MiR-484靶向HIPK1促进肝纤维化发生发展的研究

发布时间：2018-03-09 23:29

本文选题：肝纤维化　切入点：miR-484　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景肝纤维化是指当肝脏受到各类有害因素的损伤时,细胞外基质(extracellular matrix,ECM)沉积而导致肝脏正常结构和功能发生改变的病理过程。多种慢性肝病均可经此过程并最终进展为预后极差的肝硬化和肝癌。所以,针对肝纤维化的早期诊断和治疗成为研究的重中之重。而目前为止,肝穿刺仍为诊断肝纤维化的金标准,但因其有创性而得不到广泛、及时实施,在一定程度上耽误了患者的早期诊治。虽已有肝纤维化无创性早期诊断的方法,但均不十分理想,新的更有效的手段亟待被发现,这就有必要对肝纤维化发生发展的分子机制行进一步深入研究。miRNA(micro RNA)是一段大小约20~25个核苷酸序列的小分子片段,其在真核细胞中广泛存在,可通过负向调控其靶基因参与多种疾病的发生和进展,且在内分泌、心血管和肿瘤等多种研究领域受到广泛关注。目前亦有多种研究表明miRNA与肝纤维化发生密切相关,其可通过影响肝星状细胞(hepatic stellated cell,HSC)的活化、增殖、凋亡、迁移等细胞学功能,在肝纤维发生及进展过程中起促进或抑制作用。本课题组多年来一直从事miRNAs与肝纤维化发生发展的分子机制研究,前期工作发现:miR-484在DMN诱导的大鼠肝纤维化模型中呈显著性差异表达,且相关文章已经发表。本研究为进一步探讨miR-484是否对HSC细胞功能学产生影响,通过提取原代HSC,检测到miR-484在HSC自然活化过程中表达呈上升趋势。并通过生物信息学和双荧光素酶报告基因实验筛选并验证得知HIPK1(homeodomain interacting protein kinase 1,同源结构域交互蛋白激酶1)为miR-484的靶基因。已有研究表明,HIPK1可调控细胞增殖、分化、凋亡等多种生物过程。本研究中同样发现miR-484可通过靶向HIPK1促进HSC活化并抑制其凋亡,且在此过程中伴有Wnt/β-catenin信号通路的激活,若在疾病早期对miR-484的表达进行抑制,或许可阻遏肝纤维化进程。研究目的本研究在前期工作的基础上,旨在进一步探讨miR-484参与肝纤维化形成的具体分子机制。共包括三个部分:(一)检测miR-484在大鼠原代HSC自然活化过程中的表达变化;(二)miR-484靶基因的筛选及验证;(三)miR-484对HSC活化和凋亡的信号通路机制研究。研究方法一、检测miR-484在大鼠原代HSC自然活化过程中的表达变化(一)提取大鼠原代hsc,以自发荧光实验鉴定所提细胞的纯度;将培养2天内的细胞作为静止态,培养至14天的细胞作为完全活化态,并用免疫荧光实验鉴定细胞状态;(二)qrt-pcr检测原代细胞不同时期mir-484的表达水平。二、mir-484靶基因的筛选及验证(一)以mir-484为关键词,在microrna.org、mirbase、targetscan等网站进行了靶基因预测,并对感兴趣的靶基因进行geneontology(go)分析,发掘其可能参与的功能;(二)双荧光素酶报告基因验证mir-484与hipk1之间是否存在直接靶向关系。(三)利用hsc-t6细胞株进行转染实验,将mir-484inhibitor/mimics及其阴性对照转染入细胞48h后,提取细胞总蛋白。利用westernblot技术检测hipk1是否被mir-484负向调控;三、mir-484对hsc活化和凋亡的信号通路机制研究(一)在hsc-t6细胞株中转染mir-484inhibitor后,qrt-pcr和westernblot检测不同分组之间肝纤维化相关指标α-平滑肌肌动蛋白(α-smoothmuscleactin,α-sma)和?型胶原(type?collagen,col?)表达变化;(二)在hsc-t6细胞株中转染mir-484inhibitor后,利用qrt-pcr和westernblot技术检测wnt-3a、wnt-5a、β-catenin表达是否发生相应改变,以检测mir-484对hsc细胞功能的调控,是否通过wnt/β-catenin信号通路实现;(三)annexinv-fitc/pi双染法用以检测不同分组之间细胞凋亡的差异。四、统计学方法:计量资料用均数±标准差表示;两组数据之间比较采用t检验;统计软件采用spss21.0;统计学显著性定义为p0.05。研究结果一、mir-484在大鼠原代hsc自然活化过程中的表达变化(一)自发荧光实验鉴定结果提示所提原代hsc的纯度可达90%以上,2天内静止态的细胞呈椭圆形,脂质含量丰富,在328nm波长紫外光照射下,可发射出黄绿色荧光;培养至14天达到完全活化状态,细胞延展生长,呈星状;免疫荧光实验显示活化态细胞中α-sma表达量明显升高;(二)qrt-pcr结果提示,相对于静止态,活化态hsc中mir-484表达呈显著性升高(p0.05)。二、mir-484靶基因的筛选与验证(一)生物信息学分析结果提示,hipk1与mir-484在3’utr区域存在两个结合位点;(二)双荧光素酶报告基因验证miR-484与HIPK1之间确实存在靶向关系;(三)Western Blot结果表明,在HSC-T6细胞株中转染miR-484 inhibitor后,HIPK1在蛋白水平表达明显上调;而转染miR-484 mimics后,HIPK1在蛋白水平表达明显下调。三、miR-484对HSC活化和凋亡的信号通路机制研究(一)相对于阴性对照组,转染miR-484 inhibitor后48h,HSC-T6细胞株中α-SMA和col?在mRNA和蛋白水平表达均明显下降(P0.05);而空白对照组与阴性对照组表达则无明显差异(P0.05);(二)转染miR-484 inhibitor后,Wnt/β-catenin信号通路相关分子Wnt-3a、Wnt-5a、β-catenin表达下降(P0.05);(三)人为下调miR-484后,流式细胞仪检测得HSC-T6细胞凋亡率明显升高(P0.05)。结论一、miR-484在原代HSC自然活化过程中表达升高;二、HIPK1是miR-484的直接靶基因;三、miR-484靶向HIPK1促进HSC活化并抑制其凋亡,使得细胞外基质合成增多,促进肝纤维化形成及进展;四、miR-484靶向HIPK1对HSC细胞功能的调控,可能是通过Wnt/β-catenin信号通路实现的。
[Abstract]:The research background of liver fibrosis is when the liver caused by various harmful factors of injury, extracellular matrix (extracellular matrix ECM) deposition caused the pathological process of the normal structure and function of liver changes. Chronic liver diseases may pass through this process and ultimately progress to prognosis of cirrhosis and liver cancer. So, according to becometop Study on the early diagnosis and treatment of liver fibrosis. Liver biopsy is still so far, the gold standard for diagnosis of liver fibrosis, but because it is invasive and not widely implemented in a timely manner, to a certain extent delayed the early diagnosis and treatment