Alteration Of Cx43Expression In Bone Marrow Mesenchymal Stem Cells Induced By Myeloma Cells And Its Role In Pathogenesis Of Multiple Myeloma

Multiple myeloma (MM) is a neoplasm characterized by the clonal expansion ofmalignant plasma cells that accumulate mainly in the bone marrow (BM) and are closelyrelated to the surrounding stromal microenvironment. The interaction of MM cells with theBM microenvironment, either directly via adhesion molecules or indirectly via thestimulation of autocrine/paracrine production of cytokines, activates a broad range ofproliferative and anti-apoptotic signaling pathways.It has been found that alterations in thelocal microenvironment are not only supportive of tumor growth but also required fortumorigenesis. During the process of MM development, BM stromal cells graduallydevelop special characteristics that support MM cells. For example, BM mesenchymalstem cells(BMSCs) can increase MM cell adhesion to BM, protecting the cells fromchemotherapy and aiding their accumulation within the BM. Meanwhile, during theprocess of MM cell migration and homing to the BM, MM cells remodel the BMmicroenvironment and make it a place that is suitable for the survival and proliferation ofMM cells. At present, the detailed mechanisms involved in this process have not beencompletely elucidated.The gap junction (GJ) is a common type of cell-cell junction and is involved in theregulation of many physiological process, such as cell proliferation and differentiation. Theexchange of information and energy between adjacent cells occurs via GJ-mediatedintercellular communication(GJIC). Connexin-43(Cx43) is the major component of GJs inhuman tissue, and it is expressed in many cell types, including hematopoietic cells andstromal cells. Dysregulation of Cx43expression and dysfunction of GJIC are related touncontrolled proliferation and malignant phenotypes and may be two of the genetic eventsinvolved in tumorigenesis. GJs and connexins may be new therapeutic targets in cancer. The aim of this study was to assess the alteration of Cx43expression in BMSCs,evaluate the interactions between BMSCs and MM cells in coculture systems. We alsoaimed to determine the effects of altered Cx43expression on the migration and adhesion ofMM cells.Part1Cx43Expression In Myeloma Cells And Bone MarrowMesenchymal Stem Cells And Its Biological FunctionObjective To analyse Cx43expression in multiple myeloma (MM) cells and bonemarrow mesenchymal stem cells (BMSCs) and investigate the role of gap junction playedin the process of BMSCs induced MM cells migration and adhesion and SDF-1α secretionof BMSCs.Methods BMSCs were isolated and cultured with adherent culture method. CD138magnetic beads and midi MACs system were employed to isolate primary MM cells. Theimmunophenotye of BMSCs and primary MM cells were detected by Flowcytometry.Cx43expression in MM cells and BMSCs were analysed by westernblot andimmunofluorescence. The influence of GJ inhibitor18α-GA on MM cells and BMSCsproliferation was determined by CCK-8. Transwell was applied to study the effect of18α-glycyrrhetinic acid（18α-GA）on MM cells transmigrion induced by BMSCs. SDF-1αsecretion was detected by ELISA.Results1) MM cell lines RPMI8266、U266and one of three primary MM cellsexpressed Cx43at moderate and low levels. Cx43was not expressed in XG-4、XG-7celllines but highly expressed in BMSCs. Cx43expression in BMSCs from patients newlydiagnosed with MM was stronger than that in BMSCs from healthy donors.2)18α-GA hadlittle effects on the proliferation of RPMI8226cells or BMSCs.3) SDF-1α concentrationin the supernatant of BMSCs cultured alone was237.84±9.23pg/ml, which decreased to94.31±6.44pg/mL（P<0.01）when the cells were incubated with18α-GA.4)18α-GAinhibited the migration of MM cells induced by BMSCs. The migration rate of ofRPMI8226and XG-7cells were （8.0±0.673）%、（0.88±0.036）%, respectively, whichdecreased to （4.82±0.186）%（P<0.01）、（0.58±0.020）%（P<0.05）after BMSCs were incubated with18α-GA.5)18α-GA