ZNF469 is a novel pro-fibrotic regulator of extracellular matrix in hepatic stellate cells. ZNF469 is a novel pro-fibrotic regulator of extracellular matrix in hepatic stellate cells

Activation of quiescent hepatic stellate cells (HSCs) into proliferative myofibroblasts drives extracellular cellular matrix (ECM) accumulation and liver fibrosis; nevertheless, transcriptional network that promotes such the process remains elusive. ZNF469, a putative C2H2 zinc finger protein, is found to be upregulated upon HSC activation; however, the molecular function of ZNF469 is completely unknown. Here, we show that knockdown of ZNF469 in primary human HSCs impaired proliferation, migration, and collagen production. Conversely, overexpression of ZNF469 in HSCs yielded the opposite results. TGFb promoted expression of ZNF469 in a Smad3-dependent manner where the binding of Smad3 was confirmed at the ZNF469 promoter. RNA-seq data of ZNF469-knockdown HSCs revealed the ECM-receptor interaction as the top affected pathway, and significant downregulation of various collagen and proteoglycan genes was observed. To explore the function of ZNF469, we cloned a full-length open reading frae (ORF) of ZNF469 with an epitope tag and identified a nuclear localization of the protein. ChIP assays revealed the presence of ZNF469 at the promoter of ECM genes, supporting its function as a transcription factor. Analysis of human fibrotic and cirrhotic tissues showed increased expression of ZNF469 and a positive correlation between expression levels of ZNF469 and ECM genes. Moreover, this observation was similar in other fibrotic organs, including heart, lung, and skin. Together, this study is the first to reveal the roles of ZNF469 as a pro-fibrotic factor in HSCs and suggests ZNF469 as a novel target for antifibrotic therapy. Overall design: Primary human hepatic stellate cells with control and ZNF469 knockdown with short-hairpin RNAs. Three technical replicates were performed for each group.

静息肝星状细胞（hepatic stellate cells, HSCs）激活为增殖性肌成纤维细胞的过程，可驱动细胞外基质（extracellular cellular matrix, ECM）沉积与肝纤维化的发生；然而，介导该过程的转录调控网络至今仍未明确。ZNF469作为一种推定的C2H2型锌指蛋白，在HSC激活过程中表达上调，但其分子功能目前仍完全未知。本研究发现，在原代人肝星状细胞中敲低ZNF469，会抑制细胞的增殖、迁移能力并减少胶原合成；反之，在HSCs中过表达ZNF469则会得到相反的实验结果。转化生长因子β（TGFβ）可通过Smad3依赖的方式促进ZNF469的表达，且Smad3在ZNF469启动子区域的结合已得到验证。对ZNF469敲低的HSCs进行RNA测序（RNA-seq）分析显示，细胞外基质-受体互作通路是受影响最显著的信号通路，同时多种胶原及蛋白聚糖基因的表达显著下调。为探究ZNF469的功能，我们克隆了带有表位标签的ZNF469全长开放阅读框（open reading frame, ORF），并确认该蛋白定位于细胞核。染色质免疫沉淀实验（ChIP assays）显示，ZNF469可结合至细胞外基质基因的启动子区域，佐证了其作为转录因子的功能。对人纤维化及肝硬化组织的分析表明，ZNF469的表达水平升高，且ZNF469与细胞外基质基因的表达量呈正相关。此外，这一现象在心脏、肺、皮肤等其他纤维化器官中也同样存在。本研究首次揭示了ZNF469作为肝星状细胞中促纤维化因子的作用，并提示ZNF469可作为抗纤维化治疗的新型靶点。实验整体设计：通过短发夹RNA（short-hairpin RNAs, shRNA）对原代人肝星状细胞进行ZNF469敲低处理并设置对照组；每组均开展3次技术重复实验。