miR-216a-5p promotes mesangial cell proliferation by targeting FoxO1 in diabetic nephropathy

Abstract

Background: Diabetic nephropathy (DN) is a leading cause of end-stage renal disease worldwide. microRNAs (miRNAs) have been reported to play essential roles in DN progression. However, the mechanism of miR-216a-5p on DN progression is still unclear.

Methods: A DN model was established in human mesangial cells (HMC) by high glucose treatment. Cell proliferation was investigated using the cell counting kit-8 (CCK-8) assay. The cell cycle was measured through a propidium iodide (PI) cell cycle kit with flow cytometry. The interaction between miR-216a-5p and forkhead boxO1 (FoxO1) was probed by a bioinformatics analysis and luciferase activity assay. The expression of miR-216a-5p was detected using a quantitative real-time polymerase chain reaction (qRT-PCR). The abundances of FoxO1 and cell cycle-related cyclinD1, cyclin-dependent kinase 4 (CDK4), CDK6 and p27 were examined by qRT-PCR and Western blots (WB).

Results: miR-216a-5p was up-regulated while FoxO1 was down-regulated in DN tissues. Moreover, miR-216a-5p promoted cell proliferation by regulating the cell cycle in high glucose-treated HMC cells. Notably, FoxO1 was a direct target and negatively correlated with miR-216a-5p. In addition, miR-216a induced cyclinD1, CDK4 and CDK6 but inhibited p27 expressions at the mRNA and protein levels. Furthermore, FoxO1 restoration reversed the regulatory effect of miR-216a on the cell cycle by regulating cyclinD1, CDK4, CDK6 and p27 abundances at the mRNA and protein levels.

Conclusion: miR-216a-5p is ectopic in DN and it promotes cell proliferation through regulating the cell cycle by targeting FoxO1 in high glucose-stimulated HMC cells, indicating it may serve as a novel biomarker for DN treatment.

Keywords: Diabetic nephropathy; FoxO1; cell cycle; mesangial cells; miR-216a-5p; proliferation.

Figure 1

miR-216a-5p was highly expressed while FoxO1 was reduced in DN. A. The expression of miR-216a-5p was detected in renal biopsies of patients with or without DN. B. The FoxO1 mRNA level was measured in renal tissues by qRT-PCR. C, D. The abundance of the FoxO1 protein was examined by WB. E. The expression of miR-216a-5p in serum was measured by qRT-PCR. *P < 0.05.

Figure 2

The addition of miR-216a-5p promoted cell proliferation in HMC cells. A. The effect of miR-216a-5p on cell proliferation was investigated in HMC cells cultured with high glucose or normal medium after transfection at 24, 48 or 72 h. B, C. The effect of miR-216a-5p on the cell cycle was evaluated in HMC cells with transfection for 72 h by flow cytometry. *P < 0.05.

Figure 3

FoxO1 was a target of miR-216a-5p. A. The potential binding sites of FoxO1 and miR-216a-5p were predicted by sequence alignment. B. The luciferase activity was investigated in HMC cells co-transfected FoxO1-wt or FoxO1-mut with miR-216a-5p or miR-216a-5p NC. C. The effect of miR-216a-5p on FoxO1 mRNA expression was measured in HMC cells with both high and normal glucose treatment. D, E. The FoxO1 protein abundance was measured in the transfected HMC cells. *P < 0.05.

Figure 4

miR-216a-5p regulated the expression of the cell-cycle-related biomarkers in HMC cells. A. The expression of miR-216a-5p was measured in the HMC cells after transfection with miR-216a-5p or anti-miR-216a-5p. B-E. The mRNA expressions of cyclinD1, CDK4, CDK6 and p27 were detected in the HMC cells. F-J. The protein abundances of cyclinD1, CDK4, CDK6 and p27 were examined in HMC cells. *P < 0.05.

Figure 5

FoxO1 reversed the regulatory effect of miR-216a-5p on the cell cycle in HMC cells. A-E. The effect of FoxO1 on the mRNA expressions of FoxO1, cyclinD1, p27, CDK4 and CDK6 was investigated in miR-216a-5p-transfected HMC cells. F-K. The effect of FoxO1 on the protein levels of FoxO1, cyclinD1, p27, CDK4 and CDK6 was evaluated in cells with miR-216a-5p transfection. *P < 0.05.