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.

Vitamin D deficiency inhibits microRNA-196b-5p which regulates ovarian granulosa cell hormone synthesis, proliferation, and apoptosis by targeting RDX and LRRC17

“…In this study, a mouse model of VDD was established as previously described (19), and transcriptome sequencing analysis revealed that miR-196b-5p was abnormally downregulated in the ovarian tissues of VD-deficient mice, which was further confirmed using RT-qPCR of the extracted ovarian tissues from the mice. Functional experiments further demonstrated that overexpression of miR-196b-5p mimics facilitated cell proliferation and inhibited apoptosis in KGN cells.…”

“…Determination of serum levels of follicle stimulating hormone (FSH), LH, estradiol, and testosterone was performed using commercial ELISA kits (Tongwei Bio, Shanghai, China). Ovarian tissues were collected in 4% formalin for hematoxylin and eosin (H&E) staining or RNAlater (Invitrogen, USA) for RNA sequencing by Genewiz, Inc. (Suzhou, China), as described previously (19).…”

Vitamin D can ameliorate premature ovarian failure by inhibiting neutrophil extracellular traps: A review

“…Experimental studies have shown that VD can promote follicle development and maturation by regulating oxidative stress and steroid production pathways in the ovarian granulosa cells. [ 36 , 37 ] VD plays an important role in the antiinflammatory, immunosuppressive, antioxidant, and antitissue fibrosis aspects of the body. Therefore, VD may interfere with the pathogenesis of POF by suppressing inflammation, immune response, oxidative stress, and tissue fibrosis to improve POF.…”