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. 2019 Aug;44(2):608-616.
doi: 10.3892/ijmm.2019.4224. Epub 2019 May 31.

A microRNA‑24‑to‑secretagogin regulatory pathway mediates cholesterol‑induced inhibition of insulin secretion

Jing Yang  1 , Yuncheng Lv  2 , Zhibo Zhao  1 , Wu Li  1 , Sunmin Xiang  1 , Lingzhi Zhou  3 , Anbo Gao  2 , Bin Yan  1 , Lingling Ou  1 , Hong Ling  4 , Xinhua Xiao  1 , Jianghua Liu  1
Affiliations

A microRNA‑24‑to‑secretagogin regulatory pathway mediates cholesterol‑induced inhibition of insulin secretion

Jing Yang et al. Int J Mol Med. 2019 Aug.

Abstract

Hypercholesterolemia is a key factor leading to β‑cell dysfunction, but its underlying mechanisms remain unclear. Secretagogin (Scgn), a Ca2+ sensor protein that is expressed at high levels in the islets, has been shown to play a key role in regulating insulin secretion through effects on the soluble N‑ethylmaleimide‑sensitive factor attachment receptor protein complexes. However, further studies are required to determine whether Scgn plays a role in hypercholesterolemia‑associated β‑cell dysfunction. The present study investigated the involvement of a microRNA‑24 (miR‑24)‑to‑Scgn regulatory pathway in cholesterol‑induced β‑cell dysfunction. In the present study, MIN6 cells were treated with increasing concentrations of cholesterol and then, the cellular functions and changes in the miR‑24‑to‑Scgn signal pathway were observed. Excessive uptake of cholesterol in MIN6 cells increased the expression of miR‑24, resulting in a reduction in Sp1 expression by directly targeting its 3' untranslated region. As a transcriptional activator of Scgn, downregulation of Sp1 decreased Scgn levels and subsequently decreased the phosphorylation of focal adhesion kinase and paxillin, which is regulated by Scgn. Therefore, the focal adhesions in insulin granules were impaired and insulin exocytosis was reduced. The present study concluded that a miR‑24‑to‑Scgn pathway participates in the mechanism regulating cholesterol accumulation‑induced β‑cell dysfunction.

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Figures

Figure 1
Figure 1
Changes in lipid accumulation and miR-24 expression in CHO-treated MIN6 cells. (A) MIN6 cells were treated with 0, 2.5, 5.0 or 10.0 mM cholesterol for 12 h and the intracellular cholesterol was observed using oil red O staining (original magnification, ×400). (B) The intracellular cholesterol content was analyzed using a cholesterol assay kit, as described in the methods. (C) Expression of miR-24 in MIN6 cells treated with increasing concentrations of CHO. **P<0.01 vs. the 0 mM CHO-treated group. CHO, cholesterol; miR, microRNA.
Figure 2
Figure 2
miR-24 is involved in the CHO-mediated inhibition of insulin secretion in MIN6 cells. (A) Treatment with 5.0 mM CHO alone or in combination with miR-24 mimic significantly decreased the expression of Ins1 mRNA and the decrease was rescued by the miR-24 inhibitor, whereas the expression of the Ins2 mRNA was not altered. (B) Treatment with CHO alone or in combination with the miR-24 mimic for 12 h decreased glucose-stimulated insulin secretion in MIN6 cells, while the miR-24 inhibitor rescued the reduction in insulin secretion. The insulin content in MIN6 cells was analyzed using (C) an ELISA and (D) immunofluorescence staining 12 h after treatment (scale bars, 50 μm). **P<0.01 vs. the Scr-treated group. Scr, scrambled miRNA sequence; CHO, cholesterol; Mim, miR-24 mimic; Inh, miR-24 inhibitor; miR, microRNA.
Figure 3
Figure 3
Effect of the CHO treatment on the levels of the Scgn mRNA and protein in MIN6 cells. Levels of the Scgn (A) mRNA and (B) protein were measured using reverse transcription quantitative PCR or western blotting, respectively, in MIN6 cells treated with 5.0 mM CHO alone or in combination with 40 nM miR-24 mimic/80 nM miR-24 inhibitor for 12 h. **P<0.01 vs. the Scr-treated group. Scr, scrambled miRNA sequence; CHO, cholesterol; Mim, miR-24 mimic; Inh, miR-24 inhibitor; Scgn, secretagogin.
Figure 4
Figure 4
Bioinformatics prediction and experimental verification that miR-24 directly targets the Sp1 3′UTR. (A) The highly evolutionary conserved miR-24 targeting sequences from different species. (B) The free energy scores (in RNAhybrid) for miR-24 hybridization to the human and mouse Sp1 sequence. (C) The putative miR-24 binding sites within the human and mouse Sp1 3′UTR and the mutant sequences in the luciferase reporter plasmid are shown. (D) Luciferase activity assays in 293T cells cotransfected with the control luciferase reporter plasmid and a recombinant plasmid containing the WT or Mut Sp1 3′UTR and miR-24 mimic for 24 h. *P<0.05 vs. the control; #P<0.05 vs. the Sp1-WT 3′UTR group. UTR, untranslated region; WT, wild type; Mut, mutant; miR, microRNA.
Figure 5
Figure 5
CHO affects the levels of proteins involved in the Sp1/Scgn-FAK signaling pathway mediating insulin trafficking. Changes in the levels of the Sp1, FAK, p-FAK, paxillin and p-paxillin proteins in MIN6 cells treated with 5.0 mM CHO alone or in combination with 40 nM miR-24 mimic/80 nM miR-24 inhibitor for 12 h were measured using western blotting assay. β-Actin served as the loading control. (A) Sp1, (B) FAK, (C) p-FAK (D) paxillin and (E) p-paxillin. **P<0.01 vs. the Scr-treated group. Scr, scrambled miRNA sequence; CHO, cholesterol; Mim, miR-24 mimic; Inh, miR-24 inhibitor; Scgn, secretagogin; p-FAK, phosphorylated-focal adhesion kinase; miR, microRNA.
Figure 6
Figure 6
Schematic summarizing the potential pathogenic mechanism underlying CHO-mediated inhibition of insulin secretion. Intracellular CHO accumulation induces miR-24 expression in MIN6 cells and then decreases the expression of Sp1. The levels of Scgn and its downstream insulin granule trafficking-related proteins are decreased and insulin secretion is subsequently decreased. CHO, cholesterol; ↑, induction; ↓, inhibition. SCGN, secretagogin; p-FAK, phosphorylated-focal adhesion kinase; miR, microRNA; LDL-R, low-density lipoprotein receptor.
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