Epigenetic mechanisms, including DNA methylation, histone modifications, and chromatin remodeling, are highly involved in the regulation of hepatocyte viability, proliferation, and plasticity. We have previously demonstrated that repression of H3K27 methylation in differentiated hepatic HepaRG cells by treatment with GSK-J4, an inhibitor of JMJD3 and UTX H3K27 demethylase activity, changed their phenotype, inducing differentiated hepatocytes to proliferate. In addition to the epigenetic enzymatic role in the regulation of the -differentiation process, emerging evidence indicate that microRNAs (miRNAs) are involved in controlling hepatocyte proliferation during liver regeneration. Hence, the aim of this work is to investigate the impact of H3K27 methylation on miRNAs expression profile and its role in the regulation of the differentiation status of human hepatic progenitors HepaRG cells. A miRNA-sequencing was carried out in differentiated HepaRG cells treated or not with GSK-J4. Target searching and Gene Ontology analysis were performed to identify the molecular processes modulated by differentially expressed miRNAs. The biological functions of selected miRNAs was further investigated by transfection of miRNAs inhibitors or mimics in differentiated HepaRG cells followed by qPCR analysis, albumin ELISA assay, CD49a FACS analysis and EdU staining. We identified 12 miRNAs modulated by GSK-J4; among these, miR-27a-3p and miR- 423-5p influenced the expression of several proliferation genes in differentiated HepaRG cells. MiR-27a-3p overexpression increased the number of hepatic cells reentering proliferation. Interestingly, both miR-27a-3p and miR-423-5p did not affect the expression levels of genes involved in the differentiation of progenitors HepaRG cells. Modulation of H3K27me3 methylation in differentiated HepaRG cells, by GSK-J4 treatment, influenced miRNA’ s expression profile pushing liver cells towards a proliferating phenotype. We demonstrated the involvement of miR-27a-3p in reinducing proliferation of differentiated hepatocytes suggesting a potential role in liver plasticity.

中文翻译：

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miRNA-27a-3p参与分化肝细胞的可塑性

表观遗传机制，包括 DNA 甲基化、组蛋白修饰和染色质重塑，高度参与肝细胞活力、增殖和可塑性的调节。我们之前已经证明，通过用 JMJD3 和 UTX H3K27 去甲基酶活性抑制剂 GSK-J4 处理，抑制分化肝 HepaRG 细胞中的 H3K27 甲基化，改变其表型，诱导分化肝细胞增殖。除了表观遗传酶在分化过程调节中的作用外，新出现的证据表明 microRNA (miRNA) 还参与肝再生过程中肝细胞增殖的控制。因此，本工作的目的是研究H3K27甲基化对miRNA表达谱的影响及其在调节人肝祖细胞HepaRG细胞分化状态中的作用。在用或未用 GSK-J4 处理的分化 HepaRG 细胞中进行 miRNA 测序。进行目标搜索和基因本体分析来识别差异表达 miRNA 调节的分子过程。通过在分化的 HepaRG 细胞中转染 miRNA 抑制剂或模拟物，然后进行 qPCR 分析、白蛋白 ELISA 测定、CD49a FACS 分析和 EdU 染色，进一步研究所选 miRNA 的生物学功能。我们鉴定了 12 个受 GSK-J4 调节的 miRNA；其中，miR-27a-3p和miR-423-5p影响分化的HepaRG细胞中多个增殖基因的表达。 MiR-27a-3p 过表达增加了肝细胞重新增殖的数量。有趣的是，miR-27a-3p和miR-423-5p都不影响祖细胞HepaRG细胞分化相关基因的表达水平。通过 GSK-J4 处理，对分化的 HepaRG 细胞中 H3K27me3 甲基化的调节影响了 miRNA 的表达谱，从而推动肝细胞走向增殖表型。我们证明了 miR-27a-3p 参与分化肝细胞的重新增殖，表明其在肝脏可塑性中具有潜在作用。