The Molecular Mechanism Of Hh-TWIST1-Arl13b Signaling Loop Promotes Proliferation And Migration Of Glioma

BackgroundNeurogliocytoma known as glioma,accounts for about 30%-40% of all primary tumors in the human central nervous system,although there is a lower overall incidence of glioma,the mortality rate of glioma patients ranks tenth in the world.especially,glioblastoma(GBM)is most malignant and the prognosis of patients is very poor.the median survival time of GBM patients is about 15 months,and the 5-year survival rate is about 5%.Despite maximum surgical resection and subsequent chemoradiotherapy,tumor recurrence and drug resistance occurrred in almost all patients,indicating that glioma pose a serious threat to people’s lives and health.In recent years,studies have shown that Arl13 b is abnormally high expressed in gliomas,and promotes angiogenesis and glioma growth by activating the VEGFA-VEGFR2 pathway.However,it’s still unclear about the molecular mechanism of Arl13 b is aberrantly overexpressed in glioma.The aim of this study to elucidate the molecular mechanisms of Hh-TWIST1-Arl13 b signaling loop maintains high expression of Arl13 b to promote proliferation and migration of glioma cells,which provide a potential molecular therapeutic target.Purpose of the study1.To verify Arl13 b promotes glioma cells growth.2.To identified whether Hh signaling regulates the expression of transcription factor TWIST1.3.To confirm how TWIST1 regulate the expression of Arl13 b.4.To elucidate the molecular mechanism by which the Hh-TWIST1-Arl13 b signaling loop maintains high expression of Arl13 b in glioma,thereby promoting malignant proliferation of glioma cells.Research methodsPart I: Arl13 b promotes glioma growth1.Bioinformatics analysis of the relationship between the expression of Arl13 b in glioma clinical samples and patient prognosis(1)The clinical samples of glioma in the TCGA database were analyzed by bioinformatics technology to confirm the expression level of Arl13 b in glioma.(2)The correlation between Arl13 b expression level and patient prognosis was analyzed by Kaplan-Meier test.2.In vitro cell function experiments verified that Arl13 b promoted glioma cell proliferation and migration(1)Arl13b was overexpressed in glioma cell lines U87 through lentivirus infection,and the ability of tumor proliferation,migration and invasion was verified by plate cloning,Ed U labeling,soft agar cloning,cell migration and invasion experiments.(2)A cell line with stable interfere with Arl13 b expression was constructed in U118,and determine the ability of tumor proliferation,migration and invasion by plate cloning,Ed U labeling,soft agar cloning,cell migration and invasion experiments.(3)The expression level of protein related to tumor cell proliferation was explored by Western Blot.Part II: To explore the molecular mechanism of Hh-TWIST1-Arl13 b signaling loop promote proliferation and migration of glioma1.Hh signaling regulates TWIST1(1)To confirm that the Hedgehog signaling pathway regulates TWIST1 by overexpressing Gli1 or Gli2 in HEK293 T,we analyzed changes in m RNA levels and protein levels of TWIST1 by q PCR and Western Blot.(2)Glioma cells U87 and U118 are treated with drugs such as SAG/GANT61 by activating/interfering with Hh signaling,using q PCR and Western Blot to detect TWIST1,E-cadherin and Vimentin,and Arl13 b.2.TWIST1 regulates Arl13b(1)The mRNA level of TWITS1 in the TCGA database was analyzed by bioinformatics,and the correlation between the expression level of TWIST1 and the prognosis of glioma patients was analyzed by Kaplan-Meier test,and correlation analysis of TWIST1 and Arl13 b in glioma patients.(2)Overexpressed/interference with TWIST1,using Western Blot to analyze effect on Arl13 b protein levels.(3)When it is proved that TWIST1 regulates Arl13 b,we designed the DualLuciferase Reporter Assay to confirm the binding site of TWIST1 regulate the Arl13 b specific promoter sequence.Research resultsPart I: Arl13 b promotes glioma growth1.Bioinformatics analysis resultsThe results of bioinformatics analysis showed that Arl13 b was highly expressed in glioma and correlated with poor prognosis in patients.The higher the expression of Arl13 b,the shorter the overall survival of patients.2.Cell function experiments verified that Arl13 b promotes gliomagenesis and progression(1)Overexpressed Arl13 b in U87 cell lines,and the proliferation,migration and invasion ability of glioma cells in the overexpressed group were significantly improved compared with the control group.And the detection of migration-related proteins with Western Blot also showed that protein levels were also significantly increased.(2)Knocking down Arl13 b in U118 cell lines significantly inhibited the proliferation,migration and invasion ability of tumor cells in the experimental group,and the level of protein associated with cell migration was also significantly reduced.Part II: The Hh-TWIST1-Arl13 b signaling loop maintains high level of Arl13 b in glioma cells1.Hh signaling regulates TWIST1(1)Overexpression of Gli1 and Gli2 in HEK293 T,q PCR and Western Blot results confirmed that Gli1 upregulated the m RNA and protein levels of TWIST1,but overexpression of Gli2 didn’t,illustrating that Gli1 regulates the expression of TWIST1.(2)SAG treated U87 cells,and the results showed TWIST1,E-cadherin and Vimentin protein levels were upregulated.GANT61 treated U118 cells,TWIST1,Ecadherin and Vimentin protein levels decreased.(3)SAG treated U87/U118 cells,and the protein level of Arl13 b was upregulated;When U87/U118 were treated with GANT61,the protein levels of Arl13 b decreased.2.TWIST1 binds to the Arl13 b promoter region and regulates Arl13b(1)Bioinformation analysis showed that the transcription factor TWIST1 was abnormally high expressed in glioma,and the survival time of patients with high expression of TWIST1 was significantly shorter than that of the low expression group.(2)Overexpression of TWIST1,Arl13 b protein levels were increased,knocking down TWIST1,downregulated the protein level of Arl13 b.(3)Dual-luciferase reporter assay confirmed that TWIST1 can bind to the promoter region(-2000～-1000)of Arl13 b.Conclusion1.Arl13 b is highly expressed in glioma and associated with poor prognosis in patients2.Arl13 b promotes glioma proliferation,migration and invasion.3.The Hh-TWIST1-Arl13 b signaling loop maintains the high expression of Arl13 b in glioma cells,thereby promoting the occurrence and development of glioma.