Mechanism Of STAT3 Activation Mediation Mediating MSCs Malignant Transformation In C6 Glioma Microenvironment

Chapter Ⅰ The isolation,culture and identification of rat bone mesenchymal stem cellsObjective: To isolate,culture and identify rat bone marrow mesenchymal stem cells(BMSCs)in vitro,so as to prepare for the safe application of BMSCs in tumor microenvironment.Methods: 1.Isolation and culture of rat bone marrow mesenchymal stem cells: the experimental animals were sacrificed by cervical vertebra bleaching,and bone marrow cavities were exposed by removing the hind leg tibia,femoral muscle and cartilage at both ends.DMEM/F12 medium was aspirated with a sterile syringe to repeatedly flush the marrow cavity,and the collected marrow cavity washing solution was placed in a horizontal centrifuge at 1000 rpm for 5min.When the cell were adhered to 80% of culture bottle,the passage was carried out in a 2:1 ratio,and p3-p4 generation cells were saved for subsequent experiments.2.Identification of rat bone marrow mesenchymal stem cells: the expression of MSCs markers(CD34,CD90,CD45,CD71)on the surface of P3 generation cells detected by immunofluorescence and the expression of these markers(CD34,CD90,CD105)on the surface of P3 generation cells detected by flow cytometry.Meanwhile,osteogenesis and lipogenic differentiation of P3 generation cells were detected.Osteogenic induction: calcium nodules were observed after 20 days,and the osteogenic differentiation potential was identified by alizarin red staining.Lipogenic induction: observe the presence of lipid droplets after day 1,and identify the potential of lipogenic differentiation with oil red O.Results: 1.Morphology under inverted microscope: the cell growth was uniform,the morphology was spindle-like,there was no obvious morphological change in continuous passage,and the cell itself showed no aging trend.2.Indirect immunofluorescence results showed that CD90 and CD105 were expressed in primary cells,while CD34 was not.Flow detection results showed that the expression rates of CD71,CD90,CD34 and CD45 on cell surface antigens were 94.0%,100.0%,7.2% and 4.7%,respectively,meeting the requirements of MSCs cell surface antigens and allowing follow-up experiments.The rust-red nodules and red-stained lipid droplets were observed in the induction of osteogenesis and lipogenesis.Conclusion: Rat bone marrow mesenchymal stem cells can be isolated by differential adherent culture,and can be cultured with high purity and multidirectional differentiation potential.Chapter Ⅱ IL-22 furthers malignant transformation of rat mesenchymal stem cells,possibly in association with IL22RA1/STAT3 signalingObjective: To explore the mechanism of IL-22 inducing malignant transformation of rat BMSCs through IL22RA1/STAT3 pathway in C6 glioma microenvironment.Method: 1.MSCs were indirectly co-cultured with glioma cells using Transwell chambers.A total of 3×105 C6 cells(in 200 μl DMEM/F12 supplemented with 10% FBS)were seeded into the upper chamber of the Transwell plate(0.4 mm).An equal number of MSCs(in 200 μl DMEM/F12 supplemented with 10% FBS)was seeded in the lower part of the chamber.After 7 days of indirect co-culture with C6 cells,the MSCs were collected for analysis.MSCs cultured alone served as the control.The cells were imaged under a confocal microscope.2.Normal bone marrow mesenchymal stem cells of rats were used as the control group to detect the changes in cell morphology,cell cycle,proliferation,invasion and migration ability after indirect co-culture with C6 glioma cells.The expression of IL-22 in the culture supernatant was detected by ELISA.The expression of IL22RA1 in bone marrow mesenchymal stem cells was detected by quantitative fluorescence PCR and Western blot.Meanwhile,bioinformatics was used to study the expression of IL-22 and IL22RA1 in glioma and normal cells.3.Exogenous IL-22(20ng/ml)was added to the culture medium of bone marrow mesenchymal stem cells,and changes in cell proliferation,invasion and migration ability were detected after 24 and 48 hours of culture.Meanwhile,changes of STAT3 and p-stat3 in MSCs were detected.4.Transfection of si-STAT3 inhibited the expression of STAT3,and the changes of proliferation,invasion and migration of MSCs cells induced by IL-22 were observed.Results: 1.After co-culture with C6 glioma cells for 1 month,the morphology of MSCs changed significantly,presenting glioma cell-like changes,cytoplasm contracted to the nucleus,became thinner and longer,cell nuclei became smaller,cells were arranged closely,and the ratio of nucleus to cytoplasm increased.2.The proliferation capacity of MSCs was significantly increased after co-culture with C6 glioma cells,and the ratio of G2/S phase was significantly higher than that in the MSCs culture group alone.At the same time,the invasion and migration ability of MSCs co-cultured with C6 glioma cells were significantly increased,suggesting that MSCs co-cultured with C6 glioma stem cells have the tendency of malignant transformation.3.UCSC was used to analyze the expression profile of IL-22 and IL22RA1 in tcga-gbm cancer genome browser,and it was found that the expression of IL22RA1 in primary and recurrent glioblastoma was higher than that in normal tissues,while the expression of IL-22 in glioblastoma and normal tissues was lower.4.No IL-22 expression was found in the supernatant of C6 glioma cells and mesenchymal stem cells by ELISA.The m RNA and protein expression of IL22RA1 in MSCs co-cultured with C6 glioma cells were significantly increased.5.At 24 h and 48 h after the action of IL-22,the activity of MSCs cells was significantly higher than that of the MSCs culture group alone.Bcl-xl expression was significantly increased.The invasion and migration ability of MSCs were significantly increased.6.IL-22 significantly increased the expression of STAT3 and p-stat3,and up-regulated the proportion of phosphorylated and non-phosphorylated