Effect And Mechanism Of Autocrine Motility Factor On Proliferation And Migration Of Human Keloid Fibroblast

Background & objective:Keloid is one of common pathological scars with the characters of fibroblast overgrowth and exceedingly collagen synthesis,during the process of wound healing in human skin.It is seriously affected in human physical and psychological health with disfigurement and joint disfunction.Since no certain effective treatment of keloid for clinical doctors to use,so far,that lead to a high recurrence rate.With the difference of other scars,keloid have many significant biological characteristics like malignant tumor,such as,no self-limited growth,invasiveness out of wound margin,rich blood flow,close relationships between keloid pathogenesis and tumor-associated genes and cytokines,effectivity by the treatment of malignant tumor,and so on.It has important guiding significance to explore keloid pathogenesis and enhance differential diagnosis and therapeutic effect.Autocrine motility factor/autotaxin(AMF/ATX)is a cytokine with molecular weight of 60 kD in reducing status and 55 kD in no-reducing status,separated and purified recently from malignant tumor cell medium,such as,hepatic carcinoma,lung carcinoma,fibrosarcoma,mastocarcinoma and melanoma.After combination of AMF and AMF receptor(AMFR),a glycoprotein of cell surface with molecular weight of 78 kD,the migration,invasion and adhesive ability of tumor cell are enhancd,tumor blood vessels are promoted.In recent years,RNA interference(RNAi)is a new gene technology which takes the synthetic RNA into cells by outside intervention,inhibiting the target gene expression,so as to the gene therapy.It is widespread used in regulation of gene expression.We had found abnormal expression of AMF in keloid,however,it is unclear that the effect and mechanism of AMF on pathogenesis in keloid.Now,we aimed to confirm the abnormal expression of AMF in keloid and fibroblast,observe effect and mechanism of AMF on proliferation and migration in keloid fibroblast.We examined the change of keloid fibroblast biological behaviour,after silenced AMF gene expression by AMF siRNA lentiviral vector,and confirmed by animal model.We hoped to contribute to study of keloid pathogenesis and provide theoretical basis for the therapy of keloid.Methods:1.Expression and distribution of AMF in human keloid,hypertrophic scar and normal dermis by organization immunofluorescence staining method.After primary culture of fibroblasts in the three kinds of tissues,expression of AMF in fibroblasts by RT-PCR and Western blot.2.Effect of AMF on proliferation and migration in human keloid fibroblast was examined by MTT and Transwell assay,and effect on RhoA and JNK1 protein was tested by Western blot.After blocking RhoA and JNK pathways by Y-27632 and SP60012,the change of proliferation and migration was examined by MTT and Transwell assay,the expression of vascular endothelial growth factor(VEGF)and collagen?was examined by Western blot.3.Constructed FIV lentivirus vector transduct AMF siRNA into human keloid fibroblast from primary culture.proliferation was examined by MTT,cell cycle by flow cytometry,and migration by Transwell assay.Forming nude mouse model of keloid,transplantation was observed by General observation,HE and Masson stain.Results:1.The immunohistochemical staining results: Positive results could be seen in everywhere of cells.Positive results were stronger in keloid than other two kinds of tissues.AMF expression was higher than other cells in keloid fibroblast by RT-PCR and Western blot.2.The cell number significantly increased with respective addition of 10ng/ml,20ng/ml and 30ng/ml AMF,compared with control group.12 h after addition of 10ng/ml AMF,cell number increased,and 24 h,48h and 72 h later,it increased significantly(P<0.001).The migration capacity of human keloid fibroblast was increased with 10ng/ml AMF added,while significantly increased with 20ng/ml and 30ng/ml AMF.Activity of RhoA and expression of JNK1 were rised added AMF.Cell number,migration capacity and expression of VEGF and collage?decreased significantly after RhoA and JNK pathway were blocked by Y-27632 and SP60012.3.FIV lentivirus vector was constructed to transduct AMF siRNA into human keloid fibroblast from primary culture.Cell proliferation falled slowly,ratio of G0/G1 cell and apoptosis proportion of increased,and migration ability was weaken in AMF siRNA group.Forming nude mouse model of keloid,transplantation was smaller,blood vessels were fewer and the collagen?was looser in AMF siRNA group,compared with control group.Conclusions:1.The aberrant expression of AMF was confirmed in human keloid and keloid fibroblast in our study.2.The aberrant expression of AMF in keloid was connected with dysplasia and migration of fibroblast,and also with vascular endothelial growth factor(VEGF)and collage?.The effect of AMF on keloid fibroblast was probably RhoA and JNK signaling pathways.3.Silencing expression of AMF could recede dysplasia and increase apoptosis of keloid fibroblast,growth and proliferation were arrested at G0/G1 phase.Migration was descend,also.Silencing AMF expression in keloid in nude mouse model,the growth of the transplant with decreased blood vessels and sparse collagen was slow down.