| Background Cutaneous squamous cell carcinoma(CSCC)is a skin malignancy arising from keratinocytes in epidermis and skin appendages.The incidence of CSCC is second only to basal cell carcinoma(BCC)in skin malignancies and continues to increase.Currently,the pathogenesis of CSCC is still unclear.The ID(inhibitor of DNA binding/differentiation)family,the inhibitors of basic helix-loop-helix(bHLH),regulate cell proliferation and differentiation via combining with bHLH transcription factor and inhibiting its ability to bind DNA.In recent years,the relationship between ID family and tumor has drawn increasing attention.ID4 is thought to be a key downstream gene in the TGF-β/BMP-SMAD-ID signaling pathway,which participates in tumor generation,development and metastasis.We found that ID4 gene expression was significantly reduced in CSCC compared with adjacent normal tissues.Therefore,we presume that ID4 may act as a tumor suppressor gene in skin carcinogenesis.This study aims to explore the role of ID4 gene in the generation and development of CSCC by constructing stable transgenic cell lines with ID4 gene overexpression.Methods 1.The lentivirus with ID4 overexpression was infected into A431,Colo 16,HaCaT cell lines respectively and selected with puromycin for 2 weeks.Identification of ID4 gene expression in the three cells after transfection was operated by Quantitative PCR(qPCR)and Western blot.2.Cell counting kit-8(CCK8),flow cytometry,scratch test and Transwell invasive test were used respectively in order to monitor cell proliferation,apoptosis,migration and invasion.The tumorigenic role of infected cells in nude mice was then monitored.Results Compared with the negative control group,after infected with lentivirus,ID4 mRNA and protein expression was significantly enhanced in A431,Colo 16,HaCaT cells(p<0.05),which met the requirements of the next step;2.In infected A431,significant decrease was found in cell proliferation,migration and invasion,significant increase in apoptosis,deterred tumorigenesis and smaller tumor in nude mice.3.In infected Colo 16,significant decrease was detected in cell proliferation,migration and invasion,significant increase in apoptosis,deterred tumorigenesis and smaller tumor in nude mice.4.In infected HaCaT,none significant difference was observed in cell proliferation and apoptosis,and significant decrease was found in cell migration.Conclusion ID4 over eapression inhibited the activity in proliferation,migration,invasion and enhanced the apoptosis of CSCC cells.Therefore,ID4 may act as a tumor suppressor gene in skin carcinogenesis,but the specific functions and mechanisms need to be further elucidated. |
