FGD5-AS1/miR-519d-3p轴调控高糖诱导的肾小管上皮细胞HK-2炎症因子表达和细胞凋亡

陆洁雅; 卫德强; 唐燕; 许琦

doi:10.13898/j.cnki.issn.1000-2200.2023.03.003

FGD5-AS1/miR-519d-3p轴调控高糖诱导的肾小管上皮细胞HK-2炎症因子表达和细胞凋亡

FGD5-AS1/miR-519d-3p axis regulates high glucose-induced inflammatory factor expression and apoptosis in renal tubular epithelial HK-2 cells

摘要

摘要:
目的探讨FGD5-AS1对高糖诱导的肾小管上皮细胞HK-2肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)表达、细胞凋亡的影响及其机制。
方法25 mmol/L葡萄糖诱导HK-2细胞24 h后，RT-qPCR检测细胞中FGD5-AS1和miR-519d-3p表达量。分别转染FGD5-AS1过表达载体(pcDNA-FGD5-AS1)、miR-519d-3p抑制剂或共转染pcDNA-FGD5-AS1与miR-519d-3p模拟物至HK-2细胞，用25 mmol/L葡萄糖诱导转染后的细胞24 h，ELISA法检测细胞培养上清液中TNF-α、IL-6水平，流式细胞术、Western blotting分别检测细胞凋亡率及凋亡相关蛋白cleaved-caspase3、cleaved-caspase9的表达。双荧光素酶报告基因实验验证FGD5-AS1和miR-519d-3p的调控关系。
结果与对照组比较，HK-2细胞经高糖诱导后，细胞中FGD5-AS1表达量明显降低(P < 0.01)，miR-519d-3p表达水平明显升高(P < 0.01)。上调FGD5-AS1或下调miR-519d-3p的HK-2细胞经高糖诱导后，炎症因子TNF-α与IL-6水平、细胞凋亡率、凋亡相关蛋白cleaved-caspase3及cleaved-caspase9表达均降低(P < 0.05~P < 0.01)。FGD5-AS1靶向结合miR-519d-3p，且上调FGD5-AS1的HK-2细胞中miR-519d-3p表达明显降低(P < 0.01)。过表达miR-519d-3p可逆转上调FGD5-AS1表达对高糖诱导的HK-2细胞炎症因子TNF-α与IL-6水平和细胞凋亡的影响。
结论上调FGD5-AS1可能通过靶向下调miR-519d-3p抑制高糖诱导的HK-2细胞炎症因子表达和细胞凋亡。

Abstract:
ObjectiveTo investigate the effect of FGD5-AS1 on high glucose-induced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) expression and apoptosis in renal tubular epithelial HK-2 cells and its mechanism.
MethodsAfter HK-2 cells were induced with 25 mmol/L glucose for 24 h, the expressions of FGD5-AS1 and miR-519d-3p in the cells were detected by RT-qPCR.After HK-2 cells were transfected with FGD5-AS1 overexpression vector(pcDNA-FGD5-AS1) and miR-519d-3p inhibitor, or co-transfected with pcDNA-FGD5-AS1 and miR-519d-3p mimic, respectively, they were induced with 25 mmol/L glucose for 24 h, and then the levels of TNF-α and IL-6 in the cell culture supernatant were detected by ELISA, and the apoptosis rate and the expression of apoptosis-related proteins cleaved-caspase3 and cleaved-caspase9 were detected by flow cytometry and Western blotting, respectively.The regulatory relationship between FGD5-AS1 and miR-519d-3p was verified by the dual-luciferase reporter gene assay.
ResultsCompared with the control group, the expression of FGD5-AS1 in HK-2 cells induced by high glucose significantly decreased (P < 0.01), and the expression of miR-519d-3p significantly increased (P < 0.01).After FGD5-AS1 was up-regulated or miR-519d-3p was down-regulated in HK-2 cells, and the cells were induced by high glucose, the levels of inflammatory factors TNF-α and IL-6, apoptosis rate, expression of apoptosis-related proteins cleaved-caspase3 and cleaved-caspase9 were decreased (P < 0.05 to P < 0.01).FGD5-AS1 could targetedly bind to miR-519d-3p, and the expression of miR-519d-3p was significantly decreased in HK-2 cells with up-regulated FGD5-AS1 (P < 0.01).Overexpression of miR-519d-3p reversed the effect of up-regulating FGD5-AS1 expression on the expression of inflammatory factors TNF-α and IL-6, and apoptosis in HK-2 cells induced by high glucose.
ConclusionsUp-regulation of FGD5-AS1 may inhibit high glucose-induced expression of inflammatory factors and apoptosis in HK-2 cells through targeted down-regulation of miR-519d-3p.

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