proteins.Interference with STAT3 significantly reduced the proliferation,invasion and migration of il-22 treated MSCs.Conclusion: In the C6 glioma microenvironment,in addition to malignant transformation of bone marrow mesenchymal stem cells induced by tumor cells themselves through paracrine function,immune cells can also induce malignant transformation of BMSCs through secreting IL-22 and other inflammatory factors through the STAT3 pathway.Chapter Ⅲ LncRNA MEG3 inhibits MSCs malignant transformation through STAT3 pathway in tumor microenvironmentObjective: To explore the mechanism of lnc RNA MEG inhibits the malignant transformation of rat BMSCs in the C6 glioma microenvironment through the STAT3/m TOR pathway.Methods: 1.Real-time fluorescent quantitative PCR was used to detect the expressions of lnc RNA MEG3,lnc RNA H19,lnc RNA TUG1,lnc RNA PVT1,lnc RNA salmon and lnc RNA HOTAIRM1 in rat bone marrow mesenchymal stem cells before and after co-culture with C6 glioma cells.2.Bioinformatics was used to analyze the expression of lnc RNA MEG3 in nervous system tumors.3.Impact of MEG3 on the biological characteristics of bone marrow mesenchymal stem cells after tumor formation in vitro and in vivo: flow cytometry was used to detect the impact of MEG3 on apoptosis.Transwel assay was used to detect the effect of MEG on the ability of cell invasion and migration.In vivo experiment: the growth model of subcutaneous tumorigenesis in nude mice was established to observe the effect of MEG3 on tumorigenesis of bone marrow mesenchymal stem cells in rats after tumorigenesis.4.The mechanism of MEG3 in inhibiting MSCs tumorigenesis by regulating STAT3 activity: the expression of STAT3 in MEG3 tumorigenesis MSCs transfected with lnc RNA was detected by fluorescence quantitative PCR,and the levels of STAT3 and p-stat3 were detected by Western blot and immunofluorescence.Results: 1.Realtime PCR detection found that MEG3 expression in MSCs co-cultured with C6 glioma cells was significantly decreased;2.SAGE and Oncomine were used to analyze the expression of lnc RNAs in nervous system tumors,and it was found that the expression of lnc RNA MEG3 in nervous system tumors was significantly lower than that in normal tissues,especially in glioma.2.The number of apoptotic cells and necrotic cells in the MEG3 lentivirus group transfected into rat bone marrow mesenchymal stem cells after tumorigenesis was on the rise,and the ability of proliferation,invasion and migration was significantly reduced.The weight and volume of overexpressed MEG3 MSCs were not significantly reduced and the prognosis was significantly improved in nude mice subcutaneously.3.After MEG3 lentivirus transfection,the expression of STAT3 m RNA and protein in the tumorized MSCs was significantly reduced.Conclusion: The malignant transformation of MSCs in tumor microenvironment is related to the decreased expression of MEG3,an lnc RNA.MEG3 can inhibit the malignant transformation of rat bone marrow mesenchymal stem cells in tumor microenvironment by inhibiting the STAT3 pathway.Chapter Ⅳ Mechanism of STAT3 inhibits autophagy and mediates MSCs malignant transformation in tumor microenvironment through m TOR pathwayObjective: To explore the mechanism of STAT3 inhibits autophagy and mediates MSCs malignant transformation in tumor microenvironment through m TOR pathway.Methods: 1.The expressions of autophagy related proteins(LC3B,Beclin1,P62 and m TOR)were observed by Western blot and immunofluorescence.The changes in the number of autophagy bodies in MSCs before and after tumorigenesis were observed by electron microscopy,and the changes in autophagy status before and after tumorigenesis of rat bone marrow mesenchymal stem cells were comprehensively determined.2.The expression of STAT3 was interfered by si-STAT3,and the expressions of autophagy related proteins LC3 B,Beclin1,P62 and m TOR were observed by Western blot and immunofluorescence.LC3 double-labeled adenovirus was used and laser copolymerization was used to observe the changes of autophagy in tumorized MSCs after the expression of dry STAT3.3.Using the m TOR inhibitor rapamycin,it was determined that autophagy could be restored by inhibiting the m TOR pathway,and its effects on proliferation,invasion,migration and subcutaneous tumorigenesis of MSCs after tumorigenesis were observed.Results: 1.By Western blot and immunofluorescence detection Beclin1,LC3 B,P62 and the expression of m TOR,found that after incubation with C6 glioma MSCs LC3B-Ⅱ significantly reduced,tumor of MSCs in significantly reduced autophagy level.Beclin1,an important protein involved in autophagosome formation,was also detected,and Beclin1 expression was significantly reduced in tumorized MSCs.Meanwhile,the expression of autophagy substrate protein P62 was significantly increased in MSCs after tumorigenesis,and the expression of autophagy inhibitor protein m TOR was also significantly increased,further confirming that the autophagy level of MSCs after tumorigenesis was significantly decreased.2.The number of intracellular autophagosomes decreased significantly after the tumorigenesis of stem cells was observed by electron microscopy.3.Tumoralized MSCs transfected with si-STAT3 can promote the inhibition of LC3 expression caused by tumorigenesis.At the same time,after transfection of si-STAT3,the expressions of m TOR and P62 proteins in tumorized MSCs were significantly decreased,while Beclin1 expression was significantly increased.Both autophagosomes and autophagolysosomes increased significantly after STAT3 interference,indicating that the autophagy level of tumorized MSCs recovered after STAT3 interference.4.Cell proliferation,invasion and migration were significantly reduced after the addition of mTOR inhibitor rapamycin.Conclusion: activated STAT3 in tumor microenvironment inhibits the autophagy mediated malignant transformation of bone marrow mesenchymal stem cells in rats through m TOR pathway.