Endocrinol Metab.  2022 Dec;37(6):901-917. 10.3803/EnM.2022.1565.

Inhibition of miR-146a-5p and miR-8114 in Insulin-Secreting Cells Contributes to the Protection of Melatonin against Stearic Acid-Induced Cellular Senescence by Targeting Mafa

• Su S
• Zhao Q
• Dan L
• Lin Y
• Li X
• Zhang Y
• Yang C
• Dong Y
• Li X
• Regazzi R
• Sun C
• Chu X
• Lu H

• KMID: 2537291
• DOI: http://doi.org/10.3803/EnM.2022.1565

Abstract

Background
Chronic exposure to elevated levels of saturated fatty acids results in pancreatic β-cell senescence. However, targets and effective agents for preventing stearic acid-induced β-cell senescence are still lacking. Although melatonin administration can protect β-cells against lipotoxicity through anti-senescence processes, the precise underlying mechanisms still need to be explored. Therefore, we investigated the anti-senescence effect of melatonin on stearic acid-treated mouse β-cells and elucidated the possible role of microRNAs in this process.
Methods
β-Cell senescence was identified by measuring the expression of senescence-related genes and senescence-associated β-galactosidase staining. Gain- and loss-of-function approaches were used to investigate the involvement of microRNAs in stearic acid-evoked β-cell senescence and dysfunction. Bioinformatics analyses and luciferase reporter activity assays were applied to predict the direct targets of microRNAs.
Results
Long-term exposure to a high concentration of stearic acid-induced senescence and upregulated miR-146a-5p and miR- 8114 expression in both mouse islets and β-TC6 cell lines. Melatonin effectively suppressed this process and reduced the levels of these two miRNAs. A remarkable reversibility of stearic acid-induced β-cell senescence and dysfunction was observed after silencing miR-146a-5p and miR-8114. Moreover, V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) was verified as a direct target of miR-146a-5p and miR-8114. Melatonin also significantly ameliorated senescence and dysfunction in miR-146a-5pand miR-8114-transfected β-cells.
Conclusion
These data demonstrate that melatonin protects against stearic acid-induced β-cell senescence by inhibiting miR-146a- 5p and miR-8114 and upregulating Mafa expression. This not only provides novel targets for preventing stearic acid-induced β-cell dysfunction, but also points to melatonin as a promising drug to combat type 2 diabetes progression.

Keyword

Stearic acid; Melatonin; MicroRNAs; Cellular senescence; Mafa

Figure

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  Fig. 1. Long-term high-stearic-acid diet (HSD) feeding induces senescence in mouse islets. Changes in the expressions of senescence-related genes in HSD-fed mice islets (n=5 mice per group). aP<0.05, bP<0.01, compared with the control (Ctrl) group.

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  Fig. 2. Melatonin (Mel) protects mouse β-TC6 cells against stearic acid-induced senescence and dysfunction. (A) Cytotoxic effect on β-TC6 cells of treatment with different concentrations of melatonin for 24 hours, measured by cell counting kit 8 (CCK-8) assays. (B) Effect of Mel (0.1 mmol/L) on cell viability at different times. (C, D) Protective effect of Mel on stearic acid-decreased glucose-stimulated insulin secretion assay (GSIS) and cell viability. (E) Changes in mRNA levels of senescence-related genes after incubation with Mel in the presence and absence of stearic acid. Each experiment was independently repeated three times. (F) Effect of Mel on stearic acid-induced β-cell senescence measured by X-Gal staining (scale bar, 100 μm). Glu, glucose. aP<0.01, compared with 0 mmol/L; bP<0.01, compared with 0 hour; cP<0.05, dP<0.01, eP<0.001, compared with the control (Ctrl) group; fP<0.05, gP<0.01, hP<0.001, compared with the stearic acid (SA) group; iP<0.05; jP<0.01.

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  Fig. 3. Melatonin (Mel) significantly reversed the stearic acid (SA)-induced increase in the expression of miR-146a-5p and miR-8114 in β-TC6 cells and mouse islet cells. (A, B) Changes in levels of miR-146a-5p and miR-8114 in mouse islets fed with high-stearic-acid diet (HSD) (n=5 mice per group). (C, D) Expression of miR-146a-5p and miR-8114 in SA-treated β-TC6 cells in the presence or absence of Mel. Each experiment was repeated independently three (C) or four (D) times. (E, F) The miR-146a-5p and miR-8114 level in islet cells exposed to SA followed by Mel treatment. Each experiment was repeated independently five times.aP<0.05, bP<0.001, compared with the control (Ctrl) group; cP<0.05, dP<0.01, compared with the SA group; eP<0.01, compared with the Ctrl group.

