miR-152抑制Wnt10b影响人正常肝细胞和人肝癌细胞脂肪变性的分子机制研究

发布时间：2018-03-24 17:38

本文选题：miR-152　切入点：非酒精性脂肪肝病　出处：《浙江大学》2015年硕士论文

【摘要】：研究背景非酒精性脂肪肝病(NAFLD)是目前慢性肝脏疾病首要病因,其疾病谱包括单纯性脂肪肝、非酒精性脂肪肝炎、以及由其演变的肝硬化甚至肝癌。脂质代谢异常、胰岛素抵抗、线粒体功能紊乱、炎症因子、基因遗传等多种因素参与NAFLD的发生发展,然而其确切发病机制不明。MicroRNAs是真核生物体内一类大小约22nt的非编码小RNA,其成熟体通过与靶基因mRNAs的3'UTR的碱基互补配对结合,参与基因表达的转录后调控。已有研究发现miRNAs参与脂肪细胞分化、肝脏脂肪酸和胆固醇代谢、胰岛素抵抗等过程的调节,提示miRNAs很有可能参与NAFLD发生发展。miR-152是我们前期运用高脂饮食成功诱导大鼠脂肪肝并通过二代测序技术得到的在肝组织中异常高表达的microRNA.已有研究报道miR-152在酒精性脂肪肝中也呈现高表达趋势,提示我们miR-152参与脂肪肝的发病过程。近期有研究指出miR-152能直接抑制经典WNT信号通路。WNT信号通路在肝脏内广泛表达,其调控作用包括了脂质合成代谢、胰岛素抵抗、线粒体功能等过程。那么miR-152的差异表达是否是通过调控WNT信号通路直接参与了脂肪肝的发病过程? 目的发现miR-152在肝细胞脂质代谢中的作用；验证WNT是miR-152靶基因并进一步探讨miR-152抑制WNT信号通路促进肝细胞脂肪变性的分子机制。方法首先,利用慢病毒技术,构建miR-152过表达慢病毒和miR-152沉默慢病毒,分别转染人正常肝细胞和人肝癌细胞中,得到miR-152过表达组细胞(Lenti-miR-152)、miR-152沉默组细胞(Lenti-152-In)和空载对照组细胞(Lenti-NC),RT-PCR验证miR-152表达；棕榈酸和油酸(1mM)干预Lenti-miR-152、 Lenti-152-In和Lenti-NC组细胞后,采用油红O染色和甘油三酯测定试剂盒检测脂质合成情况；同时,通过构建荧光素酶报告基因质粒,结合生物信息学分析结果验证miR-152靶基因；对Lenti-miR-152、Lenti-152-In和Lenti-NC分别在mRNA水平和蛋白水平检测WNT信号通路以及下游脂质合成关键转录因子的表达水平,探讨miR-152通过抑制WNT信号通路促进肝细胞脂肪变性的分子机制。最后分别检测Lenti-miR-152、Lenti-152-In和Lenti-NC组细胞ATP含量和线粒体拷贝数,探讨miR-152对线粒体功能的调控作用。结果 RT-PCR验证miR-152表达证实成功构建miR-152过表达和miR-152沉默肝细胞。油红O染色结果显示miR-152促进肝细胞脂滴聚积；miR-152过表达组细胞甘油三酯含量高于空载组；miR-152沉默对肝细胞脂肪代谢影响不大。经生物信息学分析发现,Wnt10b是miR-152的靶基因,双荧光素酶报告基因系统结果证实miR-152直接结合于Wnt10b的3'UTR区；同时,Western blot结果显示miR-152能直接抑制Wnt10b的表达。RT-PCR与Western blot显示miR-152增加GSK-3β磷酸化水平,促进P-Catenin降解,抑制P-Catenin进入肝细胞核内,上调脂质合成关键转录因子SREBP-lc和PPARγ的表达。最后,miR-152明显降低肝细胞内ATP含量和线粒体拷贝数。结论 miR-152抑制Wnt10b,促进肝细胞发生脂肪变性和线粒体损伤,miR-152可能成为NAFLD防治的潜在的靶标。
[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.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

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