Downregulation Of BAX By MiR-365 Promotes Cutaneous Squamous Cell Carcinoma By Suppressing The Apoptosis

BackgroundNon-melanoma skin cancer（NMSC）is one of the most common malignancies in humans,including basal cell carcinoma（BBC）and squamous cell carcinoma（SCC）.SCC is a malignant tumor derived from keratinocytes in epithelial tissue,including cervical cancer,esophageal cancer and squamous cell carcinoma of skin（CSCC）and so on.CSCC is a malignant tumor that occurs in epidermal（or mucosal）epithelial cells.Its incidence is lower than that of basal cell carcinoma,but the rate of metastasis and mortality is higher,and the incidence of males is more than that of females,and forearm[1,2].MicroRNA（miRNA）is a class of non-coding single-stranded RNA small molecules encoded by endogenous genes of about 22 nucleotides.The microRNA is paired with the 3’UTR partial sequence specific base of the target mRNA,which inhibits the target gene Transcribed translations.Different microRNAs are different in different diseases and cancers.There is also a difference in the expression of different microRNAs in different cancers.For example,miR-3 65 is down-regulated in gastric cancer,non-small cell lung cancer,hepatocellular carcinoma and melanoma cell tumor.However,Cell carcinoma and primary breast cancer.Bax belongs to the BCL-2 family of pro-apoptotic proteins,BAX overexpression,activation will lead to changes in tumor biological function.The aim of this study was to investigate the biological role of miR-365 in skin squamous cell carcinoma and to further clarify the regulatory mechanism between miR-365 and BAX molecules,and to provide theoretical basis for the clinical treatment of skin squamous cell carcinoma The treatment strategy.Methods①The expressions of miR-365 and BAX in human immortalized epidermal cells and CSCC cell lines,human epidermis tissues and human CSCC tissues and nude mice transplanted tumor were detected by qPCR.Western blot was used to detect the expression of BAX at protein level.②The expression of BAX in human epidermal tissues and human CSCC tissues and nude mice transplanted tumor was detected by immunohistochemistry.③ The relationship between miR-365 and BAX was measured by colony PCR and double luciferase assay.④ MiR-365 minic and antagomiR-365 were transfected into A431 cells,respectively.The expression of miR-365 was down-regulated（P<0.001）and the expression of BAX at mRNA and protein levels were up-regulated in antagomiR-365 group（P<0.001）.⑤The changes of cell proliferation ability,wound healing ability,migration and invasion ability of A431 cells transfected with NC,siBAX₀1 and siBAX₀2 were detected by CCK8 assay,cell wound healing assay,cell migration assay and invasion assay.⑥The effect of A431 cells transfected with NC,siBAX₀1 and siBAX₀2 on cell apoptosis was detected by flow cytometry.Results①Compared with the control,miR-365 was highly expressed in CSCC cell lines（P<0.001）,BAX was lower at mRNA level and lower protein level（P<0.001）.②In CSCC tissues.The positive rate of BAX in immunohistochemistry was lower than that in normal epidermis（P<0.01）.③Double fluorescence luciferase assay showed that the fluorescence intensity of BAX-3’UTR-WT group was significantly lower than that of BAX 3’UTR-Mutant group（P<0.01）.④After transfection of NC and siBAX into A431 cells,the proliferation ability（P<0.001）,wound healing ability（P<0.05）,migration（P<0.001）and invasion ability（P<0.001）of the cells were increased compared with the control,and the anti-apoptotic ability（P<0.05）was enhanced.（P<0.05）.⑤Nude mice transplanted tumor model revealed that the growth rate of siBAX treated group was faster（P<0.05）than that of NC,and the expression of BAX was lower than that of control group at the level of mRNA and protein.In immunohistochemistry,The positive rate of BAX in siBAX treated group was lower than that in control group（P<0.05）.Conclusion①The expression of BAX in human CSCC cell lines and tumor tissues is down-regulated.②miR-365 regulates the expression of BAX directly by binding to BAX 3’UTR region.③BAX inhibits CSCC cell proliferation,migration and invasion,but promotes apoptosis.④In vivo experiments further confirmed that the reduction in BAX expression can promote the progress of CSCC.However,the miR-365-BAX control axis still needs further study.