| Objective:Glioma is the most common malignant tumor of the central nervous system,accounting for more than 60%of all nervous system tumors[1].Brain glioma can be divided into astrocytoma,ependymoma,oligodendroglioma,medulloblastoma,glioblastoma and other types according to the histological subtype and degree of malignancy[2].Despite the continuous improvement of glioma treatment in recent years,the prognosis of glioma is still not optimistic.The median survival period of patients is less than 14 months[3,4],and the 5-year survival rate is only 10%[5],which has become a big problem troubling the medical community.Therefore,the exploration of the pathological mechanism of glioma is helpful to promote the development of new targeted therapeutic drugs,which is of great significance for the effective treatment of glioma and prolonging the survival period of patients.Long non-coding RNAs（LncRNAs）are a group of non-protein-coding RNAs possessing more than 200 nucleotides in length.LncRNAs regulate their downstream gene expression through alternative splicing,transcriptional/translational regulation and epigenetics[6].It has been reported that aberrant LncRNA expression is associated with cancer carcinogenesis and metastasis[7,8].LncRNA urothelial carcinoma associated 1（UCA1）was initially discovered in human bladder carcinoma[11].It has been reported that UCA1 is upregulated in various cancers,e.g.lung cancer,colorectal cancer,gastric cancer,oral squamous cell carcinoma and renal cell carcinoma[15-19].UCA1 functions in many cellular processes,such as cell proliferation,apoptosis,metastasis and chemoresistance[25].In addition,UCA1 is also highly expressed in glioma and knockdown of UCA1 suppressed cell proliferation,migration and invasion in vitro[26,27].However,further studies are required to clarify the possible molecular mechanism of UCA1-mediated glioma progression.Epithelial-mesenchymal transition（EMT）is a cellular process that is necessary for tumor metastasis,during which epithelial cells lose cell-to-cell contacts and acquire a mesenchymal phenotype[41].Zinc-finger E-box-binding homeobox 1（ZEB1）is an important modulator of EMT and also a novel target of miR-204-5p[43,44].Lu et a1.found that miR-204/ZEB1 axis modulates EMT in nasopharyngeal carcinoma[45].Moreover,UCA1 enhances the chemosensitivities of prostate cancer cells and colorectal cancer cells through miR-204-5p and other target genes（CREB and Sirt1）[34].Based on above evidences,we speculate that UCA1 may regulate cancer progression through the miR-204-5p/ZEB1 axis in glioma.In this study,the effects of UCA1 overexpression on cell migration and invasion,as well as the expression of EMT regulators,were investigated in vitro.The involvement of the UCA1/miR-204-5p/ZEB1 axis in glioma tumorigenesis was further explored both in vitro and in vivo.Methods:1.Culture of human brain glioma cell lines U251,U87MG,SHG44 and human embryonic kidney cell line HEK293T.2.Real-time PCR was performed to detect the expression level of UCA1 in human glioma cell lines.3.Construction of UCA1 overexpressed plasmids.Vector and UCA1 overexpressed plasmids were transfected into two human glioma cells.UCA1 expression was detected by real-time PCR.4.Cell migration was detected by scratch assay.5.Transwell assay was performed to detect cell invasion.6.Transfection UCA1 expression plasmid after 48 hours,Western blot detection Fibronectin,COL5A1（collagen V）,the expression of ZEB1 levels.7.After 48 hours of transfection,immunofluorescence was used to detect the expression of Fibronectin and ZEB1 in three groups of cells of one cell line with significant changes.8.48 h after transfection with Control,Vector and UCA1 overexpressed plasmids,real-time PCR was performed to detect the expression level of miR-204-5p.9.Double luciferase assay confirmed the specific binding of miR-204-5p to UCA1.Double luciferase assay verified the specific binding of miR-204-5p and ZEB1 3’-UTR.10.After transfection with NC mimic,miR-204-5p mimic,NC inhibitor and miR-204-5p inhibitor,the expression levels of ZEB1 were detected by Western blot 48 hours after transfection.11.Vector+NC mimic,vector+miR-204-5p mimic,UCA1+NC mimic and UCA1+ miR-204-5p mimic were transfected into glioma cells.12.Changes in ZEB1 expression were detected by Western blot 48 hours after transfection.Double luciferase reporter gene assay was performed to analyze the regulatory effect of miR-204-5p and UCA1 specific binding on ZEB1 expression.13.Construction of shRNA plasmids and screening of stable transfected cells.14.A subcutaneous tumor transplantation model of nude mice silenced by UCA1 expression was constructed.15.Immunohistochemical analysis and Western blot were used to detect the expression levels of Fibronectin,COL5A1 and ZEB1 in tumor tissues.Results:1.UCA1 overexpression promoted the migration and invasion of SHG44 and U87MG glioma cells.2.Overexpression of UCA 1 results in increased expression of EMT related proteins （Fibronectin,COL5A1,ZEB1）in glioma cells.3.The expression level of miR-204-5p in overexpressed UCA1 SHG44 cells was significantly decreased.Double luciferase reporter assay confirmed the specific binding of UCA1 to miR-204-5p.4.miR-204-5p directly binds ZEB1 and negatively regulates the expression of ZEB1.5.UCA1 blocks the targeted inhibition of ZEB1 by miR-204-5p through specific binding of miR-204-5p,thereby promoting the expression of ZEB1 and the epithelial interstitial transformation of glioma cells.6.MiR-204-5p expression was increased in subcutaneous transplanted tumor tissues of nude mice with silenced UCA1 expression,while the protein expression levels of Fibronectin,COL5A1 and ZEB1 were decreased.Conclusion:In conclusion,we demonstrated that UCA1 overexpression enhanced cell migration,invasion and EMT in glioma cells.UCA1 specifically binds miR-204-5p to up-regulate the expression of ZEB1 and promote the epithelial mesenchymal transformation of glioma cells.In this study,UCA1/miR-204-5p/ZEB1was shown to be an important regulatory axis for the invasion and metastasis of glioma cells. |
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