Abstract
BackgroundThe involvement of circular RNAs (circRNAs) in diabetic nephropathy (DN) has been gradually identified. In this study, we aimed to explore the functions of circRNA F-box/WD repeat-containing protein 12 (circ-FBXW12) in DN development.MethodsReverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was performed for the levels of circ-FBXW12, FBXW12 mRNA, microRNA-31-5p (miR-31-5p) and Lin-28 homolog B (LIN28B) mRNA. RNase R assay was used to analyze the stability of circ-FBXW12. Cell Counting Kit-8 (CCK-8) assay, flow cytometry analysis and 5-ethynyl-2′- deoxyuridine (EdU) assay were employed to evaluate cell viability, cell cycle and proliferation, respectively. Enzyme linked immunosorbent assay (ELISA) was done to measure the concentrations of inflammatory cytokines. Western blot assay was conducted for protein levels. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were examined with commercial kits. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the relationships among circ-FBXW12, miR-31-5p and LIN28B.ResultsCirc-FBXW12 level was increased in DN patients’ serums and high glucose (HG)-induced human mesangial cells (HMCs). Circ-FBXW12 knockdown suppressed cell proliferation, arrested cell cycle, reduced extracellular matrix (ECM) production and oxidative stress in HG-induced HMCs. Circ-FBXW12 was identified as the sponge for miR-31-5p, which then directly targeted LIN28B. MiR-31-5p inhibition reversed circ-FBXW12 knockdown-mediated effects on cell proliferation, cell cycle process, ECM production and oxidative in HG-triggered HMCs. Moreover, miR-31-5p overexpression showed similar results with circ-FBXW12 knockdown in HG-stimulated HMC progression, while LIN28B elevation reversed the effects.ConclusionCirc-FBXW12 knockdown suppressed HG-induced HMC growth, inflammation, ECM accumulation and oxidative stress by regulating miR-31-5p/LIN28B axis.
Highlights
Diabetes mellitus is a common metabolic disease that can cause chronic renal impairment and lead to diabetic nephropathy (DN) [1, 2]
Circ‐F-box/WD repeat-containing protein 12 (FBXW12) was highly expressed in DN patients and high glucose (HG)‐induced human mesangial cells (HMCs) To explore the function of circ-FBXW12 in DN progression, the expression of circ-FBXW12 in the serums of DN patients, diabetic patients without DN (DM group) and healthy volunteers was determined by Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay
The results showed that circ-FBXW12 level was apparently increased in DN patients and DM patients in comparison with normal controls (Fig. 1A)
Summary
Diabetes mellitus is a common metabolic disease that can cause chronic renal impairment and lead to diabetic nephropathy (DN) [1, 2]. Circ_0000285 aggravated podocyte proliferation and facilitated apoptosis in DN depending on miR-654-3p/Mitogen-Activated Protein Kinase 6 (MAPK6) axis [10]. Circ_LARP4 hampered cell proliferation and fibrosis and accelerated apoptosis in HG-induced mouse mesangial cells (MMCs) by decoying miR-424 [11]. For circRNA F-box/WD repeatcontaining protein 12 (circ-FBXW12, termed as hsa_ circ_0123996), Wang et al declared that circ_0123996 knockdown suppressed MMC proliferation and fibrosis by upregulating BTB Domain And CNC Homolog 1 (Bach1) via sponging miR-149-5p [12]. We aimed to explore the functions of circRNA F-box/WD repeat-containing protein 12 (circ-FBXW12) in DN development
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