EIF3e/INT6 Gene Therapy On Cutaneous Wound Healing:An In Vivo Study

Background:The Int6 gene is a subunit of the eukaryotic translation initiation factor eIF3(eIF3e).eIF3e/INT6 plays an important role in both eukaryotic translation initiation and regulatory of protein expression.It has been reported that Int6 is a site of mouse mammary tumour virus(MMTV)integration in murine tumours.There are different types of C-terminal truncated INT6 proteins produced due to the different integrations.Recent researches suggest that INT6 protein can specifically bind to hypoxia inducible factor-2a(HIF-2a)to regulate its function.Human HIF-2a,which located on chromosome 2p21-p16,is one of the key factors of hypoxia signal pathway.The HIF-2a can form heterodimer with HIF-? and transfer into the nuclear,then band to the hypoxic response elements(HREs)which present within the regulatory regions of a variety of target genes to up-regulate their expression which include some key angiogenic factors such as vascular endothelial growth factor(VEGF),angiogenesis-1(Ang-1)and basic fibroblast growth factor(bFGF)to promote functional angiogenesis.Some studies have reported that silence Int6 by iRNA-INT6 can promote the expression and accumulation of HIF-2a,thus mediate the functional angiogenesis and promote wound healing,but,if the truncated INT6 have the similar function haven't been reported.Objective:In order to preliminary reveal the effect of INT6 on wound healing,we employed Full-thickness cutaneous defects model in mice to study the wound healing efficacy of gene therapy of INT6 with its four other truncated types.Those truncated-IINT6 are Int6-2(410bp),Int6-A(493bp),Int6-B(885bp)and Int6-C(999bp),respectively.Methods and Results:To constructe the INT6,INT6-2,INT6-A,INT6-B and INT6-C eukaryotic expression vector by RT-PCR and molecular cloning technology and verify their correctness by double enzyme digestion and sequencing.Large-scale extraction of plasmids are carried out by a strategy of SiO2 absorption followed by a endotoxin removing step using Triton X-114 isothermal extraction method.Establish Full-thickness cutaneous defects model in mice,then transfection pSecX-Int6,pSecX-Int6-2,pSecX-Int6-A,pSecX-Int6-B and pSecX-Int6-C plasmid in vivo and evaluate the effect of gene therapy respectively.The effect of gene therapy was assessed by analyzing the curative rate of re-epithelialization and vascularization of mouse cutaneous.We employed Dunnett-t(<control)test to assess the residual wound area.Results showed that on day 5,pSecX-INT6-C treated group significant differences to blank and Pluronic F-127 hydrogel control groups(p=0.002,p=0.003)and significant difference to pSecX control group(p=0.000).pSecX-INT6-B had significant difference to pSecX control group(p=0.011).pSecX-INT6-B and pSecX-INT6-C treated groups displayed completely re-epithelialization on day 7 and there are significant differences when compared with the control,pSecX and Pluronic F-127 hydrogel groups(p0.011,p=0.004,p=0.001 and p=0.004,p=0.001,p=0.000)respectively.Staistically significant difference have been showed between pSecX-INT6-C and Pluronic F-127 hydrogel treatment groups(p=0.000).The same situation occurs in pSecX-INT6-A with each control groups,the differences between pSecX-INT6-A and pSecX group is p=0.039,and Pluronic F-127 hydrogel p=0.012.Experimental results show that pSecX-INT6-B and pSecX-INT6-C treatment groups can significantly promote the cutaneous wound healing.Moreover,observation of tissue Masson staining showed that collagen depositon of pSecX-INT6-B and pSecX-INT6-C treatment groups at day 5 and day 7 are More orderly than any of the control groups,and are parallel to the epithelial which suggests that pSecX-INT6-B and pSecX-INT6-C might inhibit the deposition of collagen fibers to prevention the formation of hyperplastic scar.Conclusion:In this study,we compared the curative effect of INT6 and truncted INT6-2.INT6-A?INT6-B and INT6-C gene therapy,systematically.we found that INT6-B and INT6-C gene therapy can accelerate wound healing via promote re-epithelialization;INT6-B and INT6-C gene therapy may prevent the formation of hypertrophic scar by inhibiting collagen deposition while wound healing;INT6-B and INT6-C gene therapy has unobvious effect on vascularzation while wound healing.