Genome-wide expression profile analysis of Nicastrin gene-silenced HaCaT cells / 中华皮肤科杂志

ABSTRACT

Objective:To investigate changes of nicastrin (NCSTN) downstream molecules in signaling pathways related to cell proliferation and differentiation after silencing the expression of the NCSTN gene in the human immortalized keratinocyte cell line HaCaT.Methods:HaCaT cells were divided into 3 groups interference group transfected with a specific small interfering RNA (siRNA) targeting NCSTN (NCSTN-siRNA) , negative control group transfected with a negative control siRNA, and blank control group transfected with the equal amount of transfection reagent. Real-time PCR and Western blot analysis were performed to measure the NCSTN mRNA and protein expression in groups, in order to verify the transfection efficiency. Differences in gene expression profiles in HaCaT cells were detected between the interference group and negative control group by using Agilent whole-genome microarray, and differentially expressed genes were identified based on a fold change ≥ 2.0 with a P value ≤ 0.05. Gene ontology (GO) enrichment analysis was employed to identify the roles of the differentially expressed genes, and then to screen out significantly differentially expressed genes associated with proliferation and differentiation of keratinocytes, some of which were verified by real-time PCR. Results:The interference group showed significantly decreased mRNA and protein expression of NCSTN (0.287 ± 0.090, 0.443 ± 0.085, respectively) compared with the negative control group (0.969 ± 0.127, 1.047 ± 0.114, respectively) and blank control group (1.000 ± 0.151, 1.000 ± 0.111, F = 30.787, 31.139, respectively, both P = 0.001) . Whole genome-expression analysis using an Agilent microarray platform revealed 605 downregulated genes and 444 upregulated genes in HaCaT cells in the interference group compared with the negative control group. GO analysis showed that differentially expressed genes were enriched into 4 biological processes, including epithelial development, epithelial cell differentiation, keratinocyte differentiation and keratinization. The significantly differentially expressed genes associated with proliferation and differentiation of keratinocytes, including the Sprouty-related protein with EVH1 domain 2, fibroblast growth factor 7, insulin-like growth factor-binding protein 5, Rho-associated coiled-coil kinase 2 and bone morphogenetic protein 6 genes, were verified by real-time PCR, and the verification results were consistent with the difference trend shown by the microarray results. Conclusion:The loss of NCSTN gene function may affect the normal proliferation and differentiation of keratinocytes by regulating the expression of its downstream molecules in signaling pathways associated with cell proliferation and differentiation.