| The insulin-like growth factor binding-protein7(IGFBP7) has binding affinities to IGFs and been found to be able to etiher positively or negatively regulate the IGFs signaling pathway, and also plays a crucial role on cell growth, differentiation and development in an IGF-independent manner. It has been reported that the expression of IGFBP7in double-muscled pig RNA pool was higher than that in non-double-muscled pig RNA pool, and IGFBP7was a potential candidate gene associated for pig muscle growth and development. However, the regulatory mechanism of IGFBP7gene behind it is still unclear. In order to elucidate molecular mechanism of IGFBP7in promoting muscle growth, the genetic variations of IGFBP7and the expression pattern of IGFBP7in diverse pig breeds were investigated. Moreover the microRNAs were identified, which have IGFBP7as target gene.Firstly, the genetic structure and variation of IGFBP7gene among different pig breeds were analyzed. There were no sequences variation found in exons region but some sequences variation found in introns1,3and4. Subsequently real-time fluorescence quantitavite PCR was applied to investigate the developmental expression pattern of IGFBP7, IGF1and IGF2at the age of day1,30,90,120,150and180with longissimus muscle tissue from Yorkshire and Jinhua pigs. Results showed that IGFBP7, IGF1and IGF2mRNA expression exhibited different patterns between these two pig breeds. Furthermore, IGFBP7mRNA expression at day1,30and180was higher in Yorkshire pigs than that in Jinhua pigs, however IGFBP7mRNA expression was higher in Jinhua pigs at the rest investigated days. In Yorkshire pigs, IGFBP7expression was significantly higher at day90than at other ages. In Jinhua pigs, there were no significant differences among IGFBP7mRNA expression at day1,30and90, however, which were higher than that at day120,150,180days. The mRNA expression patterns among IGFBP7, IGF1and IGF2showed that IGFBP7may have function on muscle growth in an IGF-independent manner. The IGFBP7protein levels at day90and180were higher in Jinhua pigs than in Yorkshire pigs by western blot was inconsistent with the qPCR results, which may be due to post-transcriptional gene regulation.Sequence analysis by bioinformatics revealed that several putative transcription factors associated with muscle development existed in this region, such as MyoD, YY1. Transient transfection assay showed that the fragment at the region-333bp to+3bp exhibited the highest luciferase response and the-349～-315region might have positive elements binding sequences while the-585～-349region might have inhibitor binding sequences. The results of genetic variation analysis in the promoter region showed that a300bp-fragment insertion was identified at the-687bp site, however, which had no influence on the promoter activity. Moreover, seven genetic variations were found in IGFBP7promoter region and resulted in the change of transcription factors in the alternative alleles, which might have a role in IGFBP7transcriptional regulation. In the study, the potential alternative splicing pattern of IGFBP7gene was investigated. Two novel splice variants were identified. And the transcript was the primary transcript. The microRNA microarray was used to identify and characterize the differentially expressed miRNAs in Jinhua pigs relative to Yorkshire pigs. Four up-and sixteen down-regulated miRNAs were identified and had more than2-fold differential expression between Jinhua pigs and Yorkshire pigs (P value <0.05). Further, ssc-miR-487b and ssc-miR-142-5p were predicted targeting to IGFBP7gene and were validated by Dual-Glo Luciferase Assay System. ssc-miR-487b and ssc-miR-142-5p might regulated IGFBP7gene at post-transcriptional level. |
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