Department of Endocrinology, China-Japan Friendship Hospital, Beijing, China.
Eur Rev Med Pharmacol Sci. 2020 Jan;24(2):802-812. doi: 10.26355/eurrev_202001_20063.
Diabetic nephropathy (DN) is one of the primary complications of diabetes. Long non-coding RNA cancer susceptibility candidate 2 (CASC2) has been established to function in DN, while its role in high glucose (HG)-induced human mesangial cells (HMCs) remains limited.
The expression level of CASC2 and miR-133b was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay. Extracellular matrix (ECM) accumulation was monitored through the expression levels of collagen IV (Col IV) and fibronectin (FN) using qRT-PCR and western blot analyses. Oxidative stress was observed through the expression of NADPH oxidase2 (NOX2) and the activity of malondialdehyde (MDA) and superoxide dismutase (SOD) using western blot or corresponding detection kit. The expression of forkhead box P1 (FOXP1) at mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. The relationship between miR-133b and CASC2 or FOXP1 was predicted by online bioinformatics tools and verified by dual-luciferase reporter assay or RNA pull-down.
The expression of CASC2 was reduced in serum from DN patients and HG-induced HMCs. CASC2 upregulation inhibited HG-induced HMCs proliferation, ECM accumulation and oxidative stress. MiR-133b was a target of CASC2 with a high level in serum from DN patients and HG-induced HMCs, and its enrichment reversed the effects of CASC2 upregulation. Besides, FOXP1 was a target of miR-133b with a low level in HG-induced HMCs, and its knockdown abolished the impacts of CASC2 upregulation.
CASC2 upregulation suppressed HG-induced proliferation, ECM accumulation and oxidative stress of HMCs through miR-133b /FOXP1 regulatory axis, suggesting that CASC2 was a novel biomarker for DN treatment.
糖尿病肾病（DN）是糖尿病的主要并发症之一。长链非编码 RNA 癌症易感性候选基因 2（CASC2）已被证明在 DN 中发挥作用，但其在高葡萄糖（HG）诱导的人肾小球系膜细胞（HMC）中的作用仍有限。
通过实时定量聚合酶链反应（qRT-PCR）检测 CASC2 和 miR-133b 的表达水平。通过细胞计数试剂盒-8（CCK-8）测定细胞增殖。通过 qRT-PCR 和 Western blot 分析检测胶原 IV（Col IV）和纤维连接蛋白（FN）的表达水平来监测细胞外基质（ECM）的积累。通过 Western blot 或相应的检测试剂盒检测 NADPH 氧化酶 2（NOX2）的表达和丙二醛（MDA）和超氧化物歧化酶（SOD）的活性来观察氧化应激。通过 qRT-PCR 和 Western blot 分别测定叉头框 P1（FOXP1）在 mRNA 和蛋白水平的表达。通过在线生物信息学工具预测 miR-133b 与 CASC2 或 FOXP1 的关系，并通过双荧光素酶报告基因检测或 RNA 下拉实验验证。
DN 患者血清和 HG 诱导的 HMC 中 CASC2 的表达降低。CASC2 的上调抑制了 HG 诱导的 HMC 增殖、ECM 积累和氧化应激。miR-133b 是 CASC2 的一个靶点，在 DN 患者和 HG 诱导的 HMC 中表达水平较高，其富集逆转了 CASC2 上调的作用。此外，FOXP1 是 miR-133b 的一个靶点，在 HG 诱导的 HMC 中表达水平较低，其敲低消除了 CASC2 上调的影响。
CASC2 的上调通过 miR-133b/FOXP1 调节轴抑制 HG 诱导的 HMC 增殖、ECM 积累和氧化应激，提示 CASC2 是治疗 DN 的新型生物标志物。
