Experimental Study On The Promoter Activity Of The Human Integrin β1 In HaCaT Cells

Background:At present,the shortage of autologous skin graft don't meet the requirement of wound repair in burn patients with severe skin defects.With the development of molecular,cell and developmental biology,we have made great progress in regeneration.Keratinocyte stem cells(KSCs) possess the powerful proliferative capacity,and they also have the ability to differentiate into epidermal cells in every stage.Therefore,KSCs as "seed cells" for skin reconstruction,has become very hot in nowadays.To acquire enough amount of KSCs, and to induce KSC to differentiate into certain kind of cells is vital for the research of regulative mechanism of KSC proliferation and differentiation.Previous studies have indicated that integrinβ1 played important roles in generation and differentiation of KSCs.High expression of Integrinβ1 was found in basal body and folliculus pili of epidermal cell(EC),and in this part stem cell is rich.On surface of KSC integrinβ1 expression is 2～3 times more than that in transit amplifying cell(TAC).KSC maintain its characteristics of stem cell through adhering to basal membrane(BM).If KSC cast off,it will turn to terminal differentiation.Integrinβ1 is one of the generally accepted molecular markers for the undifferentiation of KSCs.Jones found that epidermal cells with high expression of integrinβ1 have the capacity to form more clones,and they can be passaged for more times when compared with those with low expression.In our previous study,we found that expression of integrinβ1 was down-regulated after the transfection of siIntegrinβ1 recombinant vector into KSCs.The clone formation rate of cells after transfection was lower than that without transfection,which showed that integrinβ1 paticipated in the regulation of KSC proliferation and differentiation.Piero C et al have shown that the regulatory region of theβ1-integrin gene contains two promoters,one distal and one proximal tandemly situated.The two tandemly promoter regions are very G+C-rich,and lack both a TATA box and a CAAT box.Each promoter drives the expression of a uniqueβ1 gene,and two mRNAs are synthesized that share the same coding sequence but diverge in the complete 5' untranslated region,but the regulation mechanism in transcription level was still unclear.HaCaT(Human adult skin keratinocytos,HaCaT) cell line originates from normal human epidermal cells,which have the capacity to proliferate infinitely.Research has showed that after injection of HaCaT into subcutaneous skin of the nude mice,it coμld differentiate into epithelial tissues,with expression of special markers for differentiation. In addition,our previous study indicated that expression of integrinβ1 protein was supressed by the transfection of siIntegrinβ1 vector.The growth curve of HaCaT cells after transfection was moved right forward,indicating that the proliferation of HaCaT cells was inhibited by the transfection.As described above,HaCaT cell line can provide an ideal model for the research of regulation of human epidermal cell proliferation.Objective:To explore the mechanism of regulation of integrinβ1 expression.HaCaT cells and luciferase reporter gene system containing integrinβ1 promoter were enrolled in our study to investigate the influence of integrinβ1 on the proliferation and differentiation of human epidermal cells.Methods:Dual-luciferase reporter vectors containing distal part and proximal part of integrinβ1 promoter were constructed successfully.Then they were transfected into HaCaT cells to investigate their activity after transfection.This can help us to understand the role of integrinβ1 in the transcriptive regulation of HaCaT cells.The main effect of promotor in transcriptional control was determined in according to the result in the first part.Tfsitescan software was used to analyze potential binding site of transcription factor in the promotor.Dissection and construction of mutation luciferase report gene vector with series deletion of promotor were carry out according to analytic result.After sequencing,they were transfected into HaCaT cells,then the activity of luciferase was detected,and the activity of distal promoter of integrinβ1and potential transcriptional control factors participating possibly transcriptional control were analyzed. Results:We successfully amplified the whole length of integrinβ1 promoter from human genome by PCR,and transfected the vector containing proximal,distal and full part of integrinβ1 promoter into HaCaT cells.Analysis of luciferase activity revealed that the distal part of promoter played an important role in the regulation of integrinβ1 transcripton.In addition,we successfully constructed series deletion mutation luciferase report gene vector of promotor:pGL3-1442,pGL3-840,pGL3-602,pGL3-352,pGL3-212,pGL3-270,p GL3-390,and transfected the series vector into HaCaT cells.Analysis of luciferase activity revealed that luciferase activity of pGL3-602 is highest,but pGL3-840 have no activity.Conclusion:1.The distal promoter of integrinβ1 plays an important role in the regulation of integrinβ1 transcripton in HaCaT cells.2.The part of 602 bp(—602/—1) sequence of the integrinβ1 distal promoter have the highest activity.The part of 250 bp(—602/—352) sequence of the integrinβ1 distal promoter is high transcription active zone.3.The part of 840 bp(—1442/—603)sequence of the integrinβ1 distal promoter may exist transcription inhibitor.