Research Of The Side Population Cells Isolated From Multiple Myeloma

[Objective] To identify and characterize the side population cells isolated from celllines and patients of multiple myeloma．[Methods] Side population cells of seven multiple myeloma patients and five multiplemyeloma cell lines,named NCI-H929, RPMI8226, LP-1, U266, SKO,were analyzed andisolated by fluorescence-activated cell sorting(FACS),then characterized by cell cycle,immunophenotype, colony formation assay, the expression levels of the mRNA of ABCG2.HμMan multiple myeloma (MM) cell lines NCI-H929and RPMI8226were treated with A,bortezomib, arsentic trioxide were assessed by MTT assay, the impact of these drugs on SPwere detected by FACS and colony formation assay.[Results] Side population cells were detected in all of the five hμMan multiplemyeloma cell lines and seven multiple myeloma patients, with a proportion of0.8256-1.762%and0.04-2.0%, respectively. More SP cells in the G0/Gl phase (95.47%VS49.36%, P<0．05) and fewer cells in S phase(3.27%VS40.84%, P<0．05) than MPcells. There were no difference in the expression of CD38and CD138between SP and MP,(78.5±8.5)%VS(82.0±4.0)%(,72.3±15.7)%VS(84.3±11.9)%, respectively, P>0.05.Colony formation assay showed that the colony forming efficiency of the SP cels washigher than the MP cells (1392±363VS550±21,P>0.05)．The mRNA expression Ievels ofABCG2in SP cells were higher than that in MP. A, bortezomib, arsentic trioxide inhibitsthe MM cell viability and the effect was dose dependent. Compand A(4μM,8μM)candecrease the SP proportion specifically after24hours, bortezomib(10nM,25nM), arsentictrioxide(1.25μM,2.5μM)can decrease the the total cell after24hours, nonspecifically.But lenalidomide have no effect on SP after24hours. bortezomib(10nM) can decrease thecolony forming efficiency of the SP cells and MP cells.[Conclusion] Side population cells could be detected in the cell lines and patients of multiple myeloma．There were notably difference in the cell cycle, immunophenotype,colony formation assay and the expression levels of the mRNA expression,but nodifference in the expression of CD38and CD138between SP and MP. A, bortezomib,arsentic trioxide inhibits the MM cell viability and the effect was dose dependent. A candecrease the SP proportion specifically, bortezomib and arsentic trioxidecan decrease thethe total cell, nonspecifically. But lenalidomide has no effect on SP. Bortezomib and A candecrease the colony forming efficiency of the SP cells and MP cells. [Objective] To determine the diference of microRNA expression profiles betweenSP cells and MP cells so as to lay necessary basis for research on the function of miRNA inmultiple myeloma stem cell.[Methods] Side population cells of multiple myeloma cellS were isolated byfluorescence-activated cell sorting(FACS), the microarray detection system was employedto analyze whether there was difference in miRNA expression profile between SP and MPcells．realtime-PCR verified the results of microarray, the related pathways and targetgenes of SP cells was analysised by bioinformatics technology. The differences ofp-Erk1/2,p-S6,p-S6K1,p-Src,but not TSC1expression level between SP and MP cellswas detected by Western blotting.[Results] A total of43differentially expressed miRNA were found including10over-expression and33low-expression miRNA in SP cells. Realtime-PCR revealed thatmiR-144,miR-451expression level is higher, miR-150expression level is lower in SP cells than that in MP cells, consistented with the microarray. bioinformatics technologyfound30pathway have statistically significance(P<0.01). the pathway involved in cancerstem cells and MM-onset include:Pathways in cancer, Focal adhesion, Endocytosis,MAPK signaling pathway, ErbB signaling pathway, Wnt signaling pathway, mTORsignaling pathway, Ubiquitin mediated proteolysis, TGF-beta signaling pathway. Westernblotting revealed that p-Erk1/2,p-S6,p-S6K1expression level is lower in SP cells thanthat in MP cells.[Conclusion] A total of43differentially expressed miRNA were found including10over-expression and33low-expression miRNA in SP cells. Nine pathways are involved inSP cells. p-Erk1/2,p-S6,p-S6K1,p-Src expression level is lower in SP cells than that inMP cells.