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. 2020 Feb 10:2020:8708236.
doi: 10.1155/2020/8708236. eCollection 2020.

Metformin Reduces the Senescence of Renal Tubular Epithelial Cells in Diabetic Nephropathy via the MBNL1/miR-130a-3p/STAT3 Pathway

Xue Jiang  1 , Xue-Lei Ruan  2   3   4 , Yi-Xue Xue  2   3   4 , Shuang Yang  1 , Mai Shi  1 , Li-Ning Wang  1
Affiliations

Metformin Reduces the Senescence of Renal Tubular Epithelial Cells in Diabetic Nephropathy via the MBNL1/miR-130a-3p/STAT3 Pathway

Xue Jiang et al. Oxid Med Cell Longev. .

Abstract

Senescence of renal tubular epithelial cells plays an important role in diabetic nephropathy, but the mechanism is unknown. Metformin may alleviate diabetic nephropathy by reducing this senescence. This study is aimed at clarifying the effects and mechanism of metformin on the senescence of renal tubular epithelial cells in diabetic nephropathy. We found that metformin reduced the expression of senescence-associated gene P21 in high-glucose-induced (30 mmol/L) renal tubular epithelial cells and decreased the β-galactosidase positive staining rate (decreased 16%, p < 0.01). Metformin was able to reduce senescence by upregulating the expression of RNA-binding protein MBNL1 and miR-130a-3p and reducing STAT3 expression. MBNL1 prolonged the half-life of miR-130a-3p, and miR-130a-3p could negatively regulate STAT3 by binding to its mRNA 3'UTR. In db/db diabetic mice, we found an enhanced senescence level combined with low expression of MBNL1 and miR-130a-3p and high expression of STAT3 compared with db/m control mice during nephropathy development. Meanwhile, metformin (200 mg/kg/day) could increase the expression of MBNL1 and miR-130a-3p and decreased STAT3 expression, thus reducing this senescence in db/db mice. Our results suggest that metformin reduces the senescence of renal tubular epithelial cells in diabetic nephropathy via the MBNL1/miR-130a-3p/STAT3 pathway, which provided new ideas for the therapy of this disease.

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Conflict of interest statement

All authors have read and approved the final manuscript. And there is no conflict of interest. All authors declare of no financial interest. Our manuscript has not been published previously.

