Supplementary Material for: LncRNA Dlx6os1 Accelerates Diabetic Nephropathy Progression by Epigenetically Repressing SOX6 via Recruiting EZH2

<b><i>Introduction:</i></b> Diabetic nephropathy (DN) is the leading cause of kidney failure worldwide. To explore the pathogenesis and effective biological target of DN is beneficial to seeking novel treatment strategies. <b><i>Objective:</i></b> This study aimed to investigate the role of the lncRNA Dlx6os1/SOX6/EZH2 axis in DN progression. <b><i>Methods:</i></b> PAS staining was performed to evaluate extracellular matrix accumulation; ELISA was carried out to assess the levels of urine microalbumin and blood glucose concentration; RT-qPCR was carried out to detect the levels of lncRNA Dlx6os1, TNF-α, IL-1β, IL-6, SOX6, and EZH2. Western blot was performed to assess the levels of Col-IV, FN, TGF-β1, and SOX6 proteins. RIP assay was carried out to verify the interaction between lncRNA Dlx6os1 and EZH2. ChIP-qPCR was conducted to verify the interaction between EZH2 and SOX6 promoter. <b><i>Results:</i></b> Our results illustrated that lncRNA Dlx6os1 was highly expressed in DN mice and HG-induced SV40 MES13 cells. LncRNA Dlx6os1 knockdown inhibited HG-induced SV40 MES13 cell proliferation, fibrosis, and inflammatory cytokine release. LncRNA Dlx6os1 inhibited SOX6 expression by recruiting EZH2 in HG-SV40 MES13 cells, and SOX6 mediated the effects of lncRNA Dlx6os1 on proliferation, fibrosis, and inflammatory factor release of HG-induced SV40 MES13 cells. <b><i>Conclusion:</i></b> LncRNA Dlx6os1 accelerates the progression of DN by epigenetically repressing SOX6 via recruiting EZH2.

<b><i>引言：</i></b> 糖尿病肾病（Diabetic nephropathy, DN）是全球范围内导致肾衰竭的首要病因。探究糖尿病肾病的发病机制与有效生物学靶点，有助于探索新型治疗策略。<b><i>目的：</i></b> 本研究旨在探讨长链非编码RNA（long non-coding RNA, lncRNA）Dlx6os1/SOX6/EZH2轴在糖尿病肾病进展中的调控作用。<b><i>方法：</i></b> 采用过碘酸-雪夫染色（periodic acid-Schiff staining, PAS染色）评估细胞外基质蓄积情况；通过酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）检测尿微量白蛋白与血糖浓度；采用实时定量聚合酶链式反应（real-time quantitative polymerase chain reaction, RT-qPCR）检测lncRNA Dlx6os1、肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）、白细胞介素-1β（interleukin-1β, IL-1β）、白细胞介素-6（interleukin-6, IL-6）、SOX6及EZH2的表达水平；采用蛋白质印迹法（Western blot）检测IV型胶原（Collagen IV, Col-IV）、纤连蛋白（fibronectin, FN）、转化生长因子-β1（transforming growth factor-β1, TGF-β1）及SOX6蛋白的表达水平；开展RNA免疫沉淀实验（RNA immunoprecipitation assay, RIP assay）验证lncRNA Dlx6os1与EZH2的相互作用；采用染色质免疫沉淀定量PCR（chromatin immunoprecipitation coupled with quantitative PCR, ChIP-qPCR）验证EZH2与SOX6启动子的结合相互作用。<b><i>结果：</i></b> 本研究结果显示，lncRNA Dlx6os1在糖尿病肾病小鼠及高糖（high glucose, HG）诱导的SV40 MES13细胞中呈高表达状态。敲低lncRNA Dlx6os1可抑制高糖诱导的SV40 MES13细胞增殖、纤维化进程以及炎症细胞因子的释放。在高糖处理的SV40 MES13细胞中，lncRNA Dlx6os1通过招募EZH2抑制SOX6的表达；且SOX6介导了lncRNA Dlx6os1对高糖诱导的SV40 MES13细胞增殖、纤维化及炎症因子释放的调控作用。<b><i>结论：</i></b> 长链非编码RNA Dlx6os1通过招募EZH2表观遗传抑制SOX6的表达，进而加速糖尿病肾病的进展。