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Methylation of RCAN1.4 mediated by DNMT1 and DNMT3b enhances hepatic stellate cell activation and liver fibrogenesis through Calcineurin/NFAT3 signaling.
Theranostics ( IF 12.4 ) Pub Date : 2019-01-01 , DOI: 10.7150/thno.32710
Xue-Yin Pan 1, 2, 3 , Hong-Mei You 1, 2, 3 , Ling Wang 1, 2, 3 , Yi-Hui Bi 1, 2, 3 , Yang Yang 1, 2, 3 , Hong-Wu Meng 1, 2, 3 , Xiao-Ming Meng 1, 2, 3 , Tao-Tao Ma 1, 2, 3 , Cheng Huang 1, 2, 3 , Jun Li 1, 2, 3
Affiliation  

Background: Liver fibrosis is characterized by extensive deposition of extracellular matrix (ECM) components in the liver. RCAN1 (regulator of calcineurin 1), an endogenous inhibitor of calcineurin (CaN), is required for ECM synthesis during hypertrophy of various organs. However, the functional role of RCAN1 in liver fibrogenesis has not yet been addressed. Methods: We induced experimental liver fibrosis in mice by intraperitoneal injection of 10 % CCl4 twice a week. To investigate the functional role of RCAN1.4 in the progression of liver fibrosis, we specifically over-expressed RCAN1.4 in mice liver using rAAV8-packaged RCAN1.4 over-expression plasmid. Following the establishment of the fibrotic mouse model, primary hepatic stellate cells were isolated. Subsequently, we evaluated the effect of RCAN1.4 on hepatic fibrogenesis, hepatic stellate cell activation, and cell survival. The biological role and signaling events for RCAN1 were analyzed by protein-protein interaction (PPI) network. Bisulfite sequencing PCR (BSP) was used to predict the methylated CpG islands in the RCAN1.4 gene promoter. We used the chromatin immunoprecipitation (ChIP assay) to investigate DNA methyltransferases which induced decreased expression of RCAN1.4 in liver fibrosis. Results: Two isoforms of RCAN1 protein were expressed in CCl4-induced liver fibrosis mouse model and HSC-T6 cells cultured with transforming growth factor-beta 1 (TGF-β1). RCAN1 isoform 4 (RCAN1.4) was selectively down-regulated in vivo and in vitro. The BSP analysis indicated the presence of two methylated sites in RCAN1.4 promoter and the downregulated RCAN1.4 expression levels could be restored by 5-aza-2'-deoxycytidine (5-azadC) and DNMTs-RNAi transfection in vitro. ChIP assay was used to demonstrate that the decreased RCAN1.4 expression was associated with DNMT1 and DNMT3b. Furthermore, we established a CCl4-induced liver fibrosis mouse model by injecting the recombinant adeno-associated virus-packaged RCAN1.4 (rAAV8-RCAN1.4) over-expression plasmid through the tail vein. Liver- specific-over-expression of RAN1.4 led to liver function recovery and alleviated ECM deposition. The key protein (a member of the NFAT family of proteins) identified on PPI network data was analyzed in vivo and in vitro. Our results demonstrated that RCAN1.4 over-expression alleviates, whereas its knockdown exacerbates, TGF-β1-induced liver fibrosis in vitro in a CaN/NFAT3 signaling-dependent manner. Conclusions: RCAN1.4 could alleviate liver fibrosis through inhibition of CaN/NFAT3 signaling, and the anti-fibrosis function of RCAN1.4 could be blocked by DNA methylation mediated by DNMT1 and DNMT3b. Thus, RCAN1.4 may serve as a potential therapeutic target in the treatment of liver fibrosis.

中文翻译:

DNMT1和DNMT3b介导的RCAN1.4的甲基化通过Calcineurin / NFAT3信号传导增强了肝星状细胞的活化和肝纤维化。

背景:肝纤维化的特征是肝内细胞外基质(ECM)成分大量沉积。RCAN1(钙调神经磷酸酶1的调节剂),钙调神经磷酸酶(CaN)的内源性抑制剂,是各种器官肥大过程中ECM合成所必需的。但是,RCAN1在肝纤维化中的功能作用尚未得到解决。方法:我们每周两次腹膜内注射10%CCl4诱导小鼠实验性肝纤维化。要研究RCAN1.4在肝纤维化进展中的功能,我们使用rAAV8包装的RCAN1.4过表达质粒在小鼠肝脏中特异性过表达RCAN1.4。建立纤维化小鼠模型后,分离出原代肝星状细胞。随后,我们评估了RCAN1.4对肝纤维化的影响,肝星状细胞的活化和细胞的存活。通过蛋白质-蛋白质相互作用(PPI)网络分析了RCAN1的生物学作用和信号传导事件。亚硫酸氢盐测序PCR(BSP)用于预测RCAN1.4基因启动子中的甲基化CpG岛。我们使用染色质免疫沉淀(ChIP测定)来研究DNA甲基转移酶,该酶诱导肝纤维化中RCAN1.4的表达降低。结果:在CCl4诱导的肝纤维化小鼠模型和用转化生长因子β1(TGF-β1)培养的HSC-T6细胞中表达了RCAN1蛋白的两种同工型。RCAN1亚型4(RCAN1.4)在体内和体外选择性下调。BSP分析表明,RCAN1.4启动子中存在两个甲基化位点,而5-CANA-2'可以恢复RCAN1.4表达水平的下调。-脱氧胞苷(5-azadC)和DNMT-RNAi体外转染。使用ChIP分析来证明降低的RCAN1.4表达与DNMT1和DNMT3b相关。此外,我们通过尾静脉注射重组腺相关病毒包装的RCAN1.4(rAAV8-RCAN1.4)过表达质粒,建立了CCl4诱导的肝纤维化小鼠模型。RAN1.4的肝特异性过度表达导致肝功能恢复并减轻了ECM沉积。在体内和体外分析了在PPI网络数据上鉴定的关键蛋白质(NFAT蛋白质家族的成员)。我们的结果表明,RCAN1.4的过表达减轻了,而其敲低加剧了TGF-β1在体外以CaN / NFAT3信号依赖的方式诱导的肝纤维化。结论:RCAN1。4可以通过抑制CaN / NFAT3信号来减轻肝纤维化,而DNMT1和DNMT3b介导的DNA甲基化可以阻断RCAN1.4的抗纤维化功能。因此,RCAN1.4可以作为治疗肝纤维化的潜在治疗靶标。
更新日期:2019-01-01
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