of patients. Although liver fibrosis non-invasive methods for early diagnosis. But they are not very ideal, a new and more effective instrument to be found, it is necessary for the development of liver fibrosis. The molecular mechanism for the further research on.MiRNA (micro RNA) is a size of about 20~25 Small molecular fragment nucleotide sequences, which exists widely in eukaryotic cells, occurrence and progress can be involved in a variety of diseases to regulate the target gene by negative, and in many research fields of endocrine, cardiovascular and cancer received extensive attention. At present, there are many studies show that miRNA and liver fibrosis is closely related to the the effect of hepatic stellate cells (hepatic stellated cell, HSC) activation, proliferation, apoptosis, migration and other cellular functions, in the process and progress of liver fibrosis in promoting or inhibiting effect. Molecular mechanism of the group has been engaged in miRNAs and liver fibrosis occurrence and development of this topic, previous work found that miR-484 was significantly the difference in rat liver fibrosis model induced by DMN expression, and related articles have been published. This study to further investigate the effect of miR-484 on HSC cell function, provided by The primary HSC, detected miR-484 activation increased expression in the process of HSC. And through bioinformatics and dual luciferase reporter assay to screen and verify that the HIPK1 (homeodomain interacting protein kinase 1 homeodomain interacting protein kinase 1) is the target gene of miR-484. Studies have shown that HIPK1 can regulate cell the proliferation, differentiation, apoptosis and other biological processes. This study also found that miR-484 can promote HSC activation and inhibit its apoptosis by targeting HIPK1 activation, and in the process with Wnt/ beta -catenin signaling pathway, if early in the disease on the expression of miR-484 was inhibited, or permit repression of hepatic fibrosis. This study on the basis of previous work, to further investigate the molecular mechanism of miR-484 involved in the formation of liver fibrosis. Consists of three parts: (a) the detection of miR-484 in primary rat HSC natural expression changes in the process; (two) screening and identification of target genes of miR-484; (three) research on the mechanism of HSC signaling pathway activation and apoptosis of miR-484. The method of detection of miR-484 activation, natural expression change in the process of primary rat HSC (1) extract in primary rat HSC, the experimental identification of the autofluorescence of cell purity; the culture within 2 days of the cell as a stationary state, after 14 days of culture the cells as a fully activated state, and cells were identified by immunofluorescence; (two) the expression of qRT-PCR was detected in different periods of mir-484 primary cells. Two, screening and verification mir-484 target gene (1) with mir-484 as key words in microrna.org, miRBase, targetscan and other sites of target genes, and target genes of interest were geneontology (go) analysis, explore the possible involvement of function; (two) dual luciferase substrate Because mir-484 and hipk1 verify whether there is a direct relationship between the target. (three) using HSC-T6 cell transfection experiments, mir-484inhibitor/mimics and negative control were transfected into 48h cells after extraction of total cellular