Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
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Advances in Clinical and Experimental Medicine

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doi: 10.17219/acem/147891

Publication type: original article

Language: English

License: Creative Commons Attribution 3.0 Unported (CC BY 3.0)

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MiR-218 promotes oxidative stress and inflammatory response by inhibiting SPRED2-mediated autophagy in HG-induced HK-2 cells

Lanfang Fu1,A,D,F, Xinxin Huang1,B,C, Juyun Zhang1,C,E, Zhu Lin1,C,E, Guijun Qin2,A,E,F

1 Department of Endocrinology, Haikou Affiliated Hospital of Central South University, Xiangya School of Medicine, China

2 Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University, China

Abstract

Background. Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus (DM). MicroRNA (miR)-218 is associated with the development of diabetes. Besides, sprouty-related EVH1 domain containing 2 (SPRED2), the downstream target of miR-218, is involved in insulin resistance and inflammation.
Objectives. Since inflammation plays a key role in DN, and SPRED2 is known to facilitate cell autophagy, the present study aimed to investigate the role and molecular mechanism of miR-218 and SPRED2-mediated autophagy in high glucose (HG)-induced renal tubular epithelial cells using an in vitro model.
Material and Methods. The HK-2 cells were cultured in 5.5 mM or 30 mM D-glucose medium. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-218 and SPRED2. Western blotting was performed to calculate the levels of SPRED2, inflammatory cytokines, autophagy-related and apoptosis-related proteins. Reactive oxygen species (ROS) level was evaluated using cellular ROS assay kit, superoxide dismutase (SOD) activity was detected using SOD activity assay kit, and malondialdehyde (MDA) content was measured using lipid peroxidation. The levels of interleukin (IL)-1β, IL-6, IL-4, and tumor necrosis factor alpha (TNF-α) were detected with enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was evaluated using flow cytometry analysis. The targeting relationship between miR-218 and SPRED2 was identified with a luciferase reporter. The LC3-II expression was detected with immunofluorescence.
Results. The miR-218 expression was upregulated and SPRED2 expression was downregulated in HG-induced HK-2 cells. The miR-218 was proven to target SPRED2 and negatively regulate SPRED2 expression. Besides, downregulated miR-218 alleviated inflammatory response, oxidative stress and cell apoptosis, but aggravated autophagy. We also showed that downregulated SPRED2 reversed the effect of miR-218 on inflammation, cell apoptosis and autophagy in HG-induced HK-2 cells.
Conclusion. The miR-218 can promote oxidative stress and inflammatory response in HG-induced renal tubular epithelial cells by inhibiting SPRED2-mediated autophagy. This study might bring novel understanding for molecular mechanism of DN.

Key words

miR-218, renal tubular epithelial cell, autophagy, SPRED2, high glucose

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