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Wnt antagonist, secreted frizzled-related protein 1, is involved in prenatal skeletal muscle development and is a target of miRNA-1/206 in pigs.

Yang Y, Sun W, Wang R, Lei C, Zhou R, Tang Z, Li K - BMC Mol. Biol. (2015)

Bottom Line: The expression level of the SFRP1 was significantly higher in the embryonic skeletal compared with postnatal skeletal muscle, whereas miR-206 showed the inverse pattern of expression.Dual luciferase assay and Western-blot results demonstrated that SFRP1 was a target of miR-1/206 in porcine iliac endothelial cells.Our results indicate that the SFRP1 gene is regulated by miR-1/206 and potentially affects skeletal muscle development.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, P.R. China. yangyalan1988@126.com.

ABSTRACT

Background: The Wnt signaling pathway is involved in the control of cell proliferation and differentiation during skeletal muscle development. Secreted frizzled-related proteins (SFRPs), such as SFRP1, function as inhibitors of Wnt signaling. MicroRNA-1/206(miRNA-1/206) is specifically expressed in skeletal muscle and play a critical role in myogenesis. The miRNA-mRNA profiles and bioinformatics study suggested that the SFRP1 gene was potentially regulated by miRNA-1/206 during porcine skeletal muscle development.

Methods: To understand the function of SFRP1 and miRNA-1/206 in swine myogenesis, we first predicted the targets of miRNA-1/206 with the TargetScan and PicTar programs, and analyzed the molecular characterization of the porcine SFRP1 gene. We performed a temporal-spatial expression analysis of SFRP1 mRNA and miRNA-206 in Tongcheng pigs (a Chinese indigenous breed) by quantitative real-time polymerase chain reaction, and conducted the co-expression analyses of SFRP1 and miRNA-1/206. Subsequently, the interaction between SFRP1 and miRNA-1/206 was validated via dual luciferase and Western blot assays.

Results: The bioinformatics analysis predicted SFRP1 to be a target of miRNA-1/206. The expression level of the SFRP1 was highly varied across numerous pig tissues and it was down-regulated during porcine skeletal muscle development. The expression level of the SFRP1 was significantly higher in the embryonic skeletal compared with postnatal skeletal muscle, whereas miR-206 showed the inverse pattern of expression. A significant negative correlation was observed between the expression of miR-1/206 and SFRP1 during porcine skeletal muscle development (p <0.05). Dual luciferase assay and Western-blot results demonstrated that SFRP1 was a target of miR-1/206 in porcine iliac endothelial cells.

Conclusions: Our results indicate that the SFRP1 gene is regulated by miR-1/206 and potentially affects skeletal muscle development. These findings increase understanding of the biological functions and the regulation of the SFRP1 gene in mammals.

Show MeSH
The miRNA-1 and miRNA-206 regulateSFRP1at the protein level. (A) Overexpression of porcine SFRP1 in PIEC cells. Histogram indicates overexpression of SFRP1 48 h after transfection. (B) miRNA-1 and miRNA-206 down-regulated the SFRP1 at the protein level. The expression of SFRP1 was normalized against β-actin.
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Fig9: The miRNA-1 and miRNA-206 regulateSFRP1at the protein level. (A) Overexpression of porcine SFRP1 in PIEC cells. Histogram indicates overexpression of SFRP1 48 h after transfection. (B) miRNA-1 and miRNA-206 down-regulated the SFRP1 at the protein level. The expression of SFRP1 was normalized against β-actin.

Mentions: Then, we determined whether SFRP1 was affected by miRNA-1/206 at the protein level. The overexpression vector of SFRP1 was constructed and transfected into PIECs, and the quantitative real-time PCR (qPCR) results showed that the expression of SFRP1 was increased approximately 16-fold compared with the NC group. This result indicated that the SFRP1 overexpression vector was successful (Figure 9A). The porcine SFRP1-CDS-3′-UTR-psiCHECK-2 vector was constructed, and it was co-transfected with miRNA-1 and miRNA-206 mimics in PIECs. The Western blot results showed that the protein level of SFRP1 in the groups containing miRNA-1/206 mimics was decreased compared with the NC group (Figure 9B). These results suggested that the SFRP1 gene was a target of miRNA-1/206.Figure 9


Wnt antagonist, secreted frizzled-related protein 1, is involved in prenatal skeletal muscle development and is a target of miRNA-1/206 in pigs.

