FTO介导m6A修饰的PRKD2调节SIRT1/HIF-1α通路抑制糖尿病肾病足细胞损伤的机制研究OACSTPCD
Mechanism of FTO-mediated and m6A-modified PRKD2 Inhibiting Podocyte Injury in Diabetic Kidney Disease through the SIRT1/HIF-1 Pathway
目的 探究脂肪含量和肥胖相关蛋白(fat mass and obesity-associated protein,FTO)和丝氨酸-苏氨酸激酶蛋白激酶D2(serine-threonine kinase protein kinase D2,PRKD2)在糖尿病肾病(diabetic kidney disease,DKD)进展中的调控作用和调节机制.方法 采用 35 mmol/L葡萄糖对足细胞(MPC5 细胞)进行高糖刺激 24h构建DKD体外模型.采用FTO过表达载体(pcDNA-FTO)和PRKD2 过表达载体(pcDNA-PRKD2),或空载体(vector)转染高糖诱导的MPC5 细胞.通过RT-qPCR检测FTO和PRKD2 过表达效率;MeRIP检测PRKD2 mRNA的N6-甲基腺苷(N6-methyladenosine,m6A)修饰水平;ELISA检测Caspase-3 活性、IL-6,TNF-α和单核细胞趋化蛋白-1(monocyte chemotactic protein-1,MCP-1)分泌量;流式细胞术分析细胞凋亡率;Western blot评估FTO和PRKD2 蛋白水平,以及SIRT1/HIF-1α通路关键蛋白表达水平;Pearson分析FTO和PRKD2 水平的相关性.结果 与无高糖诱导对照组比较,高糖诱导的足细胞中FTO蛋白(0.51±0.04 vs 1.00±0.03)和PRKD2 蛋白(0.45±0.03 vs 1.01±0.04)水平显著下调,差异具有统计学意义(t=13.17,16.76,均P＜0.001).高糖诱导的足细胞中FTO蛋白水平和PRKD2 蛋白水平呈正相关(r2=0.705 1,P＜0.001).与vector组相比,pcDNA-FTO组PRKD2 mRNA的m6A水平(0.56±0.09 vs 1.01±0.13)降低,PRKD2 mRNA水平(3.16±0.14 vs 1.03±0.02)显著升高,差异具有统计学意义(t=51.37,11.82,均P＜0.001).与control组(IL-6:512.76±61.85 pg/ml,TNF-α:28.17±2.83 pg/ml,MCP-1:157.31±17.69 pg/ml)和vector组(IL-6:498.41±87.51 pg/ml,TNF-α:26.35±5.47 pg/ml,MCP-1:165.52±16.87 pg/ml)比较,pcDNA-PRKD2 组IL-6(301.86±21.85 pg/ml),TNF-α(11.06±4.12 pg/ml),MCP-1 分 泌 量(81.45±9.03pg/ml)显著减少,差异具有统计学意义(F=7.51,10.47,61.97,均P＜0.01).与control组(Caspase-3:689.65±79.5 U/L,细胞凋亡率:22.31%±2.69%)和vector组(Caspase-3:715.91±113.58 U/L,细胞凋亡率:21.07%±3.28%)比较,pcDNA-PRKD2组Caspase-3活性(437.64±104.76 U/L)和细胞凋亡率(8.41%±3.15%)下降,差异具有统计学意义(F=2.35,79.13,均P＜0.01).与control组(SIRT1:1.01±0.05,HIF-1α:1.03±0.07)和vector组(SIRT1:0.97±0.05,HIF-1α:1.02±0.03)相比,pcDNA-PRKD2组SIRT1蛋白(3.51±0.15)水平升高,HIF-1α蛋白(0.37±0.07)水平降低,差异具有统计学意义(F=31.54,8.31,均P＜0.01).结论 FTO介导m6A修饰的PRKD2 通过SIRT1/HIF-1α通路抑制高糖诱导的足细胞炎症反应和细胞凋亡.