inhibited MM cells adhesion to BMSCs. The adhesionrates of RPMI8226and XG-7cells were16.967±1.55%and9.5±1.323%, respectively,which decreased to11.1±0.819%（P<0.05）and6.63±0.551%（P<0.05）after incubationwith18α-GA.Conclusions BMSCs and proportion of MM cells express CX43. GJ inhibitor candownregulate SDF-1α secretion of BMSCs and inhibit the migration and adhesion of MMcells induced by BMSCs. Part2Construction Of three Dimentional Cultured Bone MarrowMesenchymal Stem Cells Spheroids And Investigation Of TheirCharacteristicsObjective To establish the method of three dimentional culture of BMSCs，andinvestigate the characteristics of BMSCs spheroids，and observe MM cells migration in theBMSCs spheroids.Methods Three dimentional cultured BMSCs spheroids （3D-BMSCs） werecultured in the agarose coated96well round bottom plates. The apperance of BMSCsspheroids were examined under an inverted microscope and HE staining and ScanningElectronic Microscopy. Cx43mRNA、SDF-1α mRNA expression of3D-BMSCs andcommon2dimentional cultured BMSCs （2D-BMSCs） were analysed with reversetranscription polymerase chain reaction （RT-PCR）. The effects of18α-GA on themigration of MM cells in3D-BMSCs spheroids were detected by immunofluorescence.Results After4days, BMSC formed spheroid structures of approximately450mmdiameters in the agarose coated96well round bottom plates. Scanning electronicmicroscopy showed that cells with numerous filopodia-like projections contactedneighboring cells and formed a complex three-dimensional network.3D-BMSCs expressedhigher SDF-1α mRNA compared to2D-BMSCs. Cx43mRNA in3D-BMSCs and 2D-BMSCs had no difference.18α-GA inhibited MM cells migrated into BMSCsspheroid.Conclusion3D-BMSCs spheroid can mimic BM microenvionment, and its Cx43expression plays an important role in MM cells migration. Part3Alteration Of Cx43Expression And Its Biological Effects in TheCoculture Systems Of MM cells And BMSCsObjective To investigate the interplay of MM cells and BMSCs and alteration ofCx43expression in coculture systems.Methods Westernblot、qRT-PCR and immunofluorescence were employed todetect the alteration of Cx43expression and distribution in BMSCs directly and indirectlycocultured with myeloma cells. Lucifer yellow dye spread was utilised to evaluate GJICbetween BMSCs. SDF-1α secretion was detected by ELISA. Westernblot were employedto detect beclin、LAMP、LC3expression in BMSCs after cocultured with RPMI8226withor without autophgy inhibitor3-MA. Vonkossa staining were used to determine osteogenicdifferentiation of BMSCs.Results1) The expression of Cx43mRNA in BMSCs were both upregulated aftercoculture directly and indirectly with RPMI8226，1.36and2.1times that of BMSCscultured alone respectively.2) Westernblot analysis showed that Cx43protein expressionin BMSCs was also upregulated after coculture with RPMI8226, and the increased Cx43was mainly distributed in cytoplasm.3) Lucifer yellow dye spread showed that GJIC wasupregulated in BMSCs cocultured with RPMI8226.4) Cx43expression in RPMI8226cellswas downregulated after directly coculture with BMSCs and had no marked alteration afterindirectly coculture with BMSCs.5) SDF-1α concentration in supernant of BMSCsdirectly and indirectly cocultured with RPMI8226were（373.02±10.11）pg/ml and（309.71±10.71）pg/ml respectively, which were both higher than that of BMSCs cultured alone （237.84±9.23）pg/ml（P<0.01, P<0.05）, and could be inhibited by18α-GA to(126.01±4.80）and（106.99±3.39）pg/ml, respectively（P<0.001, P<0.01）.6) Autophagyassociated protein beclin、LAMP、LC3expression in BMSCs were upregulated aftercoculture with RPMI8226, which could be inhibited by3-MA.7) Vonkossa stainingshowed that RPMI8226inhibited osteogenic differentiation of BMSCs, which could beimproved by3-MA.Conclusion The Cx43expression in BMSCs is upregulated after directly andindirectly coculture with MM cells, which can improve SDF-1α secretion of BMSCs. MMcells can inhibit osteogenic differentiation of BMSCs via upregulating the autophgy ofBMSCs.