Circ-FBXW12 aggravates the development of diabetic nephropathy by binding to miR-31-5p to induce LIN28B

Abstract

Background: The 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.

Methods: Reverse 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.

Results: Circ-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.

Conclusion: Circ-FBXW12 knockdown suppressed HG-induced HMC growth, inflammation, ECM accumulation and oxidative stress by regulating miR-31-5p/LIN28B axis.

Keywords: Circ-FBXW12; DN; HMCs; LIN28B; miR-31-5p.

Fig. 1

Circ-FBXW12 was increased in DN patients and HG-treated HMCs. A The expression of circ-FBXW12 in the serums of DN patients, diabetic patients without DN and normal controls was detected by RT-qPCR assay. B The expression of circ-FBXW12 in HG-stimulated HMCs was detected by RT-qPCR assay. C The levels of circ-FBXW12 and FBXW12 in HMCs treated with or without RNase R were determined using RT-qPCR assay. D The expression of circ-FBXW12 in the cytoplasm and nucleus of HMCs was examined by RT-qPCR assay. **P < 0.01, ****P < 0.0001

Fig. 2

Circ-FBXW12 silencing reversed HG-mediated effects on cell growth, inflammation, cell cycle, ECM production and oxidative stress in HMCs. HMCs were assigned to Control, HG, HG + si-NC and HG + si-circ-FBXW12 groups. A The expression of circ-FBXW12 in HMCs was determined by RT-qPCR assay. B The viability of HMCs was assessed by CCK-8 assay. C The concentrations of IL-6 and TNF-α in HMCs were examined with ELISA kits. D The cell cycle process in HMCs was analyzed by flow cytometry analysis. E The proliferation of HMCs was evaluated by EdU assay. F–H The protein levels of CyclinD1, P21, collagen I, collagen IV and TGF-β1 in HMCs were measured via western blot assay. I and J The activity of SOD and the level of MDA in HMCs were examined with specific commercial kits. **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Circ-FBXW12 directly interacted with miR-31-5p. A The binding sites between circ-FBXW12 and miR-31-5p. B The expression of miR-31-5p in HMCs transfected with miR-NC or miR-31-5p was detected by RT-qPCR assay. C and D The interaction between miR-31-5p and circ-FBXW12 was demonstrated by dual-luciferase reporter assay and RIP assay. E and F The expression of miR-31-5p in DM, DN patients’ serums and HG-stimulated HMCs was determined through RT-qPCR assay. G The linear correlation between the levels of circ-FBXW12 and miR-31-5p in the serums of DN patients was analyzed by Spearman’s correlation coefficient analysis. H The expression of circ-FBXW12 in HMCs transfected with pCD5-ciR or circ-FBXW12 in HG condition was detected by RT-qPCR assay. I After HMCs were transfected with si-NC, si-circ-FBXW12, pCD5-ciR or circ-FBXW12 in HG condition, the expression of miR-31-5p was detected by RT-qPCR assay. ***P < 0.001, ****P < 0.0001

Fig. 4

Circ-FBXW12 regulated cell proliferation, inflammation, cell cycle, ECM production and oxidative stress in HG-treated HMCs by targeting miR-31-5p. A The expression of miR-31-5p in HMCs transfected with anti-miR-NC or anti-miR-31-5p was determined by RT-qPCR assay. (B-I) HMCs cells were transfected with si-NC, si-circ-FBXW12, si-circ-FBXW12 + anti-miR-NC or si-circ-FBXW12 + anti-miR-31-5p under HG condition. B The expression of miR-31-5p in HMCs was detected by RT-qPCR assay. C HMC viability was assessed by CCK-8 assay. D The concentrations of IL-6 and TNF-α in HMCs were examined with ELISA kits. E The cell cycle process in HMCs was analyzed by flow cytometry analysis. F The proliferation of HMCs was assessed by EdU assay. G–I The protein levels of CyclinD1, P21, collagen I, collagen IV and TGF-β1 in HMCs were measured via western blot assay. J and K SOD activity and MDA level in HMCs were detected with specific kits. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

MiR-31-5p directly targeted LIN28B. A LIN28B contained miR-31-5p binding sites. B and C Dual-luciferase reporter assay and RIP assay were conducted to analyze the interaction between miR-31-5p and LIN28B. D The mRNA level of LIN28B in the serums of DM, DN patients and healthy volunteers was detected by RT-qPCR assay. E The correlation between the levels of mIR-31-5p and LIN28B in DN patients’ serums was analyzed by Spearman’s correlation coefficient analysis. F The protein level of LIN28B in HG-treated HMCs was measured via western blot assay. G The protein level in HG-treated HMCs transfected with miR-NC, miR-31-5p, anti-miR-NC or anti-miR-31-5p was measured through western blot assay. ***P < 0.001, ****P < 0.0001

Fig. 6

LIN28B knockdown suppressed cell viability, inflammation, cell cycle process, ECM production and oxidative stress in HG-treated HMCs. HMCs were transfected with si-con or si-LIN28B under HG condition. A The protein level of LIN28B in HMCs was measured via western blot assay. B The viability of HMCs was evaluated by CCK-8 assay. C The levels of IL-6 and TNF-α in HMCs were determined by ELISA kits. D The cell cycle process in HMCs was analyzed by flow cytometry analysis. E The proliferation of HMCs was tested by EdU assay. F–H The protein levels of CyclinD1, P21, collagen I, collagen IV and TGF-β1 in HMCs were measured via western blot assay. I and J The activity of SOD and the level of MDA in HMCs were measured by relevant kits. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 7

Overexpression of miR-31-5p directly targeted LIN28B to repress the development of HG-stimulated HMCs. A The protein level of LIN28B in HMCs transfected with pcDNA or LIN28B was measured via western blot assay. B–I HMCs were transfected with miR-NC, miR-31-5p, miR-31-5p + pcDNA or miR-31-5p + LIN28B in HG condition. B The protein level of LIN28B in HMCs was measured using western blot assay. C HMC viability was assessed by CCK-8 assay. D The concentrations of IL-6 and TNF-α in HMCs were examined with ELISA kits. E Cell cycle in HMCs was analyzed by flow cytometry analysis. F HMC proliferation was evaluated by EdU assay. G–I The protein levels of CyclinD1, P21, collagen I, collagen IV and TGF-β1 in HMCs were measured by western blot assay. J and K The activity of SOD and the level of MDA in HMCs were examined with commercial kits. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 8

Circ-FBXW12 regulated LIN28B expression by targeting miR-31-5p. A and B After HMCs were transfected with si-NC, si-circ-FBXW12, si-circ-FBXW12 + anti-miR-NC or si-circ-FBXW12 + anti-miR-31-5p in HG condition, the mRNA and protein levels of LIN28B were determined by RT-qPCR assay and western blot assay, respectively. ***P < 0.001, ****P < 0.0001