The Interaction Of CLIC1 And Its Truncated Mutants With RACK1 & The Mechanism Of ACNT4 In Melanoma Cells With Resistance To BARF Inhibitors

Objective To investigate the interaction of chloride intracellular channel 1(CLIC1)and its truncated mutants CLIC1(1-90aa),CLIC1(91-241aa)with activated protein kinase C receptor 1(RACK1)in eukaryotic cells.Methods The eukaryotic expression plasmids of pc DNA3.1-CLIC1(1-90)-FLAG and pc DNA3.1-CLIC1(91-241)-FLAG were constructed using the full-length c DNA sequence of CLIC1 as a template;the plasmids were transiently transfected into mammalian fibroblast cell line COS7,respectively,and then their localization was analyzed by immunofluorescence(IF);the eukaryotic expression plasmids were transiently transfected into human renal epithelial cell line HEK 293 T,respectively,and then their expression were checked by Western blot;co-immunoprecipitation(co-IP)was conducted to probe the interaction of RACK1 with CLIC1 and its truncated mutants as well.Results After analysis by double restriction enzyme digestion,the positive clones were chosen and sent to the biotechnology company for sequencing,the two truncated plasmids of CLIC1 were successfully constructed.By fluorescence microscopy,it was found that CLIC1(1-90)-FLAG was mainly localized to cytoplasm,while CLIC1(91-241)-FLAG was mostly localized to nucleus;In COS7 cells,RACK1-GFP co-colazised with CLIC1(1-90)-FLAG and CLIC1(91-241)-FLAG in cytoplasm,respectively.In HEK 293 T cells,co-IP experiments showed that RACK1 interacted with both CLIC1(1-90)-FLAG and CLIC1(91-241)-FLAG,respectively.Conclusion The wild type RACK1 and the two truncated mutants of CLIC1,CLIC1(1-90)and CLIC1(91-241),co-localized to cytoplasm,respectively.And co-IP experiments further confirmed that RACK1 interact with both CLIC1(1-90)and CLIC1(91-241),respectively.Objective The purpose of this study was to investigate the role of alpha-actinin-4(ACTN4)in the malignant progression of melanoma,and to determine the downstream effector of ACTN4-mediated invasion of melanoma cells and the effect of ACTN4 on BRAF inhibitors.Methods ACTN4 sh RNA plasmids and lentivirus packaged plasmids were co-transfected into HEK 293 T cells by lipofectamine transfection.After the release of virus particles into the medium,the lentiviral supernatant was collected to infect target melanoma cells,and the stable ACTN4 sh RNA knockdown cell lines were obtained.The stable ACTN4 sh RNA melanoma cells with BRAFV600 Ewere treated with BRAF inhibitors such as PLX4720 or PLX4032,and cell pellets were collected.The effectors of ACTN4 knockdown on melanoma cells were probed and then checked by Western blot.Results ACTN4 sh RNA plasmids were successfully constructed to obtain a stable melanoma cell line with low expression of ACTN4.Knockdown of ACTN4 inhibited the growth of melanoma cells.In the melanoma cells with stable ACTN4 sh RNA knockdown after the use of BRAF inhibitors,the protein expression of MAPK,AKT and NF-κB signaling pathway were decreased.Conclusion Down-regulation of ACTN4 inhibited the growth of melanoma cells.ACTN4 plays a role in the malignant progression of melanoma by regulating MAPK,AKT and NF-κB signaling pathways.