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Dynamic changes in the functions of Klf5 in maturing mouse corneas revealed by the changes in its target genes at postnatal days 11 and 56

ABSTRACT: Purpose: Klf5 plays a critical role in the mouse ocular surface (Kenchegowda et al., 2011. Dev Biol. 356:5-18). Here, we compare wild-type (WT) and Klf5-conditional null (Klf5CN) corneal gene expression at postnatal day-11 (PN11) and PN56 to identify the Klf5-target genes. Methods: Gene expression was compared using Affymetrix microarrays with QPCR validation. Transient transfection assays examined the effect of Klf5 on selected target gene promoter activities. Whole-mount corneal immunofluorescent staining examined neovascularization and CD45+ macrophage influx. Results: Expression of 714 and 753 genes was increased, and 299 and 210 genes decreased in PN11 and PN56 Klf5CN corneas, respectively, with 366 concordant increases, 72 concordant decreases and 3 discordant changes. Canonical pathway analysis identified 35 and 34 significantly (p<0.001) enriched pathways at PN11 and PN56, respectively, with 24 common pathways. PN56 Klf5CN corneas shared 327 increases and 91 decreases with the previously described Klf4CN corneas (Swamynathan et al., 2008. IOVS 49:3360-70). Angiogenesis and immune response-related genes were affected consistent with lymphangiogenesis and macrophage influx in Klf5CN corneas, respectively. Expression of 1574 genes was increased and 1915 decreased, in the WT PN56 compared with PN11 corneas. Expression of many collagens, matrix metalloproteinases and other extracellular matrix associated genes decreased in WT corneas between PN11 and PN56, while that of solute carrier family members increased. Conclusions: Differences in PN11 and PN56 corneal Klf5-target genes reveal dynamic changes in Klf5 functions during corneal maturation. Klf4- and Klf5-target genes do not overlap, consistent with their non-redundant roles in the mouse cornea.

ORGANISM(S): Mus musculus

PROVIDER: GSE36229 | GEO | 2012/12/25

SECONDARY ACCESSION(S): PRJNA153137

REPOSITORIES: GEO

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Project description:Purpose: Klf5 plays a critical role in the mouse ocular surface (Kenchegowda et al., 2011. Dev Biol. 356:5-18). Here, we compare wild-type (WT) and Klf5-conditional null (Klf5CN) corneal gene expression at postnatal day-11 (PN11) and PN56 to identify the Klf5-target genes. Methods: Gene expression was compared using Affymetrix microarrays with QPCR validation. Transient transfection assays examined the effect of Klf5 on selected target gene promoter activities. Whole-mount corneal immunofluorescent staining examined neovascularization and CD45+ macrophage influx. Results: Expression of 714 and 753 genes was increased, and 299 and 210 genes decreased in PN11 and PN56 Klf5CN corneas, respectively, with 366 concordant increases, 72 concordant decreases and 3 discordant changes. Canonical pathway analysis identified 35 and 34 significantly (p<0.001) enriched pathways at PN11 and PN56, respectively, with 24 common pathways. PN56 Klf5CN corneas shared 327 increases and 91 decreases with the previously described Klf4CN corneas (Swamynathan et al., 2008. IOVS 49:3360-70). Angiogenesis and immune response-related genes were affected consistent with lymphangiogenesis and macrophage influx in Klf5CN corneas, respectively. Expression of 1574 genes was increased and 1915 decreased, in the WT PN56 compared with PN11 corneas. Expression of many collagens, matrix metalloproteinases and other extracellular matrix associated genes decreased in WT corneas between PN11 and PN56, while that of solute carrier family members increased. Conclusions: Differences in PN11 and PN56 corneal Klf5-target genes reveal dynamic changes in Klf5 functions during corneal maturation. Klf4- and Klf5-target genes do not overlap, consistent with their non-redundant roles in the mouse cornea. Wild type and Klf5-conditional null mouse corneal gene expression at postnatal day-11 and -56 was compared by Affymetrix mouse whole genome 430 2 arrays. Four age-matched PN56 WT and Klf5CN mice, and 3 age-matched PN11 WT and Klf5CN mice each were used for comparison of corneal gene expression by microarrays. Two dissected corneas from each mouse were pooled for isolation of total RNA using the RNeasy Mini kit (Qiagen, Germantown, MD). The quality and integrity of the isolated total RNA was confirmed using an Agilent Bioanalyzer. Total RNAs were amplified and labeled using a 3' IVT Express Kit (Affymetrix Inc., Santa Clara, CA), and hybridized to Affymetrix MG 430 2 chips following the protocol suggested by the manufacturer.

Project description:Purpose: We compared the levels of miRNA specific for DEGs in isograft corneas with those in normal corneas, as well as the levels of miRNA specific for DEGs in allograft corneas with those in isograft corneas, to gain a better understanding of molecular variables that affect corneal graft rejection pathways. Methods: Illumina Hiseq 2500 deep sequencing was used to screen for differentially expressed genes (DEGs) in matched pairs of isograft corneas and normal corneas, allograft corneas and isograft corneas. Potential target genes among the DEGs were predicted using target prediction software (TargetScan, Miranda, miRDB, and CLIP), and the overlay portion was analyzed using the Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG). An analysis of the interactions between DEG proteins (PPI analysis) and a MetaCore software analysis. Results: Our results showed that 22 miRNAs were significantly upregulated and 4 were significantly downregulated in the isograft group when compared with the control group (P < 0.01), while 17 miRNAs were significantly upregulated and 3 were significantly downregulated in the allograft group when compared with the isograft group (P < 0.01). Among the miRNAs with altered expression levels, miR-155-5p, miR-142-3p, miR-142-5p, and miR-223-3p displayed simultaneous changes in the above two comparisons. Potential target genes among the DEGs were predicted using target prediction software, and the overlay portion was analyzed using the Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG). GO and KEGG analyses showed that the DEGs were mainly involved in metabolic pathways, cytokine secretion, and tumor immunity functions. An analysis of the interactions between DEG proteins (PPI analysis) and a MetaCore software analysis of 4 key DEGs revealed that the genes regulated by miR-155-5p played important roles in the miRNA-mRNA regulatory network. Furthermore, the MetaCore analysis identified C/EBP beta, p53, and sp1 as key transcription factors in that network. Conclusions: Our study identified transplanted corneas-specific miRNA in matched pairs of isograft corneas and normal corneas, allograft corneas and isograft corneas. Furthermore, bioinformatics analysis of the key miRNA regulatory network revealed the molecular mechanisms, which suggests miRNAs may as new molecular targets for treating corneal injuries and corneal transplant rejection

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Dynamic changes in the functions of Klf5 in maturing mouse corneas revealed by the changes in its target genes at postnatal days 11 and 56

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