Screening Differentially Expressed Genes Of GJB2 Mutant By Gene Chip And Functional Verification Of Candidate Genes

Background Vohwinkel syndrome(VS)is a refractory rare hereditary skin disease.According to different mutant genes and clinical manifestations,it is divided into two types,typical and variant types.Studies have confirmed that GJB2 gene mutations can cause typical VS,but there is no relevant functional research at home and abroad to clarify its specific mechanism.Recently,with the rapid development of gene expression profiling technology,gene chip plays an increasingly important role in screening and discovering disease-related genes,exploring the occurrence and development of diseases,and seeking precise treatment target sites.In order to more comprehensively understand the differentially expressed gene(DEG)in VS,this study will screen the gene chip of the diseased cell lines,so as to find and analyze the specific functions of DEG in the occurrence of diseases more accurately.Objective This study constructed stable strains transfected with the HaCaT cell line carrying lentiviral vectors of two GJB2 mutant genes(G130V,D66H)of VS controlled by the Tet-on system.Using flow cytometry to detect cell apoptosis,combined with human gene expression profiling technology(Affymetrix expression profiling),and then study the mechanism of GJB2 gene mutants may cause special clinical symptoms of VS.Methods By using PCR technology to amplify human GJB2 gene mutants(G130V,D66H),clone into the lentiviral plasmid regulated by Tet-on system,and carry out the identification and sequencing of the recombinant plasmid to obtain two positive lentiviruses whose quality meets the standard.Two positive lentiviruses were transfected into HaCaT cells,and after puromycin and DOX double screening,HaCaT cell lines that over expressed GJB2 gene mutants(G130V,D66H)were cultured.And RT-PCR was used to detect the expression of GJB2 gene mRNA in HaCaT cell lines stably over expressing GJB2 gene mutants(G130V,D66H).Flow cytometry was used to detect the effect of overexpression of GJB2 gene mutations(G130V,D66H)on HaCaT cell apoptosis.At the same time,two Affymetrix expressions profiling chips,G130V vs.NC and D66H vs.NC,were produced respectively(NC is control group).And the differentially expressed genes of the two gene chips were screened out after data analysis.combined with Ingenuity Pathway Analysis(IPA)analysis technology,in-depth bioinformatic analysis of DEGs was screened out.At the same time,genes and signal pathway closely related to the symptoms and pathogenesis of VS were screened out of the DEGs.Finally,using RT-PCR technology to verify the candidate genes.Results(1)After induced by tetracycline,the expression levels of HaCaT cell lines stably transfected with GJB2 gene mutants D66H and G130V increased significantly,indicating that the stable target two gene transfection HaCaT cell lines were successfully constructed.(2)The results of flow cytometry found that both D66H and G130V groups increased the apoptosis rate compared with the NC group,and the difference was statistically significant(P<0.05).(3)G130V vs.NC and D66H vs.NC gene chip results data screening results:Compared with the NC group,the number of up-regulated genes in the G130V group was 251 and the number of down-regulated genes was 273;the number of up-regulated genes in the D66H group was 1072 and the number of down-regulated genes was 701(P<0.05 and the difference was more than 1.5 times).Based on the results of the gene chip and IPA analysis,in the G130V vs.NC group,a total of 21 DEGs were screened based on the results of the gene chip and IPA analysis,and the RT-PCR verification results were consistent with the chip predictions.In the D66H vs.NC group,A total of 23 DEGs were screened in the D66H and NC groups.RT-PCR verification found that the mRNA expression of the 4 DEGs except MAPK8 was contrary to the prediction of the chip results,and the other results were consistent with the chip prediction.(4)At the same time,we also detected genes of related to skin keratinization(FLG and LOR)both in G130V vs.NC group and D66H vs.NC group.As a result,compared with NC group,the expression levels of FLG gene mRNA in both groups were up-regulated,to our surprise,the expression level of LOR gene mRNA was down-regulated in the D66H group but G130V group had no expression.(5)Experimental verification found that the differential genes were related to the AHR,IFN,JAK/STAT signaling pathway,and JNK/AP1 signaling pathway.At the same time,different GJB2 gene mutations may have different signaling pathways involved in the pathogenesis of VS.Conclusions(1)This project found that the JNK/AP1 signaling pathway is involved in the regulation of VS caused by different GJB2 mutations.(2)Different mutations in the GJB2 gene may involve different signal transmission and participate in abnormal pathogenesis.(3)The upstream network involved in the regulation of different GJB2 mutations is different,and the regulation of downstream functions is more complicated.(4)The research used gene chip technology in combination with traditional laboratory research methods to discover for the first time multiple abnormal differential genes in typical VS,which enriches the knowledge of related abnormal signal transmission pathways.Further study of the pathogenic mechanism laid the foundation,and provided more specific research directions and ideas.