MicroRNA‑378d inhibits Glut4 by targeting Rsbn1 in vitamin D deficient ovarian granulosa cells

Abstract

Vitamin D (VD) is not only associated with bone growth and development, but is also closely associated with numerous other pathological conditions. The present study aimed to investigate the effect of microRNA (miRNA/miR)‑378d on ovarian granulosa cells by regulating the round spermatid basic protein 1 (Rsbn1) in the absence of VD. The abnormal expression of miRNAs in ovarian tissues of the VD‑deficient mouse was analyzed using transcriptome sequencing. miR‑378d, glucose transporter 4 (Glut4) and aromatase (Cyp19a) expression levels were examined via reverse transcription‑quantitative (RT‑q)PCR and western blotting. The expression levels of Rsbn1, Glut4 and Cyp19a were detected in transfected mouse ovarian granulosa cells. The targeting regulation between miR‑378d and Rsbn1 was verified using double reporter gene assay and functional rescue experiments. Among the 672 miRNAs that were differentially expressed, cluster analysis revealed that 17 were significantly upregulated and 16 were significantly downregulated. Moreover, miR‑378d showed significant upregulation, which was further verified via RT‑qPCR. It was identified that the protein expression level of Rsbn1 was significantly downregulated. Furthermore, Glut4 mRNA expression was significantly decreased in the mimic group but markedly increased in the inhibitor group. By contrast, the mRNA expression levels of Rsbn1 and Cyp19a did not demonstrate any significant difference. The western blotting results indicated that the protein expression levels of Rsbn1 and Glut4 were decreased and increased, respectively, while Cyp19a did not show any significant change. In addition, the double reporter gene experiments confirmed that Rsbn1 was the target gene of miR‑378d. Collectively, the present results demonstrated that miR‑378d was abnormally overexpressed in the ovarian tissues of the VD‑deficient mice, and that miR‑378d could inhibit Glut4 production by targeting Rsbn1, which may lead to insulin resistance.

Keywords: microRNA‑378d; vitamin D; round spermatid basic protein 1; ovarian granulosa cells.

Figure 1.

Transcriptome sequencing analysis of ovary tissues from VD- and control mice. (A) miRNA clustering analysis of VD deficient mice using a heat map to show the significantly downregulated gene number. Differential miRNA clustering diagram. The log2 TPM value was used for clustering. Red indicated highly expressed miRNAs, while green indicated lowly expressed miRNAs. (B) DESeq test results were screened according to the significant difference criteria (differential miRNA expression changes were >2 times and false discovery rate ≤0.05), and the significant downregulation of miRNA expression was measured. (C) Scatter plot of KEGG enrichment of differential miRNA target genes. VD, vitamin D; miRNA, microRNA; Con, control.

Figure 2.

Expression levels of miR-378d and Rsbn1 in VD-deficient mouse ovaries. (A) miR-378d expression levels were significantly upregulated in the VD-deficiency group compared with the control group. (B) Relative expression of Rsbn1 mRNA in mouse ovaries, compared with control group. (C) Western blot analyses of Rsbn1 and GAPDH expression levels in ovary tissue of VD-group and control group. (D) The optical density of Rsbn1 was quantified by ImageJ of (C). ***P<0.001 vs. control. ns, non-significant; VD, vitamin D; miR, microRNA; Rsbn1, round spermatid basic protein 1.

Figure 3.

miR-378d post-transcriptionally regulates Rsbn1 in ovaries. (A) Reverse transcription-quantitative PCR results showed that mir-378d was significantly upregulated in the mimic group and significantly downregulated in the inhibitor group. (B) Glut4 mRNA expression was decreased significantly in the mimic group and increased significantly in the inhibitor group, while Rsbn1 and Cyp19a mRNA expression levels showed no significant differences. (C) Western blot analyses of Rsbn1, Glut4 and Cyp19a expression levels in mouse granulosa cells transfected with miR-378d mimics or inhibitors. (D) Expression levels of Rsbn1 and Glut4 were decreased after mir-378d overexpression, and the expression levels of Rsbn1 and Glut4 were increased after mir-378d inhibition, while Cyp19a showed no significant change. ***P<0.001 vs. respective NC. ns, non-significant; VD, vitamin D; miR, microRNA; Rsbn1, round spermatid basic protein 1; NC, negative control; Glut4, glucose transporter 4; Cyp19a, aromatase.

Figure 4.

Luciferase reporter gene activity shows that miR-378d directly targets Rsbn1 in mouse granulosa cells. (A) Predicted target site of miR-378d in 3′UTR of Rsbn1 mRNA of mice. (B) A luciferase activity assay was performed in the presence of WT Rsbn1 3′UTR or MUT compared with the control. (n=3). ***P<0.001. WT, wild-type; MUT, mutant; UTR, untranslated region; miR, microRNA; Rsbn1, round spermatid basic protein 1; NC, negative control.

Figure 5.

Rsbn1 is a functional target of miR-378d in mouse granulosa cells. (A) Western blotting results showed that siRNA 1 interference was the most efficient. (B) Western blotting results demonstrated that Rsbn1 expression was upregulated in the overexpression group. (C) Expression levels of Rsbn1, Glut4 and Cyp19a were detected via western blotting after overexpression or knockdown of miR-378d. (D) mRNA expression levels of Rsbn1, Glut4 and Cyp19a were detected via reverse transcription-quantitative PCR after miR-378d interference. ***P<0.001. miR, microRNA; Rsbn1, round spermatid basic protein 1; NC, negative control; Glut4, glucose transporter 4; Cyp19a, aromatase; OE, overexpression; siRNA, small interfering RNA; ns, non-significant.