Role Of STAT3 In Malignant Transformation Of Bone Marrow Mesenchymal Stem Cells Under Tumor Microenvironment

Background: Tumor is a serious threat to human life and health.It’s also a problem that we persist in trying to overcome.Mesenchymal stem cells(MSCs) are more easily obtained,strongly self-renewal,especially they have capacity to home sites of inflammation,manifesting immunosuppressive properties,called as Medicinal Signaling Cells or “ambulatory cells”.Tumor microenvironment is very complicated,tumor cells may influence the phenotype of MSCs through reciprocal crosstalk,MSCs could be greatly influenced by the transcription factors and nucleic acids,thus they may unexpectedly undergo malignant transformation.The activation of Signal Transducer and Activator of Transcription 3(STAT3) is under strict control in normal cells.However,the process is out of control,stem cells may result in tumor-like lesion and participate in each step of tumor development.micro RNAs(miRNAs) are small noncoding RNAs with a length of 18～22 nucleotides and can bind to 3’-UTR of the targeted message RNA,thus inhibiting translation or promoting RNA degradation without changing the nucleotide sequence of the gene.Mi RNAs are implicated in physiological and pathological processes,especially in tumor pathogenesis,and can act as tumor suppressors or oncogenes.In this study,we hypothesize that miRNA-134/STAT3 pathway plays an important role in promoting the malignant transformation of BMSCs.This novel insight may help us to better understand the pathogenesis of malignant transformation and cancer prevention.Purpose: To test this hypothesis,we established a co-cultural system to mimic tumor microenvironment.Hanging transwell chamber was inserted into the 6-well plate,wherein the MSCs and C6 were separately seeded at a small chamber.We intend to determine the role of STAT3 on oncological differention of BMSCs in tumor microenvironment,and study the exact regulatory mechanism of the effect of miRNAs on STAT3.Methods: Part Ⅰ: BMSCs were indirect co-cultured with C6 glioma cells 2 weeks,the changes of cell proliferation capacity were detected by cck-8 and FCM.Colony-Forming Assay in Soft Agar was used to measure the ability of cells in culture to grow and divide into groups,any cell that has the potential to form a colony may also have the potential to cause cancer.BMSCs that has been malignantly transformed in vitro tumor microenvironment,were injected into immunodeficient mice subcutaneously.After 4-8 weeks,the survival rate of mice should be observed and the tissue of transplanted site should be analyzed.Real-time quantitative polymerase chain reaction(RT-qPCR) method was used to analyze the expression of STAT3 mRNA,and western blot was used to analyze the changes of STAT3 protein expression.Part Ⅱ: Targetscan and miRanda were used to predict the miRNAs that target STAT3 gene.Differentially expressed miRNAs were verified by RT-qPCR.And miRNA-134-5p were selected for further study.Part Ⅲ: miRNA-134-5p mimics、mimics NC、miRNA-134-5p inhibitor and inhibitor NC were transfected into each groups,The protein expression level of STAT3 was detected by Western blot,RT-qPCR was used to detect the mRNA expression of STAT3.Luciferase reporter plasmids GP-miRGLO-STAT3-WT/GP-miRGLO-STAT3-MUT containing the miR-134-5p putative binding site or mutation site in the STAT3 3’-UTR,and miRNA-134-5p mimics、mimics NC was co-transfected into HEK293.The putative binding site of miR-134-5p in the 3’-UTR of STAT3 mRNA was analyzed by dual luciferase reporter assay.Results: Part Ⅰ: Co-cultured with C6 glioma cells,the proliferative activity of the transformed BMSCs had a increasing trend tested by CCK-8,the proportion of cells in S and M phase were increased,while the proportion of cells in G1 phase in malignant transformed cells was decreased(p<0.05) .Colony-Forming Assay in Soft Agar experiments showed that the co-cultured BMSCs were cloned and counted after Crystal violet staining for 14 days.Co-cultured BMSCs were injected into immunodeficient mice subcutaneously,approximately 4 to 8 weeks later,neoplasm could be observed in the imbedded locus.The expression of STAT3 in co-cultured group was remarkable higher when compared with blank group.Part Ⅱ: We use miRanda and Targetscan to predict miRNAs that may target STAT3 gene,and it was miR-134-5p that be found.The problem that whether miR-134-5p regulates STAT3 has not been reported in previous studies,so it was initially identified a further study subject.Part Ⅲ: RT-qPCR results showed that STAT3 mRNA expression was inhibited by miR-134-5p transfection.Western blot results showed that the protein expression of STAT3 was suppressed by miR-134-5p.Compared with the miRNA-134-5p mimics NC group,the luciferase activity of the GP-miRGLO-STAT3-WT vector was decreased in the miRNA-134-5p mimics group,while miRNA-134-5p mimics had no effect on the luciferase activity of the GP-miRGLO-STAT3-MUT vector.Conclusion: Indirect co-cultured with C6 glioma cells,BMSCs could unexpectedly undergo malignant transformation and STAT3 was significantly higher expression,Further researches revealed that miR-134-5p may play an important role by targeting the inhibition of STAT3.