MiR-145靶向ZEB2抑制肝星状细胞活化增殖

发布时间：2018-05-12 09:20

本文选题：MiR-145 + 肝纤维化　；参考：《安徽医科大学》2017年硕士论文

【摘要】：肝纤维化是指一种或多种慢性致病因子致使肝细胞弥漫性损伤,进而使肝星状细胞(hepatic stellate cell,HSC)活化增殖并导致细胞外基质(extracellular matrix,ECM)大量沉积的一种病理过程。HSC的活化增殖是肝纤维化发生发展的中心环节,抑制HSC的活化增殖可减缓或阻止肝纤维化的发生。研究发现在机体多种组织或器官的纤维化疾病进程中,都发现有microRNA(miRNA)的调控作用。Mi RNA能调控多种促肝纤维化或抑肝纤维化相关因子,反过来,肝纤维化的发生又能使多种mi RNA表达发生变化。近年也有研究发现miR-145与多种纤维化的发生发展密切相关,但是miR-145在肝纤维化以及肝星状细胞活化过程中的作用鲜有报道,这需要进一步的研究。本实验主要观察miR-145在肝纤维化发生过程中和HSC活化过程中的表达变化,并进一步探讨miR-145对转化生长因子β1(transforming growth factor-β1,TGF-β1)诱导的HSC活化增殖的影响及其可能的作用分子机制。研究内容主要概况如下:(1)ZEB2和miR-145在肝纤维化过程中的表达体内我们采用四氯化碳(carbon tetrachloride,CCl4)皮下注射4周建立小鼠肝纤维化模型,取肝组织进行HE和Masson染色,同时检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和I型胶原(Collagen type1,Colα1)的蛋白表达鉴定模型构建成功。通过qPCR技术检测肝组织和原代HSC中ZEB2和miR-145的表达,同时采用免疫荧光双染技术观察ZEB2是否表达于肝星状细胞中。体外我们采用不同浓度TGF-β1分别对大鼠肝星状细胞HSC-T6和人肝星状细胞lx-2进行刺激,24h后检测ZEB2和mir-145的表达变化。结果发现在ccl4诱导的肝纤维化小鼠的肝组织和原代HSC中以及体外TGF-β1处理的HSC中,mir-145的表达均是降低的,而ZEB2表达水平升高。ZEB2和α-sma双重染色显示,ZEB2主要表达于α-sma阳性的细胞即HSC中。(2)mir-145对TGF-β1诱导的HSC-T6细胞活化增殖的影响在HSC-T6中,采用mir-145mimics和inhibitor建立过表达和沉默mir-145细胞模型,同时采用TGF-β1(10ng/ml,24h)进行刺激,westernblot检测处理后的HSC-T6细胞中colα1和α-sma的蛋白表达,流式细胞术检测细胞周期情况。结果显示:过表达mir-145能抑制colα1和α-sma的蛋白表达,同时使TGF-β1引起的HSC-T6增殖明显减少;沉默mir-145,使TGF-β1诱导的HSC-T6增殖增多,同时colα1和α-sma的蛋白表达水平进一步升高。(3)ZEB2对TGF-β1诱导的HSC-T6细胞活化增殖的影响在HSC-T6细胞中,采用ZEB2小干扰rna(ZEB2sirna)建立沉默ZEB2细胞模型,同时采用TGF-β1(10ng/ml,24h)进行刺激,westernblot检测处理后colα1和α-sma蛋白表达,流式细胞术检测细胞周期情况。结果显示:沉默ZEB2能抑制colα1和α-sma的蛋白表达,同时使TGF-β1诱导的HSC-T6增殖明显减少。(4)mir-145与ZEB2之间靶向关系的研究使用生物信息学软件分析了解到mir-145同ZEB2之间存在可能的靶向关系。在HSC-T6细胞中,我们采用mir-145mimics和inhibitor建立过表达和沉默细胞模型,westernblot检测ZEB2mrna和蛋白表达情况。结果显示:过表达mir-145能抑制ZEB2mrna和蛋白表达,而沉默mir-145能升高ZEB2mrna和蛋白表达。进一步,本课题组采用双荧光素(萤火虫和海肾)酶报告基因实验确认两者的靶向关系,发现共转染mir-145mimics和ZEB2的3’端非编码区(3’untranslatedregion,3’-UTR)质粒能抑制HSC-T6细胞中ZEB2 3’-UTR的相对荧光值表达。(5)miR-145和ZEB2对Wnt/β-catenin信号通路的影响在HSC-T6中,采用miR-145 mimics、inhibitor和ZEB2 siRNA进行转染以后,采用Western blot技术检测β-catenin及其下游靶蛋白cyclinD1和c-Myc的表达情况。结果显示:过表达miR-145和沉默ZEB2能抑制β-catenin及其相关靶蛋白的表达,而沉默miR-145则能促进β-catenin及其相关靶蛋白的表达。上述结果提示,miR-145可能通过靶向ZEB2来负调控Wnt/β-catenin信号通路进而影响HSC-T6细胞的活化增殖。
[Abstract]:Liver fibrosis refers to one or more chronic pathogenic factors that cause diffuse damage to the liver cells, and then the hepatic stellate cell (HSC) is activated and proliferated and the extracellular matrix (extracellular matrix, ECM) is deposited in a large number of pathological processes. The activation and proliferation of.HSC is the central link in the development of liver fibrosis, and the inhibition of HSC is the central part of the liver fibrosis. It is found that in the process of fibrosis of various tissues or organs of the body, the regulatory role of microRNA (miRNA),.Mi RNA, can regulate a variety of liver fibrosis or liver fibrosis related factors. In turn, the occurrence of liver fibrosis can also cause a variety of MI RNA expression. Recent studies have also found that miR-145 is closely related to the development of multiple fibrosis, but the role of miR-145 in liver fibrosis and the activation of hepatic stellate cells is rarely reported. This need further study. This experiment mainly observed the expression of miR-145 during the activation of Cheng Zhonghe HSC in liver fibrosis, and the results were also observed. One step is to investigate the effect of miR-145 on the activation and proliferation of HSC induced by transforming growth factor beta 1 (transforming growth factor- beta 1, TGF- beta 1) and its possible molecular mechanism. The main contents are as follows: (1) we use carbon tetrachloride (carbon tetrachloride, CCl4) subcutaneous injection for the expression of ZEB2 and miR-145 in the process of liver fibrosis. The liver fibrosis model of mice was established for 4 weeks. The