circPRKDC在新发2型糖尿病患者血清中的表达及对棕榈酸诱导胰岛细胞损伤的保护作用

Expression of circPRKDC in serum of patients with new-onset type 2 diabetes mellitus and its protective effect on palmitate-induced islet cell injury

目的探讨circPRKDC在新发2型糖尿病(T2DM)患者血清中的表达和对棕榈酸诱导的胰岛细胞损伤的保护作用及作用机制。方法选择50例新发T2DM患者和50例同期体检健康者,实时定量聚合酶链反应(qRT-PCR)检测血清中circPRKDC表达量。加入110μmol/L棕榈酸钠处理小鼠胰岛β细胞系MIN6细胞24 h,建立细胞损伤模型。分别感染阴性对照慢病毒或circPRKDC过表达慢病毒,即对照组和circPRKDC组。流式细胞术测定各组细胞的凋亡情况和细胞内活性氧(ROS)水平。生物信息学软件预测circPRKDC具有结合位点的miRNA。双荧光素酶报告基因实验验证circPRKDC与下游miRNA的直接结合。qRT-PCR检测各组细胞中miRNA的表达。Western blot检测各组细胞中凋亡相关因子(Bax、Bcl-2、caspase 8)和炎症相关蛋白(NF-κB、p65)的表达。结果与体检健康者比较,T2DM患者血清中circPRKDC表达明显降低(P<0.01)。与对照组比较,circPRKDC组细胞中circPRKDC表达上升(P<0.01),细胞凋亡水平降低(P<0.01),细胞内ROS含量降低(P<0.01)。circPRKDC直接结合miR-375(P<0.01)。与对照组比较,circPRKDC组细胞中miR-375表达降低(P<0.01),Bcl-2蛋白表达上调(P<0.01),Bax、caspase 8、NF-κB、p65蛋白表达下调(均P<0.01)。结论新发T2DM患者血清中circPRKDC表达下调,其通过靶向miR-375降低棕榈酸诱导的胰岛细胞氧化应激,从而抑制细胞凋亡和炎症反应。

Objective To investigate the expression of circular RNA(circRNA)circPRKDC in serum of patients with new-onset type 2 diabetes and its protective effect on palmitic acid-induced islet cell injury and its mechanism.Methods Fifty patients with new-onset type 2 diabetes and 50 healthy individuals who underwent physical examination during the same period were selected,and the expression of circPRKDC in serum was detected by real-time quantitative polymerase chain reaction(qRT-PCR).The mouse pancreaticβcell line MIN6 was treated with 110μmol/L palmitic acid for 24 h to establish cell injury model.Negative control lentivirus or circPRKDC overexpression lentivirus were transfected,namely control group and circPRKDC group,respectively.Flow cytometry was used to determine the apoptosis and intracellular reactive oxygen species(ROS)levels of cells in each group.Bioinformatics software predicted miRNA with binding sites for circPRKDC.The dual-luciferase reporter gene experiment verified the direct binding of circPRKDC to downstream miRNA.The expression of miRNA in each group of cells was detected by qRT-PCR.Western blot was used to detect the expressions of apoptosis-related factors(Bax,Bcl-2,caspase 8)and inflammation-related proteins(NF-κB,p65)in each group of cells.Results Compared with healthy subjects,the expression of circPRKDC in serum of patients with type 2 diabetes was significantly lower(P<0.01).Compared with the control group,the expression of circPRKDC in the circPRKDC group increased(P<0.01),the level of apoptosis decreased(P<0.01),and the content of intracellular ROS decreased(P<0.01).circPRKDC directly bound miR-375(P<0.01).Compared with the control group,the expression of miR-375 in cells in the circPRKDC group decreased(P<0.01),the protein expression of Bcl-2 was up-regulated(P<0.01),and the protein expressions of Bax,caspase 8,NF-κB and p65 were down-regulated(all P<0.01).Conclusion The expression of circPRKDC is down-regulated in the serum of patients with new-onset type 2 diabetes,and it reduces palmitic acid-induced oxidative stress in islet cells by targeting miR-375,thereby inhibiting apoptosis and inflammatory responses.