Limits...
Mest but Not MiR-335 Affects Skeletal Muscle Growth and Regeneration.

Hiramuki Y, Sato T, Furuta Y, Surani MA, Sehara-Fujisawa A - PLoS ONE (2015)

Bottom Line: When skeletal muscle fibers are injured, they regenerate and grow until their sizes are adjusted to surrounding muscle fibers and other relevant organs.In addition to reduced body weight in Mest+/-; DMD- mice, decreased muscle growth was observed in Mest+/- mice during cardiotoxin-induced regeneration, suggesting roles of Mest in muscle regeneration.Thus, Mest likely mediates muscle regeneration through regulation of imprinted gene networks in skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

ABSTRACT
When skeletal muscle fibers are injured, they regenerate and grow until their sizes are adjusted to surrounding muscle fibers and other relevant organs. In this study, we examined whether Mest, one of paternally expressed imprinted genes that regulates body size during development, and miR-335 located in the second intron of the Mest gene play roles in muscle regeneration. We generated miR-335-deficient mice, and found that miR-335 is a paternally expressed imprinted microRNA. Although both Mest and miR-335 are highly expressed during muscle development and regeneration, only Mest+/- (maternal/paternal) mice show retardation of body growth. In addition to reduced body weight in Mest+/-; DMD- mice, decreased muscle growth was observed in Mest+/- mice during cardiotoxin-induced regeneration, suggesting roles of Mest in muscle regeneration. Moreover, expressions of H19 and Igf2r, maternally expressed imprinted genes were affected in tibialis anterior muscle of Mest+/-; DMD- mice compared to DMD- mice. Thus, Mest likely mediates muscle regeneration through regulation of imprinted gene networks in skeletal muscle.

No MeSH data available.


Related in: MedlinePlus

Generation of miR-335 deficient mice.(A) Design of constructs used for generation of miR-335 deficient mice. The miR-335 genomic locus was replaced by a floxed neomycin-resistance cassette (loxP-Neo-loxP) to obtain miR-335+/Neo mice. miR-335+/- mice (+/-) were generated by crossing male miR-335+/Neo mice with female CAG-cre transgenic mice. (B) Southern blot analysis of WT and G418 resistant ES clones with 5’ and 3’ probes. †: Non-specific band. (C and D) PCR analysis for targeted allele with genomic DNA in tails of WT (+/+), miR-335+/Neo (+/Neo), and miR-335+/- mice (+/-). In (C), an insertion and a deletion of a floxed neomycin-resistance cassette in genomic DNA of miR-335+/Neo (+/Neo) and miR-335+/- mice (+/-), respectively, were detected with PCRs amplified with primers shown in Fig 2A (Orange and Green arrows). In (D), a PCR analysis to distinguish alleles for WT (+/+), miR-335+/- (+/-), and miR-335-/- mice (-/-) was shown. The WT allele-specific (280 bp) and the mutant allele-specific (306 bp) bands were amplified with the primers shown in Fig 2A (Green arrow). (E, F and G) qRT-PCR for miR-335 was performed in TA muscles isolated from WT, miR-335+/Neo, and miR-335+/- or miR-335-/+ mice (n = 3 per genotype). (H and I) qRT-PCR for Mest mRNA was performed in TA muscles of WT, miR-335+/-, and miR-335+/Neo mice (n = 3 per genotype). Expression of Mest mRNA and that of miR-335 are normalized to Gapdh and snoRNA-202, respectively. Error bars indicate the s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4476715&req=5

