本文選題：miR-152　切入點：非酒精性脂肪肝病　出處：《浙江大學(xué)》2015年碩士論文

【摘要】：研究背景 非酒精性脂肪肝病(NAFLD)是目前慢性肝臟疾病首要病因,其疾病譜包括單純性脂肪肝、非酒精性脂肪肝炎、以及由其演變的肝硬化甚至肝癌。脂質(zhì)代謝異常、胰島素抵抗、線粒體功能紊亂、炎癥因子、基因遺傳等多種因素參與NAFLD的發(fā)生發(fā)展,然而其確切發(fā)病機制不明。MicroRNAs是真核生物體內(nèi)一類大小約22nt的非編碼小RNA,其成熟體通過與靶基因mRNAs的3'UTR的堿基互補配對結(jié)合,參與基因表達的轉(zhuǎn)錄后調(diào)控。已有研究發(fā)現(xiàn)miRNAs參與脂肪細胞分化、肝臟脂肪酸和膽固醇代謝、胰島素抵抗等過程的調(diào)節(jié),提示miRNAs很有可能參與NAFLD發(fā)生發(fā)展。miR-152是我們前期運用高脂飲食成功誘導(dǎo)大鼠脂肪肝并通過二代測序技術(shù)得到的在肝組織中異常高表達的microRNA.已有研究報道m(xù)iR-152在酒精性脂肪肝中也呈現(xiàn)高表達趨勢,提示我們miR-152參與脂肪肝的發(fā)病過程。近期有研究指出miR-152能直接抑制經(jīng)典WNT信號通路。WNT信號通路在肝臟內(nèi)廣泛表達,其調(diào)控作用包括了脂質(zhì)合成代謝、胰島素抵抗、線粒體功能等過程。那么miR-152的差異表達是否是通過調(diào)控WNT信號通路直接參與了脂肪肝的發(fā)病過程? 目的 發(fā)現(xiàn)miR-152在肝細胞脂質(zhì)代謝中的作用；驗證WNT是miR-152靶基因并進一步探討miR-152抑制WNT信號通路促進肝細胞脂肪變性的分子機制。 方法 首先,利用慢病毒技術(shù),構(gòu)建miR-152過表達慢病毒和miR-152沉默慢病毒,分別轉(zhuǎn)染人正常肝細胞和人肝癌細胞中,得到miR-152過表達組細胞(Lenti-miR-152)、miR-152沉默組細胞(Lenti-152-In)和空載對照組細胞(Lenti-NC),RT-PCR驗證miR-152表達；棕櫚酸和油酸(1mM)干預(yù)Lenti-miR-152、 Lenti-152-In和Lenti-NC組細胞后,采用油紅O染色和甘油三酯測定試劑盒檢測脂質(zhì)合成情況；同時,通過構(gòu)建熒光素酶報告基因質(zhì)粒,結(jié)合生物信息學(xué)分析結(jié)果驗證miR-152靶基因；對Lenti-miR-152、Lenti-152-In和Lenti-NC分別在mRNA水平和蛋白水平檢測WNT信號通路以及下游脂質(zhì)合成關(guān)鍵轉(zhuǎn)錄因子的表達水平,探討miR-152通過抑制WNT信號通路促進肝細胞脂肪變性的分子機制。最后分別檢測Lenti-miR-152、Lenti-152-In和Lenti-NC組細胞ATP含量和線粒體拷貝數(shù),探討miR-152對線粒體功能的調(diào)控作用。 結(jié)果 RT-PCR驗證miR-152表達證實成功構(gòu)建miR-152過表達和miR-152沉默肝細胞。油紅O染色結(jié)果顯示miR-152促進肝細胞脂滴聚積；miR-152過表達組細胞甘油三酯含量高于空載組；miR-152沉默對肝細胞脂肪代謝影響不大。經(jīng)生物信息學(xué)分析發(fā)現(xiàn),Wnt10b是miR-152的靶基因,雙熒光素酶報告基因系統(tǒng)結(jié)果證實miR-152直接結(jié)合于Wnt10b的3'UTR區(qū)；同時,Western blot結(jié)果顯示miR-152能直接抑制Wnt10b的表達。RT-PCR與Western blot顯示miR-152增加GSK-3β磷酸化水平,促進P-Catenin降解,抑制P-Catenin進入肝細胞核內(nèi),上調(diào)脂質(zhì)合成關(guān)鍵轉(zhuǎn)錄因子SREBP-lc和PPARγ的表達。最后,miR-152明顯降低肝細胞內(nèi)ATP含量和線粒體拷貝數(shù)。 結(jié)論 miR-152抑制Wnt10b,促進肝細胞發(fā)生脂肪變性和線粒體損傷,miR-152可能成為NAFLD防治的潛在的靶標。
[Abstract]:Research background
Nonalcoholic fatty liver disease (NAFLD) is currently the leading cause of chronic liver disease, including nonalcoholic fatty liver disease spectrum, nonalcoholic steatohepatitis, as well as the evolution of liver cirrhosis and even hepatocellular carcinoma. The abnormal lipid metabolism, insulin resistance, mitochondrial dysfunction, inflammatory factors, genetic and other factors involved in the occurrence and development of NAFLD however, its exact pathogenesis is unknown.MicroRNAs non small RNA encoding eukaryotic organisms within a class size of about 22nt, the mature body with target gene mRNAs 3'UTR base pairing with transcription gene expression regulation. It has been found that miRNAs involved in adipocyte differentiation, hepatic fatty acid and cholesterol metabolism. Regulation of insulin resistance process, suggesting that miRNAs may be involved in the occurrence and development of NAFLD.MiR-152 is our early use of high fat diet induced fatty liver in rats and The two generation sequencing technology in the liver tissue of abnormal high expression of microRNA. has been reported miR-152 also showed high expression trend in alcoholic fatty liver, suggesting that we miR-152 to participate in the pathogenesis of fatty liver. Recent studies suggest that miR-152 can directly inhibit the classical WNT signaling pathway.WNT signaling pathway is widely expressed in the liver. The regulation including lipid metabolism, insulin resistance, mitochondrial function and process. Then the differences in miR-152 expression is regulated by WNT signaling pathway in the pathogenesis of fatty liver were directly involved in?
