MicroRNA-148a Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting PTEN

MicroRNAs (miRNAs) have been found to be involved in lipid deposition and metabolism. However, there have been no reports on the roles of miR-148a in the proliferation and adipogenesis of preadipocytes in sheep. In this study, the expression of miR-148a was profiled in the eight tissues of Tibetan ewes and differentiated preadipocytes, and the role of miR-148a in differentiation and proliferation of ovine preadipocytes was investigated using Oil Red O staining, CCK-8, EdU staining, cell cycle detection, and RT-qPCR. The effect of PTEN on the differentiation of ovine preadipocytes was also investigated. The miR-148a was widely expressed in the eight tissues investigated and had significantly increased expression in liver, spleen and subcutaneous adipose tissues, and the heart. The expression of miR-148a continued to increase with the differentiation of ovine preadipocytes. The over-expression of miR-148a significantly promoted differentiation but inhibited the proliferation of ovine preadipocytes. The inhibition of miR-148a had the opposite effect on the differentiation and proliferation of ovine preadipocytes with over-expressed miR-148a. The results from the dual luciferase reporter assays showed that miR-148a mimic significantly decreased the luciferase activity of PTEN-3′UTR dual luciferase reporter vector, suggesting that PTEN is a target gene of miR-148a. In over-expressed-PTEN preadipocytes, the number of lipid droplets remarkably decreased, and the expression levels of adipogenesis marker genes PPARγ, FASN, FATP4, GLUT4, C/EBPβ and LPL were also significantly down-regulated. These results suggest that miR-148a accelerated the adipogenic differentiation of ovine preadipocytes by inhibiting PTEN expression, and also inhibited the proliferation of ovine preadipocytes.

circBIRC6 regulates cisplatin resistance of choroidal melanoma via positively modulating ERK by sponging miR-503-3p

Background/AimscircRNA plays a key regulatory role in various human tumors. This study was carried out to investigate the molecular mechanism of circBIRC6 in the regulation of choroidal melanoma cisplatin (DDP) resistance. MethodsThe expression of circBIRC6 in choroidal melanoma tissues and cells (MUM-2B, MUM-2B / DDP) was detected by RT-qPCR. The function of circBIRC6 in DDP resistance of choroidal melanoma was analyzed by functional loss and overexpression experiments. DDP inhibition rate was detected by MTT method. Western blot was used to analysis the protein levels of P-gp, MRP1 and ERK. The relationship between circBIRC6, miR-503-3p and ERK was analyzed by CircRIP and luciferase reporter vector assay.ResultcircBIRC6 was up-regulated in choroidal melanoma, and it was significantly raised in choroidal melanoma tissues and cells that are resistant to DDP. CircBIRC6 regulated DDP resistance of choroidal melanoma cells by binding to miR-503-3p. ERK was directly targeted by miR-503-3p, and ERK inhibited miR-503-3p-induced DDP resistance. Finally, the expression of circBIRC6 was positively correlated with ERK, and it was verified that circBIRC6 regulated ERK by targeting miR-503-3p.ConclusionCircBIRC6 regulated the expression of ERK choroidal melanoma cells and DDP resistance via miR-503-3p. These results suggested that the knockout of circBIRC6 may provide an effective treatment strategy for choroidal melanoma.

Upregulated microRNA-106a Promotes Porcine Preadipocyte Proliferation and Differentiation by Targeting Different Genes

Adipose tissue is one of the main organs for the energy storage and supply of organisms. Adipose deposition and metabolism are controlled by a cascade of transcription factors and epigenetic regulatory mechanisms. Previous studies have also shown that miR-106a plays a considerable role in the development of organisms. The regulatory mechanism of miR-106a on porcine preadipocytes is still not clear. In this study, preadipocytes were isolated from the neck subcutaneous deposits of 3–5-day old Chinese native Guanzhong black pigs using 5-ethynyl-20-deoxyuridine (EdU) staining and a CCK-8 assay to detect the number of proliferous cells and real-time qPCR (RT-qPCR) and western blot analysis to detect gene expression, as well as Oil Red O and BODIPY staining dye lipid droplets and flow cytometry (FCM) to detect cell cycles. We also used the double luciferase method to detect the relative luciferase activities. Upregulated miR-106a increased the number of proliferous cells and enhanced the expression of cell proliferation-related genes in porcine adipocytes. The double luciferase reporter vector confirmed that p21 was a target gene of miR-106a in the cell proliferation phase. miR-106a upregulation increased the number of lipid droplets and the expression of lipogenic genes and directly targeted BMP and activin membrane-bound inhibitor (BAMBI) in the process of differentiation. Our results indicated that miR-106a promotes porcine preadipocyte proliferation and differentiation by targeting p21 and BAMBI.

