背景:糖尿病肾病(DN)是糖尿病的常见并发症,在高葡萄糖(HG)诱导的肾小管上皮细胞(TECs)中,脯氨酸4-羟化酶亚基β(P4HB)的表达增加。但其在HG诱导的TECs中的作用仍有待阐明。
方法:使用HG诱导HK-2细胞并用SiRNA-P4HB转染。DCFH-DA染色用于检测ROS的细胞水平。采用WB和免疫荧光检测P4HB的表达,上皮-间质转化(EMT),纤维化,HK-2细胞中TGFβ/SMAD3相关蛋白的表达。在线数据库用于预测P4HB的相互作用目标,和免疫沉淀(IP)实验用于验证P4HB与靶标的结合。用SiRNA和靶基因过表达载体验证P4HB的作用机制。
结果:HG诱导P4HB和TGFβ的表达增加,p-SMAD3和HK-2细胞中的ROS。此外,HG下调E-cadherin的表达,上调N-cadherin的表达,Vimentin,α-SMA,纤连蛋白,胶原蛋白IV,蜗牛,和SLUG在HK-2细胞中。干扰P4HB显著逆转了这些蛋白的表达。数据库预测和IP实验表明,P4HB与PRMT1相互作用,在HG诱导的HK-2细胞中PRMT1的表达增加。干扰PRMT1抑制HG诱导的EMT和纤维化相关蛋白表达的变化。然而,PRMT1的过表达削弱了P4HB干扰对EMT的调节作用,纤维化,HK-2细胞中TGFβ/SMAD3相关蛋白的表达。
结论:P4HB通过PRMT1调节TGFβ/SMAD3信号通路,从而参与HG诱导的HK-2细胞EMT和纤维化。
BACKGROUND: Diabetic nephropathy (DN) is a common complication of diabetes mellitus, and Prolyl 4-Hydroxylase Subunit Beta (P4HB) expression is increased in high glucose (HG)-induced renal tubular epithelial cells (TECs). But it\'s role in HG-induced TECs remains to be elucidated.
METHODS: The HK-2 cells were induced using HG and transfected with SiRNA-P4HB. DCFH-DA staining was utilized for the detection of cellular levels of ROS. WB and immunofluorescence were utilized to detect the expression of P4HB, epithelial-mesenchymal transition (EMT), fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells. Online databases were utilized for predicting the interaction target of P4HB, and immunoprecipitation (IP) experiments were employed to validate the binding of P4HB with the target. SiRNA and overexpression vectors of target gene were used to verify the mechanism of action of P4HB.
RESULTS: HG induced an increase in the expression of P4HB and TGFβ, p-SMAD3, and ROS in HK-2 cells. Furthermore, HG downregulated the expression of E-cadherin and upregulated the expression of N-cadherin, Vimentin, α-SMA, Fibronectin, Collagen IV, SNAIL, and SLUG in HK-2 cells. Interfering with P4HB significantly reversed the expression of these proteins. Database predictions and IP experiments showed that P4HB interacts with PRMT1, and the expression of PRMT1 was increased in HG-induced HK-2 cells. Interfering with PRMT1 inhibited the changes in expression of EMT and fibrosis related proteins induced by HG. However, overexpression of PRMT1 weakened the regulatory effect of P4HB interference on the EMT, fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells.
CONCLUSIONS: P4HB regulated the TGFβ/SMAD3 signaling pathway through PRMT1 and thus participates in HG-induced EMT and fibrosis in HK-2 cells.