Objective To explore the regulatory role of fat mass and obesity-associated protein(FTO)and serine-threonine kinase protein kinase D2(PRKD2)in progression of diabetic kidney disease(DKD)and its regulatory mechanisms.Methods DKD model in vitro was constructed by podocytes(MPC5 cells)treated with high glucose(HG,35 mmol/L glucose)for 24 h.HG-induced MPC5 cells were transfected with FTO overexpression vector(pcDNA-FTO)and PRKD2 overexpression vector(pcDNA-PRKD2),or empty vector.The overexpression efficiency of FTO and PRKD2 were detected with RT-qPCR.MeRIP was used to detect the N6-methyladenosine(m6A)modification level of PRKD2 mRNA.The activity of Caspase-3 and the secretion of IL-6,TNF-α and monocyte chemotactic protein-1(MCP-1)were detected by ELISA.Cell apoptosis rate was analyzed by flow cytometry.The protein levels of FTO and PRKD2,as well as the key proteins in SIRT1/HIF-1α pathway,were evaluated by Western blot.Pearson analysis was used to analyze the correlation between FTO levels and PRKD2 levels.Results Compared with the control group without HG-induction,the protein expression of FTO(0.51±0.04 vs 1.00±0.03)and PRKD2(0.45±0.03 vs 1.01±0.04)was significantly down-regulated in HG-induced podocytes,and the differences were statistically significant(t=13.17,16.76,all P＜0.001).FTO protein levels were positively correlated with PRKD2 protein levels in HG-induced podocytes(r2=0.705 1,P＜0.001).Compared with the vector group,the m6A levels of PRKD2 mRNA(0.56±0.09 vs 1.01±0.13)in the pcDNA-FTO group were decreased,and the mRNA levels of PRKD2(3.16±0.14 vs 1.03±0.02)were increased,with significant differences(t=51.37,11.82,all P＜0.001).Compared with the control group(IL-6:512.76±61.85 pg/ml,TNF-α:28.17±2.83 pg/ml,MCP-1:157.31±17.69 pg/ml)and the vector group(IL-6:498.41±87.51 pg/ml,TNF-α:26.35±5.47 pg/ml,MCP-1:165.52±16.87 pg/ml),the secretion of IL-6(301.86±21.85 pg/ml),TNF-α(11.06±4.12 pg/ml)and MCP-1(81.45±9.03 pg/ml)were significantly decreased in the pcDNA-PRKD2 group,and the differences were statistically significant(F=7.51,10.47,61.97,all P＜0.01).Compared with the control group(caspase-3 activity:689.65±79.5 U/L,cell apoptosis:22.31%±2.69%)and the vector group(Caspase-3 activity:715.91±113.58 U/L,cell apoptosis:21.07%±3.28%),Caspase-3 activity(437.64±104.76 U/L)and the rate of apoptosis(8.41%±3.15%)were significantly decreased in the pcDNA-PRKD2 group,and the differences were statistically significant(F=2.35,79.13,all P＜0.01).Compared with the control group(SIRT1:1.01±0.05,HIF-1α:1.03±0.07)and the vector group(SIRT1:0.97±0.05,HIF-1α:1.02±0.03),SIRT1 protein levels(3.51±0.15)were increased and HIF-1α protein levels(0.37±0.07)were decreased in the pcDNA-PRKD2 group,and the differences were statistically significant(F=31.54,8.31,all P＜0.01).Conclusion FTO-mediated and m6A-modified PRKD2 suppresses inflammation and apoptosis in HG-induced podocytes through the SIRT1/HIF-1 pathway.
李亚宁;李成乾
青岛大学附属医院内分泌科,山东青岛 264200
临床医学
糖尿病肾病足细胞N6甲基腺苷修饰脂肪和肥胖相关蛋白丝氨酸-苏氨酸激酶蛋白激酶D2
diabetic kidney diseasepodocytesN6-methyladenosine modificationfat mass and obesity-associated proteinserine-threonine kinase protein kinase D2
《现代检验医学杂志》 2024 (001)
5-9,22 / 6
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