RESUMEN
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 VDdeficient mouse was analyzed using transcriptome sequencing. miR378d, glucose transporter 4 (Glut4) and aromatase (Cyp19a) expression levels were examined via reverse transcriptionquantitative (RTq)PCR and western blotting. The expression levels of Rsbn1, Glut4 and Cyp19a were detected in transfected mouse ovarian granulosa cells. The targeting regulation between miR378d 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, miR378d showed significant upregulation, which was further verified via RTqPCR. 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 miR378d. Collectively, the present results demonstrated that miR378d was abnormally overexpressed in the ovarian tissues of the VDdeficient mice, and that miR378d could inhibit Glut4 production by targeting Rsbn1, which may lead to insulin resistance.
Asunto(s)
Transportador de Glucosa de Tipo 4/genética , Proteínas de Homeodominio/genética , MicroARNs/genética , Proteínas de Plasma Seminal/genética , Deficiencia de Vitamina D/genética , Animales , Femenino , Células de la Granulosa/metabolismo , Células de la Granulosa/patología , Humanos , Resistencia a la Insulina/genética , Ratones , Síndrome del Ovario Poliquístico/genética , Síndrome del Ovario Poliquístico/metabolismo , Síndrome del Ovario Poliquístico/patología , Transducción de Señal/genética , Vitamina D/genética , Vitamina D/metabolismo , Deficiencia de Vitamina D/patologíaRESUMEN
Mycobacterium tuberculosis (M. tb) can survive in the hostile microenvironment of cells by escaping host surveillance, but the molecular mechanisms are far from being fully understood. MicroRNAs might be involved in regulation of this intracellular process. By RNAseq of M. tb-infected PMA-differentiated THP-1 macrophages, we previously discovered down-regulation of miR-378d during M. tb infection. This study aimed to investigate the roles of miR-378d in M. tb infection of THP-1 cells by using a miR-378d mimic and inhibitor. First, M. tb infection was confirmed to decrease miR-378d expression in THP-1 and Raw 264.7 macrophages. Then, it was demonstrated that miR-378d mimic promoted, while its inhibitor decreased, M. tb survival in THP-1 cells. Further, the miR-378d mimic suppressed, while its inhibitor enhanced the protein production of IL-1ß, TNF-α, IL-6, and Rab10 expression. By using siRNA of Rab10 (siRab10) to knock-down the Rab10 gene in THP-1 with or without miR-378d inhibitor transfection, Rab10 was determined to be a miR-378d target during M. tb infection. In addition, a dual luciferase reporter assay with the Rab10 wild-type sequence and mutant for miR-378d binding sites confirmed Rab10 as the target of miR-378d associated with M. tb infection. The involvement of four signal pathways NF-κB, P38, JNK, and ERK in miR-378d regulation was determined by detecting the effect of their respective inhibitors on miR-378d expression, and miR-378d inhibitor on activation of these four signal pathways. As a result, activation of the NF-κB signaling pathway was associated with the down-regulation of miR-378d. In conclusion, during M. tb infection of macrophages, miR-378d was down-regulated and functioned on decreasing M. tb intracellular survival by targeting Rab10 and the process was regulated by activation of the NF-κB and induction of pro-inflammatory cytokines IL-1ß, TNF-α, IL-6. These findings shed light on further understanding the defense mechanisms in macrophages against M. tb infection.