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TLR4/NF-κB-responsive microRNAs and their potential target genes: a mouse model of skeletal muscle ischemia-reperfusion injury.

Yang JC, Wu SC, Rau CS, Chen YC, Lu TH, Wu YC, Tzeng SL, Wu CJ, Hsieh CH - Biomed Res Int (2015)

Bottom Line: Immunoprecipitation with an argonaute 2- (Ago2-) specific monoclonal antibody followed by whole genome microarray was performed to identify mRNA associated with the RNA-silencing machinery.The combined in silico and whole genome microarray approaches identified 5, 4, and 20 potential target genes for miR-15a, miR-744, and miR-1196, respectively.Among the 3 genes (Zbed4, Lrsam1, and Ddx21) regulated by at least 2 of the 3 upregulated miRNAs, Lrsam1 and Ddx21 are known to be associated with the innate immunity pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic and Reconstructive Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, No. 123, Ta-Pei Road, Niao-Song District, Kaohsiung City 833, Taiwan.

ABSTRACT

Background: The aim of this study was to profile TLR4/NF-κB-responsive microRNAs (miRNAs) and their potential target genes in the skeletal muscles of mice following ischemia-reperfusion injury.

Methods: Thigh skeletal muscles of C57BL/6, Tlr4(-/-), and NF-κB(-/-) mice isolated based on femoral artery perfusion were subjected to ischemia for 2 h and reperfusion for 0 h, 4 h, 1 d, and 7 d. The muscle specimens were analyzed with miRNA arrays. Immunoprecipitation with an argonaute 2- (Ago2-) specific monoclonal antibody followed by whole genome microarray was performed to identify mRNA associated with the RNA-silencing machinery. The potential targets of each upregulated miRNA were identified by combined analysis involving the bioinformatics algorithm miRanda and whole genome expression.

Results: Three TLR4/NF-κB-responsive miRNAs (miR-15a, miR-744, and miR-1196) were significantly upregulated in the muscles following ischemia-reperfusion injury. The combined in silico and whole genome microarray approaches identified 5, 4, and 20 potential target genes for miR-15a, miR-744, and miR-1196, respectively. Among the 3 genes (Zbed4, Lrsam1, and Ddx21) regulated by at least 2 of the 3 upregulated miRNAs, Lrsam1 and Ddx21 are known to be associated with the innate immunity pathway.

Conclusions: This study profiled TLR4/NF-κB-responsive miRNAs and their potential target genes in mouse skeletal muscle subjected to ischemia-reperfusion injury.

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Related in: MedlinePlus

Immunoblotting analysis of muscle lysates before and after Ago2 immunoprecipitation. Total lysates (−) and precipitates with the Ago2 antibody (+) and control IgG (IgG IP) were separated by SDS-PAGE and probed with the Ago2 antibody to determine the presence of Ago2 proteins.
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fig3: Immunoblotting analysis of muscle lysates before and after Ago2 immunoprecipitation. Total lysates (−) and precipitates with the Ago2 antibody (+) and control IgG (IgG IP) were separated by SDS-PAGE and probed with the Ago2 antibody to determine the presence of Ago2 proteins.

Mentions: RIP-chip is a high-throughput method to identify mRNAs that are targeted by RNA-binding proteins (RBP) or ribonucleoproteins (RNP), such as RNA-induced silencing complex (RISC), based on immunoprecipitation (IP) of the RBP, or RNP with associated mRNAs followed by microarray [37, 39]. The protein of interest is immunoprecipitated, and the identity and relative amount of mRNA associated with it are measured on microarrays. Since miRNA function is mediated by argonaute 2 (Ago2) proteins in the RISC, an anti-Ago2 antibody was used to isolate global miRNA targets from the muscle sample under different experimental conditions, which we identified using a genome-wide comparative hybridization microarray. Immunoblotting with antibodies against Ago2 showed the presence of Ago2 proteins in immunocomplexes following immunoprecipitation (Figure 3); without immunoprecipitation, the Ago2 levels in the muscle specimens were below the limit of detection of immunoblotting. In addition, the absence of Ago2 in the negative-control IgG immunoprecipitates demonstrated the specificity of Ago2 precipitation with the anti-Ago2 antibody. Expression profiling of 2 replicates of array data of the IRI muscle samples against those of sham control mice was performed using the whole genome microarray, which showed 881 significantly (2-fold or greater) downregulated gene transcripts in the muscles of mice after ischemia and reperfusion for 1 d. In addition, the Ago2 RIP-chip assay showed 1433 significantly upregulated gene transcripts in the Ago2-pull down mRNA pool in the IRI muscles compared to those from the sham control mice.


