背景:糖尿病肾病(DN)是由糖尿病引起的并发症。环状RNA(circularRNAs,circRNAs)是一类具有封闭环状结构的RNA,它具有很高的稳定性,并参与许多疾病相关的过程。circRNATAO激酶1(circTAOK1)在DN发生发展中的作用机制尚不清楚。
方法:CircTAOK1,microRNA(miR)-142-3p,通过实时定量聚合酶链反应(RT-qPCR)分析性别决定区Y-box转录因子6(SOX6)的mRNA水平。细胞计数试剂盒-8(CCK8)和5-乙炔基-2'-脱氧尿苷(EdU)测定用于分析细胞增殖。流式细胞术检测细胞周期分布。Westernblot检测B细胞淋巴瘤2(Bcl-2),Bcl-2相关X(Bax),裂解的半胱天冬酶3和纤连蛋白(FN),胶原蛋白I(ColI),和胶原蛋白IV(ColIV)的蛋白质水平。ELISA法检测白细胞介素1β(IL-1β),白细胞介素6(IL-6),和肿瘤坏死因子(TNF-α)水平。通过相应的试剂盒评估活性氧(ROS)和丙二醛(MDA)水平以及超氧化物歧化酶(SOD)活性。miR-142-3p与circTAOK1或SOX6的相关性通过双荧光素酶报告基因检测得到证实,RNA免疫沉淀测定和RNA下拉测定。
结果:CircTAOK1和SOX6表达水平上调,而miR-142-3p在DN血清和HG处理的HK-2细胞中表达下调。敲除circTAOK1可以抑制HG诱导的HK-2细胞的细胞损伤。通过miR-142-3p下调,circTAOK1敲低对HG诱导的HK-2细胞损伤的抑制作用得以恢复。CircTAOK1充当miR-142-3p的海绵,SOX6被miR-142-3p靶向。SOX6的过表达可以恢复miR-142-3p过表达对HG诱导的HK-2细胞损伤的影响。CircTAOK1通过靶向miR-142-3p调节SOX6的表达。
结论:CircTAOK1敲低通过miR-142-3p/SOX6轴抑制HG诱导的DNHK-2细胞损伤。
BACKGROUND: Diabetic nephropathy (DN) is a complication caused by diabetes. Circular RNAs (circRNAs) are a kind of RNA with a closed circular structure, which has high stability and is involved in many disease-related processes. The mechanism of circRNA TAO kinase 1 (circTAOK1) in the pathogenesis and development of DN is unclear.
METHODS: CircTAOK1, microRNA (miR)-142-3p, and sex-determining region Y-box transcription factor 6 (
SOX6) mRNA levels were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 (CCK8) and 5-ethynyl-2\'-deoxyuridine (EdU) assays were used to analyze cell proliferation. Cell cycle distribution was detected by flow cytometry. Western blot assay was performed to test B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax), cleaved-caspase 3, and fibronectin (FN), collagen I (Col I), and collagen IV (Col IV) protein levels. ELISA assay was used to measure interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor (TNF-α) levels. The reactive oxygen species (ROS) and malondialdehyde (MDA) levels and the superoxide dismutase (SOD) activity were assessed by the corresponding kits. And the correlation between miR-142-3p and circTAOK1 or
SOX6 was confirmed by dual luciferase reporter assay, RNA immunoprecipitation assay and RNA pull down assay.
RESULTS: CircTAOK1 and
SOX6 expression levels were up-regulated, while miR-142-3p expression was down-regulated in DN serum and HG-treated HK-2 cells. Knockdown of circTAOK1 could inhibit cell injury of HG-induced HK-2 cells. The inhibitory effect of circTAOK1 knockdown on HG-induced HK-2 cell injury was restored by miR-142-3p downregulation. CircTAOK1 acted as a sponge for miR-142-3p, and
SOX6 was targeted by miR-142-3p. The overexpression of SOX6 could recover the effect of miR-142-3p overexpression on HG-induced HK-2 cell injury. CircTAOK1 regulated the expression of
SOX6 by targeting miR-142-3p.
CONCLUSIONS: CircTAOK1 knockdown inhibited HG-induced HK-2 cell damage in DN by the miR-142-3p/SOX6 axis.