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  Fig. 4. Inhibition of miR-146a-5p alleviated stearic acid-induced cell senescence and glucose-stimulated insulin secretion assay (GSIS) in β-TC6 cells. (A) Change in miR-146a-5p expression after transfection of stearic acid-treated β-TC6 cells with anti-miRNA oligonucleotides (AMO)-146a-5p. (B, C) Effect of AMO-146a-5p on cell viability and GSIS in the presence and absence of stearic acid, respectively. (D) Alterations in senescence-related gene expression in stearic acid-treated β-TC6 cells transfected with AMO-146a-5p. (E) Inhibition of miR- 146a-5p reversed stearic acid-reduced β-gal activity. Each experiment was repeated independently three times (scale bar, 100 μm). Glu, glucose. aP<0.05, bP<0.01, compared with control (Ctrl)+AMO-negative control (NC) group; cP<0.05, dP<0.01, eP<0.001, compared with the stearic acid (SA)+AMO-NC group; fP<0.05; gP<0.01.

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  Fig. 5. Silencing miR-8114 improved stearic acid (SA)-induced cell senescence and glucose-stimulated insulin secretion assay (GSIS) in β-TC6 cells. (A) Change in miR-8114 level after knockdown of miR-8114 in the presence and absence of SA. (B, C) Silencing miR-8114 improved SA-impaired cell viability and GSIS, respectively. (D) Effect of anti-miRNA oligonucleotides (AMO)-8114 on senescence-related gene expression in SA-treated β-TC6 cells. Each experiment was repeated independently three times. (E) Transfection of SA-treated β-TC6 cells with AMO-8114 reversed the decrease in β-gal activity (scale bar, 100 μm). aP<0.05, bP<0.01, cP<0.001, compared with control (Ctrl)+AMO-negative control (NC) group; dP<0.05, eP<0.01, fP<0.001, compared with SA+AMO-NC group; gP<0.05.

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  Fig. 6. V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) is a direct target of miR-146a-5p and miR-8114. (A) Effect of melatonin on Mafa protein levels in stearic acid (SA)-treated β-TC6 cells. (B, C) Predicted binding sites between miR-146a/miR-8114 and Mafa, respectively. (D, E) Changes in levels of Mafa mRNA and Mafa protein after transfection of anti-miRNA oligonucleotides (AMO)- 146a-5p in SA-treated β-TC6 cells, respectively. (F, G) Mafa mRNA and Mafa protein levels after inhibition of miR-8114 in the presence of SA, respectively. (H, I) Overexpression of miR-146a-5p decreased Mafa mRNA and Mafa protein levels in the absence of SA, respectively. (J, K) Transfection of miR-8114 mimic alone inhibited the expression of Mafa mRNA and Mafa protein, respectively. (L) Luciferase activity between Mafa and miR-146a-5p or miR-8114, respectively. Each experiment was repeated independently three (A-K) or four (L) times. UTR, untranslated region; WT, wild-type; MUT, mutant; NS, no significance. aP<0.05, bP<0.01, compared with control (Ctrl)/Ctrl+AMO-negative control (NC)/mimic-NC group; cP<0.05, dP<0.01, compared with SA/SA+AMO-NC/miR-146a-5p mimic (mimic-146a-5p)/8114 group.

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  Fig. 7. Melatonin (Mel) rescued miR-146a-5p mimic-induced senescence and dysfunction in β-TC6 cells. (A) Change in miR-146a-5p expression after exposure to Mel with a miR-146a-5p mimic. (B, C, D, E, F) Effect of Mel on cell viability, glucose-stimulated insulin secretion assay (GSIS), senescence gene expression, and V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) protein levels after overexpression of miR-146a-5p (D: scale bar, 100 μm). Each experiment was repeated independently three times. Glu, glucose. aP<0.05, bP<0.01, cP<0.001, compared with the control (Ctrl)+mimic-negative control (NC) group; dP<0.05, eP<0.01, fP<0.001, compared with the miR-146a-5p mimic (mimic-146a-5p) group; gP<0.05.

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  Fig. 8. Melatonin (Mel) blocked miR-8114 overexpression-induced β-cell senescence and dysfunction in β-TC6 cells. (A) Change in miR- 8114 expression after incubation with Mel after miR-8114 mimic transfection. (B, C, D, E, F) Effects of Mel on cell viability, glucose-stimulated insulin secretion assay (GSIS), senescence gene expression and V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) protein levels after overexpression of miR-8114 (D: scale bar, 100 μm). Each experiment was repeated independently three times. aP<0.05, bP<0.01, cP<0.001, compared with the control (Ctrl)+mimic-negative control (NC) group; dP<0.05, eP<0.01, fP<0.001, compared with the miR-8114 mimic (mimic-8114) group; gP<0.05.

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  Fig. 9. Schematic diagram of the molecular mechanisms underlying the protective effect of melatonin (Mel) on stearic acid (SA)-induced β-cell senescence through miR-146a-5p and miR-8114. ORF, Open Reading Frame; Mafa, V-maf musculoaponeurotic fibrosarcoma oncogene homolog A.

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