Data Sheet 1_Loss of a primary cilia protein ARL13B promotes TGFβ-1 induced EMT of RPE in proliferative vitreoretinopathy via increasing Smad3 expression.pdf

BackgroundProliferative vitreoretinopathy (PVR) is a major complication of rhegmatogenous retinal detachment. Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) plays a central role in PVR pathogenesis. This study aims to investigate the effect of ADP-ribosylation factor-like 13B (ARL13B) on RPE EMT in PVR.
MethodsThe expression of ARL13B in PVR specimens was analyzed by immunofluorescence (IF) staining. The effect of ARL13B on RPE EMT was assessed by IF staining and Western blot. The proliferation and migration of RPE were measured with EdU and transwell and scratch assays, respectively. The EMT-related transcriptome was analyzed by bulk RNAseq. An intravitreal injection mouse model of PVR was used to investigate the role of ARL13B in PVR formation.
ResultsImmunofluorescence revealed significantly reduced ARL13B levels in α-SMA-positive cells as compared with Pan-CK-positive cells in an epiretinal membrane derived from retinal tears. During EMT, TGFβ1 treatment remarkably reduced ARL13B expression and shortened the length of cilia in RPE cells. In line with this, ARL13B knockdown (KD) decreased the length of cilia and enhanced TGFβ1-induced EMT, evidenced by morphology change and a globally upregulated EMT-related gene expression in RPEs. Moreover, ARL13B KD enhanced TGFβ1-induced RPE proliferation and migration. Consistently, ARL13B KD promoted PVR formation in vivo. Mechanistically, ARL13B KD enhanced TGFβ1 signaling by increasing the phosphorylation and expression of Smad3.
ConclusionThis study demonstrated a crucial role of ARL13B on TGFβ1-induced RPE EMT, highlighting the importance of ARL13B in PVR formation.

**背景** 增生性玻璃体视网膜病变（Proliferative vitreoretinopathy, PVR）是孔源性视网膜脱离的主要并发症。视网膜色素上皮（Retinal Pigment Epithelial, RPE）的上皮间质转化（Epithelial-Mesenchymal Transition, EMT）在PVR的发病机制中居于核心地位。本研究旨在探讨ADP核糖基化因子样13B（ADP-ribosylation factor-like 13B, ARL13B）对PVR中RPE EMT的影响。
**方法** 采用免疫荧光（Immunofluorescence, IF）染色分析ARL13B在PVR标本中的表达水平。通过免疫荧光染色与Western blot评估ARL13B对RPE EMT的调控作用。分别借助EdU实验、Transwell小室实验及划痕实验检测RPE的增殖与迁移能力。通过bulk RNAseq分析EMT相关转录组特征。构建PVR玻璃体内注射小鼠模型，探究ARL13B在PVR形成过程中的作用。
**结果** 免疫荧光检测结果显示，在视网膜裂孔来源的视网膜前膜中，α-平滑肌肌动蛋白（α-SMA）阳性细胞内的ARL13B水平显著低于广谱细胞角蛋白（Pan-CK）阳性细胞。在EMT进程中，转化生长因子β1（TGFβ1）处理可显著下调RPE细胞中ARL13B的表达，并缩短纤毛长度。与之相符的是，ARL13B敲低（KD）可缩短纤毛长度，并增强TGFβ1诱导的EMT，该效应可通过RPE细胞的形态变化及全局上调的EMT相关基因表达得以验证。此外，ARL13B敲低还可促进TGFβ1诱导的RPE增殖与迁移。体内实验同样证实，ARL13B敲低可加速PVR的形成。机制研究表明，ARL13B敲低可通过提升Smad3的磷酸化水平与表达量，增强TGFβ1信号通路活性。
**结论** 本研究证实了ARL13B在TGFβ1诱导的RPE EMT中的关键调控作用，揭示了ARL13B在PVR形成中的重要意义。