EZH2 Plays A Role In Liver Fibrosis And Its Relevant Mechanism

Fibrosis of the liver occurs as a common pathological response to chronic liver disease, and is characterized by increased and altered deposition of extracellular matrix(ECM) components. Activation of hepatic stellate cells(HSCs) is now known as a critical step involved in the development and maintenance of liver fibrosis. Therefore, inhibition of HSC activation and its related subsequent events, such as increased production of ECM components and enhanced proliferation, are crucial goals for intervention in the hepatic fibrogenesis cascade. To explore the cellular and molecular mechanisms of liver fibrosis, we choose HSC as a target for the pharmacological, molecular, and other novel therapeutics for hepatic fibrosis. Investigations suggest that histone methylation plays an important role in the process of HSC activation and proliferation. To further investigate the mechanism of histone methylation in liver fibrosis, we detecte histone methylation-associated protein in vitro and in vivo, and the influence on HSCs proliferation and activation, and the mechanisms was discussed. The main contents are divided into four sections, as follows: 1. Expressions of EZH2 and Dkk1 in CCl4-induced rat liver fibrosisLiver fibrosis was generated by 12-weeks treatment of adult male Sprague-Dawley(200-220g) rats with CCL4(CCl4/olive oil, 1:1(vol/vol) per kg body weight by intraperitoneal injection twice weekly) as previously described. After 12-Weeks, the liver tissues were used for hematoxylin and eosin(H&E) and Masson staining. The expression of EZH2, Dkk1 and α-SMA in rat liver tissues was measured by Immunohistochemistry, Real-time PCR and Western blotting respectively. Experimental results showed that the expression of EZH2 was up-regulated and the expression of Dkk1 was down-regulated in the liver fibrogenesis. 2. Expressions of EZH2 and Dkk1 in HSCs treated by TGF-β1Rat hepatic stellate cell line HSC-T6 was studied, and treated by 10ng/ml TGF-b1 for 24 h and 48 h, then harvested. The expression of EZH2, Dkk1 and α-SMA in TGF-β1-induced HSC-T6 cells by Real-time PCR and Western Blot. Experimental results showed that the expression of EZH2 was up-regulated and the expression of Dkk1 was down-regulated in TGF-b1-induced activated HSC-T6 cells. 3. Inhibition of EZH2 prevents TGF-β1-mediated HSCs proliferation and activation1) Rat hepatic stellate cell line HSC-T6 was studied, and were divided into normal group, model group, DZNep 6h, 12 h, 24 h and 48 h groups. Then cell viability was assessed by MTT. Experimental result showed that DZNep suppressed cell growth of HSC-T6 cells in dose-dependent manner. Rat hepatic stellate cell line HSC-T6 was studied, and were divided into normal group, model group, negative control group and EZH2-siRNA group. Experimental result showed that si-EZH2 suppressed cell growth of HSC-T6 cells. These suggested that DZNep and si-EZH2 can suppress the proliferation of HSC-T6 cells.2) Rat hepatic stellate cell line HSC-T6 was studied, when treated with TGF-b1 for 30 min, 1ng/ml DZNep was added for 24 h. The expression of EZH2 and α-SMA were detected by Real-time PCR and Western Blot. Experimental results showed that DZNep suppressed the activation of HSC-T6 cells. According to the nucleotide sequence of EZH2, small interfering RNA was designed and synthesized, and transfected into HSC-T6 cells by LipofectamineTM 2000. The expression of EZH2 and α-SMA were detected by Real-time PCR and Western Blot. Experimental results showed that the activation of TGF-β1-induced HSC-T6 cells was suppressed by siEZH2. These suggested that inhibiting EZH2 expression can suppress the proliferation and activation of TGF-β1-induced HSC-T6 cells. 4. The mechanisms of HSCs proliferation and activation by inhibiting the expression of EZH21) Rat hepatic stellate cell line HSC-T6 was studied and small interfering RNA of Dkk1 was designed and synthesized according to the nucleotide sequence of EZH2, then transfected into HSC-T6 cells by LipofectamineTM 2000. The mRNA expression of Dkk1 and α-SMA were detected by Real-time PCR, and the protein expression of Dkk1, β-catenin, C-myc, CyclinD1 and α-SMA were detected by Western Blot. Experimental results showed that the activation of TGF-β1-induced HSC-T6 cells was promoted by siDkk1. DZNep and siEZH2 were taken to inhibit the expression of EZH2, the mRNA expression of Dkk1 was detected by Real-time PCR, and the protein expression of Dkk1 and H3K27me3 were detected by Western Blot. Experimental results showed that the expression of Dkk1 was regulated by epigenetic. DZNep and siEZH2 were taken to inhibit the expression of EZH2, the protein expression of β-catenin, C-myc and CyclinD1 were detected by Western Blot. Experimental results showed that epigenetic repression of Dkk1 contributes to the activation of Wnt/β-catenin pathway. HSC-T6 cells induced by TGF-β1 were cultured with the siRNA of Dkk1 with or without DZNep. The expression of Dkk1 and α-SMA in TGF-β1-induced HSC-T6 cells by Real-time PCR, and the protein expression of Dkk1, β-catenin, C-myc, CyclinD1 and α-SMA were detected by Western Blot. Experimental results showed the Essential role of Dkk1 in EZH2-mediated activation HSCs. These suggested that EZH2 promotes the activation of Wnt/β-catenin signaling pathway by down-regulating Dkk1 expression, and finally leaded to the proliferation and activation of HSCs.2) Rat hepatic stellate cell line HSC-T6 was studied, and DZNep and siEZH2 were taken to inhibit the expression of EZH2. The protein expression of p-ERK, p-AKT and α-SMA were detected by Western Blot. Experimental results showed that EZH2 promoted the proliferation and activation of HSCs by activating the ERK and PI3K/AKT pathway.