Figures

Figure 1
Figure 1
Metformin reduced HK-2 cell senescence that was induced by HG. (a–c) Western blot was used to determine the expression of P21 in HK-2 cells that were treated with NG, MA, and HG at different times. (d) The percentage of SA-β-gal-positive cells was detected in HK-2 cells that were exposed to NG, MA, and HG for 72 h. (e) P21 protein expression levels and (f) the percentage of SA-β-gal-positive cells were detected in HK-2 cells that were exposed to NG, NG+Met, HG, and HG+Met for 72 h. The data are expressed as the mean ± SD (n = 3/group). ∗∗p < 0.01, vs. the NG group; ##p < 0.01, vs. HG group. Scale bars = 50 μm.
Figure 2
Figure 2
MBNL1 was downregulated in senescent HK-2 cells, and metformin reduced cell senescence in HK-2 cells by upregulating MBNL1. (a) qRT-PCR was performed to detect expression levels of MBNL1 in HK-2 cells that were cultured in NG and HG for 72 h. (b) MBNL1 protein expression levels in HK-2 cells that were cultured in NG and HG for 72 h. The data are expressed as the mean ± SD (n = 3/group). ∗p < 0.05, vs. the NG group; ∗∗p < 0.01, vs. the NG group. (c) The expressions of MBNL1, miR-130a-3p, and STAT3 were detected in HK-2 cells with MBNL1 overexpression or inhibition by qRT-PCR. (d) The protein expression levels of MBNL1, STAT3, and P21 were detected to determine the effect of MBNL1 on senescence in HK-2 cells. The data are expressed as the mean ± SD (n = 5/group). ∗p < 0.05, vs. the MBNL1(+)-NC group; ∗∗p < 0.01, vs. the MBNL1(+)-NC group; #p < 0.05, vs. the MBNL1(-)-NC group; ##p < 0.01, vs. MBNL1(-)-NC group. (e) The expressions of MBNL1, miR-130a-3p, and STAT3 were detected in HK-2 cells treated with metformin by qRT-PCR. (f) The protein expression levels of MBNL1, STAT3, and P21 were detected in HK-2 cells that were treated with metformin. The data are expressed as the mean ± SD (n = 3/group). ∗p < 0.05, vs. the control group; ∗∗p < 0.01, vs. the control group; #p < 0.05, vs. the Met group; ##p < 0.01, vs. the Met group.
Figure 3
Figure 3
miR-130a-3p was downregulated in senescent HK-2 cells, and metformin reduced cell senescence in HK-2 cells by upregulating miR-130a-3p. (a) qRT-PCR was used to detect expression levels of miR-130a-3p in HK-2 cells that were cultured in NG and HG for 72 h. The data are expressed as the mean ± SD (n = 3/group). ∗∗p < 0.01, vs. NG group. (b) The RNA expression levels of miR-130a-3p and STAT3 were detected in HK-2 cells after miR-130a-3p overexpression or knockdown. (c) The protein expression levels of STAT3 and P21 were detected in HK-2 cells. The data are expressed as the mean ± SD (n = 5/group). ∗∗p < 0.01, vs. the miR-130a-3p(+)-NC group; #p < 0.05, vs. the miR-130a-3p(-)-NC group; ##p < 0.01, vs. the miR-130a-3p(-)-NC group. (d) The RNA expression levels of miR-130a-3p and STAT3 were detected in HK-2 cells after treatment with metformin. (e) The protein expression levels of STAT3 and P21 were detected in HK-2 cells after treatment with metformin. The data are expressed as the mean ± SD (n = 3/group). ∗p < 0.05, vs. the control group; ∗∗p < 0.01, vs. the control group; #p < 0.05, vs. the Met group; ##p < 0.01, vs. the Met group.
Figure 4
Figure 4
MBNL1 bound with miR-130a-3p, and metformin reduced cell senescence in HK-2 cells by upregulating MBNL1/miR-130a-3p signaling. (a) miR-130a-3p was identified in the MBNL1 complex. miR-130a-3p enrichment was measured using qRT-PCR. The data are expressed as the mean ± SD (n = 3/group). ∗p < 0.05, vs. the anti-IgG group; ∗∗p < 0.01, vs. the anti-IgG group; ##p < 0.01, vs. the NG group. (b) The levels of MBNL1 and GAPDH proteins that immunoprecipitated with miR-130a-3p were evaluated by Western blot. The expression levels of MBNL1 and GAPDH proteins are shown. (c) The Cell-Light EU Apollo567 RNA Stain Kit was used to label and capture newly synthesized RNA. (d) Relative levels of miR-130a-3p at different actinomycin D treatment times in the control group, MBNL1(+)-NC group, and MBNL1(+) group. (e) The expression levels of STAT3 and P21 were detected to determine the effect of MBNL1 and miR-130a-3p on senescence in HK-2 cells. The data are expressed as the mean ± SD (n = 3/group). ∗p < 0.05, vs. the control group; ∗∗p < 0.01, vs. the control group; #p < 0.05, vs. the MBNL1(+) group; ##p < 0.01, vs. the MBNL1(+) group; &p < 0.05, vs. the MBNL1(-) group; &&p < 0.01, vs. the MBNL1(-) group; ∧p < 0.05, vs. the miR-130a-3p(+) group; ∧∧p < 0.01, vs. the miR-130a-3p(+) group; %p < 0.05, vs. the miR-130a-3p(-) group. (f) STAT3 and P21 protein expression levels were detected in HK-2 cells after treatment with metformin. The data are expressed as the mean ± SD (n = 3/group). ∗∗p < 0.01, vs. the control group; #p < 0.05, vs. the Met group; ##p < 0.01, vs. the Met group.
