Screening Differentially Expressed Genes Of GJB6 Mutant By Gene Chip

Background Hidrotic ectodermal dysplasia(HED)or Clouston syndrome is a rare autosomal dominant disorder.It is characterized by the triad of nail dystrophy,alopecia and palmoplantar hyperkeratosis.To date,all mutations have been involving in five codons:G11R?A88V?V37E?D50N and N14S in the connexin 30(Cx30)gene have been shown to cause this disorder.At present,there is little research on the pathogenesis of HED,and the pathological process of clinical phenotype,which is the function of Cx30,is a huge challenge in this field.Thanks for the successful completion of human genome project,the gene expression profile chip has become one of the main tools to study the differential expression of genes.Gene chip technology has the advantages of high throughput,low cost,automation,pollution prevention and other aspect.Our study group has successfully constructed lentiviral vector containing human wild-type GJB6 gene and its mutation(A88V)stably expressing by Tet-on system on HaCaT cell lines,which provided a stable experimental model for the following experiment.In this study,we try to construct the HaCaT cell line GJB6 gene stably expressing G11R gene,and make the Affymetrix expression profile chip to explore the possible signal pathway and mechanism of GJB6 gene.Objective To screen the differentially expressed genes in HaCaT cell line stably expressing human GJB6 gene by gene chip and explore the possible signal pathway and mechanism of the mutant gene.Methods To construct lentiviral vector containing human GJB6 gene mutation(G11R)stably expressing by Tet-on system on HaCaT cell lines.After extraction of total RNA and quality control,the RNA was processed for fluorescent labeling,hybridization,washing,scanning and signal digitizing.Then the differentially expressed genes were screened by using Affymetrix microarray chip.Then the screened gene were analyzed on expression and function by IPA(Ingenuity Pathway Analysis),the differentially expressed genes with higher correlation were validated by using the WES Simple Western technology.Results The result of total RNA quality identification showed that all samples were high-quality.(RIN>= 9.3,28S/18S>= 1.5,A260/A280 were between 1.99 and 2.06).Microarray analysis revealed that the OE group(target group)and the NC group(negative control)were differentially expressed,of which 546 genes significantly up-regulated and 926 genes significantly down-regulated(p<0.05 and differences were more than 2 times).The differentially expressed genes were rich in many diseases and functions,in which the apoptosis,cell differentiation,epithelial cell differentiation,dermal cell differentiation,epidermal cell differentiation,epithelial tissue differentiation and neoplasia of epithelial tissue may be closely related to the function of GJB6 gene.WES Simple Western analysis revealed that MKI67 was down-regulated by 73.65%,the PLK1 was down-regulated by 48.41%and the BCL2L11 was up-regulated by 147.21%,which were consistent with the results of chip screening.Conclusions Microarray analysis can screen the differentially expressed genes in GJB6 gene rapidly and efficiently,which play an important role by regulating multiple signaling pathways.Its final goal is to lay a solid theoretical foundation for further study of the pathogenesis of HED.