| Tim-1 is a susceptibility gene for asthma and allergies.It is preferentially expressed in Th2 cells and is a co-stimulatory molecule for T cell activation.In recent years,abnormal expression of Tim-1 has been associated with many cancers,for example,the expression of Tim-1 is abnormal in non-small cell lung cancer(NSCLC).After knocking down Tim-1 in NSCLC cell lines,it can significantly inhibit cell viability,cell migration and invasion by down-regulating PTEN / AKT pathway.At present,Tim-1 is thought to promote the secretion of cytokines such as IL-10 and TGF-? in lymphocytes,and plays an important role in regulating immune cell activity.TGF-? is closely related to the carcinogenicity of gliomas,and its high expression also indicates a worse prognosis for glioma patients.Studies have found that Tim-1 molecules are expressed in the central nervous system,such as oligodendrocytes.And Tim-1 also has abnormal expression in primary central nervous system lymphoma,which is positively correlated with the expression of cytokine IL-10.Combined with the tumor microenvironment mechanism of gliomas,these studies suggested that Tim-1 may play an important role in the secretion of cytokines in the glioma microenvironment,and may therefore be involved in the occurrence and development of gliomas.Here,we investigated the expression of Tim-1 in human gliomas and its significance.The effect of Tim-1 on the biological function of glioma cells was analyzed by molecular biology methods,and the related mechanism was analyzed in depth to provide a theory for immunotherapy of gliomas targeting Tim-1.This study was constituted as the following three parts:Part ? Study of Tim-1 expression in clinical glioma tumor tissues and cell lines Objective: To investigate the expression of Tim-1 in clinical glioma tumor tissues and cell lines,and to construct glioblastoma cell lines that knocked down the expression of Tim-1.Methods: The differentially expressed genes in normal and glioma tissues were detected by microarray analysis.Western Blot,Immunohistochemistry,immunofluorescence staining,real-time quantitative polymerase chain reaction(q RT-PCR)and other methods were used to analyze: 1)Differences in Tim-1expression between glioma clinical specimens and normal brain tissues;2)Tim-1expression in glioblastoma cell lines(U87 and U251)and normal glial cell line(HEB).Transfect U87 and U251 cells lines with Tim-1 sh RNA lentivirus,construction of stable transfected cell lines after Tim-1 knockdown,and use Western Blot and q RT-PCR to verify the virus transfection efficiency.Results: 1)The expression of Tim-1 was significantly increased in high-grade gliomas compared with the normal brain tissue,and the expression level of Tim-1was positively correlated with the malignant grade of gliomas.2)Tim-1 expression in U87 and U251 cell lines was significantly higher than that of in HEB.3)The efficiency of knockdown of Tim-1 by lentivirus is obvious,and U87 and U251 cell lines with low expression of Tim-1 were successfully constructed.Conclusion: Compared with the normal brain tissue and HEB,Tim-1 expression was significantly increased in high-grade glioma and glioblastoma cell lines U87 and U251.Part ?The role of Tim-1 in the proliferation and invasion of glioblastoma Objective: To explore the effect of Tim-1 on the proliferation,invasion and apoptosis of glioblastoma cells.Methods: CCK-8 proliferation assay was used to detect the effects of knockdown of Tim-1 on cell proliferation in vitro.The effects of Tim-1 knockdown on glioma cell migration and invasion were tested by the Scratch and Transwell experiments.Apoptosis of glioblastoma cells was detected by flow cytometry after Tim-1knockdown.The expression of cleaved-caspase3,Bax,Bcl-2 and cell invasion related proteins MMP-2 and MMP-9 were examined by Western Blot.Subcutaneous tumor formation experiments in nude mice were performed to verify the effect of Tim-1 knockdown cell lines on tumor development in vivo.Results: 1)Tim-1 knockdown can significantly inhibit tumor cell proliferation,migration and invasion.2)Apoptosis of U87 and U251 cells did not change significantly after Tim-1 knockdown.3)The levels of MMP-2 and MMP-9 proteins in U87 and U251 cells were significantly reduced after Tim-1 knockdown,but apoptosis-related indicators cleaved-caspase3,Bax,Bcl-2 protein levels did not change significantly.4)The subcutaneous tumor model constructed by U87 cells after knocking down Tim-1 significantly inhibited tumor growth and progression in tumor-forming models.Conclusion: In gliomablastoma cell lines U87 and U251,the knockdown of Tim-1can significantly inhibit the proliferation,migration and invasion of glioblastoma cells,but does not affect the apoptosis of glioblastoma cells.Part ? Tim-1 mediates TGF-?-micro RNA-133a-TGFBR1 signaling axis regulates the proliferation and invasion of glioblastoma Objective: To study the effect of knockdown of Tim-1 on the expression of autocrine cytokine TGF-? in glioblastoma cells and the regulatory effect of signal transduction mechanism on the proliferation and invasion of glioblastoma cells.Methods: The expression levels of cytokines IL-4 and TGF-?1 in U87 and U251 cells after Tim-1 knockdown were measured by enzyme-linked immunosorbent assay(ELISA).RNA-seq technology was used to analyze the significantly differentially expressed genes in U87 and U251 cells after Tim-1 knockdown,and bioinformatics analysis of differential pathways.The expression of Stat3 and TGFBR1 in U87 and U251 cells after Tim-1 knockdown were determined by Western Blot.The expression of micro RNA-133 a in U87 and U251 cells after Tim-1 knockdown was determined by fluorescent quantitative polymerase chain reaction.The RNA-IP co-precipitation(RNA-IP)experiment was used to analyze the binding of micro RNA-133 a to the3?UTR region of TGFBR1 and its effect on the expression of TGFBR1 protein.Intervention of the expression levels of micro RNA-133 a and TGFBR1 in U87 and U251 cells and detection the reversed effect of effect of tumour cell proliferation,migration and invasion after Tim-1 knockdown by the CCK-8 proliferation test,Transwell test,and Scratch test,to determine the effect of Tim-1 on the TGF-?-micro RNA-133a-TGFBR1 signal axis was determined.The effect of Tim-1knockdown on MEF2 C protein expression was confirmed by Western Blot experiments,to determine whether Tim-1 knockdown can increase micro RNA-133 a expression by up-regulating MEF2 C activity.Results: 1)Tim-1 knockdown significantly inhibited the expression of TGF-?1 in glioma cells.2)Tim-1 knockdown significantly reduced Stat3 activation and caused down-regulation of Stat3 and TGFBR1 protein expression.3)Tim-1 knockdown can significantly increase the expression of micro RNA-133 a.4)micro RNA-133 a can target the 3?UTR region of TGFBR1,disrupt TGFBR1 m RNA stability and inhibit TGFBR1 protein expression.5)Inhibition of micro RNA-133 a can reverse the decrease in glioma cell proliferation,migration and invasion ability caused by Tim-1knockdown.6)Overexpression of TGFBR1 can reverse the reduction of glioma cell proliferation,migration and invasion ability caused by Tim-1 knockdown.7)Tim-1knockdown can increase MEF2 C activation by increasing the level of MEF2 C phosphorylation modification.Conclusion: Tim-1 knockdown inhibit the expression of TGF-?1 in glioblastoma cells and suppress the expression of TGFBR1 by up-regulating the expression of micro RNA-133 a,which is further involved in the effects on the proliferation,migration and invasion of glioblastoma through the TGF-?-MEF2C-micro RNA-133a-Stat3/ TGFBR1 signal axis. |
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