The Research On Avoiding STAT3Overactivation During MSCs Tumor-like Transformation

ObjectiveTo explore the relationship between MSCs (mesenchymal stem cells,MSCs) tumor-like conversion and STAT3(Signal Transducer and theActivator of Transcription3, STAT3) overexpression in the umormicroenvironment; Using a STAT3-specific inhibitor STA-21treatment toclear inhibition of STAT3overexpression whether reduce the risk ofMSCs tumor-like transformation in the tumor microenvironment. In orderto provide a scientific basis for Circumvent the malignant transformation ofMSCs and make it more safely used in clinical.Methods1. Cell culture: Human breast cancer cell line MCF-7B,rat C6glioma cellsand primary culture MSCs were cultured in DMEM/F12completemedium;.2. Experimental groups: The experiment is divided into two parts.The firstpart of the experiment is diveded into5groups:Blank group: culturedalone MCF-7B;Control group:0.1%DMSO in the medium added with MCF-7B;Experimental group: with the control group, it is divided intothree subgroups according to the STA-21concentration in the medium.The second part of the experiment is still set to5groups.The blank group:cultured MSCs alone;Control group: the use of the transwellinsert-culture dish with a6-well plate combined together to build ofMSCs and C6cells indirect co-culture system;Experimental group: withthe control group, according to the STA-21concentrations were dividedinto three subgroups.3. Experimental methods:(1) To observation the cells changes from themicroscopic by using an inverted microscope;(2) Flow cytometry detectcell cycle and apoptosis；(3) MTT assay of cell proliferation;(4)Real-time PCR detection of tumor-related genes of MDM2, STAT3,CyclinD1, Bcl-xl mRNA relative expression level;(5) Western blotanalysis groups MCF-7B P-STAT3, STAT3, CyclinD1and Bcl-xl proteinexpression, detection groups MSCs of MDM2, STAT3, P-STAT3, CyclinD1and Bcl-xl protein expression after co-culture for7d.Results1. Cell morphologyWith the prolongation of the STA-21increase in drug concentration andduration of action, MCF-7B appear to inhibition of cell proliferation andapoptosis increased shrinkage, cell morphology microscope, a largenumber of deaths MCF-7B cells; indirect co-culture of MSCs with C6 for7d, the morphology of the control group changed significantlysmaller nucleus, cytoplasm to the nucleus of the central narrow cells intolong spindle. Cells of the experimental group as the STA-21concentration of the drug increases, MSCs morphology morehomogeneous, and cell morphology is approximated with the blankgroup.2. MTT assay the effect of cell proliferationMTT assay showed that MCF-7B cells after different concentration ofSTA-21affecting, MCF-7B cells of the control group and the blankgroup showed a significant difference (P <0.01); proliferation inhibitionrate was close to blank group (P>0.05); the control group proliferationof MSCs was higher than blank group (P <0.05), the experimental group,MSCs significantly inhibited MSCs the role of various concentrations ofSTA-217d excessive proliferation of, and with the increase in theconcentration of STA-21role, inhibiting the effects of the proliferation ofMSCs are also growing, showing a concentration-dependent.3. Flow cytometry analysis cell cycleCell cycle analysis showed that with the increase in the concentration ofSTA-21affecting, MCF-7B gradually increased the proportion of cells inG1phase, the control group was significantly higher (P <0.05). Showedthat STA-21can cause MCF-7B cell cycle arrest at the G1phase,resulting in a reduced ability of the cell proliferation. 4. Real-time PCR detection of five groups MSCs tumor gene MDM2,STAT3, CyclinD1, Bcl-xl mRNA relative expression levelThe results showed that MSCs compared with the control group, thecontrol group, high expression of MDM2, STAT3, CyclinD1, Bcl-xlmRNA (P <0.05); experimental group STA-2110group of MDM2,CyclinD1, Bcl-xl mRNA expression levels compared with blankgroupincreased (P <0.05); rest of the experimental group MDM2,CyclinD1, Bcl-xl mRNA expression levels close to the blank controlgroup (P>0.05); of STAT3mRNA expression levels in threeexperimental groups were horizontal approaches (P>0.05).5. Western blot analysisMCF-7B cells with different concentration of STA-21after24h oftreatment compared with the control group STA-21can significantlyinhibit MCF-7B cells P-STAT3protein expression (P <0.01), downCyclinD1Bcl-xl protein expression, significant differences of the threegroups (P <0.05); CyclinD1, Bcl-xl protein expression levels of thecontrol group and blank group approaches (P>0.05);cells STAT3proteinexpression level of approximation (P>0.05); blank group MSCsexpression of STAT3, low expression of MDM2, CyclinD1, Bcl-xl, didnot express P-STAT3high expression of MDM2; All protein exceptSTAT3in Control group were significantly higher than the blank group(P <0.05); experimental group, MSCs by10,20,30μM STA-21 treatment compared with the control group, can significantly reduce ofMDM2, P-STAT3, CyclinD1and reduce the amount of the protein withthe STA-21concentration increased expression of Bcl-xl white, whichare20μm the STA-21treatment change significantly, similar to theblank control group (P>0.05); close three groups of cells STAT3protein(P>0.05).ConclusionMSCs in vitro to be tumor microenvironment in the tumor-liketransformation and persistent activation of STAT3in STAT3specificinhibitor STA-21intervention of MSCs in a certain extent, tumor-liketransformation for MSCs clinical Security applications providecertaintheoretical basis.