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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
Validating SFRP1 as a positive target for miRNA-1 and miRNA-206. Cotransfection of porcine pre-miRNA-1 (A) and pre-miRNA-206 (B) or control and porcine SFRP1 UTR-derived psiCHECK-2 construct or mutant in PIEC cells. Renilla activity at 48 h post-transfection shows a significant decrease in normalized values compared with the control and mutant. Three replicates were performed for each group. **Indicates a p-value of less than 0.01 in Student’s t-test.
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Fig8: Validating SFRP1 as a positive target for miRNA-1 and miRNA-206. Cotransfection of porcine pre-miRNA-1 (A) and pre-miRNA-206 (B) or control and porcine SFRP1 UTR-derived psiCHECK-2 construct or mutant in PIEC cells. Renilla activity at 48 h post-transfection shows a significant decrease in normalized values compared with the control and mutant. Three replicates were performed for each group. **Indicates a p-value of less than 0.01 in Student’s t-test.

Mentions: To validate whether SFRP1 was directly targeted by miRNA-1/206 in pigs, we constructed the psiCheck2-SFRP1-3′-UTR , a luciferase reporter vector. Subsequently, miRNA-1and miRNA-206 mimics and a normal control (NC) were co-transfected into PIECs, and luciferase activity was detected. The miRNA-1 mimic-transfected group exhibited 68.29% less luciferase activity compared with the NC group (p < 0.01) and the miRNA-206 mimic-transfected group exhibited 71.25% less luciferase activity compared with the control (p < 0.01) (Figure 8). To further validate the specific target site, the binding region of the SFRP1 3′-UTR was mutated by bridge PCR (Figure 6B). The luciferase activity of the psiCHECK-2-SFRP1–3′-UTR (mut) was not significantly decreased by both the miRNA-1 and miRNA-206 mimics (12.39% of control for the miRNA-1 mimic-transfected group and 16.76% of control for the miRNA-206 mimic-transfected group) (p >0.05) (Figure 8).Figure 8


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)

Validating SFRP1 as a positive target for miRNA-1 and miRNA-206. Cotransfection of porcine pre-miRNA-1 (A) and pre-miRNA-206 (B) or control and porcine SFRP1 UTR-derived psiCHECK-2 construct or mutant in PIEC cells. Renilla activity at 48 h post-transfection shows a significant decrease in normalized values compared with the control and mutant. Three replicates were performed for each group. **Indicates a p-value of less than 0.01 in Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig8: Validating SFRP1 as a positive target for miRNA-1 and miRNA-206. Cotransfection of porcine pre-miRNA-1 (A) and pre-miRNA-206 (B) or control and porcine SFRP1 UTR-derived psiCHECK-2 construct or mutant in PIEC cells. Renilla activity at 48 h post-transfection shows a significant decrease in normalized values compared with the control and mutant. Three replicates were performed for each group. **Indicates a p-value of less than 0.01 in Student’s t-test.
Mentions: To validate whether SFRP1 was directly targeted by miRNA-1/206 in pigs, we constructed the psiCheck2-SFRP1-3′-UTR , a luciferase reporter vector. Subsequently, miRNA-1and miRNA-206 mimics and a normal control (NC) were co-transfected into PIECs, and luciferase activity was detected. The miRNA-1 mimic-transfected group exhibited 68.29% less luciferase activity compared with the NC group (p < 0.01) and the miRNA-206 mimic-transfected group exhibited 71.25% less luciferase activity compared with the control (p < 0.01) (Figure 8). To further validate the specific target site, the binding region of the SFRP1 3′-UTR was mutated by bridge PCR (Figure 6B). The luciferase activity of the psiCHECK-2-SFRP1–3′-UTR (mut) was not significantly decreased by both the miRNA-1 and miRNA-206 mimics (12.39% of control for the miRNA-1 mimic-transfected group and 16.76% of control for the miRNA-206 mimic-transfected group) (p >0.05) (Figure 8).Figure 8

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