| ObjectiveThis study was aimed to investigate the effects of proteasome inhibitor bortezomiband bone marrow mesenchymal stem cells (MSC) on the cell viability, migratoryability and the chemokines receptors mRNA expressions such as C-C chemokinereceptor type1(CCR1）、C-C chemokine receptor type2（CCR2)、CC chemokinereceptor type3（CCR3）、stromal cell derived factor-1receptor（CXCR4)、CCchemokine receptortype6（CCR6) and hepatocyte growth factor receptor（HGFR/c-Met)of multiple myeloma (MM)cell line U266, which can be used toexplore the impact of MM cell chemotaxis on MM pathological process.MethodThe inhibitory and apoptosis rate of U266cells after treated with differentconcentrations of bortezomib were evaluated by MTT and flow cytometry（FCM）assay．The mRNA expression levels of CCR1，CCR2，CCR3，CXCR4，CCR6，c-Met in U266cells were measured by SYBR Green I dye-based fluorescentquantitative real-time polymerase chain reaction (RQ-PCR). Transwell assay wasemployed to evaluate the migratory capacity of U266cellsin the presence of stromalcell derived factor-1（SDF-1)，monocyte chemotactic protein-1（MCP-1),macrophageinflammatory protein1-alpha（MIP-1α）, MSC supernatant from multiple myelomaand normal individuals in vitro before and after treated with2.5nmol/L bortezomibfor48hours. MSC derived from bone marrow sample of newly diagnosed multiple myeloma patients (MM-MSC,n=10) and normal individuals (N-MSC,n=7) wereisolated using density gradient centrifugation,then cultured and expanded invitro.MSC on passage2to4were used to be directly co-cultured with U266cells inthe absence or presence of2.5nmol/L bortezomib for48hours. Four groups wereassigned:U266and MM-MSC co-culture groups(U266+MM-MSC),U266andMM-MSC co-culture in the presence of bortezomib groups (U266+MM-MSC+bortezomib),U266and N-MSC co-culture groups(U266+N-MSC),U266andN-MSC co-culture system in the presence of bortezomib groups(U266+MM-MSC+bortezomib).Then U266cells were collected and the mRNA expression levels ofCCR1,CCR2,CCR3,CXCR4,CCR6and c-Met were further assayed by real-timequantitative PCR.Transwell assay was also employed to evaluate the migratorycapacity of U266cells from forementioned groups.Results1. Demonstrated by both MTT and FCM assays, bortezomib was shown to promoteapoptosis and decrease the cell viability of U266cells, which was in correlation to atime-and dose-dependant manners.2. The migratory ability of U266cells can be enhanced in the presence of SDF-1,MCP-1, MIP-1α, the supernatant of MM-MSC and N-MSC in all groups(P<0.05),which is more obvious in MM-MSC supernatant and N-MSC supernatantgroups(P<0.05).However, bortezomib was shown to inhibit the transwell migratorycapacity of U266cells even in the presence ofSDF-1and MIP-1α, MM-MSC andN-MSC supernatant.Besides,mRNA expression levels of CCR1,CCR2,CCR3,CCR6and c-Met in U266cells were also shown to be down-regulated by bortezomib(P<0.05).3. After co-cultured with either MM-MSC or N-MSC, the mRNA expression levels of CCR1，CCR2and CCR6in U266cells were much higher (P<0.05) than those ofcultured alone groups,and the mRNA expression levels of CCR3were alsoup-regulated in MM-MSC co-cultured groups.Interestingly, the mRNA expressionlevels of CCR1,increased most significantly in MM-MSC groups(P<0.05), while themRNA expression of CXCR4wasd own-regulated in both MM-MSC and N-MSCgroups(P<0.05).The migratory capacity of U266cells in MSC co-cultured groupsoralone groups measured by transwell assay was no significant difference (P>0.05).4.MSC derived from either MM or normal subjects were shown to compromise theinhibitory effect of bortezomib on the expression of CCR1and CCR6in U266cells(P<0.05)and this effect is more pronounced for MM-MSC. However, the mRNAexpression of CXCR4and c-Met in U266cells were much lower in the N-MSCco-cultured groups rather than those bortezomib alone groups (P<0.05).ConclusionThe transwell migratory capacity of U266cells was shown to be enhanced by SDF-1,MCP-1, MIP-1, the MM-MSC andN-MSC supernatant, which is more significantlyin MM-MSC and N-MSC supernatant.Bortezomib not only was shown to inhibit theproliferation rate and also promote apoptosis rate of U266cells, it was also shown tosuppress the migratory capacity and down-regulate the mRNA expression levels ofCCR1、CCR2、CCR3、CCR6and c-Met of U266cells. There seemed to be nosignificant difference between co-cultured groups and control groups, however,themRNA expression levels of CCR1,CCR2,CCR3andCCR6of U266cells wereenhanced after being co-cultured with MM-MSC. Besides, MSC derived from eitherMM or normal subjects were shown to compromise the inhibitory effect ofbortezomib on the expression of CCR1and CCR6in U266cells and this effect ismore pronounced for MM-MSC. In conclusion, our study suggested that MM-MSC provide survival support and reduce the sensitivity ofU266cells to bortezomib andplay an important role in the resistance and disease progression of MM. ObjectiveTo investigate the dysregulated genes of normal mesenchymal stem cells (N-MSC)after interacting with myeloma cells by microarray assay.Method1. An indirect co-culture system of N-MSC with mixed myeloma cell line U266and8226were employed by transwell.Approximately1×104N-MSC were placed on themembrane insert and1×105MM cells were placed in the lower chamber.Co-cultureswere maintained in L-DMEM with10%FBS for7days,at the same time N-MSCcultured alone was the control groups.2. At the end of co-cultured, the total RNA of N-MSC was obtained by theone-stepmethod and inversely transcribed into double-chain cDNA．With separatelymarking by Cy5-dCTP and Cy3-dCTP，the cDNAprobes of N-MSC were crossedwith chips and strictly cleaned．Then we analyzed the data after scanning byRoche-Nimble Gen, MS200scanner chip using Gene Pix Pro4.0software.Using thetwo-folds of difference as the threshold，that wasCy5/Cy3>2meant the up-regulatedgene，Cy5/Cy3<0.5meant the down-regulated gene.The differential expressed geneswere selectedand further categorized according to differentiation, apoptosis andchemotactic function.Thenwe elected four genes (transglutaminase2(TGM2）,C-Cmotif chemokine7(CCL7）,microfibri associated protein5(MFAP5）and fibroblast growth factor receptor2(FGFR2))of them to verify by real-time quantitative PCR.Results1. After co-cultured of N-MSC with MM cells for7days,N-MSC were harvested andthen proceeded to microassay assay for total mRNA expression. Among total10001genes,we filtered a total of837differential representing genes(837/10001,8.37%)，there were472up-regulated genes(472/837,56.39%) and365genes weredown-regulated(365/837,43.61%).Among the837differential representing genes,many genes were involved in cell growth and differentiation, signal transduction,immune regulation and inflammatory response,chemotaxis, anti-apoptosis and otherbiological functions.2. The Real-time quantitative PCR results confirmed that TGM2and CCL7geneexpression levels in N-MSC after co-cultured with myeloma cells were significantlyhigher than control cohorts, and MFAP5, FGFR2expression levels were lower thanthat in control, which are consistented with cDNA microarray results.ConclusionA variety of biological functions are changed of N-MSC after co-cultured with MMcells.This variations suggested that the MSC play an important role in thepathogenesis of MM. |
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