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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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Related in: MedlinePlus

Transfection analysis of miR-143 and miR-145.(A–D) Human P2 CEPCs transfected with (A and B) Lenti-miR-143 and (C and D) Lenti-miR-145. (A, C) Phase-contrast images; (B, D) live GFP imaging. (E) Overexpression levels of miR-143 and 145 in transfected CEPCs by qPCR analysis. Amplification signals from cells with scrambled sequences (Scm) were indicated. Immunofluorescence of (F–H) cytokeratin-3/12 and (I–K) connexin-43 in CEPCs transfected with (F, I) scrambled sequence, (G, J) Lenti-miR-143 and (H, K). (L) Western blotting and densitometry analysis of connexin-43 (Cnx43) and GAPDH in CEPCs transfected with Lenti-miR-145 or scrambled sequences. (M) RT-PCR result of integrin β8 (ITGB8), cytokeratin-3 (CK3), ABCG2, β-actin and GAPDH in different primary CEPCs (at P2) transfected with Lenti-miR-145 or scrambled sequences. (N) Immunofluorescence of miR-145 (revealed by GFP), p63α and nuclear DAPI stain in P2 CEPCs after Lenti-miR-145 transfection. Scale bars: (A–D) 100 µm; (F–K, N) 10 µm.
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pone-0021249-g003: Transfection analysis of miR-143 and miR-145.(A–D) Human P2 CEPCs transfected with (A and B) Lenti-miR-143 and (C and D) Lenti-miR-145. (A, C) Phase-contrast images; (B, D) live GFP imaging. (E) Overexpression levels of miR-143 and 145 in transfected CEPCs by qPCR analysis. Amplification signals from cells with scrambled sequences (Scm) were indicated. Immunofluorescence of (F–H) cytokeratin-3/12 and (I–K) connexin-43 in CEPCs transfected with (F, I) scrambled sequence, (G, J) Lenti-miR-143 and (H, K). (L) Western blotting and densitometry analysis of connexin-43 (Cnx43) and GAPDH in CEPCs transfected with Lenti-miR-145 or scrambled sequences. (M) RT-PCR result of integrin β8 (ITGB8), cytokeratin-3 (CK3), ABCG2, β-actin and GAPDH in different primary CEPCs (at P2) transfected with Lenti-miR-145 or scrambled sequences. (N) Immunofluorescence of miR-145 (revealed by GFP), p63α and nuclear DAPI stain in P2 CEPCs after Lenti-miR-145 transfection. Scale bars: (A–D) 100 µm; (F–K, N) 10 µm.

Mentions: Freshly isolated human CEPCs were cultured in CnT20 medium and cells from primary holoclones were pooled and plated as passage 2 (P2) for transfection with lenti-miR expression plasmid pMIRH143PA-1 or pMIRH145PA-1 at a ratio of 3 µl Lipofectamine 2000 per µg DNA. Over-expressions of miR-143 and miR-145 were shown by GFP live imaging (Figs. 3A–B, miR-143; 3C–D, miR-145) and qPCR (Fig. 3E). The transfected cells were kept in CnT50 medium with low bovine pituitary extract (15 µg/ml) for optimal corneal epithelial cell growth for 2 days followed by expression analysis. By immunofluorescence, both miR-143 and 145-transfected cells had increased CK3/12 expression (Figs. 3G and H), when compared to cells transfected with scrambled sequences (Fig. 3F). This was also detected by western blotting (Fig. 3N, second panel). On the other hand, miR-145-transfected cells showed relatively stronger Cnx-43 expression (Fig. 3K), which was mild in cells transfected with scrambled sequences (Fig. 3I) or miR-143 (Fig. 3J). Western blot analysis showed Cnx-43 upregulation in CEPCs transfected with miR-145 by about 15 folds more than those with scrambled sequences (Fig. 3L). In addition, these cells had reduced ABCG2 (Fig. 3M, bottom panel) and p63α expression (Fig. 3N) as revealed by RT-PCR and immunofluorescence, respectively. This expression pattern of corneal differentiation markers, i.e., reduced ABCG2 and p63α expression, and its expression in parabasal layers of limbal epithelium, indicated that miR-145 might be involved in corneal epithelial differentiation. We corroborated this supposition by a three-dimensional corneal epithelial organotypic assay. Human P2 CEPCs transfected with miR-143, miR-145 or scrambled sequences were expanded to monolayer cell sheet on denuded AM in submerged culture, followed by air-lifting to induce cell stratification. The composites were harvested for morphological examination. The number of epithelial layer was quantified in 15 random sites along the composite to obtain the epithelium forming efficiency. CEPCs without transfection (Fig. 4D), with lipofectamine only (Fig. 4E) or transfected with scrambled sequences (Fig. 4A), generated thicker epithelia. They had typical epithelial morphology with basal cuboidal-like cells next to the basement membrane. The cells were packed and appeared squamous in shape at the superficial layers (non-transfected: 12.5±1.5 layers; lipofectamine-only; 9.9±2.3 layers; transfected with scrambled sequence: 10.2±2.4 layers) (Fig. 4F). However, this was not observed in the epithelia generated from miR-145-transfected CEPCs (Fig. 4B). The epithelium was degraded, thin (5.6±1.3 layers) and loosened with reduced cell density. Few cuboidal cells were found in the basal layer and cells were tends to be flatten or squamous in shape. The epithelium generated from miR-143-transfected CEPCs had morphology and compactness intermediate between control and miR-145 epithelia (8.3±1.6 layers) (Fig. 4C). The same results were obtained in duplicated experiments.


