Effect Of The New Candidate Tumor Suppressor Gene SLC8A2in Glioma Cell Line U87and Its Molecular Mechanisms

Part ITo explore the effect of SLC8A2in glioma cell line U87via in vitroand in vivo experimentsObjective: To construct the U87cells stably transfected with SLC8A2and explorethe effect of SLC8A2in glioma cell line U87through in vitro and in vivo experimentsMethods: The lentivirus plasmid containing SLC8A2（Lenti-SLC8A2-IRES-EGFP）and negative control（Lenti-EGFP）were transfected into U87cells. The cells stablytransfected with SLC8A2and negative control called U87-SLC8A2and U87-NCrespectively were constructed; Real-time PCR and western blot were carried out to detectthe expression of SLC8A2in transfected cells;The cell invasion and migration ability weredetected by Transwell assay;The cell proliferation was detected by CCK-8assay;The cellcycle and apoptosis were detected by flow cytometry;The nude mouse xenograft modelwas used to examine the effect of SLC8A2on the growth of glioma cell line U87in vivo.Results: The cells stably transfected with SLC8A2were well constructed; SLC8A2markedly inhibited the invasion and migration of U87cells,but had no effect on cellproliferation or cell cycle.No mouse developed tumor in U87-SLC8A2cell group, while allfive mice developed tumors in U87-NC and U87group.Conclusions: The invasion, migration and in vivo xenograft growth of glioma cellline U87were notably inhibited by SLC8A2, suggesting that SLC8A2may be a noveltherapeutic target protein against glioma. Part IIMechanisms of the invasion and migration inhibition by SLC8A2in glioma cell line U87Objective: To investigate the related molecular mechanisms of the invasion andmigration inhibition by SLC8A2in glioma cell line U87.Methods: RT-PCR is carried out to detect the mRNA expression levels of MMP-2,MMP-3, MMP-7, MMP-9,MT1-MMP,uPAand uPAR; Western blot is carried out to detectthe protein level of MMP-2; The enzymatic activity of MMP-2secreted into culturemedium was determined by Gelatin Zymography assay;The levels of proteins related toNF-κB, MAPK andAKT pathways was determined by Western blot.Results: The mRNA levels of MMP-2, MMP-3, MMP-7, MMP-9, MT1-MMP, aswell as the protein level and enzymatic activity of MMP-2were dramaticlly downregulatedin U87-SLC8A2cells compared with U87-NC and U87cells. And furthermore,the activityof NF-kB and ERK1/2pathways is inhibited,which are generally considerned to be theupstream regulator of MMPs and uPA/uPAR system.Conclusions: SLC8A2dramaticlly inhibited the invasion and migration of U87cellsperhaps through the ERK-NF-κB-MMPs/uPA/uPAR system.