ZEB1 Promotes Glioma Cell Proliferation And Migration Through Transcriptional Way Regulating Expression Of Arl13b

Background:Features of gliomas include ongoing angiogenesis,tissue invasion and metastasis,metabolic reorganisation or alteration,abnormal immune regulation and promoting the formation of tumour microenvironment.These make gliomas highly resistant to radiotherapy and chemotherapy.Moreover,the overall prognosis of patients is poor.These make glioma treatment a significant challenge to clinical practice.Therefore,the search for effective drugs and therapeutic targets for glioma is urgent.The Hh signalling pathway is an evolutionary highly conserved signalling pathway that runs from the cell membrane to the nucleus and plays an important role in embryonic development.Arl13b is a ciliary protein in the ADP ribosylation factor family and the Ras superfamily of GTPases that is highly aggregated within cilia and is an extracellular signalling hub.Arl13b plays a key role as a ciliary protein involved in ciliogenesis,protein and membrane transport and signal transduction under normal physiological and pathological conditions.Arl13b has also been shown to be associated with Hh activation.Arl13b has been reported to be highly expressed in gliomas,but the mechanisms regulating its expression in gliomas are unclear.The transcription factor ZEB1 influences tumourigenesis from the initial stage and plays an important role in regulating cell differentiation,survival and proliferation.Therefore,ZEB1 is often considered to be an activator that promotes tumour development.Like Hh signalling pathway,ZEB1 is also thought to be involved in embryogenesis and is required for embryonic development.It has been reported that ZEB1 is required for the differentiation of radial glial-like stem cells,the formation of neuronal-like stem cells and astrocytes.Increased ZEB1 leads to the development of gliomas and is associated with reduced survival time in patients with glioblastoma.We found a significant positive correlation between the expression levels of the transcription factor ZEB1 and Arl13b,as predicted by software.In contrast,the effect of ZEB1 on Arl13b has not been reported in gliomas.In this paper,we explored the effects of ZEB1 transcriptional regulation of Arl13b activation of the Hh signalling pathway on glioma cell proliferation and migration through experimental approaches such as transcription factor prediction,ZEB1 gene disruption or overexpression,and cell function.Purpose:1.Detect the expression level of Arl13b in glioma.2.To explore the molecular mechanisms underlying the abnormally high expression of Arl13b in gliomas.3.Verify whether ZEB1 activates the Hh signalling pathway.4.Probe the physiological function of ZEB1 and glioma cell growth.Methods:1.The expression of Arl13b in glioma tissues and normal tissues and the correlation between the expression level of Arl13b and the prognosis of glioma patients were analysed using public databases;glioma tissues of different lesion stages were collected and the protein expression level of Arl13b in glioma tissues of different lesion stages was detected by Western blot.2.The correlation between ZEB1 and Arl13b was analyzed by public database,and the correlation between ZEB1 and Arl13b in HEK293T cells and glioma cell lines that stably overexpressed or interfered with ZEB1 was detected by western blotting and qRT-PCR.The software was used to predict the ZEB1 binding site in the Arl13b promoter regulatory region and construct a segmented plasmid in the Arl13b promoter region.The double luciferase experiment was used to find the promoter-binding region of ZEB1 in Arl13b.The two ZBS sites in the+1～+500bp fragment of the Arl13b promoter region were mutated,and the double luciferase experiment was used to find the binding site of ZEB1 in the promoter region of Arl13b.3.qRT-PCR and Western blot were used to verify the changes in mRNA and protein expression of Hh downstream target genes in glioma cell lines with stably overexpressing or disrupting ZEB 1.4.The effect of ZEB 1 on glioma cell proliferation and migration was investigated by CCK-8 and Transwell assays in glioma cell lines with stably overexpressing or disrupting ZEB 1.Results:1.Database analysis revealed that Arl13b expression was significantly higher in glioma tissues than in normal tissues and that high Arl13b expression was associated with poor prognosis of glioma patients;results from clinical tissue samples showed that Arl13b was highly expressed in glioma tissues with higher degree of lesions and higher expression in grade Ⅳ than in grade Ⅱ.2.The database showed a significant positive correlation between ZEB1 and Arl13b;qRT-PCR and Western blot results showed that overexpression of ZEB1 in HEK293T cells and glioma cell lines upregulated the mRNA and protein levels of Arl13b;while interference with ZEB1 in HEK293T cells and glioma cell lines inhibited the mRNA and protein levels of Arl13b.The software predicted that there is a ZEB1 potential binding site(ZBS)for ZEB1 at-2000-bp～+500-bp in the Arl13b promoter region;luciferase reporter analysis showed that ZEB1 binds to a+1-to+500-bp fragment in the Arl13b promoter region;luciferase analysis reported that ZEB1 binds mainly to ZBS9 and regulates Ar13b transcription.3.Overexpression of ZEB1 upregulates mRNA and protein expression of target genes downstream of Hh,whereas interference with ZEB1 suppresses mRNA and protein expression of target genes downstream of Hh.4.Overexpression of ZEB1 enhances the proliferation and migration of glioma cells,while interference with ZEB1 inhibits the proliferation and migration of glioma cells.Conclusion:1.Arl13b expression levels are high in glioma tissue and correlate with patient prognosis.2.ZEB1 can transcriptionally regulate the expression of Arl13b and is a direct transcriptional activator of Arl13b.3.ZEB1 can activate the Hh signalling pathway.4.ZEB1 activates the Hh signalling pathway by transcriptionally regulating the expression of Arl13b,which in turn affects the proliferation and migration of glioma cells.