Downregulation of miR‑214-3p attenuates mesangial hypercellularity by targeting PTEN‑mediated JNK/c-Jun signaling in IgA nephropathy

Abstract

Mesangial cell (MC) proliferation and matrix expansion are basic pathological characteristics of IgA nephropathy (IgAN). However, the stepwise mechanism of MC proliferation and the exact set of related signaling molecules remain largely unclear. In this study, we found a significant upregulation of miR-214-3p in the renal cortex of IgAN mice by miRNA sequencing. In situ hybridization analysis showed that miR-214-3p expression was obviously elevated in MCs in the renal cortex in IgAN. Functionally, knockdown of miR-214-3p alleviated mesangial hypercellularity and renal lesions in IgAN mice. In vitro, the inhibition of miR-214-3p suppressed MC proliferation and arrested G1-S cell cycle pSrogression in IgAN. Mechanistically, a luciferase reporter assay verified PTEN as a direct target of miR-214-3p. Downregulation of miR-214-3p increased PTEN expression and reduced p-JNK and p-c-Jun levels, thereby inhibiting MC proliferation and ameliorating renal lesions in IgAN. Moreover, these changes could be attenuated by co-transfection with PTEN siRNA. Collectively, these results illustrated that miR-214-3p accelerated MC proliferation in IgAN by directly targeting PTEN to modulate JNK/c-Jun signaling. Therefore, miR-214-3p may represent a novel therapeutic target for IgAN.

Keywords: IgA nephropathy; JNK/c-Jun signaling; PTEN; mesangial cell proliferation; miR‑214-3p.

Figure 1

miR-214-3p expression was upregulated in IgAN mesangial cells (MCs). (A) Immunofluorescence staining of IgA in the glomeruli. Original magnification, 400x. (B-C) Representative images of hematoxylin and eosin (HE) staining and periodic acid-Schiff (PAS) staining in control and IgAN mice. Original magnification, 400x. (D) Proteinuria was reported as the ratio of urinary albumin to urinary creatinine (ACR). (E-F) Clustering map of miRNA expression in the renal cortex from control and IgAN mice (P < 0.05). (G) The differential expression of miR-214-3p in the renal cortex of control and IgAN mice was validated by real-time PCR. The relative level of miR-214-3p was normalized to the level of U6, and the ratio of control mice was arbitrarily set as 1. (H) In situ hybridization analysis showed that miR-214-3p was significantly elevated in MCs from the mouse renal cortex following IgAN. Original magnification, 400x. (I) The differential expression of miR-214-3p in MCs from the control and IgAN groups was validated by real-time PCR. The relative level of miR-214-3p was normalized to the expression of U6, and the ratio of control mice was arbitrarily set as 1. All data are expressed as the mean ± SD. *P < 0.05, **P < 0.01.

Figure 2

Knockdown of miR-214-3p alleviated renal lesions in IgAN mice. IgAN mice were subjected to tail intravenous injection of 20 mg/kg miR-214-3p antagomir or negative control (antagomir NC) every two weeks for 3 consecutive days from the sixth week to the eleventh week. (A) The expression of miR-214-3p in the renal cortex. The relative level of miR-214-3p was normalized to the expression of U6, and the ratio of control mice was arbitrarily set as 1. (B) Immunofluorescence staining of IgA in the glomeruli. Original magnification, 400x. (C-D) Representative images of hematoxylin and eosin (HE) staining and periodic acid-Schiff (PAS) staining. Original magnification, 400x. (E) Proteinuria was reported as the ratio of urinary albumin to urinary creatinine (ACR). (F) Immunoblot analyses and (G) quantitative determination of PCNA and cyclinD1 protein levels in the renal cortex. For densitometry, the signals of the target proteins were normalized to the β-actin signal of the same samples to determine the ratios. All data are expressed as the mean ± SD. *P < 0.05, **P < 0.01.

