circPRKCI靶向miR-448对高脂高糖诱导胰岛β细胞损伤的影响  

Effect of circPRKCI on the isletβcell damage induced by high fatty acid and high glucose via targeting miR-448

作　　者：王梅 谭金枚 WANG Mei;TAN Jinmei(Department of Endocrinology,People’s Hospital of Dongxihu District,Hubei,Wuhan 430040,China;不详)

机构地区：[1]湖北省武汉市东西湖区人民医院内分泌科,430040 [2]长江航运总医院内分泌科

出　　处：《河北医药》2023年第20期3045-3049,3055,共6页Hebei Medical Journal

基　　金：武汉市医学科研项目(编号:WX20D48)。

摘　　要：目的考察环状RNA(circRNA)circPRKCI是否靶向miR-448调节高脂高糖诱导的胰岛β细胞损伤。方法胰岛β细胞被分为Con组(正常对照,11.1 mmol/L葡萄糖)、Model组(高脂高糖模型,33.3 mmol/L葡萄糖+0.25 mmol/L棕榈酸)、Model+pcDNA组(高脂高糖模型+转染pcDNA)、Model+pcDNA-circPRKCI组(高脂高糖模型+转染pcDNA-circPRKCI)、Model+anti-miR-NC组(高脂高糖模型+转染anti-miR-NC)、Model+anti-miR-488组(高脂高糖模型+转染anti-miR-488)、Model+pcDNA-circPRKCI+miR-NC组(高脂高糖模型+转染pcDNA-circPRKCI+miR-NC)、Model+pcDNA-circPRKCI+miR-448组(高脂高糖模型+转染pcDNA-circPRKCI+miR-448)。采用qRT-PCR检测circPRKCI、miR-448、肿瘤坏死因子-α(TNF-α)mRNA和白细胞介素-1β(IL-1β)mRNA表达,流式细胞术检测细胞凋亡,Western blot检测活化-含半胱氨酸的天冬氨酸蛋白水解酶(cleaved-cysteinyl aspartate specific proteinase,cleaved-caspase)3和cleaved-caspase9蛋白表达。运用荧光素酶报告分析circPRKCI与miR-448的靶向结合。结果与Con组比较,Model组胰岛β细胞中circPRKCI表达量减少,miR-488表达量、凋亡率、cleaved-caspase3、cleaved-caspase9蛋白表达量、TNF-αmRNA和IL-1βmRNA表达量增加(P<0.05)。与Model+pcDNA组比较,Model+pcDNA-circPRKCI组胰岛β细胞中circPRKCI表达量增加,凋亡率、cleaved-caspase3、cleaved-caspase9蛋白表达量、TNF-αmRNA和IL-1βmRNA表达量减少(P<0.05)。circPRKCI靶向调控miR-448的表达。抑制miR-448表达后,Model+anti-miR-488组胰岛β细胞的miR-488表达量、凋亡率、cleaved-caspase3、cleaved-caspase9蛋白、TNF-αmRNA和IL-1βmRNA表达量均比Model+anti-miR-NC组降低(P<0.05)。上调miR-448表达后,Model+pcDNA-circPRKCI+miR-448组胰岛β细胞的miR-488表达量、凋亡率、cleaved-caspase3、cleaved-caspase9蛋白、TNF-αmRNA和IL-1βmRNA表达量均比Model+pcDNA-circPRKCI+miR-NC组提升(P<0.05)。结论circPRKCI通过靶向miR-448,减少细胞凋亡和炎性因子表达,从而保护高脂高糖诱导Objective To investigate whether circular RNA(circRNA)circPRKCI regulates pancreaticβcell damage induced by high fatty acid and high glucose via targeting miR-448.Methods Isletβcells were divided into Con group(normal control,11.1mmol/L glucose),Model group(high fatty acid and high glucose model,33.3mmol/L glucose+0.25mmol/L palmitic acid),Model+pcDNA group(high fatty acid and high glucose model+transfection with pcDNA),Model+pcDNA-circPRKCI group(high fatty acid and high glucose model+transfection with pcDNA-circPRKCI),Model+anti-miR-NC group(high fatty acid and high glucose model+transfection with anti-miR-NC),Model+anti-miR-488 group(high fatty acid and high glucose model+transfection anti-miR-448),Model+pcDNA-circPRKCI+miR-NC group(high fatty acid and high glucose model+transfection pcDNA-circPRKCI+miR-NC),Model+pcDNA-circPRKCI+miR-448 group(high fatty acid and high glucose model+transfection pcDNA-circPRKCI+miR-448).The mRNA levels of circPRKCI,miR-448,tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were detected by quantitative real-time polymerase chain reaction(qRT-PCR).Flow cytometry was performed to detect cell apoptosis,and Western blot was applied to determine the protein expressions of activation-cleaved-cysteinyl aspartate specific proteinase(cleaved-caspase)3 and cleaved-caspase 9.Dual-luciferase reporter assay was performed to validate the targeted binding of circPRKCI and miR-448.Results Compared with the Con group,pancreatic isletβcells in the Model group showed significantly downregulated circPRKCI,upregulated miR-448,TNF-α,IL-1β,cleaved caspase 3 and cleaved caspase 9,and higher apoptotic rate(P<0.05).Compared with the Model+pcDNA group,pancreatic isletβcells in the Model+pcDNA-circPRKCI group showed significantly upregulated circPRKCI,downregulated TNF-α,IL-1β,cleaved caspase 3 and cleaved caspase 9,and lower apoptotic rate(P<0.05).CircPRKCI targeted miR-448.After knockdown of miR-448,significantly lower levels of miR-448,TNF-α,IL-1β,cleaved caspase 3 and cleaved caspase

关 键 词：糖尿病 circPRKCI 胰岛Β细胞 miR-448 高脂高糖 凋亡 炎症 

分 类 号：R587.1[医药卫生—内分泌]