抑制作用。5.肝癌荷瘤鼠模型上,fxr可顯著誘導(dǎo)mir-122的表達(dá),并抑制其靶基igf-1r和cycling1的表達(dá),進(jìn)而抑制腫瘤生長。結(jié)論:FXR通過直接結(jié)合于miR-122啟動(dòng)子區(qū)的DR2(-338到-325)反應(yīng)元件,促進(jìn)mi R-122的表達(dá),進(jìn)而抑制腫瘤的生長,提示FXR/miR-122通路可能是防治HCC的新靶點(diǎn)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)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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