Abstract

MiR-429 was reported to be downregulated in contrast-induced acute kidney injury (CI-AKI). However, whether miR-429 is functionally relevant with CI-AKI needs further investigation. Human renal tubular epithelial cell (HK-2) cells were stimulated with contrast media iodixanol to establish in vitro CI-AKI model. Cell Counting Kit-8 (CCK-8) was applied to access cell viability. Flow cytometry was performed to determine apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to evaluate level of programmed cell death 4 (PDCD4) mRNA and miR-429 while western blot was applied to evaluate level of proteins including PDCD4, B-cell leukaemia/lymphoma 2 (Bcl-2), BCL2-associated X protein (Bax), cleaved caspase 3, cleaved caspase 9, p65, phosphorylated p65. Dual luciferase assay was used to validate miR-429 targeting PDCD4. MiR-429 was downregulated whereas PDCD4 was upregulated in contrast media iodixanol-stimulated HK-2 cells. MiR-429 overexpression elevated cell viability and attenuated cell apoptosis. Moreover, the activation of nuclear factor kappa-B (NF-κB) signalling was suppressed after miR-429 overexpression, while PDCD4 overexpression reversed these effects. MiR-429 directly targeted PDCD4 and negatively regulated its expression. CI-AKI induced NF-κB signalling activation and PDCD4 overexpression further promoted NF-κB signalling activation. However, the treatment of BAY11-7082 reversed above results. Overexpression of miR-429 attenuated apoptosis and elevated cell viability in a CI-AKI cell model via targeting PDCD4 and thus restraining NF-κB signalling.

摘要

据报道 MiR-429 在造影剂诱导的急性肾损伤 (CI-AKI) 中下调。然而，miR-429 是否与 CI-AKI 功能相关需要进一步研究。用造影剂碘克沙醇刺激人肾小管上皮细胞 (HK-2) 细胞在体外建立CI-AKI 模型。Cell Counting Kit-8 (CCK-8) 用于获取细胞活力。进行流式细胞术以确定细胞凋亡。定量实时聚合酶链反应 (qRT-PCR) 用于评估程序性细胞死亡 4 (PDCD4) mRNA 和 miR-429 的水平，而蛋白质印迹用于评估蛋白质水平，包括 PDCD4、B 细胞白血病/淋巴瘤 2 (Bcl-2)、BCL2 相关 X 蛋白 (Bax)、切割的 caspase 3、切割的 caspase 9、p65、磷酸化 p65。双荧光素酶测定用于验证靶向 PDCD4 的 miR-429。MiR-429 在造影剂碘克沙醇刺激的 HK-2 细胞中下调，而 PDCD4 上调。MiR-429 过表达提高细胞活力并减弱细胞凋亡。此外，核因子 kappa-B (NF-κB) 信号的激活在 miR-429 过表达后被抑制，而 PDCD4 过表达则逆转了这些影响。MiR-429 直接靶向 PDCD4 并负调控其表达。CI-AKI 诱导 NF-κB 信号激活，PDCD4 过表达进一步促进 NF-κB 信号激活。然而，BAY11-7082 的处理逆转了上述结果。在 CI-AKI 细胞模型中，miR-429 的过表达减弱了细胞凋亡并提高了细胞活力通过靶向 PDCD4 从而抑制 NF-κB 信号传导。