FOXP3 Promote Choroidal Melanoma Cell Migration And Invasion By Wnt5a/CaMKⅡ Signaling Pathway

Background and objective: As an important transcription factor,FOXP3 plays a important role in many tumors.At present,a study has shown that FOXP3 level is higher in metastatic uveal melanoma than in primary uveal melanoma,but the specific role and mechanism of FOXP3 in uveal melanoma have no report.Therefore,this study explored the role of FOXP3 in the proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of choroidal melanoma(CM)which is the most common tumor in uveal melanoma and the effect of FOXP3 on Wnt5a/CaMKⅡ signaling pathway,providing a new target for studying CM.Methods: The effects of FOXP3 on OCM-1 and C918 of CM cell lines and its related mechanisms were studied.The plasmid of overexpression of FXOP3 and the CRISPR Cas9 lentivirus of knockdown of FOXP3 were transfected into OCM-1 and C918 cells,and the expression of FOXP3 in the transfected cells was verified by Western blotting.CCK-8 cell activity assay,monoclonal assay,migration and invasion assays were used to measure the effects of overexpression or knockdown of FOXP3 on cell proliferation,migration and invasion.Transcriptome m RNA sequencing was used to screen the pathways related to FOXP3 acting on CM,and Western blotting was used to verify the gene expressions,at the same time,the effect of overexpression or knockdown of FOXP3 on markers of EMT was detected.The effect of FOXP3 on CM growth was further explored.Xenograft tissues protein was extracted and the effects of knockdown of FOXP3 on EMT markers and Wnt5a/CaMKⅡ signaling pathway in xenograft were detected.Results: In the function study of OCM-1 and C918 cells by FOXP3,CCK-8 cell activity assay,monoclonal colony formation assay,migration and invasion assays showed that overexpression of FOXP3 can promote cell proliferation,migration and invasion(P<0.05),Monoclonal colony formation assay showed that knockdown of FOXP3 could reduce the size and number of OCM-1 and C918 cell masses,thereby inhibiting the cell proliferation(P<0.05).The results of migration and invasion assays showed that knockdown of FOXP3 could inhibit cell proliferation,migration and invasion(P<0.05).After overexpression of FOXP3,pseudopods of OCM-1 and C918 cells became longer,and the cells changed from aggregate condition to dispersed condition.In addition,the expression of E-cadherin,a marker of EMT epithelial cell,was decreased(P<0.05).The expressions of mesenchymal cell markers N-cadherin and Snail proteins were up-regulated(P<0.05).Knockdown of FOXP3 up-regulated the expression of E-cadherin and down-regulated the levels of N-cadherin,Snail and matrix metalloproteinase-9(MMP9)(P<0.05).These results suggest that FOXP3 can induce EMT.In the study of signaling pathways,overexpression of FXOP3 up-regulated the expression of Wnt5a/CaMKⅡ signaling pathway in OCM-1 and C918 cells(P<0.05),conversely,knockdown of FOXP3 could down-regulate the Wnt5a/CaMKⅡ signaling pathway(P<0.05).In C918 cells,silence of Wnt5 a reduced the promotion effects of overexpression of FOXP3 on cell migration,invasion and EMT mesenchymal marker expressions(P<0.05),and enhanced the inhibitory effects of knockdown of FOXP3 on cell migration,invasion and EMT mesenchymal marker expressions(P<0.05).Nude mouse model assay showed that knockdown of FOXP3 can slow down the growth rate of xenografts and inhibit the growth ability(P<0.05).Knockdown of FOXP3 up-regulated the expression of E-cadherin in xenograft(P<0.05)and down-regulated the Wnt5a/CaMKⅡ signaling pathway(P<0.05).Conclusion: 1.FOXP3 can promote the proliferation,migration and invasion activities of CM cells,and induce EMT;In addition,FOXP3 can promote the growth ability of CM transplanted tumors in mouse.2.FOXP3 can promote the migration and invasion of CM cells by up-regulating Wnt5a/CaMKⅡ signaling pathway.