| Objective: Bladder cancer is one of the ten most common cancer worldwide,which can be divided into muscle and non-muscle-invasive bladder cancer.Despite treatment was improved,the diagnostic yield,outcomes,and five-year survival rate have hardly changed.BLCA carries the highest recurrence rate;50%–70% of patients with incomplete cystectomy relapse within five years of treatment.Therefore,further exploration of the deep mechanism of the occurrence and development of bladder cancer will help to improve the diagnosis rate and treatment strategy so as to improve the prognosis of patients.LncZEB1-AS1 is highly expressed in various tumors and can promote tumor proliferation and metastasis.Therefore,it is of great significance to further explore the mechanism of LncZEB1-AS1 promoting proliferation and metastasis in bladder cancer,and it may become a potential therapeutic target.Methods: 1.Bioinformatics analysis was conducted using the information in TCGA and UCSC Xena public databases.The correlation between LncZEB1-AS1 and clinical parameters of patients was investigated by bioinformatics methods.Real-time quantitative PCR(qRT-PCR)was used to investigate the expression level of LncZEB1-AS1 in the tissues of patients and bladder cancer cells.The effects of LncZEB1-AS1 on the proliferation,migration and invasion ability of bladder cancer cells were investigated in vitro,and the effects of LncZEB1-AS1 on important marker proteins in epithelialmesenchymal transformation were investigated by immunoblotting assay.Gene enrichment analysis(gene set enrichment analysis,GSEA)was used to explore the potential mechanism LncZEB1-AS1 and immunoblotting assay was used to validate it.The effect of LncZEB1-AS1 on the proliferation of bladder cancer cells in nude mice was investigated in vivo.2.Pull-down assay and LC-MS/MS analysis were conducted to find proteins that LncZEB1-AS1 acts on.The binding of LncZEB1-AS1 to protein UBA52 was investigated by RNA immunoprecipitation(RIP)assay.Fluorescence in situ hybridization(FISH)and immunofluorescence(IF)experiments(RNA-protein co-localization)were conducted to investigate the localization of LncZEB1-AS1 and UBA52 in cells.The effect of LncZEB1-AS1 on UBA52 mRNA was investigated by qRT-PCR.The effect of LncZEB1-AS1 on protein UBA52 was investigated by immunoblotting assay.3.Immunoblotting assay was used to investigate the expression level of UBA52 in the tissues of patients and bladder cancer cells.The effects of UBA52 on the proliferation,migration and invasion ability of bladder cancer cells were investigated in vitro,and the effects of UBA52 on important marker proteins in epithelial-mesenchymal transition were investigated by immunoblotting assay.GSEA was used to explore the potential mechanism LncZEB1-AS1 and immunoblotting assay was used to validate it.4.Rescue experiments:Immunoblotting assay and in vitro experiments were conducted to investigate the effect of down-regulation of the downstream protein UBA52 on the changes of JAK/STAT signaling pathway and the changes in bladder cancer cell proliferation,migration and invasion ability mediated by down-regulation of LncZEB1-AS1.Results: 1.In the public database,the expression of LncZEB1-AS1 is related to the grade,stage,T stage and subtype of bladder cancer patients.LncZEB1-AS1 was significantly upregulated in bladder cancer tissues compared with the paired adjacent normal tissues.After knockdown and over-expression of LncZEB1-AS1,the proliferation,migration and invasion of bladder cancer cells decreased and increased,respectively.After knockdown and over-expression of LncZEB1-AS1,the expression of N-cadherin and Vimentin decreased and increased,respectively.After knockdown of LncZEB1-AS1,the expression of Phospho-JAK2 and Phospho-STAT3,which are key proteins in JAK/STAT pathway,was decreased,while the expression of Phospho-JAK3 and Phospho-STAT1 was increased.Compared with the negative control group,the subcutaneous proliferation rate of bladder cancer cells with stable knockdown LncZEB1-AS1 was significantly slowed in nude mice.2.Pull-down assay and LC-MS/MS analysis showed that LncZEB1-AS1 might act on Protein UBA52,which was confirmed by RIP experiment.The co-localization of LncZEB1-AS1 and UBA52 was mostly in the cytoplasm.After knockdown or overexpression of LncZEB1-AS1,the mRNA expression of UBA52 was unchanged,but the protein expression was increased or decreased,respectively.3.UBA52 was significantly down-regulated in bladder cancer tissues compared with the paired adjacent normal tissues.After knocking down UBA52 expression,the proliferation,migration and invasion of bladder cancer cells increased.After UBA52 was knocked down,the expression of Ncadherin and Vimentin increased.After knockdown of UBA52,the expression of PhosphoJAK2,Phospho-STAT1,Phospho-JAK3 and Phospho-STAT3,which are key proteins in JAK/STAT pathway,was increased.4.The changes mediated by down-regulation of LncZEB1-AS1 in JAK2/STAT3 signaling pathway and the proliferation,migration and invasion ability of bladder cancer cells can be partially rescued by knocking down the downstream protein UBA52.Conclusion: 1.The increased expression of LncZEB1-AS1 is related to the occurrence and development of bladder cancer.LncZEB1-AS1 is up-regulated in bladder cancer tissues and can promote EMT,proliferation,migration and invasion of bladder cancer cells.2.LncZEB1-AS1 can directly interact with the protein UBA52.3.UBA52 is up-regulated in adjacent normal tissues compared with the bladder cancer tissues and can inhibit EMT,proliferation,migration and invasion of bladder cancer cells.4.LncZEB1-AS1 can promote EMT,proliferation,migration and invasion of bladder cancer cells,and JAK2/STAT3 signaling pathway by down-regulating UBA52. |
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