protein. Westernblot was used to detect whether hipk1 is mir-484 negative regulation; three, study on the mechanism of HSC signaling pathway activation and apoptosis mir-484 (a) in HSC-T6 cells after transfection of mir-484inhibitor, qRT-PCR and Westernblot detection between the different groups related to liver fibrosis of alpha smooth muscle actin (alpha -smoothmuscleactin, alpha -sma) and collagen (type?? collagen, col?) expression; (two) in HSC-T6 cells after transfection of mir-484inhibitor, detected by Wnt-3a, qRT-PCR and Westernblot Wnt-5a, beta -catenin expression is changed, in order to detect mir-484 on HSC cell function regulation, whether through wnt/ beta -cate NIN signal pathway; (three) annexinv-fitc/pi double staining method used to detect the differences between the different groups of apoptosis. Four, statistical methods: measurement data with standard deviation; between the two groups of data were compared using t test using spss21.0 statistical software;; statistical significance is defined as the results of p0.05., mir-484 expression change in the process of primary rat HSC (1) autofluorescence experiment results suggest that the primary identification of the purity of HSC can reach more than 90%, within 2 days of resting cells were oval, lipid content at the wavelength of 328nm under UV light can emit yellow green fluorescence; cultured for 14 days to complete the activation of cell spreading, growth, are star shaped; immunofluorescence experiments showed increased expression of activation of alpha -sma cells; (two) the results of qRT-PCR showed that, relative to a stationary state, the activation of mir-484 expression in HSC state Was significantly increased (P0.05. Two), screening and identification of mir-484 target genes (1) bioinformatics analysis showed that hipk1 and mir-484 in the presence of the 3 'UTR region of two binding sites; (two) there is relationship between the targeted dual luciferase reporter gene verification of miR-484 and HIPK1; (three) Western Blot the results showed that in HSC-T6 cells transfected with miR-484 inhibitor, the expression of HIPK1 is upregulated at the protein level; and miR-484 mimics after transfection, the expression of HIPK1 at protein level was significantly reduced. Three. Study on the mechanism of HSC signaling pathway activation and apoptosis of miR-484 (a) compared with the negative control group, transfected with miR-484 inhibitor 48h, alpha -SMA Col and HSC-T6 cell lines? Decreased expression at mRNA and protein level (P0.05); and the blank control group and negative control group was no significant difference (P0.05); (two) miR-484 inhibitor after transfection, Wnt/ beta -catenin signal Pathway related molecules Wnt-3a, Wnt-5a, -catenin beta expression decreased (P0.05); (three) artificially lowered after miR-484, flow cytometry was used to detect HSC-T6 cell apoptosis rate increased significantly (P0.05). Conclusion the increased expression of miR-484 in a process of activation of primary HSC; two, HIPK1 is a direct target gene of miR-484; three, miR-484 targeting HIPK1 to promote HSC activation and apoptosis, the increased synthesis of extracellular matrix, and promote the progress of hepatic fibrosis; four, miR-484 targeting HIPK1 on the regulation of HSC cell function, may be achieved through Wnt/ beta -catenin signaling pathway.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2

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