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MicroRNA-145 regulates human corneal epithelial differentiation.

Lee SK, Teng Y, Wong HK, Ng TK, Huang L, Lei P, Choy KW, Liu Y, Zhang M, Lam DS, Yam GH, Pang CP - PLoS ONE (2011)

Bottom Line: As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.We found expression of miR-143/145 cluster in human corneal epithelium.Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.

Methodology/principal findings: Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.

Conclusion/significance: We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.

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Validation of miR-143 and miR-145 expression in limbal epithelium.By qPCR analysis, (A) miR-143 and (B) miR-145 was up-regulated (compared to U6 expression) in LPC epithelia, when compared to CC (P<0.001, Mann Whitney U-test). Red lines indicated mean CT value. In situ hybridization showed (C) miR-143 and (E) miR-145 in limbal epithelium, compared to (G) scrambled sequences and (H) U6. At higher magnification, (D) miR-143 and (F) miR-145 were present in parabasal layers. Scale bars: 150 µm (C, E, G, H); 40 µm (D, F).
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pone-0021249-g002: Validation of miR-143 and miR-145 expression in limbal epithelium.By qPCR analysis, (A) miR-143 and (B) miR-145 was up-regulated (compared to U6 expression) in LPC epithelia, when compared to CC (P<0.001, Mann Whitney U-test). Red lines indicated mean CT value. In situ hybridization showed (C) miR-143 and (E) miR-145 in limbal epithelium, compared to (G) scrambled sequences and (H) U6. At higher magnification, (D) miR-143 and (F) miR-145 were present in parabasal layers. Scale bars: 150 µm (C, E, G, H); 40 µm (D, F).

Mentions: Elevated expression of miR-143 and miR-145 in LPC epithelia was validated by qPCR on additional 11 pairs of human LPC and CC epithelia. After normalization with the respective U6, ΔCT of miR-143 was 5.9±0.8 in LPC and 11.1±0.9 in CC epithelia (P = 0.0006, Mann Whitney U-test) (Fig. 2A). Similarly, ΔCT of miR-145 was 4.5±0.7 in LPC and 10.2±0.7 in CC epithelia (P = 0.0004, Mann Whitney U-test) (Fig. 2B). The smaller the ΔCT values relative to U6, the higher the expression. With about 88% efficiency in our PCR amplification system, LPC had miR-143 and miR-145 expressions 26.6-fold and 36.5-fold higher than CC epithelia respectively. Such levels were comparable to the array results described earlier.


MicroRNA-145 regulates human corneal epithelial differentiation.

Lee SK, Teng Y, Wong HK, Ng TK, Huang L, Lei P, Choy KW, Liu Y, Zhang M, Lam DS, Yam GH, Pang CP - PLoS ONE (2011)

Validation of miR-143 and miR-145 expression in limbal epithelium.By qPCR analysis, (A) miR-143 and (B) miR-145 was up-regulated (compared to U6 expression) in LPC epithelia, when compared to CC (P<0.001, Mann Whitney U-test). Red lines indicated mean CT value. In situ hybridization showed (C) miR-143 and (E) miR-145 in limbal epithelium, compared to (G) scrambled sequences and (H) U6. At higher magnification, (D) miR-143 and (F) miR-145 were present in parabasal layers. Scale bars: 150 µm (C, E, G, H); 40 µm (D, F).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3119052&req=5

pone-0021249-g002: Validation of miR-143 and miR-145 expression in limbal epithelium.By qPCR analysis, (A) miR-143 and (B) miR-145 was up-regulated (compared to U6 expression) in LPC epithelia, when compared to CC (P<0.001, Mann Whitney U-test). Red lines indicated mean CT value. In situ hybridization showed (C) miR-143 and (E) miR-145 in limbal epithelium, compared to (G) scrambled sequences and (H) U6. At higher magnification, (D) miR-143 and (F) miR-145 were present in parabasal layers. Scale bars: 150 µm (C, E, G, H); 40 µm (D, F).
Mentions: Elevated expression of miR-143 and miR-145 in LPC epithelia was validated by qPCR on additional 11 pairs of human LPC and CC epithelia. After normalization with the respective U6, ΔCT of miR-143 was 5.9±0.8 in LPC and 11.1±0.9 in CC epithelia (P = 0.0006, Mann Whitney U-test) (Fig. 2A). Similarly, ΔCT of miR-145 was 4.5±0.7 in LPC and 10.2±0.7 in CC epithelia (P = 0.0004, Mann Whitney U-test) (Fig. 2B). The smaller the ΔCT values relative to U6, the higher the expression. With about 88% efficiency in our PCR amplification system, LPC had miR-143 and miR-145 expressions 26.6-fold and 36.5-fold higher than CC epithelia respectively. Such levels were comparable to the array results described earlier.

Bottom Line: As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.We found expression of miR-143/145 cluster in human corneal epithelium.Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.

Methodology/principal findings: Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.

Conclusion/significance: We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.

Show MeSH
Related in: MedlinePlus