Figure 3

Inhibition of miR-214-3p suppressed mesangial cell (MC) proliferation. MCs were transfected with miR-214-3p inhibitor or negative control (inhibitor NC) and then treated with 25 µg/mL p-IgA1 for 24 hours. (A) The expression of miR-214-3p in MCs. The relative level of miR-214-3p was normalized to the expression of U6, and the ratio of control mice was arbitrarily set as 1. (B) Cell viability was measured by CCK8 assay. (C-D) Cell cycle analysis was performed by staining DNA with propidium iodide prior to flow cytometry. The proliferation index (PI) was calculated as the sum of the percentage of cells in S phase and G2/M phase. (E) Immunoblot analyses and (F) quantitative determination of PCNA and cyclinD1 protein levels in MCs. For densitometry, the signals of the target proteins were normalized to the β-actin signal of the same samples to determine the ratios. All data are expressed as the mean ± SD. *P < 0.05, **P < 0.01.

Figure 4

The effect of miR-214-3p on PTEN expression in IgAN. (A) PTEN mRNA levels in the mouse renal cortex were determined by real-time PCR. PTEN levels were normalized to GAPDH, and the ratio of control mice was arbitrarily set as 1. (B) Immunoblot analyses and (C) quantitative determination of PTEN protein expression in the renal cortex. For densitometry, the PTEN protein signal was normalized to the GAPDH signal of the same sample to determine the ratio. (D) Immunohistochemical staining illustrated the repressive effect of miR-214-3p on PTEN expression in glomeruli. (E) PTEN mRNA levels in mesangial cells (MCs) were determined by real-time PCR. PTEN levels were normalized to GAPDH, and the ratio of the control group was arbitrarily set as 1. (F) Immunoblot analyses and (G) quantitative determination of PTEN protein expression in MCs. For densitometry, the PTEN protein signal was normalized to the GAPDH signal of the same sample to determine the ratio. (H) Immunofluorescence illustrated the repressive effect of miR-214-3p on PTEN expression in MCs. All data are expressed as the mean ± SD. *P < 0.05, **P < 0.01.

Figure 5

miR-214-3p regulated activation of the JNK/c-Jun pathway. (A) Immunoblot analyses and (B) quantitative determination of p-JNK and p-c-Jun protein levels in the renal cortex. (C) Immunoblot analyses and (D) quantitative determination of p-JNK and p-c-Jun protein levels in mesangial cells. For densitometry, the signals of phosphorylated protein were normalized to the corresponding total protein signals of the same samples to determine the ratios. All data are expressed as the mean ± SD. *P < 0.05, **P < 0.01.

Figure 6

miR-214-3p targeted PTEN to promote mesangial cell (MC) proliferation through the JNK/c-Jun pathway in IgAN. MCs were co-transfected with miR-214-3p mimic or negative control (mimic NC) and PTEN siRNA or control siRNA for 6 hours and then treated with 25 µg/ml p-IgA1 for 24 hours. (A, C) Immunoblot analyses and (B, D) quantitative determination of PTEN, p-JNK, p-c-Jun, PCNA and cyclinD1 protein levels. For densitometry, the PTEN protein signal was normalized to the GAPDH signal, the phosphorylated protein signal was normalized to the corresponding total protein signal, and the PCNA and cyclinD1 signals were normalized to the β-actin signal. (E) Cell viability was measured by CCK8 assay. (F-G) Cell cycle analysis was performed by staining DNA with propidium iodide prior to flow cytometry. The proliferation index (PI) was calculated as the sum of the percentage of cells in S phase and G2/M phase. All data are expressed as the mean ± SD. *P < 0.05, **P < 0.01.

Figure 7

miR-214-3p is involved in mesangial cell proliferation by targeting PTEN‑mediated JNK/c-Jun signaling in IgA nephropathy. p-IgA1 upregulated the expression of miR-214-3p, which targets PTEN to activate JNK/c-Jun signaling, thereby promoting mesangial cell proliferation by accelerating G1-S cell cycle progression.