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)

Transfection analysis of miR-143 and miR-145.(A–D) Human P2 CEPCs transfected with (A and B) Lenti-miR-143 and (C and D) Lenti-miR-145. (A, C) Phase-contrast images; (B, D) live GFP imaging. (E) Overexpression levels of miR-143 and 145 in transfected CEPCs by qPCR analysis. Amplification signals from cells with scrambled sequences (Scm) were indicated. Immunofluorescence of (F–H) cytokeratin-3/12 and (I–K) connexin-43 in CEPCs transfected with (F, I) scrambled sequence, (G, J) Lenti-miR-143 and (H, K). (L) Western blotting and densitometry analysis of connexin-43 (Cnx43) and GAPDH in CEPCs transfected with Lenti-miR-145 or scrambled sequences. (M) RT-PCR result of integrin β8 (ITGB8), cytokeratin-3 (CK3), ABCG2, β-actin and GAPDH in different primary CEPCs (at P2) transfected with Lenti-miR-145 or scrambled sequences. (N) Immunofluorescence of miR-145 (revealed by GFP), p63α and nuclear DAPI stain in P2 CEPCs after Lenti-miR-145 transfection. Scale bars: (A–D) 100 µm; (F–K, N) 10 µm.
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getmorefigures.php?uid=PMC3119052&req=5

pone-0021249-g003: Transfection analysis of miR-143 and miR-145.(A–D) Human P2 CEPCs transfected with (A and B) Lenti-miR-143 and (C and D) Lenti-miR-145. (A, C) Phase-contrast images; (B, D) live GFP imaging. (E) Overexpression levels of miR-143 and 145 in transfected CEPCs by qPCR analysis. Amplification signals from cells with scrambled sequences (Scm) were indicated. Immunofluorescence of (F–H) cytokeratin-3/12 and (I–K) connexin-43 in CEPCs transfected with (F, I) scrambled sequence, (G, J) Lenti-miR-143 and (H, K). (L) Western blotting and densitometry analysis of connexin-43 (Cnx43) and GAPDH in CEPCs transfected with Lenti-miR-145 or scrambled sequences. (M) RT-PCR result of integrin β8 (ITGB8), cytokeratin-3 (CK3), ABCG2, β-actin and GAPDH in different primary CEPCs (at P2) transfected with Lenti-miR-145 or scrambled sequences. (N) Immunofluorescence of miR-145 (revealed by GFP), p63α and nuclear DAPI stain in P2 CEPCs after Lenti-miR-145 transfection. Scale bars: (A–D) 100 µm; (F–K, N) 10 µm.
Mentions: Freshly isolated human CEPCs were cultured in CnT20 medium and cells from primary holoclones were pooled and plated as passage 2 (P2) for transfection with lenti-miR expression plasmid pMIRH143PA-1 or pMIRH145PA-1 at a ratio of 3 µl Lipofectamine 2000 per µg DNA. Over-expressions of miR-143 and miR-145 were shown by GFP live imaging (Figs. 3A–B, miR-143; 3C–D, miR-145) and qPCR (Fig. 3E). The transfected cells were kept in CnT50 medium with low bovine pituitary extract (15 µg/ml) for optimal corneal epithelial cell growth for 2 days followed by expression analysis. By immunofluorescence, both miR-143 and 145-transfected cells had increased CK3/12 expression (Figs. 3G and H), when compared to cells transfected with scrambled sequences (Fig. 3F). This was also detected by western blotting (Fig. 3N, second panel). On the other hand, miR-145-transfected cells showed relatively stronger Cnx-43 expression (Fig. 3K), which was mild in cells transfected with scrambled sequences (Fig. 3I) or miR-143 (Fig. 3J). Western blot analysis showed Cnx-43 upregulation in CEPCs transfected with miR-145 by about 15 folds more than those with scrambled sequences (Fig. 3L). In addition, these cells had reduced ABCG2 (Fig. 3M, bottom panel) and p63α expression (Fig. 3N) as revealed by RT-PCR and immunofluorescence, respectively. This expression pattern of corneal differentiation markers, i.e., reduced ABCG2 and p63α expression, and its expression in parabasal layers of limbal epithelium, indicated that miR-145 might be involved in corneal epithelial differentiation. We corroborated this supposition by a three-dimensional corneal epithelial organotypic assay. Human P2 CEPCs transfected with miR-143, miR-145 or scrambled sequences were expanded to monolayer cell sheet on denuded AM in submerged culture, followed by air-lifting to induce cell stratification. The composites were harvested for morphological examination. The number of epithelial layer was quantified in 15 random sites along the composite to obtain the epithelium forming efficiency. CEPCs without transfection (Fig. 4D), with lipofectamine only (Fig. 4E) or transfected with scrambled sequences (Fig. 4A), generated thicker epithelia. They had typical epithelial morphology with basal cuboidal-like cells next to the basement membrane. The cells were packed and appeared squamous in shape at the superficial layers (non-transfected: 12.5±1.5 layers; lipofectamine-only; 9.9±2.3 layers; transfected with scrambled sequence: 10.2±2.4 layers) (Fig. 4F). However, this was not observed in the epithelia generated from miR-145-transfected CEPCs (Fig. 4B). The epithelium was degraded, thin (5.6±1.3 layers) and loosened with reduced cell density. Few cuboidal cells were found in the basal layer and cells were tends to be flatten or squamous in shape. The epithelium generated from miR-143-transfected CEPCs had morphology and compactness intermediate between control and miR-145 epithelia (8.3±1.6 layers) (Fig. 4C). The same results were obtained in duplicated experiments.

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