Construction And Identification Of Lentivirus-mediated Overexpression Of FOXO3a Gene Stable Transfected Human Keloid Fibroblasts Cell Lines

Objective:To obtain a lentiviral-mediated over-expression vector construction of FOXO3a(Forkhead box O3a)gene in keloid fibroblast cell line,which is for further investigation of FOXO3a gene in the pathology of keloid formation.Methods:Using the quantitative real-time PCR and Western blot to detect the expression of FOXO3a in human keloid tissue and the normal skin tissue.Using the Flag-FOXO3a TM plasmid as a template,the FOXO3a(2058bp)gene fragment was amplified by PCR and purified for high-quality DNA.The target DNA fragment and vector were digested with restriction enzym(BamH I and Xho I),then recovered and purified them to perform T4 enzyme ligation.The recombinant product was transformed into competent cells and at least three single colonies were obtained after plating on ampicillin-resistant LB plates,which were sent to sequence.The recombinant plasmid that was identified successfully named pLOX-FOXO3a-EF1α-EGFP-Puro.In further,the lentivirus vector containing target fragment and the lentivirus packaging plasmids were co-transfected into 293T cells that were used for virus packaging,in which pLOX-NC-EF1α-EGFP-Puro vector was as the control group.After 48h and 72h,the supernatants of transfected plates were collected,meanwhile,the expression of EGFP was observed in 293T cells under a fluorescent microscope.The supernatants containing lentivirus were used to transfect the human keloid fibroblasts until the cells with green fluorescence were captured under the fluorescence microscope.Following identification of fluorescence,puromycin was added to select the EGFP positive cells and expanding the remaining cell mass for subsequent experiments.Using the quantitative real-time PCR and Western blot to detect the expression of FOXO3a in the constructed cell line.Results:(1)The results of qPCR showed that the FOXO3a mRNA expression of the keloid tissue was lower than the normal tissue and the difference was statistically significant(P<0.05).In response,the results shown that the FOXO3a protein expression of the Keloid tissue was lower than the normal tissue and the difference was statistically significant(P<0.05).(2)The size of the amplified target gene fragment was the same as expected.(3)Under the fluorescence microscope,we observed that most of the 293T cells were positive for EGFP.(4)Under the fluorescence microscope,we found that most of the HKF cells were positive for EGFP.After implementation of puromycin,most of the cells in the normal control group were dead.(5)FOXO3a was either mRNA level(P<0.01)or protein level was markedly upregulated than control group.(P<0.01).Conclusion(1)The expression of FOXO3a in human keloid tissue is significantly lower than that in normal tissue,indicating loss of FOXO3a gene is related with keloids.(2)Lentivirus-mediated overexpression of FOXO3a gene in human keloid fibroblast cell line was constructed successfully.