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.

Background Due to the limited availability of established research models, very few studies addressed the health effects and underlying mechanisms following exposure to diesel exhaust during the initiation of pulmonary respiration. It is highly demanded to elucidate such health effects and underlying mechanisms, so as to exert protective measures during the early stages of life. Objective To evaluate the health effects of diesel exhaust very-early-in-life inhalation in hatchling chicken with a novel chicken embryo air cell inhalation exposure model, and to explore the potential roles of aryl hydrocarbon receptor signaling pathways in the observed effects with a specific aryl hydrocarbon receptor inhibitor. Methods Fertilized chicken eggs were assigned into five groups randomly (15 eggs per group): control group, air control group, aryl hydrocarbon receptor inhibitor (PDM2) group, diesel exhaust group, and diesel exhaust + aryl hydrocarbon receptor inhibitor (PDM2) group. Fertilized eggs were incubated with standard procedure. At embryonic day 17 (ED17), aryl hydrocarbon receptor inhibitor was administered to the corresponding animals. During embryonic day 18-19 (ED18-19), chicken embryos were exposed to diesel exhaust via air cell inhalation, then placed back to incubator until hatch. The air control group received clean air infusion during ED18-19, while the control group did not receive any treatment. Within 24 h post-hatch, 26 hatchling chickens were anesthetized with sodium pentobarbital, subjected to electrocardiography, and sacrificed to harvest tissue samples of heart and lung. Cardiopulmonary toxicities were evaluated by histopathology, and potential changes in the protein expression levels of aryl hydrocarbon receptor pathway molecule cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and fibrosis-related pathway molecule phosphorylated SMAD family member 2 (pSMAD2) were assessed by Western blotting. The remaining 29 hatchling chickens were reared until two weeks old, and then subjected to identical treatments. Results The inhalation exposure to diesel exhaust at initiation of pulmonary respiration resulted in thickened right ventricular wall (by 220.3% relative to the control group, same hereafter) and elevated heart rate (17.4%) in one-day-old hatchling chickens. Although no remarkable fibrotic lesions were observed at this point, the expression levels of CYP1A1 and phosphorylation levels of SMAD2 in the lung tissues significantly increased (by 81.3% and 71.6%, respectively). Such changes were effectively abolished by the aryl hydrocarbon receptor inhibitor PDM2 pretreatment. In the two-week-old animals, the thickened right ventricular wall (by 339.3%) and elevated heart rate (by 18.9%) persisted, and significant fibrotic lesions were observed in the lung tissue samples under Masson staining. Again, the aryl hydrocarbon receptor inhibitor PDM2 pretreatment effectively abolished such changes. In addition, no statistically significant changes in CYP1A1 expression levels were observed in the two-week-old chicken lung samples, and a remarkable down-regulation of SMAD2 phosphorylation was observed. The aryl hydrocarbon receptor inhibitor PDM2 pretreatment independently decreased the phosphorylation levels of SMAD2 in the two-week-old chicken lung samples. Conclusion Inhalation exposure to diesel exhaust at initiation of pulmonary respiration could result in persistent cardiopulmonary injury in hatchling chickens, and the underlying mechanism might be associated with the regulation of pSMAD2 by the aryl hydrocarbon receptor signaling pathway.