| Objective:Bladder cancer is one of the most common type of cancers in the population worldwide,and more common in males than femles.There are 2 major types of bladder cancer:Non-muscle invasive bladder cancer(NMIBC)and muscle-invasive bladder cancer(MIBC).Bladder cancer exhibits a high frequency of relapse and a poor clinical outcome once the tumors progress to muscle-invasive disease.Patients with MIBC have a poor prognosis due to the aggressive nature of the tumor and its resistance to chemo-and radiotherapy.Furthermore,for patients with MIBC,the risk of developing lymph node metastases is increased and chemotherapy is less effective in comparison with patients with NMIBC.Therapies and prognosis for this type of cancer depend on the clinical information and individual tumor pathology.However,in certain cases,even when tumors present with similar histology,they respond differently to the same treatment,resulting in different survival outcomes for the patients.Protein-coding genes constitute only 2%of the total genome sequence;the remaining sequences produce various classes of functional non-coding RNAs.There are two major classes of non-coding RNAs,including small and long non-coding RNAs(lncRNAs).The difference between lncRNAs and small non-coding RNAs is primarily their size,with the length of lncRNAs defined as being>200 nucleotides.Similar to other non-coding RNAs,lncRNA transcripts are associated with a wide range of cancer types,including bladder cancer.Alterations in the expression levels of lncRNAs have been demonstrated to be associated with a number of important cellular functions,and may promote the migration and invasion of cancer cells.Non-coding RNAs have attracted increasing attention in previous years regarding their role in bladder cancer,with the number of studies on this topic increasing considerably.The combination of traditional methods of treatment with the use of non-coding RNAs may provide improved therapy for patients.The accumulation of data in this field may assist in elucidating the molecular profiles of patients with bladder cancer and contribute to the use of non-coding RNAs as tools for precision medicine to target critical genes in bladder cancer.LINC00460 is a human lncRNA gene,transcribed from chromosome 13,measuring935 bp;its function is poorly understood.Recent studies have demonstrated that LINC00460 is associated with kidney cancer,nasopharyngeal carcinoma and head and neck squamous cell carcinoma.A previous study demonstrated,using a bioinformatics approach,that LINC00460 may serve an important role in tumorigenesis and metastasis through the regulation of the cell cycle and cell death.During the analysis of the differential expression of lncRNAs in bladder cancer tissues in the present study,it was identified that LINC00460 was highly upregulated compared with the normal adjacent tissue.Furthermore,the upregulation of LINC00460 was demonstrated to be associated with the poor survival of patients.We hypothesized that this lncRNA may serve an important role in the regulation of biological processes in bladder urothelial carcinoma.However,the precise function and the underlying molecular mechanisms remain to be elucidated.The main purpose of this study is:1.To explore the basic expression of LINC00460 in bladder cancer tissues and cell lines and its corelation with prognosis of patients.2.The influence of LINC00460 on proliferation and migration of cells in bladder urothelial carcinoma.3.The functional mechanism of LINC00460 on proliferation and migration of bladder cancer cells.Methods:1.Gene expression data obtained by RNA sequencing and the corresponding clinical data for 412 patients(including 413 samples)with bladder urothelial carcinoma were downloaded from The Caner Genome Atlas(TCGA).All RNA expression levels of the samples were normalized.The edgeR Bioconductor package was used to analyze P-values and fold-change(FC)using R software.The expression of LINC00460 in baldder cancer cell lines(T24,J82,TCCSUP and UM-UC-3)and the normal bladder epithelial SV-HUC-1 cell line were explored using reverse transcription-quantitative polymerase chain reaction(RT-qPCR).2.The effective shRNA targeting LINC00460was transfected into T24 and 5637 cells.The pcDNA-LINC00460 was transfected into T24 and 5637 cells to overexpress LINC00460.We determined the efficiency of transfection according to the observation of green fluorescent protein(GFP)and results of RT-qPCR after 48 h.Ability of cell proliferation and migration were evaluated using a Cell Counting Kit-8(CCK-8)and wound healing assays respectively.The best concentration of MA2 used was selected by using CCK-8 assay to detect the cell viability.We predicted the combination sites of FTO mRNA and LINC00460 with microRNAs on the website of starBase v2.0.Double luciferase reporter gene assay was used to verify the binding sites and binding effects between LINC00460 the predicted miRNA.3.SPSS23.0 software(IBM,USA)was used to perform statistical analysis.The differential expression levels of LINC00460 and FTO mRNA between the cancerous and adjacent tissues were analyzed with t-tests.The differences in LINC00460 expression between bladder cancer 5637 and T24 cell lines and the normal bladder epithelial SV-HUC-1 cell line was assessed using a one-way analysis of variance with Tamhane's post-hoc test.The correlation between LINC00460 and FTO mRNA was analyzed by Spearman's rank correlation analysis following logarithmic(log10)conversion of the original data.For the survival analysis,a Kaplan-Meier plot was generated using the survival package in R(version 3.4.0)with log-rank tests.P<0.05 was considered to indicate a statistically signifiant difference.Results:1.The expression of LINC00460 was signifiantly upregulated in bladder urothelial carcinoma tissues compared with the normal controls(P<0.0001).An increase in LINC00460 expression was observed in bladder cancer 5637 and T24 cell lines compared with the normal bladder epithelial SV-HUC-1 cell line(P<0.05).2.High levels of LINC00460 were signifiantly associated with a decreased survival time in male patients(P=0.03229).There was a similar association observed in the overall group(P=0.04678),but not among the female patients(P=0.42445).3.Downregulation of LINC00460 inhibits the proliferation and migration of bladder urothelial carcinoma cells.On the other hand,up-regulation of LINC00460 could promote the proliferation and migration of bladder urothelial carcinoma cells.4.Expression of FTO mRNA is positively correlated with the expression of LINC00460 in tissues of bladder urothelial carcinoma(P<0.0001;r=0.73)and high levels of FTO mRNA were signifiantly associated with a decreased survival time in total patients(P=0.003).5.Silencing of LINC00460 could inhibit the expression of both FTO mRNA and protein in bladder cancer cells,while silencing of FTO didn't influence the expression of LINC00460.6.Influence of LINC00460 overexpression on proliferation and migration of bladder cancer cells could be rescued by FTO inhibitor(MA2).7.LINC00460 up-regulated FTO expression by competitively binding to miR-320a and promoted the proliferation and migration of bladder cancer cells.Conclusion:The relative expression of LINC00460 in both bladder cancer tissues and cell lines are up-regulated than the normal controls.In addition,the survival of patients with higher expression of LINC00460 was shorter,which is a clinically promising biomarker.After down-regulation of LINC00460 expression,the ability of proliferation and migration of T24 and 5637 cells were inhibited,while up-regulation of LINC00460could promote the proliferation and migration of bladder urothelial carcinoma cells.Another finding is that the expression of FTO mRNA in patients with bladder cancer could predict the prognosis.In recent years,FTO is proved to play an oncogenic role in both acute myeloid leukemia and glioblastoma as a N~6-methyladenosine(m~6A)demethylase of eukaryotic messenger RNA(mRNA).LINC00460 up-regulated FTO expression by competitively binding to miR-320a and promoted the proliferation and migration of bladder cancer cells,which may provide novel insights into molecular therapy for bladder cancer. |
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