Different expression profiles of micro-RNA in epidermal cells of different developmental stages

Abstract

Objective To investigate the difference in expression profiles of micro-RNA (miR-NA) between human epidermal stem cells and epidermal keratinocytes.Methods (1) Human primary epidermal stem cells and keratinocytes were obtained with enzyme digestion method and type Ⅳ collagen coated chosen method.Growth of cells cultured in vitro was observed by inverted microscope.Monoclonal antibody of integrinβ1,keratin 1 (CK1),CK10,and CK19 were detected by immunocytochemical staining.(2)Total RNA was respectively isolated from epidermal stem cells and epidermal keratinocytes by Trizol-based single-step procedure,detected by formaldehyde denaturing gel electrophoresis,purified by mirVanaTM miRNA isolation kit,and then labeled and hybridized by miRNA labeling and hybridization kit.Images of hybridization were analyzed using the Feature Extraction (Version 10.7).Data normalization and difference analysis were performed with GeneSpring (GX 10.0).Moreover,miRNA microarray results were confirmed by RT-PCR.(3) Target genes of differently expressed miRNA were predicted.Results Epidermal stem cells exhibited rapid adherence to type Ⅳ collagen and formed distinct clones after 3 days of culture; expressions of integrinβ1 and CK19 were positive.Keratinocytes could not adhere rapidly to type Ⅳ collagen and formed few clones after 3 days of culture ; expressions of CK1 and CK10 were positive.(2) Of the epidermal stem cells,31 miRNAs were up-regulated and 153 down-regulated.Besides,significantly up-regulated miRNAs were hsa-miRNA-125b-3p,hsa-miRNA-197-5p,and hsa-miRNA-376a-3p,while significantly down-regulated miRNAs were hsa-miRNA-203,hsa-miRNA-29b-3p,and hsa-miRNA-34a-3p.Findings of RT-PCR on hsa-miRNA-197-5p and hsa-miR-29b-3p revealed high concordance with the microarray results.(3) Some miRNAs target genes indicated that miRNA related to cell proliferation,differentiation,apoptosis,aging and other biological characteristics.Conclusion Distinct differences in miRNA expression profiles are detected between human epidermal stem cells and keratinocytes and this may correlate closely with their different biological characteristics such as proliferation and differentiation ability. Key words: Epidermis;  Stem cells;  Gene expression profiling