Study Of Keloid Pathogenesis: Inhibition Of PTGS2 Gene Expression In Fibroblasts By Vector-based RNAi

Objection: To explore the pathogenesis of keloid and to find out novel candidate keloid related genes which may take part in the pathogenesis of keloid and as targets of therapy reserch. 1.To validate the difference of expression of specific target genes in keloid and normal skin fibroblast by real-time RT-PCR.2. To detect the effects of RNAi-mediated target gene silencing in fibroblast of normal skin by real-time RT-PCR and Western blotting. 3. After transfection of the fibroblast with recombinant vector, cDNA microarray was used to measure genes expression in transfected fibroblast vs control. A literature mining method was used to annotate these different expression genes to explain the relationships among these genes and keloid.Methods: The cDNA microarray comprised of 21,552 clones were purchased from Beijing CapitalBio Corp.To test the results of gene chips, real-time RT-PCR was used to confirm the differential expression of NFKBIA, FDPS, CCNA2, MIF, EGFR and PTGS2. After normal skin fibroblast was transfected with recombinant vector, the interference effects were detected by real-time RT-PCR and western blotting. And we used cDNA microarray to measure genes expression in transfected fibroblast vs control. Annotating different expression genes with literature mining method.Result: Comparing with former data of microarray , trend of most real-time RT-PCR results were same with microarray. The differential expression of PTGS2 as downregulated gene was obvious in keliod. The results of real-time RT-PCR and western blotting indicated that siRNA could knock down the transcription and expression of PTGS2 gene. After fibroblast was transfected with recombinant vector, there were 10,682 efficient genes data in cDNA microarray according to the designed data filtered standards. Among these genes,189 genes were differently expressed genes between transfected fibroblast and control, of which 115 genes were up-regulated and 74 genes were down-regulated. These differently expressed genes may play an important role in keloid scar pathogenesis and thus may become prospective targets of keloid scar therapy.Conclusion: The results of real-time RT-PCR not only verified the dependences of microarray data but also implied that PTGS2 may play important roles in initiation of keloid. The study provided some material for studing the function of PTGS2 gene and indicated that the PTGS2 may be a new target of gene therapy on keloid.