The microRNA, miR-29c, participates in muscle development through targeting the <i>YY1</i> gene and is associated with postmortem muscle pH in pigs

doi:10.15302/J-FASE-2015075

Front. Agr. Sci. Eng.

2015, Vol. 2

Issue (4) : 311-317 https://doi.org/10.15302/J-FASE-2015075

RESEARCH ARTICLE

The microRNA, miR-29c, participates in muscle development through targeting the YY1 gene and is associated with postmortem muscle pH in pigs

Weiya ZHANG¹,Wei WEI^1,²,Yuanyuan ZHAO¹,Shuhong ZHAO¹,Xinyun LI^1,^*(

)

1. Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China
2. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

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Abstract

Previous studies indicated that miR-29c is important for muscle development in mice and human, but its role in pigs is unknown. In this study, we detected the expression of miR-29c in Meishan longissimus lumborum (LL) muscle. The results showed that miR-29c was gradually upregulated during development of skeletal muscle in pig. Moreover, the expression of YY1 and Akt3 genes, which were confirmed to be targeted by miR-29c in mice, was decreased along with muscle development. Furthermore, the expression level of miR-29c was significantly higher in adult Meishan pigs than Large White pigs, while the expression of YY1 and Akt3 genes was significantly lower in Meishan pigs. These results indicated that the expression pattern of miR-29c was opposite to that of YY1 and Akt3 genes in pigs. Also, the luciferase assay indicated that miR-29s can target the YY1 gene in pigs. In addition, we identified a T to C mutation in the primary transcript of miR-29c, which was associated with the postmortem muscle pH in pigs. Based on these results, we concluded that miR-29c is also important in skeletal muscle development of pigs.

Keywords pig miR-29c skeletal muscle expression SNP

Corresponding Author(s): Xinyun LI

Just Accepted Date: 24 November 2015 Online First Date: 16 December 2015 Issue Date: 19 January 2016

Cite this article:

Weiya ZHANG,Wei WEI,Yuanyuan ZHAO, et al. The microRNA, miR-29c, participates in muscle development through targeting the YY1 gene and is associated with postmortem muscle pH in pigs[J]. Front. Agr. Sci. Eng. , 2015, 2(4): 311-317.

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https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2015075
https://academic.hep.com.cn/fase/EN/Y2015/V2/I4/311

Fig.1 Identification of the target genes of miR-29c. (a) The potential binding site of ssc-miR-29 in YY1 3′UTR, and the investigation of the conservation of this binding site in different species (http://www.targetscan.org); (b) identification of the target of YY1 gene using dual luciferase reporter system. The 756 bp 3′UTR of YY1 gene was first inserted into the psiCHECK-2 luciferase vector, and then co-transfected into PK-15 cells together with miR-29a, miR-29b or miR-29c, and finally the luciferase activity was detected at 24 h post-transfection; (c) luciferase activity was detected when the mutant miR-29 (2 nt substitution was introduced into the seed sequence of miR-29), pooled miR-29 or NC (negative control) were co-transfected with the luciferase vector inserted with the 765bp 3′UTR of YY1 gene. Results are shown as mean±S.E.M, means are of measurements from three independent individuals. ** Indicates a significant difference (P<0.01); *** indicates a significant difference (P<0.001).

Fig.2 Expression profiles of miR-29c, YY1 and Akt3 during muscle development. (a) The expression of miR-29c in LL muscle of Meishan pigs at E50d, E95d, and adult stage (12 month) was detected by qPCR; (b) the expression YY1 genes in LL muscle of Meishan at three stages was detected by qPCR; (c) the expression Akt3 genes in LL muscle of Meishan at three stages was detected by qPCR. The expression level of E50d was set as 1. Expression of miR-29c was normalized to U6. Expression of Akt3 and YY1 genes were normalized to b-Tubulin. Results are shown as mean±S.E.M., means are of three independent individuals. * Indicates a significant difference (P<0.05); ** indicates a significant difference (P<0.01); *** indicates a significant difference (P<0.001).

Fig.3 Expression profiles of miR-29c, YY1 and Akt3 genes in different breeds. (a) The expression of miR-29c in LL muscle of Meishan and Large White at the adult stage (12 months); (b) the expression of YY1 genes in LL muscle of Meishan and Large White at the adult stage (12 months); (c) the expression of Akt3 genes in LL muscle of Meishan and Large White at the adult stage (12 months).The expression level of Large White was set as 1. Expression of miR-29c was normalized to U6. Expression of YY1 and Akt3 genes was normalized to b-Tubulin. Results are shown as mean±S.E.M., means are of three independent individuals. * Indicates a significant difference (P<0.05); ** indicates a significant difference (P<0.01).

Fig.4 SNP identification in the pre-miR-29b-2/c cluster. (a) The scheme of the SNP site in the pre-miR-29b-2/c cluster on SSC9; (b) the sequence of the T/C SNP. The SNP is labeled by an arrow; (c) the gel photo of the genotype digested using restriction enzyme NcoI. The CC homozygote had one band of 752 bp; the TT homozygote had two bands of 631 and 121 bp; TC heterozygote had bands of 752, 631 and 121 bp. L indicates the DNA ladder; (d) the expression level of miR-29c in TT and CC homozygote. The expression level of miR-29c in CC homozygote was set as 1.0,expression of miR-29c was normalized to U6. * indicates a significant difference (P<0.005)

Tab.1 Association study of the T/C SNP in the miR-29b-2/c cluster and meat quality traits

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