C-FLIP Over-expression Leads To Aberrant Activation Of β-catenin/TCF Pathway In Melanoma

Partâ… Expression of c-FLIP and nuclear or cytoplasmicβ-catenin andthe relationship between themObjective: To study expression of c-FLIP,β-catenin in melanoma, nevus, psoriasis andacrobystia and explore the relationship between them. To investigate the correlationbetween histopathological characteristics and expression of those protein in melanoma.Methods: The protein of c-FLIP,β-catenin and cyclinD1 were detecded byimmunohistochemistry and quantitative analysis the relationship among c-FLIP, nuclear orcytoplasmicβ-catenin and cyclinD1.Results: In melanoma, positive expression of c-FLIP, nuclear or cytoplasmicβ-catenin andcyclinD1 were observed in 65.0% (50 case), 32.5% (25 case) and 31.2%(24 case), respectively,and the positive relationship were observed among c-FLIP and nuclear or cytoplasmicβ-catenin,cyclinD1. Increased those protein expression in melanoma lesion were significantly associatedwith histological type. In nevus, the expression of c-FLIP, nuclear or cytoplasmicβ-catenin andcyclinD1 were lower than that in melanoma. In psoriasis, the expression of c-FLIP was increased,and nearly no expression of nuclear or cytoplasmicβ-catenin.Conclusions: Our data suggested that increased c-FLIP, nuclear or cytoplasmicβ-catenin and cyclinD1 protein in melanoma may contribute to the development ofmelanoma. The increased expression of c-FLIP maybe have relationship with theexpression of nuclear or cytoplasmicβ-catenin, and then induce the expression ofdownstream genes ofβ-catenin/T Cell Factor(β-catenin/TCF), such as, cyclinD1. Partâ…¡Construction and expression of pCMV-Tag2B-FLIP_L/P43/S andc-FLIP-shRNA expression vectorObjective: To construct pCMV-Tag2B-c-FLIP_L/P43/S eukaryotic expression vector,transfect into melanoma cells and investigate the expression. Construct c-FLIP-shRNAeukaryotic expression vector, transfect into melanoma cells and explore the effect ofinterference for c-FLIP.Methods: Extracted total RNA from Jurkat cells and then isolated mRNA, and thenreverse transcripted to obtain cDNA. Three pairs of PCR primers were designed and used toamplify the c-FLIP_L, c-FLIP_P43 and c-FLIP_S gene respectively. Two restrictionendonuclease sites xho I and EcoRI were introduced. The c-FLIP_L or c-FLIP_P43 or c-FLIP_Sgene was inserted into the pGME-T vector. The recombinant plasmid was transfomed intoE. coli DH5α. Positive cell clones were indentified by restriction enzyme digestion andsequenced. The c-FLIP_L or c-FLIP_P43 or c-FLIP_S gene was subcloned into the eukaryoticfusion protein expression vector pCMV-Tag-2B to obtain pCMV-Tag-2B-c-FLIP_L/P43/S.After identification, pCMV-Tag-2B-c-FLIP_L/P43/S plasmid was transfected into A375 cellsand get the stable transfectants of A375/c-FLIP_L/P43/S. Dectected the expression ofc-FLIP_L/P43/S in the A375/c-FLIP_L/P43/S. To synthesize the sequence target human c-FLIPgene, c-FLIP-shRNA, and insrted into pSilencer 3.1-H1 neo vetor by BamHâ… and Hindâ…¢restriction endonuclease sites to get pSilencer-c-FLIP. After identification, pSilencer-c-FLIPplasmid was transfected into A375 cells and observe the c-FLIP_L/P43/S expression.Results: The c-FLIP_L/P43/S gene was identified by sequencing, and the A375/c-FLIP_L/P43/Sand pSilencer-c-FLIP were identified by western blot and flow cytometry(FCM).Conclusions: The pCMV-Tag-2B-c-FLIP_L/P43/S and pSilencer-c-FLIP eukaryotic expressionvector were successfully constructed . The expression of c-FLIP_L/P43/S protein could beregulated by those vectors. Those vectors could be used in future research for melanoma. Partâ…¢c-FLIP_L/P43 activateβ-catenin/TCF pathway and induce theexpression of cyclinD1Objective: To observe whether the change of c-FLIP protein expression could lead toaberrant activation of beta-catenin/TCF pathway in melanoma and induce gene expressionof the downstream. Explore the function of c-FLIP_L, c-FLIPP₄₃ and c-FLIP_S during theprocess above.Methods: To detecte the expression of cytoplasm or nuclearβ-catenin protein andcyclinD1 protein in stable clone A375/c-FLIP_L/P43/S by western blot. To observe the locationofβ-catenin in stable clone A375/c- FLIP_L/P43/S by cell immunofluorescence, and toobserve the activity of transcription factor T cell factor by TOP/FOP Flash. Compare thedifference of function among A375/c- FLIP_L/P43/S. To reduce the expression of c-FLIP_L/P43by pSilencer-c-FLIP vector, and then observe the effect during the process above.Results: In stable clone A375/c-FLIP_L/P43, we observed the expression of cytoplasm andnuclearμ-catenin was increased. At the same time, we found in those cells, the expressionof cyclinD1 was increased. By the TOP/FOP Flash, we dectected the activition of TCF wasup-regulated. However, we did not found the same results in the A375/c-FLIPs cells.Couclusions: Overexpression of c-FLIP_L/P43 in A375 cell could promote the nulcear/cytoplasm accumulation ofβ-catenin and the increase transcriptional activity ofβ-catenin/TCF pathway and then induce the downstream gene expression, such as,cyclinD1. Partâ…£The effects of c-FLIP on bionomics of melanoma cell A375Objective: observe the effects of three type of c-FLIP on bionomics of melanoma cellA375, such as, growth, proliferation, apoptosis and transformation activity.Methods: To count cells of A375/c-FLIP_L/P43/S and obtain the cells' growth curve, andthen to compare the difference among those type of cells. To detecet the proliferationalactivity of A375/ c-FLIP_L/P43/S by MTT; to observe the rate of apoptosis by by flowcytometry(FCM) using annexin V and propidium iodide(PI) double staining; to detect thetransformation activity of those cells by soft agar assay. At the same time, suppress theexpression of c-FLIP by RNAi and then observe the effects on A375.Results: In comparison to control group, c-FLIP_L/P43/S over-expression promote thegrowth and proliferation activity of A375 cells, c-FLIP_L/P43 have more remarkable effect onA375 cells' activity of growth and proliferation than that of c-FLIP_S. Compare to the controlcells, c-FLIP _L/P43/S can resist the apoptosis. Sofa agar assay indicate c-FLIP _L/P43/Sover-expression promote the transformation activity of A375. c-FLIP _L/P43 have more effectson transformation activity of A375 than that of c-FLIP_S. Using siRNA specifically knockingdown the expression of c-FLIP, those effects above have been reversed partly.Conclusions: The three subtypes of c-FLIP can promote the activity of growth,proliferation and transformation. As to resist to apoptosis, c-FLIP_L/P43 have more effectsthan that of c-FLIP_S.