pone.0130436.g002: Generation of miR-335 deficient mice.(A) Design of constructs used for generation of miR-335 deficient mice. The miR-335 genomic locus was replaced by a floxed neomycin-resistance cassette (loxP-Neo-loxP) to obtain miR-335+/Neo mice. miR-335+/- mice (+/-) were generated by crossing male miR-335+/Neo mice with female CAG-cre transgenic mice. (B) Southern blot analysis of WT and G418 resistant ES clones with 5’ and 3’ probes. †: Non-specific band. (C and D) PCR analysis for targeted allele with genomic DNA in tails of WT (+/+), miR-335+/Neo (+/Neo), and miR-335+/- mice (+/-). In (C), an insertion and a deletion of a floxed neomycin-resistance cassette in genomic DNA of miR-335+/Neo (+/Neo) and miR-335+/- mice (+/-), respectively, were detected with PCRs amplified with primers shown in Fig 2A (Orange and Green arrows). In (D), a PCR analysis to distinguish alleles for WT (+/+), miR-335+/- (+/-), and miR-335-/- mice (-/-) was shown. The WT allele-specific (280 bp) and the mutant allele-specific (306 bp) bands were amplified with the primers shown in Fig 2A (Green arrow). (E, F and G) qRT-PCR for miR-335 was performed in TA muscles isolated from WT, miR-335+/Neo, and miR-335+/- or miR-335-/+ mice (n = 3 per genotype). (H and I) qRT-PCR for Mest mRNA was performed in TA muscles of WT, miR-335+/-, and miR-335+/Neo mice (n = 3 per genotype). Expression of Mest mRNA and that of miR-335 are normalized to Gapdh and snoRNA-202, respectively. Error bars indicate the s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: To distinguish roles of Mest from those of miR-335, we generated miR-335 deficient mice by homologous recombination in embryonic stem (ES) cells. The miR-335 sequence was replaced with a neomycin resistance cassette flanked by loxP sites (Fig 2A). Targeted ES clones were confirmed by Southern blotting using 5’ and 3’ probes against mouse genomic DNA digested with BamHI and HindIII, respectively (Fig 2B). The absence of random integration was verified with a neomycin probe (data not shown). Removal of the neomycin resistance cassette by mating male miR-335+/Neo mice to female CAG-cre transgenic mice resulted in the deletion of short flanking sequences together with the neomycin cassette (miR-335+/- mice) (Fig 2C and 2D). Mest is an imprinted gene that is expressed essentially from the paternal allele during development [2]. We investigated whether miR-335 is also paternally expressed. qRT-PCR analysis revealed that miR-335+/Neo (maternal/paternal) mice obtained by crossing male miR-335+/Neo mice with female WT mice scarcely expressed miR-335 in skeletal muscle (Fig 2E). Similarly, miR-335+/- (maternal/paternal) mice obtained by crossing male miR-335+/- mice with female WT mice scarcely expressed miR-335 in skeletal muscle (Fig 2F). On the contrary, miR-335-/+ (maternal/paternal) mice obtained by crossing male WT mice with female miR-335+/- mice expressed miR-335 in skeletal muscle comparable to WT mice (Fig 2G). Thus, these results indicate that miR-335 is a paternally expressed imprinted miRNA. In addition, while the expression of Mest was decreased in miR-335+/- mice, it was not altered in miR-335+/Neo mice (Fig 2H and 2I), suggesting that the region surrounding the neomycin resistance cassette flanked by loxP sites, but not the miR-335 itself, is involved in transcriptional regulation of Mest. To evaluate roles of miR-335, further analyses were mainly performed with miR-335+/Neo mice.


Mest but Not MiR-335 Affects Skeletal Muscle Growth and Regeneration.

Hiramuki Y, Sato T, Furuta Y, Surani MA, Sehara-Fujisawa A - PLoS ONE (2015)

Generation of miR-335 deficient mice.(A) Design of constructs used for generation of miR-335 deficient mice. The miR-335 genomic locus was replaced by a floxed neomycin-resistance cassette (loxP-Neo-loxP) to obtain miR-335+/Neo mice. miR-335+/- mice (+/-) were generated by crossing male miR-335+/Neo mice with female CAG-cre transgenic mice. (B) Southern blot analysis of WT and G418 resistant ES clones with 5’ and 3’ probes. †: Non-specific band. (C and D) PCR analysis for targeted allele with genomic DNA in tails of WT (+/+), miR-335+/Neo (+/Neo), and miR-335+/- mice (+/-). In (C), an insertion and a deletion of a floxed neomycin-resistance cassette in genomic DNA of miR-335+/Neo (+/Neo) and miR-335+/- mice (+/-), respectively, were detected with PCRs amplified with primers shown in Fig 2A (Orange and Green arrows). In (D), a PCR analysis to distinguish alleles for WT (+/+), miR-335+/- (+/-), and miR-335-/- mice (-/-) was shown. The WT allele-specific (280 bp) and the mutant allele-specific (306 bp) bands were amplified with the primers shown in Fig 2A (Green arrow). (E, F and G) qRT-PCR for miR-335 was performed in TA muscles isolated from WT, miR-335+/Neo, and miR-335+/- or miR-335-/+ mice (n = 3 per genotype). (H and I) qRT-PCR for Mest mRNA was performed in TA muscles of WT, miR-335+/-, and miR-335+/Neo mice (n = 3 per genotype). Expression of Mest mRNA and that of miR-335 are normalized to Gapdh and snoRNA-202, respectively. Error bars indicate the s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476715&req=5

