Analysis Of The Differential Whole Proteins And Phosphoproteins On Plasma Membrane Involved In Metastasis Of Prostate Cancer Cell Mat-LyLu Mediated By Toxin Probes

Metastasis is the leading cause of death in cancer patients.Statistical data show that more than 90% of cancer patients died of tumor metastasis rather than primary tumors.The expression of voltage-gate sodium channels are considered to be related to the degree of tumor metastasis.Activate or inhibit these channels,especially Na_v1.7 subtype,could affect the migration and metastasis of tumor cells.Enhanced expression of Na_v1.7 sodium channel has been observed in the prostate cancer cell line Mat-Ly Lu and promote its metastasis,however,the mechanism underlying this process is not clarified so far.Phosphorylation and protein-protein interaction are essential for the function and regulation of sodium channels.On the one hand,phosphorylation plays an important role in cell signal transduction.The phosphorylation of Na_v1.7 could be changed dynamically when the activity of Na_v1.7 was affected.On the other hand,changes in Na_v1.7 activity can be performed by interacting with different types of proteins.To clarify the interacting protein and phosphorylation of Na_v1.7 during cell metastasis could provide the theoretical basis for the molecular mechanism underlying Na_v1.7 regulates the metastasis of Mat-LyLu.Therefore,in the first part,we treated rat prostate cancer Mat-Ly Lu cells with two peptide toxins which act on Na_v1.7,JZTX-I and HNTX-III,and then compared the differentially expressed phosphoproteins between JZTX-I(metastasis-enhanced group),HNTX-III(metastasis-reduced group)treated group and Control group(control)by iTRAQ labeling and TiO2 enrichment,followed by LC-MS/MS.As the resut,1919 non-redundant phosphorylated peptides were identified in total,including 3519 phosphorylation sites and corresponding to 657 non-redundant phosphoproteins.446 phosphoproteins(correspongding to 1106 phosphorylated peptides,namely iTRAQ-O)were identified in at least two iTRAQ parallel groups.These proteins mainly paticipated in cellular processes and metabolism,and function as molecular binding and transcription coactivator.1106 phosphorylated peptides in iTRAQ-O group were subjected to online motif-X software,and finally a total of 13 serine motif,4 threonine motif and 1 tyrosine motif were obtained.We collected the quantitative information of 446 overlap phosphoproteins in iTRAQ-O group,and obtained the ratios of 114/116 and 115/116 for each phosphoprotein.A ratio of ≤0.67 and a ratio of ≥1.5 was used as the threshold to select differentially expressed phosphoproteins,and a total of 120 we obtained at last.We divided the above-mentioned proteins into seven groups to make some bioinformatics analysis,and from which we found out some differentially expressed phosphoproteins associated with tumor metastasis.The protein-protein interaction network of these differentially expressed phosphoproteins were analyzed by STRING software.In the second part,2-dimension electrophoresis combine with mass spectrometry has been used to identify differentially expressed plasma membrane proteins after the Mat-LyLu cells treated with Na_v1.7 activator veratridine.As the result,29 differentially expressed proteins were identified in total,i.e.,28 were down-regulated whereas 1 were up-regulated in veratridine treated group compared with control group.We selected 4 differentially expressed proteins associated with tumor metestesis from the above 29 proteins after functional annotation analysis: Tonsoku-like protein(TONSL),Annexin A2(Anxa2),Keratin,type II cytoskeletal 1(Krt1)Beta-defensin 33(Defb33).