Long Noncoding RNA Expression Signatures Of Bladder Cancer And The Mechanism Of Its Biological Functions

Background:Long non-coding RNAs (LncRNAs) are mRNA-like transcripts ranging in length from 200 nt to 10 kb, yet are poorly conserved and do not function as templates for protein synthesis. Although initially argued to be spurious transcriptional noise of RNA polymerase Ⅱ, recent evidence suggests that lncRNAs have been associated with a spectrum of biological processes, and aberrant lncRNA expression may be a major contributor to tumorigenesis, progression and prognosis. Dysregulation of lncRNAs has been regarded as a primary feature of several human cancers. However, the genome-wide expression and functional significance of lncRNAs in bladder cancer remains unclear.Objective:To identified a collection of aberrantly expressed long noncoding RNAs (lncRNAs) in bladder cancer by microarray and explore the expression level and the functional role of LOC572558, one of the most deregulated lncRNAs, in bladder cancer.Materials and Methods:1. We present the lncRNA expression profiles in 5 pairs of bladder cancer samples and matched histologically normal urothelium samples by lncRNA microarray(Array star).2. To discover the function and associated pathways of differentially expressed mRNAs, GO and pathway analyses were performed. The lncRNA-mRNA co-expression network was constructed by Cytoscape software.3. To validate the microarray results, qPCR was performed. Four lncRNAs that were significantly deregulated (TNXA, CTA-134P22.2, CTC-276P9.1 and KRT19P3) were evaluated in the 50 patients included in this study.4. We determined the expression of LOC572558 in 50 pairs of bladder cancer tissue samples and control, as well as in bladder cancer cell line T24 and 5637 by real-time polymerase chain reaction assay. We then defined the biological functions of LOC572558 by CCK-8 assay, flow cytometry,5. Using a high-throughput phospho-proteome array, we identified proteins that were phosphorylated and dephosphorylated in bladder cancer cells where LOC572558 expression was upmodulated by shRNA. We confirmed the findings from the array by Western blotting.Results:1. We finally determined 3,324 differentially expressed human lncRNAs and 2,120 differentially expressed mRNAs (≥2-fold change). A total of 110 lncRNAs were significantly differentially expressed between the tumor and the control groups (≥8-fold change). Twenty-two lncRNAs were upregulated and 88 lncRNAs were downregulated.2. It is likely that these deregulated lncRNAs play a key or partial role in the development and/or progression of bladder cancer. KEGG annotation showed a significant association with the p53, cell cycle and propanoate metabolism pathway gene expression in the bladder cancer group compared with the normal tissue group, indicating that deregulated lncRNAs may act by regulating protein-coding genes in these pathways.3. Moreover, ectopic expression of LOC572558 inhibited cell proliferation, induced a S phase arrest in cell cycle, inhibit cell invasion and migration and promoted cell apoptosis in T24 and 5637 bladder cancer cell lines.4. We further verified that over-expression of LOC572558 was associated with dephosphorylation of cAMP response element-binding protein (CREB) and phosphorylation of p53 protein.Conclusion:These data suggest an important role of LOC572558 in the molecular etiology of bladder cancer and implicate the potential application of LOC572558 in bladder cancer therapy.