Effect of p53 gene silencing on microRNA expression profiles in HaCaT human keratinocytes / 中华皮肤科杂志

ABSTRACT

Objective To assess differential expression profiles of microRNAs(miRNAs) in HaCaT human keratinocytes before and after p53 gene silencing,and to make a functional analysis of target genes.Methods Lentivirus-mediated RNA interference (RNAi) was used to silence p53 gene in HaCaT cells.Total RNA was extracted using Trizol reagent.Then,miRNAs were isolated by polyethylene glycol (PEG) and subjected to fluorescent labeling using T4RNA ligase followed by hybridization to a mammalian miRNA chip.Microarrays were scanned by a GenePix 4000B microarray scanner and fluorescence ratios were determined with the GenePix Pro 6.0 software.The TargetScan software was used to predict target genes of differentially expressed miRNAs (＞2-fold difference in expression level),and the top 20 target genes with the highest enrichment score were selected for each miRNA and subjected to functional analysis and pathway analysis through the KEGG signaling database.Results Totally,53 differentially expressed miRNAs,including 12 down-regulated and 41 up-regulated miRNAs,were identified in HaCaT cells after p53 silencing as compared to those before p53 silencing.Of these 53 differentially expressed miRNAs,5 (hsa-miR-141-3p,hsa-miR-15a-5p,hsa-miR-27a-3p,hsa-miR-130b-3p,hsa-miR-19a-3p) showed a more than 200-fold increase in expression,and 4 (hiv1-miR-TAR-3p,hsa-miR-630,hsa-miR-1246,hsa-miR-1275) experienced a more than 4-fold decrease in expression in HaCaT cells after p53 silencing.Functional analysis and pathway analysis revealed that some target genes of these differentially expressed miRNAs were involved in the mitogen-activated protein kinase (MAPK) signaling pathway,metabolic pathways,and tumor invasion.Conclusion Nine miRNAs,including hsa-miR-141-3p,may be involved in p53-mediated molecular regulation.