objective
We found the role of miR-152 in the lipid metabolism of hepatocytes, and verified that WNT is the target gene of miR-152, and further explore the molecular mechanism of miR-152 inhibiting WNT signaling pathway and promoting hepatic steatosis.
Method
First of all, using lentiviral technology to construct miR-152 lentivirus expressing and miR-152 silencing lentivirus were transfected into human normal liver cells and hepatoma cells, miR-152 cells (Lenti-miR-152), miR-152 group (Lenti-152-In) and silent cell load cells in the control group (Lenti-NC), the expression of RT-PCR miR-152 verification; palmitic acid oleic acid (1mM) and the intervention of Lenti-miR-152, Lenti-152-In and Lenti-NC cells after determination kit for detection of lipid synthesis by oil red O staining and triglyceride; at the same time, through the construction of luciferase report gene plasmid, combined with bioinformatics analysis results show that the target gene of miR-152; on Lenti-miR-152, Lenti-152-In and Lenti-NC respectively at mRNA and protein level detection of the WNT signaling pathway and downstream lipid synthesis of the key transcription factor expression level of miR-152 by inhibiting WNT signal transduction pathway. The molecular mechanism of hepatic steatosis. Finally, we detected the ATP content and the copy number of mitochondria in Lenti-miR-152, Lenti-152-In and Lenti-NC groups, and explored the regulation function of miR-152 on mitochondrial function.
Result
The expression of RT-PCR miR-152 verification confirmed successful construction of miR-152 overexpression and silencing of miR-152 liver cells. Oil red O staining showed that miR-152 promoted the accumulation of lipid droplets in hepatocytes; overexpression of miR-152 cells in triglyceride group was higher than that of empty vector group; effects of miR-152 silencing on hepatic fat metabolism is not large. Bioinformatics analysis shows that Wnt10b is the target gene of miR-152 the dual luciferase reporter assay results confirmed the direct binding of miR-152 to Wnt10b 3'UTR Western blot; at the same time, the results showed that miR-152 can directly inhibit the expression of Wnt10b.RT-PCR and Western blot showed that miR-152 increased GSK-3 phosphorylation level, promote the degradation of P-Catenin, inhibition of P-Catenin in liver cell nucleus, the upregulation of the expression of the key transcription factor SREBP-lc and lipid synthesis PPAR gamma. Finally, miR-152 significantly decreased hepatic cell ATP content and mitochondrial copy number.
conclusion
MiR-152 inhibits Wnt10b and promotes fatty degeneration and mitochondrial damage in hepatocytes. MiR-152 may be a potential target for the prevention and treatment of NAFLD.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.7

【參考文獻】

相關(guān)期刊論文 前4條

1 王強;江渝;;肝X受體的研究進展[J];生理科學(xué)進展;2009年02期

2 Ashley M Lakner;Herbert L Bonkovsky;Laura W Schrum;;microRNAs: Fad or future of liver disease[J];World Journal of Gastroenterology;2011年20期

3 ;Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays[J];World Journal of Gastroenterology;2012年15期

4 Ludovico Abenavoli;Mario Masarone;Valentina Peta;Natasa Milic;Nazarii Kobyliak;Samir Rouabhia;Marcello Persico;;Insulin resistance and liver steatosis in chronic hepatitis C infection genotype 3[J];World Journal of Gastroenterology;2014年41期

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本文編號：1659335