| Previous studies in our laboratory indicated that in ovo leptin administration influenced the concentration of leptin in serum and liver and the hepatic lipid metabolism in newly hatched broiler chickens. This study was conducted to investigate the effect of in ovo leptin administration on expression of genes and miRNAs involved in hepatic cholesterol metabolism of newly hatched broiler chickens.1 Effect of in ovo leptin administration on hapatic expression of genes invoved in hapatic cholesterol metabolism of newly hatched broiler chickens.Eggs purchased from Sanhuang broiler breeding farm were injected with either 0.5μg of recombinant mice leptin in 100μL of phosphate buffered saline (PBS) or 100μL PBS before incubation. Liver samples were collected at hatching (DO). Real-time PCR was performed to quantitate mRNA expression of sterol regulator element binding protein 2 (SREBP-2),3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), cholesterol-7alpha-hydroxylase (CYP7A1) and low density lipoprotein receptor (LDLR). Western blot analysis was used to determine protein content of SREBP-1, SREBP-2, HMGCR and CYP7A1. Chickens hatched from leptin-treated eggs showed up-regulated SREBP-2, HMGCR and CYP7A1 mRNA expression (P= 0.013,0.000,0.011) and SREBP-1 and SREBP-2 protein content (P= 0.030,0.000) in liver of newly hatched broiler chickens, while there was no significant differences in LDLR mRNA expression (P= 0.333), HMGCR and CYP7A1 protein content (P= 0.386,0.599) in liver of newly hatched broiler chickens between leptin-treated group and control group.2 Effect of in ovo leptin administration on hapatic expression of miRNAs invoved in hapatic cholesterol metabolism of newly hatched broiler chickens.TargetScan 5.1 (http://www.targetscan.org/) was applied to predict miRNA targeting SREBP-1 or HMGCR. Results of prediction showed that 9 miRNAs targeted SREBP-1 and 5 miRNAs targeted HMGCR, including gga-miR-200b and gga-miR-429 targeting both. Compared with control group,5 out of 14 predicted miRNA expression, gga-miR-99a, gga-miR-100, gga-miR-200a, gga-miR-200b and gga-miR-429 (P= 0.024,0.026,0.045, 0.033,0.038), were up-regulated in liver of newly hatched broiler of the leptin-treated group.3 Validation the role of microRNA in the regulation of target gene expression by using dual-luciferase reporter assay.In this part of my work, dual-luciferase reporter assay was performed to validate the role of microRNA in the regulation of target gene expression.3'UTR of SREBP-1 and HMGCR was PCR amplified from chicken genomic DNA using gene specific primers. Each PCR product was cloned into the 3'UTR of the firefly luciferase gene in the pGL3 luciferase reporter vectors at the XbaⅠrestriction sites. Pre-miRNA was cloned into pSilencer 3.0-H1 siRNA expression vectors between BamHI and HindⅢrestriction sites. Finally,3'UTR pGL3-Control firefly luciferase reporter vector and pSilencer 3.0-H1 siRNA expression vectors were cotransfected with pRL-TK renilla luciferase vector into chicken embryo fibroblast cell line DF1 cells. About 24 h post-transfection, cells were washed with PBS and lysed in passive lysis buffer. For each transfection, firefly and Renilla luciferase activities were determined using the dual-luciferase reporter assay system. Results of dual-luciferase reporter assay showed that over-expression of gga-miR-100 and gga-miR-183 repressed the expression of firefly luciferase followed by 3'UTR of SREBP-1 and over-expression of gga-miR-200b repressed the expression of firefly luciferase followed by 3'UTR of HMGCR。... |
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