RXFP1 expression is regulated by miR-144-3p in Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis

ABSTRACTRelaxin has been considered as a potential therapy for patients with pulmonary fibrosis. We have previously shown, however, that a potential limitation of relaxin-based therapy for Idiopathic Pulmonary Fibrosis (IPF) is the loss of expression of the relaxin receptor Relaxin/Insulin Like Receptor 1 (RXFP1) expression in fibroblasts. The molecular mechanism for RXFP1 down-regulation in IPF patients remains unclear. To determine whether microRNAs play a role in RXFP1 gene expression, we employed a bioinformatics approach to identify microRNAs (miRs) that are predicted to target RXFP1. By in silico analysis, we identified a putative target site in the RXFP1 mRNA for the miR-144 family. We found that miR-144-3p was upregulated in IPF fibroblasts compared to donor lung fibroblast controls. Forced miR-144-3p mimic expression reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) in donor lung fibroblasts. IPF lung fibroblasts transfected with a miR-144-3p inhibitor increased RXFP1 expression and reduced α-SMA expression. A lentiviral luciferase reporter vector carrying the WT 3’UTR of RXFP1 was repressed more in lung fibroblasts whereas vector carrying a mutated miR-144-3p binding site exhibited less sensitivity to endogenous miR-144-3p expression, suggesting that RXFP1 is a direct target of miR-144-3p. Thus, miR-144-3p is highly expressed in IPF fibroblasts and acts as a negative regulator of RXFP1 protein expression.

Methyl Methacrylate Activates the Gsta1 Promoter

Residual monomers in resin-based biomaterials cause cytotoxicity. We previously showed that methyl methacrylate (MMA) induced mRNA expression of the glutathione S-transferase alpha 1 gene ( Gsta1) located downstream of the cis-acting anti-oxidant responsive element (ARE). Herein, we tested the hypothesis that MMA activated the Gsta1 promoter through the ARE. HepG2 cells were transfected with a luciferase reporter vector containing the ARE and the Gsta1 promoter (−990 to +46 bp) and cultured for 12 hrs with MMA (initial concentration, 10 mM). Analysis of the expressed luciferase activity indicated that MMA activated the promoter 2.6-fold. MMA (from 1 to 30 mM) dose-dependently increased the promoter activity, which reached a plateau between 6 and 12 hrs. In HepG2 cells transfected with a reporter vector containing 2 AREs and a TATA-like promoter, 10 mM MMA increased the reporter expression 2.8-fold. These results suggest that MMA increases Gsta1 transcription through ARE-mediated promoter activation.

Characterization of IL-6 Responsive Elements in Hepcidin Promoters at the Molecular Level: Lessons from the Two Closely Related Promoter Sequences, Murine Hepc1 and Hepc2.

Hepcidin is an antimicrobial peptide that plays an important role in regulation of iron metabolism. There is one hepcidin peptide in humans and two hepcidin peptides in mice, hepcidin 1 and hepcidin 2, the murine mature peptides sharing 68% homology at the amino-acid level. The promoter region of these two genes is highly conserved (92% homology of 500 bp promoter sequence from the start of translation). However, regulation of transcription of these two genes is different. Human hepcidin and murine hepcidin 1 mRNA levels become elevated in response to iron excess and inflammation, while murine hepcidin 2 responds only to iron. Wrighting and Andrews (Blood Ahead of Print July 2006) demonstrated that the inflammatory pathway is mediated by STAT3 and that the binding site includes nucleotides −145 to −143 from start of translation in the human hepcidin promoter. Moreover, mutation of these nucleotides led to loss of IL-6 responsiveness. Alignment of the nucleotide sequences of hepcidin promoters revealed that there are two major differences between the murine hepcidin 2 (mHepc2) and the human hepcidin (hHEPC) and the murine hepcidin 1 (mHepc1) sequence in the proximal 140 bp promoter region. There is a substitution of T for A at −136 in the STAT3/AP1 site and an inversion of ApG to GpA at position −127–8 in the AP1 site. We cloned mHepc1 and mHepc2 promoters into a pGL3 basic luciferase reporter vector and mutated the two different sites in order to determine if creating the mHepc2 sequence in the mHepc1 promoter eliminated IL-6 responsiveness and if creating the mHepc1 sequence in the mHepc2 promoter restored IL-6 responsiveness of that promoter. We also created the equivalent A→T mutation in the STAT3/AP1 site in the hHEPC construct and the TpTpC→GpGpA mutation at −143–5, the same mutant as made by Wrighting and Andrews. Plasmids were transfected into human hepatoma HepG2 cells and human kidney HEK293T cells. As expected, the wildtype hHEPC reporter construct was induced 10 fold or more by IL-6. The T→A and TpTpC→GpGpA hHEPC mutants both displayed significantly reduced, but not absent, IL-6 responsiveness. The basal level of the native mHepc1-driven reporter was at least 10-fold higher than that of the native mHepc2. Furthermore, the mHepc1 promoter was similarly responsive to IL-6 as the hHEPC while the response of mHepc2 promoter was negligible. Interestingly, although the mHepc1 promoter-driven reporter manifests baseline expression even in cells of non-hepatic origin (kidney HEK293T cells), the ability to respond to IL-6 is restricted to cells of hepatic origin. We observed a significantly lower basal level of luciferase reporter activity in the mHepc1 promoter mutated to mHepc2 (A→ T and GpA→ApG), however, it was still IL-6 responsive. In contrast, complementary mutagenesis of mHepc2 to mHepc1 (T→ A and ApG→GpA) increased the basal level of reporter expression and significantly increased the mHepc2 promoter responsiveness to IL-6 although not to the extent seen with mHepc1 promoter. We conclude that the promoter region at nt −126 to −145 from the start of translation is important for the basal level of expression of hepcidin and is important for IL-6 responsiveness in the hHEPC promoter but there are probably additional regions responsible for IL-6 responsiveness in the mHepc1 promoter.