TLR4/NF-κB-responsive microRNAs and their potential target genes: a mouse model of skeletal muscle ischemia-reperfusion injury.

Yang JC, Wu SC, Rau CS, Chen YC, Lu TH, Wu YC, Tzeng SL, Wu CJ, Hsieh CH - Biomed Res Int (2015)

Immunoblotting analysis of muscle lysates before and after Ago2 immunoprecipitation. Total lysates (−) and precipitates with the Ago2 antibody (+) and control IgG (IgG IP) were separated by SDS-PAGE and probed with the Ago2 antibody to determine the presence of Ago2 proteins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321099&req=5

fig3: Immunoblotting analysis of muscle lysates before and after Ago2 immunoprecipitation. Total lysates (−) and precipitates with the Ago2 antibody (+) and control IgG (IgG IP) were separated by SDS-PAGE and probed with the Ago2 antibody to determine the presence of Ago2 proteins.
Mentions: RIP-chip is a high-throughput method to identify mRNAs that are targeted by RNA-binding proteins (RBP) or ribonucleoproteins (RNP), such as RNA-induced silencing complex (RISC), based on immunoprecipitation (IP) of the RBP, or RNP with associated mRNAs followed by microarray [37, 39]. The protein of interest is immunoprecipitated, and the identity and relative amount of mRNA associated with it are measured on microarrays. Since miRNA function is mediated by argonaute 2 (Ago2) proteins in the RISC, an anti-Ago2 antibody was used to isolate global miRNA targets from the muscle sample under different experimental conditions, which we identified using a genome-wide comparative hybridization microarray. Immunoblotting with antibodies against Ago2 showed the presence of Ago2 proteins in immunocomplexes following immunoprecipitation (Figure 3); without immunoprecipitation, the Ago2 levels in the muscle specimens were below the limit of detection of immunoblotting. In addition, the absence of Ago2 in the negative-control IgG immunoprecipitates demonstrated the specificity of Ago2 precipitation with the anti-Ago2 antibody. Expression profiling of 2 replicates of array data of the IRI muscle samples against those of sham control mice was performed using the whole genome microarray, which showed 881 significantly (2-fold or greater) downregulated gene transcripts in the muscles of mice after ischemia and reperfusion for 1 d. In addition, the Ago2 RIP-chip assay showed 1433 significantly upregulated gene transcripts in the Ago2-pull down mRNA pool in the IRI muscles compared to those from the sham control mice.

Bottom Line: Immunoprecipitation with an argonaute 2- (Ago2-) specific monoclonal antibody followed by whole genome microarray was performed to identify mRNA associated with the RNA-silencing machinery.The combined in silico and whole genome microarray approaches identified 5, 4, and 20 potential target genes for miR-15a, miR-744, and miR-1196, respectively.Among the 3 genes (Zbed4, Lrsam1, and Ddx21) regulated by at least 2 of the 3 upregulated miRNAs, Lrsam1 and Ddx21 are known to be associated with the innate immunity pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic and Reconstructive Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, No. 123, Ta-Pei Road, Niao-Song District, Kaohsiung City 833, Taiwan.

ABSTRACT

Background: The aim of this study was to profile TLR4/NF-κB-responsive microRNAs (miRNAs) and their potential target genes in the skeletal muscles of mice following ischemia-reperfusion injury.

Methods: Thigh skeletal muscles of C57BL/6, Tlr4(-/-), and NF-κB(-/-) mice isolated based on femoral artery perfusion were subjected to ischemia for 2 h and reperfusion for 0 h, 4 h, 1 d, and 7 d. The muscle specimens were analyzed with miRNA arrays. Immunoprecipitation with an argonaute 2- (Ago2-) specific monoclonal antibody followed by whole genome microarray was performed to identify mRNA associated with the RNA-silencing machinery. The potential targets of each upregulated miRNA were identified by combined analysis involving the bioinformatics algorithm miRanda and whole genome expression.

Results: Three TLR4/NF-κB-responsive miRNAs (miR-15a, miR-744, and miR-1196) were significantly upregulated in the muscles following ischemia-reperfusion injury. The combined in silico and whole genome microarray approaches identified 5, 4, and 20 potential target genes for miR-15a, miR-744, and miR-1196, respectively. Among the 3 genes (Zbed4, Lrsam1, and Ddx21) regulated by at least 2 of the 3 upregulated miRNAs, Lrsam1 and Ddx21 are known to be associated with the innate immunity pathway.

Conclusions: This study profiled TLR4/NF-κB-responsive miRNAs and their potential target genes in mouse skeletal muscle subjected to ischemia-reperfusion injury.

Show MeSH
Related in: MedlinePlus