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Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

ABSTRACT: To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression.cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest.The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb.Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that are potentially active in the function of corneal and conjunctival epithelia.

SUBMITTER: Turner HC

PROVIDER: S-EPMC2909883 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

Turner Helen C HC Budak Murat T MT Akinci M A Murat MA Wolosin J Mario JM

Investigative ophthalmology & visual science 20070501 5

<h4>Purpose</h4>To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression.<h4>Methods</h4>cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with th ...[more]

PMID: 17460260

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Project description:Human conjunctival cell lines are useful tools for modeling ocular surface disease and evaluation of ocular drugs and cosmetics. However, gene expression in these cells may not be comparable to primary cultured cells, raising doubts that they could be used as a substitute. We aimed to ascertain the similarities of global gene expression between commonly used cell lines and primary cells using a microarray approach. The Affymetrix U133A chip (>22,000 genes) was used to investigate conjunctival tissue (CT), primary conjunctival epithelial cells (PCEC), two conjunctival epithelial cell lines (IOBA-NHC and ChWK), and HCEC-T, a human corneal epithelial cell line (control). Using principal component analysis, the PCEC profile was clustered more closely to conjunctival tissue than either of the two cell lines. Certain extracellular matrix genes were differentially upregulated in CT compared to PCEC, suggesting presence of fibroblasts in addition to epithelial cells in CT. Overall, 67.3% (95% CI: 66.7-67.9) of transcripts in IOBA-NHC were within 1.5-fold of the corresponding transcripts in PCEC, but only 62.2% (95% CI: 61.5-62.9) in the case of ChWK. In HCEC-T, the proportion was only 58.8% (95% CI 58.1-59.4), suggesting less resemblance to PCEC than the conjunctival epithelial cell lines. The IOBA-NHC profile was more similar to PCEC than ChWK, for all genes and genes concerned with membrane association, communication, development, and regulation of metabolism, especially protein and nucleic acid metabolism. The correlation of normalized gene expression levels was high between either the IOBA-NHC or ChWK and PCEC for genes concerned with cell defense, viral life cycle, antigen presentation, antioxidation, or ubiquitin ligation. In order to evaluate the functional significance of the altered gene expression in IOBA-NHC cells, we evaluated a few proteins important for epithelial differentiation or defense, corresponding to the transcripts for S100A9, TGM2, and TLR4. Protein levels of S100A9 and TGM2 were indeed raised, and TLR4 decreased, in IOBA-NHC compared to PCEC. Gene expression in conjunctival cell lines differs from primary cells, but the profile varies according to functional gene categories. Depending on the methodology of proposed studies, if there is limited availability of PCEC, NHC-IOBA may be more suitable than ChWK, but even then, epithelial differentiation and innate immunity functions in NHC-IOBA may differ from primary cells.

Dataset Information

Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

Publications

Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

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