Yang Y, Sun W, Wang R, Lei C, Zhou R, Tang Z, Li K - BMC Mol. Biol. (2015)

The miRNA-1 and miRNA-206 regulateSFRP1at the protein level. (A) Overexpression of porcine SFRP1 in PIEC cells. Histogram indicates overexpression of SFRP1 48 h after transfection. (B) miRNA-1 and miRNA-206 down-regulated the SFRP1 at the protein level. The expression of SFRP1 was normalized against β-actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359577&req=5

Fig9: The miRNA-1 and miRNA-206 regulateSFRP1at the protein level. (A) Overexpression of porcine SFRP1 in PIEC cells. Histogram indicates overexpression of SFRP1 48 h after transfection. (B) miRNA-1 and miRNA-206 down-regulated the SFRP1 at the protein level. The expression of SFRP1 was normalized against β-actin.
Mentions: Then, we determined whether SFRP1 was affected by miRNA-1/206 at the protein level. The overexpression vector of SFRP1 was constructed and transfected into PIECs, and the quantitative real-time PCR (qPCR) results showed that the expression of SFRP1 was increased approximately 16-fold compared with the NC group. This result indicated that the SFRP1 overexpression vector was successful (Figure 9A). The porcine SFRP1-CDS-3′-UTR-psiCHECK-2 vector was constructed, and it was co-transfected with miRNA-1 and miRNA-206 mimics in PIECs. The Western blot results showed that the protein level of SFRP1 in the groups containing miRNA-1/206 mimics was decreased compared with the NC group (Figure 9B). These results suggested that the SFRP1 gene was a target of miRNA-1/206.Figure 9

Bottom Line: The expression level of the SFRP1 was significantly higher in the embryonic skeletal compared with postnatal skeletal muscle, whereas miR-206 showed the inverse pattern of expression.Dual luciferase assay and Western-blot results demonstrated that SFRP1 was a target of miR-1/206 in porcine iliac endothelial cells.Our results indicate that the SFRP1 gene is regulated by miR-1/206 and potentially affects skeletal muscle development.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, P.R. China. yangyalan1988@126.com.

ABSTRACT

Background: The Wnt signaling pathway is involved in the control of cell proliferation and differentiation during skeletal muscle development. Secreted frizzled-related proteins (SFRPs), such as SFRP1, function as inhibitors of Wnt signaling. MicroRNA-1/206(miRNA-1/206) is specifically expressed in skeletal muscle and play a critical role in myogenesis. The miRNA-mRNA profiles and bioinformatics study suggested that the SFRP1 gene was potentially regulated by miRNA-1/206 during porcine skeletal muscle development.

Methods: To understand the function of SFRP1 and miRNA-1/206 in swine myogenesis, we first predicted the targets of miRNA-1/206 with the TargetScan and PicTar programs, and analyzed the molecular characterization of the porcine SFRP1 gene. We performed a temporal-spatial expression analysis of SFRP1 mRNA and miRNA-206 in Tongcheng pigs (a Chinese indigenous breed) by quantitative real-time polymerase chain reaction, and conducted the co-expression analyses of SFRP1 and miRNA-1/206. Subsequently, the interaction between SFRP1 and miRNA-1/206 was validated via dual luciferase and Western blot assays.

Results: The bioinformatics analysis predicted SFRP1 to be a target of miRNA-1/206. The expression level of the SFRP1 was highly varied across numerous pig tissues and it was down-regulated during porcine skeletal muscle development. The expression level of the SFRP1 was significantly higher in the embryonic skeletal compared with postnatal skeletal muscle, whereas miR-206 showed the inverse pattern of expression. A significant negative correlation was observed between the expression of miR-1/206 and SFRP1 during porcine skeletal muscle development (p <0.05). Dual luciferase assay and Western-blot results demonstrated that SFRP1 was a target of miR-1/206 in porcine iliac endothelial cells.

Conclusions: Our results indicate that the SFRP1 gene is regulated by miR-1/206 and potentially affects skeletal muscle development. These findings increase understanding of the biological functions and the regulation of the SFRP1 gene in mammals.

Show MeSH