Regulating The Occurrence And Development Of Melanoma Cells Through The TGF-β Signaling Pathway

Purpose:To explore the effect of VEPH1(Ventricular zone expressed PH domaincontaining 1)on the proliferation and invasion of human cutaneous melanoma cells and its correlation with the clinical stage 、 metastasis and epithelial-mesenchymal transition of CM(Cutaneous melanoma)based on TGF-β(transforming growth factor)signaling pathway,and to provide a theoretical basis for new therapeutic targets of cutaneous melanoma.Method:Part one: Collection of medical data from 47 CM patients diagnosed by pathology in a hospital affiliated with the normal university of Hunan.Collect their CM skin tissues and normal skin tissues adjacent to cancer for histopathology and immunohistochemistry,to its pathological changes and the positive expression rate of VEPH1,to detect its pathological changes and the positive expression rate of VEPH1;Then,the m RNA and protein expression levels of TGF-β-related molecules(TGF-β,Smad4)and EMT-related molecules(E-cadherin,N-cadherin and vimentin)were detected by Real-time RT PCR and Western blotting.Part two:The model was established by cell transfection,and the experimental cells were fall into 8 categories:(1)blank controller(untreated),(2)negative control group(transfected with blank plasmid),(3)VEPH1 vector group(transfected with VEPH1 overexpression plasmid),(4)TGF-β blocking group(TGF-β receptor inhibitor was used to inhibit TGF-βsignaling pathway),(5)VEPH1 vector + TGF-β blocking group(VEPH1overexpression plasmid is treated with TGF-β signaling pathway inhibitor),(6)si-VEPH1 vector group(transfected by si-VEPH1 plasmid),(7)TGF-β vector group(transfected with TGF-β overexpression plasmid),and(8)VEPH1 + TGF-β vector group(transfected with TGF-β overexpression plasmid and VEPH1 overexpression plasmid).First,through cell line screening,the VEPH1 m RNA relative expression level was significantly lower than that of normal skin cells and the lowest expression in each group was the standard for screening experimental cells.Subsequently,the proliferation of CM cells in the selected cell lines was detected by CCK-8 kit method,and cell count was carried out on the Transwell compartment carbonate polyester membrane to determine the invasion ability of CM cells.Results:Part one :The VEPH1’s positive expression rate from CM tissue was much lower than paracancer tissue,and it was closely related to clinical stage and lymph node metastasis degree.Compared with the adjacent tissues,the m RNA and protein levels of VEPH1 and E-cadherin in CM tissues were decreased,while the m RNA and protein levels of TGF-β,Smad4,vimentin and N-cadherin were increased.Part two:In the screening of cell lines,the relative expression of VEPH1 m RNA in A375 cell line was the lowest,so A375 cells were selected for subsequent experiments.Overexpression of VEPH1 or blocking the TGF-β signaling pathway inhibited the proliferation of CM cells and down-regulated the number of invaded cells,while the result was opposite to that of the VEPH1 vector group after si-VEPH1 knockdown.Conclusion:The expression level of VEPH1 was closely related to the clinical stage and the degree of metastasis in CM patients.The m RNA and protein expression of VEPH1 and E-cadherin were decreased in CM tissues,the expressions of TGF-β,Smad4,N-cadherin and vimentin were increased,which indicated that TGF-β signal pathway and EMT were activated in CM tissue.VEPH1 inhibits the proliferation and invasion of CM cells through down-regulation of the TGF-β signaling pathway,thereby inhibiting CM.