Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct;44(4):1539-1549.
doi: 10.3892/or.2020.7718. Epub 2020 Aug 7.

miR‑30a‑5p inhibits hypoxia/reoxygenation‑induced oxidative stress and apoptosis in HK‑2 renal tubular epithelial cells by targeting glutamate dehydrogenase 1 (GLUD1)

Yangbiao He  1 , Xujun Lang  1 , Dong Cheng  1 , Ting Zhang  1 , Zhihao Yang  1 , Rongbing Xiong  1
Affiliations

miR‑30a‑5p inhibits hypoxia/reoxygenation‑induced oxidative stress and apoptosis in HK‑2 renal tubular epithelial cells by targeting glutamate dehydrogenase 1 (GLUD1)

Yangbiao He et al. Oncol Rep. 2020 Oct.

Abstract

MicroRNAs (miRNAs) are reported to be involved in renal hypoxia/reoxygenation (H/R) damage. To investigate this further, human kidney (HK‑2) cells were cultured, subjected to H/R and the function of miR‑30a‑5p and glutamate dehydrogenase 1 (GLUD1) was evaluated. The results showed that, miR‑30‑5p was downregulated and GLUD1 was upregulated in HK‑2 cells exposed to H/R. The relationship between miR‑30a‑5p and GLUD1 was determined using dual luciferase assays. Primary HK‑2 cells were cultured in H/R and transfected with negative control 1 (NC1), negative control 2 (NC2), mimic, inhibitor or GLUD1 siRNA plasmids. Reactive oxygen species (ROS) generation, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, and the rate of apoptosis in HK‑2 cells were assessed. The results showed that, miR‑30a‑5p mimic reduced the production of ROS in HK‑2 cells treated with H/R, but increased the activity of SOD, CAT and GPx. In addition, miR‑30a‑5p mimic significantly decreased H/R‑mediated apoptosis, decreased the expression of bax and activity of caspase‑3 and enhanced the expression of bcl‑2. However, miR‑30a‑5p inhibitor showed the opposite effect with regard to the degree of oxidative damage and apoptosis in H/R‑induced HK‑2 cells. Silencing GLUD1 rescued the influence of miR‑30a‑5p inhibitor on oxidative injury and apoptosis in HK‑2 cells stimulated with H/R. These results demonstrated that under H/R conditions, miR‑30a‑5p can reduce oxidative stress in vitro by targeting GLUD1, which may be a novel therapeutic target for liver failure and worth further study.

Keywords: hypoxia/reoxygenation; miRNA-30a-5p; glutamate dehydrogenase 1; human kidney cells; oxidative stress; apoptosis.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Expression of miR-30a-5p and GLUD1 in hypoxia/reoxygenation-treated HK-2 renal tubular epithelial cells. (A) Expression of miR-30a-5p detected by RT-qPCR. (B) Expression of miR-30a-5p detected by western blot analysis. (C) miR-30a-5p and GLUD1 expression transfected with miR-30a-5p mimic, inhibitor and NC and siRNA GLUD1 and NC in HK-2 renal tubular epithelial cells without hypoxia/reoxygenation. (D) miR-30a-5p and GLUD1 expression transfected with miR-30a-5p mimic, inhibitor and NC and siRNA GLUD1 and NC in HK-2 renal tubular epithelial cells after hypoxia/reoxygenation. *P<0.05 vs. control.
Figure 2.
Figure 2.
miR-30a-5p directly targets GLUD1. (A) Predicted binding site between miR-30a-5p and GLUD1 using TargetScan software. (B) GLUD1 is a target of miR-30a-5p, as shown by dual-luciferase reporter assay. (C) Expression of miR-30a-5p detected by RT-qPCR. (D) Expression of miR-30a-5p detected by western blot analysis. *P<0.05 vs. H/R + NC1, #P<0.05 vs. H/R + NC2.
Figure 3.
Figure 3.
miR-30a-5p inhibits hypoxia/reoxygenation-induced oxidative stress in HK-2 renal tubular epithelial cells. (A) The production of ROS in HK-2 cells induced by H/R. (B) The activity of SOD in HK-2 cells induced by H/R. (C) The activity of CAT in HK-2 cells induced by H/R. (D) The activity of GPx in HK-2 cells induced by H/R. *P<0.05 vs. control, #P<0.05 vs. H/R + NC1, @P<0.05 vs. H/R + NC2.
Figure 4.
Figure 4.
miR-30a-5p inhibits hypoxia/reoxygenation-induced apoptosis in HK-2 renal tubular epithelial cells. (A) Flow cytometry to detect apoptosis induced by H/R. (B) Western blot to detect the expression of proteins related to apoptosis induced by H/R. *P<0.05 vs. control, #P<0.05 vs. H/R + NC1, @P<0.05 vs. H/R + NC2.
Figure 5.
Figure 5.
Inhibition of GLUD1 rescues the effect of miR-30a-5p inhibitor on oxidative stress in HK-2 renal tubular epithelial cells. (A) The protein expression of GLUD1 in HK-2 cells detected by western blot analysis. (B) The production of ROS in HK-2 cells induced by H/R. (C) The activity of SOD in HK-2 cells induced by H/R. (D) The activity of CAT in HK-2 cells induced by H/R. (E) The activity of GPx in HK-2 cells induced by H/R. *P<0.05 vs. H/R + NC2 + si-control, #P<0.05 vs. H/R + inhibitor + si-GLUD1.
Figure 6.
Figure 6.
Inhibition of GLUD1 rescues the effect of miR-30a-5p inhibitor on apoptosis in HK-2 renal tubular epithelial cells. (A) Flow cytometry to detect apoptosis induced by H/R. (B) Western blot to detect the expression of proteins related to apoptosis induced by H/R. *P<0.05 vs. H/R + NC2 + si-control, #P<0.05 vs. H/R + inhibitor + si-GLUD1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Schiffl H. Discontinuation of renal replacement therapy in critically ill patients with severe acute kidney injury: Predictive factors of renal function recovery. Int Urol Nephrol. 2018;50:1845–1851. doi: 10.1007/s11255-018-1947-1. - DOI - PubMed
    1. Xiang HL, Xue WJ, Li Y, Zheng J, Ding C, Dou M, Wu X. C1q/TNF-related protein 6 (CTRP6) attenuates renal ischemia-reperfusion injury through the activation of PI3K/Akt signaling pathway. Clin Exp Pharmacol Physiol. 2020;47:1030–1040. doi: 10.1111/1440-1681.13274. - DOI - PubMed
    1. Feng J, Kong R, Xie L, Lu W, Zhang Y, Dong H, Jiang H. Clemaichinenoside protects renal tubular epithelial cells from hypoxia/reoxygenation injury in vitro through activating the Nrf2/HO-1 signalling pathway. Clin Exp Pharmacol Physiol. 2020;47:495–502. doi: 10.1111/1440-1681.13219. - DOI - PubMed
    1. Bai J, Zhao J, Cui D, Wang F, Song Y, Cheng L, Gao K, Wang J, Li L, Li S, et al. Protective effect of hydroxysafflor yellow a against acute kidney injury via the TLR4/NF-κB signaling pathway. Sci Rep. 2018;8:9173. doi: 10.1038/s41598-018-27217-3. - DOI - PMC - PubMed
    1. Zhou T, Chen YL. The functional mechanisms of miR-30b-5p in acute lung injury in children. Med Sci Monit. 2019;25:40–51. doi: 10.12659/MSM.911398. - DOI - PMC - PubMed

MeSH terms