P53 Deletion Promotes Myeloma Cells Invasion By Upregulating MiR19a/CXCR5

Objective:1.To investigate the invasiveness of p53-deficient myeloma cells.2.To investigate the mechanism of p53 deletion affecting the invasiveness of myeloma cells Methods:1.The p53 gene was knocked down and over-expressed in myeloma cells (NCI-H929 and U266)by cell transfection.2.Validation of transfection efficacy at the gene and protein level whether p53 was knocked down by siRNA or overexpressed by Nutlin-3 successfully.3.Transwell assay the invasive changes of knockdown or overexpression p53 in myeloma cells.4.Calcein-AM kit was used to detect the change of myeloma cells’ adhesion after transfection.5.Western blot and QT-PCR validation of EMT-like changes in myeloma cells after p53 transfection.6.The expression of CXCR5/CXCR4 in myeloma cells after p53 transfection was detected by Western blot and QT-PCR.7.Western blot and Transwell methods were used to verify whether the cells affected the CXCL13-Dependent chemotaxis8.The expression of miR19 a in myeloma cells after p53 transfection was detected by QT-PCR.9.The relationship between miR19 a and CXCR5 was detected by Western blot and QT-PCR.10.Western blot,QT-PCR and Transwell methods were used to detect the relationship between p53,CXCR5 and miR19 a.Results:1.The expression of p53 at the mRNA and protein levels was decreased after transfected by p53 siRNA in NCI-H929 myeloma cellsand overexpressed at both mRNA and protein levels after Nutlin-3 treatment,howeever,U266 showed no significant change.2.Downregulation of p53 with siRNA significantly increased the invasion ability of NCI-H929 cells,but not U266 cells.While upregulation of p53 by Nutlin-3 apparently decreased the invasion and increased the expression of p53 in NCI-H929,but not U266 cells.3.siRNA of p53 reduced adhesion to BMSCs isolated from MM patients of NCI-H929 cells but not of U266 cells..4.siRNA of p53 downregulated E-cadherin,together with upregulated Vimentin, Snail and Twist in NCI-H929 cells,but not U266 cells5.NCI-H929 cells transfected siRNA of p53,demonstrated a significant increase in CXCR5 expression at both mRNA and protein levels,respectively.The expression of CXCR4 expression at both mRNA and protein levels did not change with transfected siRNA of p53.6.The p53 gene in NCI-H929 cells after p53 transfection increased CXCL13-dependent chemotaxis;7.The expression of miR19 a was upregulated by siRNA of p53 and downregulated by Nutlin-3 in NCI-H929 cells.In contrast,the expression of miR19a in U266 cells did not change by siRNA of p53 and Nutlin-3.8.Enforced expression of miR19 a upregulates the mRNA and protein expression of CXCR5 as well as promotes the invasion of NCIH929 cells.While attenuated expression of miR-19 a inhibits the mRNA and protein expression of CXCR5 as well as the invasion of NCI-H929 cells.9.The results showed that miR19 a knockdown specifically abrogated the ability of siRNA of p53 to regulate CXCR5 expression and invasion of NCI-H929 cells,strongly suggesting that the regulation of CXCR5 by p53 was mediated by miR19 a in NCI-H929 cells.Conclusion:In patients with MM,once p53 gene is deleted/inactivated,the tumor suppressor effect is weakened and the carcinogenic effect is enhanced,which will weaken the adhesion of tumor cells with surrounding cells,enhances tumor migration,and activates the CXCR5-CXCL13 axis by increasing the expression of miR19 a and makes tumor cells more aggressive.