Role Of IL-6/STAT3Signaling Pathway In Oncological Differention Of MSCs In Tumor Microenvironment

ObjectiveTo determine the role of IL-6/STAT3signaling pathway on oncologicaldifferention of Bone marrow mesenchymal stem cells (MSCs) in tumormicroenvironment. And to provide initial experimental basis for the safeuse of mesenchymal stem cells in the bone marrow in cancer treatmentprocess and find new drugs to prevent bone marrow mesenchymal stem cellmalignant transformation.Materimal and Methods1. Experimental animalsWistar rats and newborn rats (SPF), the body weight of rats was40±10g,purchased from the Third Military Medical University, Daping MedicalExperimental Animal Center Certificate of Conformity SCXK (Army)2002-003.2. CellsC6rat giloma cells were purchased from Chongqing Medical Institute ofPediatrics Cancer Research.3.Experimental groups and Co-culture3.1Indirect co-culture systemHinging transwell chamber was inserted into the6-well plate, wherein theMSCs were seeded at a small chamber which was inserted in6-well plates. C6cells and astrocytes were seeded at6-well plates. The ratio of the twocells in the upper and lower holes was adjusted to1:1.3.2Specific experimental packetThis experiment was divided into four groups: the experimental group(MSCs and C6glioma indirect co-culture), the positive control group(conventional cultured C6glioma cells), the negative control group (MSCsand astrocytes indirect co-cultured), the blank control group (conventionalcultured MSCs).4. Experimental Method4.1Inverted phase contrast microscope was used to observe themorphological changes of the cells.4.2To detect the change of the cell cycle of cells in each group.4.3To dectct groups STAT3, GP130and IL-6R, MDM2and BCL-xlexpression by RT-qPCR and Western.4.4To detect the expression of STAT3and GP130by immunofluorescence.4.5MSCs after co-culture of each group were grown in nude mice. Thetumor growth of each group was observed by HE staining, usingimmunohistochemical methods to dected the expression of STAT3.4.6Using ELISA to detect the expression of IL-6and IL-6R in cell culturefluid.Results1．Experimental group cells showed tumor cell-like morphology.2．The expression of IL-6and IL-6R has significantly increased whenmeasured by ELISA in the cell supernatant of exprimental.3．The cell cycle of experimental MSCs has significantly enhanced whenmeasured by FCM.4．The expression of GP130, STAT3and P-STAT3in exprimental groupwas largely higher compared with control group. 5．There was not only the expression of STAT3increased but thedownstream protein of STAT3,such as MDM2and CyclinD1, inexperimental group.6．In exprimental group and the positive group, cells were deeply stained,obviously gathered and arranged in irregular and a large number ofneovascularization within the tumor.Conclusion●The oncological differention of MSCs was highly related with theoverexpression and activation of SIL-6R/GP130.●The over expression and activation of STAT3and P-STAT3is one ofthe important reason for the oncological differention of MSCs intumor microenvironment.