pone.0130436.g002: Generation of miR-335 deficient mice.(A) Design of constructs used for generation of miR-335 deficient mice. The miR-335 genomic locus was replaced by a floxed neomycin-resistance cassette (loxP-Neo-loxP) to obtain miR-335+/Neo mice. miR-335+/- mice (+/-) were generated by crossing male miR-335+/Neo mice with female CAG-cre transgenic mice. (B) Southern blot analysis of WT and G418 resistant ES clones with 5’ and 3’ probes. †: Non-specific band. (C and D) PCR analysis for targeted allele with genomic DNA in tails of WT (+/+), miR-335+/Neo (+/Neo), and miR-335+/- mice (+/-). In (C), an insertion and a deletion of a floxed neomycin-resistance cassette in genomic DNA of miR-335+/Neo (+/Neo) and miR-335+/- mice (+/-), respectively, were detected with PCRs amplified with primers shown in Fig 2A (Orange and Green arrows). In (D), a PCR analysis to distinguish alleles for WT (+/+), miR-335+/- (+/-), and miR-335-/- mice (-/-) was shown. The WT allele-specific (280 bp) and the mutant allele-specific (306 bp) bands were amplified with the primers shown in Fig 2A (Green arrow). (E, F and G) qRT-PCR for miR-335 was performed in TA muscles isolated from WT, miR-335+/Neo, and miR-335+/- or miR-335-/+ mice (n = 3 per genotype). (H and I) qRT-PCR for Mest mRNA was performed in TA muscles of WT, miR-335+/-, and miR-335+/Neo mice (n = 3 per genotype). Expression of Mest mRNA and that of miR-335 are normalized to Gapdh and snoRNA-202, respectively. Error bars indicate the s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: To distinguish roles of Mest from those of miR-335, we generated miR-335 deficient mice by homologous recombination in embryonic stem (ES) cells. The miR-335 sequence was replaced with a neomycin resistance cassette flanked by loxP sites (Fig 2A). Targeted ES clones were confirmed by Southern blotting using 5’ and 3’ probes against mouse genomic DNA digested with BamHI and HindIII, respectively (Fig 2B). The absence of random integration was verified with a neomycin probe (data not shown). Removal of the neomycin resistance cassette by mating male miR-335+/Neo mice to female CAG-cre transgenic mice resulted in the deletion of short flanking sequences together with the neomycin cassette (miR-335+/- mice) (Fig 2C and 2D). Mest is an imprinted gene that is expressed essentially from the paternal allele during development [2]. We investigated whether miR-335 is also paternally expressed. qRT-PCR analysis revealed that miR-335+/Neo (maternal/paternal) mice obtained by crossing male miR-335+/Neo mice with female WT mice scarcely expressed miR-335 in skeletal muscle (Fig 2E). Similarly, miR-335+/- (maternal/paternal) mice obtained by crossing male miR-335+/- mice with female WT mice scarcely expressed miR-335 in skeletal muscle (Fig 2F). On the contrary, miR-335-/+ (maternal/paternal) mice obtained by crossing male WT mice with female miR-335+/- mice expressed miR-335 in skeletal muscle comparable to WT mice (Fig 2G). Thus, these results indicate that miR-335 is a paternally expressed imprinted miRNA. In addition, while the expression of Mest was decreased in miR-335+/- mice, it was not altered in miR-335+/Neo mice (Fig 2H and 2I), suggesting that the region surrounding the neomycin resistance cassette flanked by loxP sites, but not the miR-335 itself, is involved in transcriptional regulation of Mest. To evaluate roles of miR-335, further analyses were mainly performed with miR-335+/Neo mice.

Bottom Line: When skeletal muscle fibers are injured, they regenerate and grow until their sizes are adjusted to surrounding muscle fibers and other relevant organs.In addition to reduced body weight in Mest+/-; DMD- mice, decreased muscle growth was observed in Mest+/- mice during cardiotoxin-induced regeneration, suggesting roles of Mest in muscle regeneration.Thus, Mest likely mediates muscle regeneration through regulation of imprinted gene networks in skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

ABSTRACT
When skeletal muscle fibers are injured, they regenerate and grow until their sizes are adjusted to surrounding muscle fibers and other relevant organs. In this study, we examined whether Mest, one of paternally expressed imprinted genes that regulates body size during development, and miR-335 located in the second intron of the Mest gene play roles in muscle regeneration. We generated miR-335-deficient mice, and found that miR-335 is a paternally expressed imprinted microRNA. Although both Mest and miR-335 are highly expressed during muscle development and regeneration, only Mest+/- (maternal/paternal) mice show retardation of body growth. In addition to reduced body weight in Mest+/-; DMD- mice, decreased muscle growth was observed in Mest+/- mice during cardiotoxin-induced regeneration, suggesting roles of Mest in muscle regeneration. Moreover, expressions of H19 and Igf2r, maternally expressed imprinted genes were affected in tibialis anterior muscle of Mest+/-; DMD- mice compared to DMD- mice. Thus, Mest likely mediates muscle regeneration through regulation of imprinted gene networks in skeletal muscle.

No MeSH data available.


Related in: MedlinePlus