| Background:Glioma(glioma)is characterized by high incidence rate,high mortality rate and high recurrence rate.At present,there is no new and effective treatment method,which brings heavy financial burden to the state,society,family and individuals.Therefore,the in-depth analysis of the pathogenesis of glioma is a great challenge and great demand for reducing incidence rate,recurrence rate and mortality rate in the future.Previous studies have shown that microenvironment plays an important role in tumor progression,and gliomas stem cells(GSCs)are important executors of tumor microenvironment.They not only take advantage of tumor microenvironment,but also have the physiological characteristics of active regulation and remodeling of microenvironment.This dynamic change of mutual utilization between microenvironment and GSCs eventually leads to the invasion and metastasis of glioma.Therefore,to explore the biological effects of GSCs in tumor microenvironment is of great clinical significance for understanding the development of glioma.Tumor epithelial mesenchymal transition(EMT)is a biological process of epithelial cells transforming into myofibroblasts in microenvironment.Previous studies have shown that GSCs secrete transforming growth factor beta-1(TGF-β1),interleukin-1(IL1),vascular endothelial growth factor(VEGF)and platelet-derived growth factor(PDGF)in microenvironment,while these cytokines can activate TGF-β1,PDGF,VEGF and other pathways.It can be seen that the over activation of various inflammatory factors and multiple signaling pathways caused by GSCs microenvironment is an important basis for the occurrence and maintenance of EMT.However,whether we can find the common target to control their activity is still a unsolved scientific problem.Six1,a member of homeobox gene family,is an important protein that affects cell differentiation,proliferation and survival.It plays an important role in the development of mammalian body and organs.At present,the homeobox gene Six1 has been deeply studied in the research of the progress,early diagnosis and prognosis of malignant tumor,especially in the search of drug resistance mechanism and the production of targeted new drugs.Therefore,we speculate that Sixl(the homeobox gene family member)may be the common target of regulating the progression of EMT induced by GSCs.Objectives:1.To clarify the effect of Sixl on glioma stem cells in microenvironment.2.Study on the molecular mechanism of EMT induced by glioma stem cells based on Six13.Analysis of Six1 is a key target for gliomas stem cells to activate multiple signaling pathways leading to EMT progression4.Making clear that Sixl is an effective index to evaluate the progression and prognosis of glioma.Methods:A large number of glioma stem cells(GSCs)were identified and amplified by suspension culture of human glioma cells(U251)using serum-free medium and immunomagnetic beads.The separation rate of CD 133+was verified by flow cytometry.Immunofluorescence was used to identify markers on the surface of stem cells.Immunofluorescence detection of SIX1 expression in primary GSCs;Real-time quantitative PCR was used to detect SIX1 expression in GSCs transfected with SIX1siRNA.The proliferation of SIX 1 protein in GSCs inhibited by SIX1siRNA was detected by cloning.ELISA method was used to detect the expression changes of TGFβ1,VEGF and PDGF in medium after SIX1siRNA inhibited SIX1 protein expression in GSCs.Using Transwell CD 133 +GSCs and U251 cell co-culture model,used a technique called RNA interference silence U251 cells SIX1 gene expression,were observed under phase contrast microscope SIX1 cell morphological changes after gene silencing,using western blot and immunofluorescence method for determination of epithelial cell phenotypic marker protein E-Cadherin and alpha SMA expression changes,Western blot assay detected the effect of SIX1 on expression of key receptor proteins and downstream phosphorylated proteins in TGFβ/smad2/3,VEGF/MAPK,PDGF/ERK signaling pathways.The expression of SIX1 in clinical samples of malignant glioma was detected by immunohistochemical method.According to the expression of SIX1 in immunohistochemistry,statistical software,basic clinical data,staging and malignant degree were used for analysis and processing.Results:1.The separation rate of CD 133+cells was 95%after flow cytometry.Immunofluorescence showed that the expression level of SIX1 in CD133+GSCs was significantly higher than that in CD133-gscs(P<0.05).The proliferation rate of CD133+GSCs increased significantly,and the proliferation rate slowed down significantly after SIX1siRNA inhibited SIX1 expression of CD133+GSCs(P<0.05).The expression levels of TGFβ1,VEGF and PDGF in CD133+GSCs were significantly up-regulated compared with cd133-gscs(P<0.05),and the expression levels of these cytokines were significantly decreased after SIX1siRNA inhibited the expression of SIX1 in CD133+GSCs(P<0.05).2.CD133+GSCs and U251 cells cultured 72 h,α-SMA expression increased significantly,and E-Cadherin expression significantly decreased in U251 cells(P<0.05),at the same time companion has raised SIX1 gene expression(P<0.05),while SIX1siRNA can significantly weaken the cell morphological changes and cell phenotype marker protein changes.To further elucidate the mechanism of SIX1siRNA inhibiting EMT,western blot assay showed that after CD133+GSCs were co-cultured with U251 cells for 72h,the expression of TGFβR,VEGFR,PDGFR and downstream phosphorylated proteins psmad2/3,MAPK and pERK on the surface of U251 cells were up-regulated,while SIX1siRNA inhibited SIX1 protein expression in U251 cells,the expression of these proteins was significantly down-regulated(P<0.05).3.Immunohistochemical test showed that the expression of SIX in clinical tissue samples of malignant glioma was significantly higher than that in normal brain tissue(P<0.05).Statistical analysis showed that the expression of SIX1 was not significantly correlated with the age and gender of the clinical data,but positively correlated with tumor stage,and the higher the expression of SIX1 was,the later the tumor stage was(P<0.05).Meanwhile,statistical analysis also found that the expression of SIX1 was significantly correlated with the survival time after diagnosis,and the higher the expression of SIX1,the shorter the survival time after diagnosis(P<0.05).Conclusion:1.Six1 can affect the proliferation and activation of glioma stem cells in microenvironment2.Six1 is an important regulatory target for the development of EMT induced by glioma stem cells3.Six1 can be used as an effective index to evaluate the malignant degree and survival prognosis of gliomas... |
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