CircACTR2调节miR-101-3p/NFAT5轴对高糖诱导的肾小管上皮细胞增殖、凋亡和上皮间质转化的影响

王晶; 黄斌芳; 周光全

doi:10.13898/j.cnki.issn.2097-5252.2025.03.001

CircACTR2调节miR-101-3p/NFAT5轴对高糖诱导的肾小管上皮细胞增殖、凋亡和上皮间质转化的影响

Effects of circACTR2 on high glucose-induced cell proliferation,apoptosis and epithelial-mesenchymal transition in renal tubular epithelial cells by regulating miR-101-3p/NFAT5 axis

摘要

摘要: 目的： 探讨环状非编码RNA(circRNA)ACTR2调节微小RNA-101-3p(miR-101-3p)/活化T细胞核因子5(NFAT5)轴对高糖诱导的肾小管上皮细胞增殖、凋亡和上皮间质转化的影响。方法： 体外培养人肾小管上皮细胞HK-2,分为对照组、高糖组(30 mmol/L葡萄糖),同时在高糖组的基础上对HK-2细胞进行转染,设置为小干扰RNA阴性对照(si NC)组、circACTR2特异性小干扰RNA(si circACTR2)组、si circACTR2+抑制物阴性对照(inhibitor NC)组、si circACTR2+miR-101-3p抑制物(miR-101-3p inhibitor)组。流式细胞术、CCK-8分别检测细胞凋亡和增殖；qRT-PCR检测细胞中circACTR2、miR-101-3p及NFAT5 mRNA表达；Western blotting检测各组细胞中增殖蛋白Ki-67、抗凋亡蛋白Bcl-2、E-钙黏附蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、NFAT5表达水平；ELISA检测细胞中白细胞介素6(IL-6)、肿瘤坏死因子-α(TNF-α)水平；双荧光素酶实验验证circACTR2、NFAT5分别与miR-101-3p的靶向关系。结果： miR-101-3p分别与circACTR2、NFAT5存在靶向关系。与对照组相比,高糖组细胞凋亡率、TNF-α、IL-6、circACTR2、NFAT5、α-SMA水平增加(P<0.05),细胞光密度(OD₄₅₀)值(24、48 h)、miR-101-3p、Bcl-2、Ki-67、E-cadherin水平降低(P<0.05)；与si NC组相比,si circACTR2组细胞凋亡率、TNF-α、IL-6、circACTR2、NFAT5、α-SMA水平下降(P<0.05),细胞OD₄₅₀值(24、48 h)、miR-101-3p、Bcl-2、Ki-67、E-cadherin水平增加(P<0.05)；与si circACTR2+inhibitor NC组相比,si circACTR2+miR-101-3p inhibitor组细胞凋亡率、TNF-α、IL-6、circACTR2、NFAT5、α-SMA水平增加(P<0.05),细胞OD₄₅₀值(24、48 h)、miR-101-3p、Bcl-2、Ki-67、E-cadherin水平降低(P<0.05)。结论： 干扰circACTR2表达可以促进高糖诱导HK-2细胞增殖、抑制其凋亡及上皮间质转化,可能与调控miR-101-3p/NFAT5轴有关。

Abstract: Objective: To investigate the effects of circular non-coding RNA (circRNA) ACTR2 on high glucose-induced cell proliferation,apoptosis and epithelial-mesenchymal transition in renal tubular epithelial cells by regulating microRNA-101-3p (miR-101-3p)/nuclear factor of activated T cells 5 (NFAT5) axis. Methods: Human renal tubular epithelial cells HK-2 were cultured in vitro and randomly divided into control group and high glucose group (30 mmol/L glucose).The cells in the high glucose group were transfected and set as small interfering RNA negative control (si NC) group,circACTR2-specific small interfering RNA(si circACTR2) group,si circACTR2+inhibitor negative control (inhibitor NC) group,and si circACTR2+miR-101-3p inhibitor (miR-101-3p inhibitor) group.Flow cytometry and CCK-8 were used to detect apoptosis and proliferation,respectively;qRT-PCR was applied to determine the mRNA expression of circACTR2,miR-101-3p and NFAT5 in cells;Western blotting was employed to analyze the expression levels of proliferation protein Ki-67,anti-apoptotic protein Bcl-2,E-cadherin,α-smooth muscle actin (α-SMA) and NFAT5;ELISA was performed to detect the level of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cells;the dual luciferase assay was used to verify the targeted relationships of circACTR2 and NFAT5 with miR-101-3p. Results: MiR-101-3p had targeted relationships with circACTR2 and NFAT5.Compared with the control group,the apoptosis rate,TNF-α,IL-6,circACTR2,NFAT5 and α-SMA levels in the high glucose group increased (P<0.05),while cell optical density (OD₄₅₀) values (24,48 hours),miR-101-3p,Bcl-2,Ki-67,and E-cadherin levels decreased (P<0.05).Compared with the si NC group,the apoptosis rate,TNF-α,IL-6,circACTR2,NFAT5 and α-SMA levels in the si circACTR2 group decreased (P<0.05),while cell OD₄₅₀ values (24,48 hours),miR-101-3p,Bcl-2,Ki-67 and E-cadherin levels increased (P<0.05).Compared with the si circACTR2+inhibitor NC group,the apoptosis rate,TNF-α,IL-6,circACTR2,NFAT5 and α-SMA levels in the si circACTR2+miR-101-3p inhibitor group increased (P<0.05),while cell OD₄₅₀ values (24,48 hours),miR-101-3p,Bcl-2,Ki-67 and E-cadherin levels decreased (P<0.05). Conclusions: Interfering circACTR2 expression can promote high glucose-induced proliferation,inhibit its apoptosis and epithelial-mesenchymal transition in HK-2 cells,which may be related to the regulation of miR-101-3p/NFAT5 axis.

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