Figure 5
Figure 5
Metformin reduced cell senescence in HK-2 cells through miR-130a-3p/STAT3 signaling. (a) qRT-PCR was used to detect expression levels of STAT3 in HK-2 cells that were cultured in NG and HG for 72 h. (b) STAT3 protein expression levels in HK-2 cells that were cultured in NG and HG for 72 h. The data are expressed as the mean ± SD (n = 5/group). ∗p < 0.05, vs. the NG group; ∗∗p < 0.01, vs. the NG group. (c) The mRNA expression of STAT3 was detected in HK-2 cells. (d) The protein expressions of STAT3 and P21 were detected to determine the effect of STAT3 on senescence in HK-2 cells. The data are expressed as the mean ± SD (n = 3/group). ∗p < 0.05, vs. the STAT3(+)-NC group; ∗∗p < 0.01, vs. the STAT3(+)-NC group; ##p < 0.01, vs. the STAT3(-)-NC group. (e) The expressions of STAT3 and P21 were detected in HK-2 cells after treatment with metformin. The data are expressed as the mean ± SD (n = 3/group). ∗∗p < 0.01, vs. the control group; #p < 0.05, vs. the Met group; ##p < 0.01, vs. Met the group. (f) Predicted miR-130a-3p binding site in STAT3 (STAT3-Wt) and mutant sequence (STAT3-Mut). The relative luciferase activity was conducted after cells were cotransfected with STAT3-WT (or STAT3-Mut) and miR-130a-3p-NC (or miR-130a-3p). The data are expressed as the mean ± SD (n = 3/group). ∗∗p < 0.01, vs. the STAT3-Wt+miR-130a-3p-NC group. (g) The expression of P21 was detected to determine the effect of miR-130a-3p, STAT3, and metformin on senescence in HK-2 cells. The data are expressed as the mean ± SD (n = 3/group). ∗p < 0.05, vs. the miR-130a-3p(+)-NC+STAT3(+)-NC group; ∗∗p < 0.01, vs. the miR-130a-3p(+)-NC+STAT3(+)-NC group; #p < 0.05, vs. the miR-130a-3p(+)+STAT3(+)-NC group; &p < 0.05, vs. the miR-130a-3p(+)+STAT3(+) group.
Figure 6
Figure 6
Metformin improved the renal function and pathological changes in db/db mice. (a) Western blot was used to determine the expressions of P-AMPK and AMPK in HK-2 cells that were treated with HG and HG+Met for 72 h. The data are expressed as the mean ± SD (n = 3/group). ∗p < 0.05, vs. the HG group; ∗∗p < 0.01, vs. the HG group. (b, c) The expressions of MBNL1 and miR-130a-3p were detected in HK-2 cells that were cultured in HG, HG+A-769662, and HG+Dorsomorphin for 72 h. (d) Fasting blood glucose was recorded every week beginning at 8 weeks of age. The data are expressed as the mean ± SD (n = 5/group). ∗∗p < 0.01, vs. the db/m group at 8 weeks; ##p < 0.01, vs. the db/db group at 8 weeks; &&p < 0.01, vs. the db/m group at 16 weeks; %%p < 0.01, vs. the db/db group at 16 weeks; ∧∧p < 0.01, vs. the db/m group at 24 weeks; ++p < 0.01, vs. the db/db group at 24 weeks. (e) Serum creatinine and (f) urine ACR were detected at different weeks. The data are expressed as the mean ± SD (n = 5/group). ∗∗p < 0.01, vs. the db/m group at 16 weeks; #p < 0.05, vs. the db/db group at 16 weeks; &&p < 0.01, vs. the db/m group at 24 weeks; %%p < 0.01, vs. the db/db group at 24 weeks. (g) HE staining, (h) PAS staining, and (i) Masson trichrome staining were performed in renal cortex sections from the db/m group, db/db group, and db/db+Met group at different weeks. Scale bars = 50 μm.
Figure 7
Figure 7
Metformin reduced the senescence of renal tubular epithelial cells in db/db mice through MBNL1/miR-130a-3p/STAT3 signaling. (a) SA-β-gal staining, (b) MBNL1 immunostaining, (c) in situ hybridization for miR-130a-3p, and (d) STAT3 immunostaining were performed in renal cortex sections from the db/m group, db/db group, and db/db + Met group at different weeks. The data are expressed as the mean ± SD (n = 5/group). ∗∗p < 0.01, vs. the db/m group at 16 weeks; #p < 0.05, vs. the db/db group at 16 weeks; ##p < 0.01, vs. the db/db group at 16 weeks; &&p < 0.01, vs. the db/m group at 24 weeks; %p < 0.05, vs. the db/db group at 24 weeks; %%p < 0.01, vs. the db/db group at 24 weeks. Scale bars = 50 μm.
Figure 8
Figure 8
The schematic summary of the regulatory mechanisms. Metformin reduces the senescence of renal tubular epithelial cells in diabetic nephropathy via the MBNL1/miR-130a-3p/STAT3 pathway.
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