Effect Of Cellular Prion Protein On Migration Of M2 Melanoma Cell Line

Objective:To investigate if cellular prion protein（Pr P）contribute to mobility of M2human melanoma cells.Also,to determine the possible regulating signal axis.Method:（1）In our study,the Crisper/Cas 9 system was used to knock out the human cellular prion protein gene（PRNP）in M2 human melanoma cells,which lacked FLNa expression.（2）The changes of M2^{Pr P-}cell phenotypes were detected by wound healing assay and MTT assay.To investigate whether the cell phenotypes were related to Pr P,Pr P-pc DNA3.1 or empty vector pc DNA3.1 were expressed in M2^{Pr P-}cell,excluding the gene off-target efforts of gene editing.（3）Immunofluorescence staining by Phalloidin showed the changes of cytoskeletal organization when PRNP was knocked out.（4）The changes of migration-related protein expression levels between M2 and M2^{Pr P-}cell and after treating with inhibitors for Akt,PKD,PKA or PKC were detected by immunoblotting.After M2 cells were directly treated with effective inhibitors,the wound healing assay was detected the changes in cell migration ability.Then q RT-PCR was used to examine the change of migration-related protein m RNA level between M2 and M2^{Pr P-}cells.（5）To investigate whether there are interactions between migration-related proteins,immunofluorescence staining and co-immunoprecipitation（Co-IP）were used.Results:（1）After human prion protein gene PRNP was knocked out in M2 melanoma cells by Crisper/Cas 9 system,the immunoblotting,immunofluorescence staining and flow cytometry were used to reveal that PRNP had been successfully eliminated in PRNP null M2(M2^{Pr P-})cells.The wound healing assay and MTT assay showed that silencing PRNP decrease the migration and proliferation of M2 cells.Since M2 cells do not express FLNa in immunoblotting,Pr P contributes to melanoma cell migration independent of its binding to FLNa.（2）In order to exclude the effects of gene off-target effects,PRNP in pc DNA3.1 or empty vector pc DNA3.1 was expressed in M2^{Pr P-}cells.Then wound healing assays showed Pr P rescued M2 cells had higher motility than the control PRNP null cells independent of binding to FLNa.（3）After staining for cytoskeletal organization in cells in the wound healing assay,the level of F-actin was greatly diminished in M2^{Pr P-}cells.In order to investigate if expression of Pr P may regulate F-actin level,M2 and M2^{Pr P-}cells was stained 14 hours after cells seeding and found that the level of Pr P negatively modified F-actin formation.This result can be rescued by re-expressing Pr P in M2^{Pr P-}cells.（4）The immunoblotting showed that phosphorylation of hsp27 was significantly decreased when PRNP was deleted.To investigate phosphorylation of ser82 of hsp27 was significantly decreased when PRNP was deleted,which kinase phosphorylates hsp27 in M2 cells,we immunoblotted cell lysates from M2 or M2 was treated with inhibitors for Akt,PKD,PKA or PKC.Only Akt inhibitor could reduce p-hsp27 level significantly.And the wound healing assay showed that inhibiting Akt activity also significantly reduced M2 cell migration.However,q RT-PCR showed that silencing Pr P did not alter Akt m RNA level significantly.（5）To investigate whether hsp27 can bind Akt in M2 cells,immunofluorescence staining and co-immunoprecipitation（Co-IP）with anti-Akt specific monoclonal antibody showed that significantly more co-localization of Akt and hsp27 was detected in M2 cells than M2^{Pr P-}cells.Conclusion:In this study,it is suggested that cellular prion protein（Pr P）could enhance cell migration by modulating the Akt—hsp27—F-actin axis.The expression of Pr P may be related to the migration of human melanoma,providing basis for melanoma biological research in the future.