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Transcriptome profiling of human keratoconus corneas through RNA sequencing identifies collagen synthesis disruption and downregulation of core elements of TGF-β, Hippo, and Wnt pathways

ABSTRACT: To understand better the factors contributing to keratoconus (KTCN), we used RNA sequencing to perform a transcriptome profile of human KTCN corneas. Over 82% of the genes and almost 75% of the transcripts detected as differentially expressed in KTCN and non-KTCN corneas were confirmed in the replication study using another set of samples. We used these differentially expressed genes to generate a network of KTCN-deregulated genes. We found an extensive disruption of collagen synthesis and maturation pathways, as well as downregulation of the core elements of the TGF-β, Hippo, and Wnt signaling pathways influencing corneal organization. We identified long noncoding RNAs (lncRNAs) and conducted a computational analysis of their potential functions, and found that lncRNAs regulated the processing and expression of the aforementioned genes. This first comprehensive transcriptome profiling of human KTCN corneas points further to a complex etiology of KTCN.

ORGANISM(S): Homo sapiens

PROVIDER: GSE77938 | GEO | 2017/02/02

SECONDARY ACCESSION(S): PRJNA312169

REPOSITORIES: GEO

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Project description:Purpose: The goals of this study are to identify differentially expressed genes between keratoconus patient and health control. Methods: We conducted a case: control RNA sequencing study of 7 African American, 12 Middle Eastern subjects, and 7 controls. Total RNA was isolated from individual corneas and sequenced at Macrogen Inc. using the TruSeq stranded RNA library kit. The samples were sequenced at 101 bp using the Illumina HiSeq. 2500 platform. Raw images were generated using the HiSeq Control software v2.2.38, base calling using an integrated primary analysis software, Real time Analysisv1.18.61.0, and converted into FASTQ files using the Illumina package bcl2fastq v1.8.4. The alignment of reads against the human reference genome hg19 was performed using the STAR 2.6.0a software. Followed by gene expression quantification using Cufflinks 2.2.1. The differential expression analysis was conducted by Cuffdiff and DESeq2 (version: 1.24.0). The criteria for significantly differential expression is adjusted p ≤ 0.01, absolute fold change ≥2, and FPKM ≥5 in at least one group. Results: We identified an overwhelming decrease in the expression of anti-oxidant genes regulated by NRF2 and those of the acute phase and tissue injury response pathways, in both patient groups. Concordantly, NRF2 immunofluorescence staining was decreased in patient corneas, while KEAP2, which helps to degrade NRF2, was increased. Diminished NRF2 signaling raises the possibility of NRF2 activators as future treatment strategies in keratoconus. Although separated by geography and ancestry, key commonalities in the two patient transcriptomes speak of disease intrinsic gene expression networks. The African American patient group also showed increases in extracellular matrix transcripts that may be due to underlying profibrogenic changes in this group. Transcripts increased across all patient samples include Thrombospondin 2 (THBS2), encoding a matricellular protein, and cellular proteins, GAS1, CASR and OTOP2, and are promising biomarker candidates. Conclusions: Our approach of analyzing transcriptomic data from different populations and patient groups will help to develop signatures and biomarkers for keratoconus subtypes. Further, RNA sequence data on individual patients obtained from multiple studies may lead to a core keratoconus signature of deregulated genes and a better understanding of its pathogenesis.

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

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.

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Transcriptome profiling of human keratoconus corneas through RNA sequencing identifies collagen synthesis disruption and downregulation of core elements of TGF-β, Hippo, and Wnt pathways

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