liver tissue was stained with HE and Masson, and the protein expression identification model of alpha smooth muscle actin (alpha -smooth muscle actin, alpha -SMA) and I collagen (Collagen Type1, Col a 1) was successfully constructed. The immunofluorescence double staining technique was used to observe whether ZEB2 was expressed in hepatic stellate cells. In vitro, different concentrations of TGF- beta 1 were used to stimulate the rat hepatic stellate cells HSC-T6 and human hepatic stellate cells lx-2. After 24h, the expression of ZEB2 and miR-145 was detected. The results were found in the liver and primary HSC induced by CCl4 in mice. And in the HSC treated with TGF- beta 1 in vitro, the expression of miR-145 was reduced, while ZEB2 expression level increased by.ZEB2 and alpha -sma double staining showed that ZEB2 was mainly expressed in alpha -sma positive cells, HSC. (2) miR-145 has an effect on the activation and proliferation of HSC-T6 cells induced by TGF- beta 1. The silent miR-145 cell model was stimulated by TGF- beta 1 (10ng/ml, 24h), and Westernblot was used to detect the protein expression of col alpha 1 and alpha -sma in the HSC-T6 cells treated with Westernblot. Flow cytometry was used to detect the cell cycle. The results showed that overexpression of miR-145 could inhibit the expression of col a 1 and alpha -sma, and the proliferation of TGF- beta 1 was significantly reduced. MiR-145, silencing miR-145, increased the proliferation of HSC-T6 induced by TGF- beta, while the protein expression level of col alpha 1 and alpha -sma increased further. (3) the effect of ZEB2 on the activation and proliferation of HSC-T6 cells induced by TGF- beta 1 in HSC-T6 cells, using ZEB2 small interference RNA (ZEB2sirna) to establish a silent cell model, and stimulated by beta 1. The expression of col alpha 1 and alpha -sma protein was detected by Westernblot, and cell cycle was detected by flow cytometry. The results showed that silent ZEB2 could inhibit the protein expression of col alpha 1 and alpha -sma, and the proliferation of HSC-T6 induced by TGF- beta 1 decreased significantly. (4) the study of the relationship between miR-145 and ZEB2 using the bioinformatics software analysis and understanding of mir-14 There is a possible targeting relationship between 5 and ZEB2. In HSC-T6 cells, we use mir-145mimics and inhibitor to establish the over expression and silencing cell model, and Westernblot to detect the expression of ZEB2mrna and protein. The results show that overexpression of miR-145 can inhibit the expression of ZEB2mrna and protein, while silence miR-145 can increase the expression of ZEB2mrna and protein. Step, we identified the target relationship between the two fluorescein (fluorescein and sea kidney) enzyme reporter gene experiment, and found that the 3 'terminal non coding region (3' untranslatedregion, 3 '-UTR) plasmids of CO transfected mir-145mimics and ZEB2 could inhibit the relative fluorescence value expression of ZEB2 3' -UTR in HSC-T6 cells. (5) miR-145 and ZEB2 pairs Wnt/ beta -catenin letter After transfection in HSC-T6, miR-145 mimics, inhibitor and ZEB2 siRNA were used to detect the expression of cyclinD1 and c-Myc of beta -catenin and its downstream target protein by Western blot technique. The results showed that the expression of beta and related target proteins could be inhibited by overexpression of miR-145 and silencing. It can promote the expression of beta -catenin and its related target proteins. These results suggest that miR-145 may negatively regulate the Wnt/ beta -catenin signaling pathway by targeting ZEB2 and then affect the activation and proliferation of HSC-T6 cells.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2

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