Human bladder cancer cell line mRNA-seq

Circular RNAs (circRNAs) have been increasingly indicated to be important participants in the development and progression of various malignant tumors. Our previous studies found that hundreds of circRNAs were aberrantly expressed in bladder cancer (BC) by high-throughput sequencing and we have confirmed that the downregulated circRNAs circHIPK3, BCRC3 and circNR3C1 played inhibitory roles in BC progression. However, the role of these upregulated circRNAs remain to be clarified. In this study, we analyzed circRNA high-throughout sequencing from human tissues and focused on the upregulated circRNAs and identified a novel circular RNA, hsa_circ_0001361 (also named bladder cancer upregulated circRNA-1, BCUC-1), as a new candidate circRNA derived from FNDC3B gene. The expression levels of circRNA and miRNAs in BC tissues and cells were detected by qRT-PCR. Transwell migration, Matrigel invasion, CCK-8, EdU, cell cycle and in vivo tumor metastasis assays were preformed to evaluate the effects of circ0001361 on BC cells. Transcriptome analysis was performed to find the potential genes that could be regulated by BCUC-1. Luciferase reporter, RNA pull-down and fluorescence in situ hybridization (FISH) assays were applied to verify the interaction between BCUC-1 and miR-491-5p. In order to screen downstream genes regulated by BCUC-1 (hsa_circ_0001361), transcriptome analysis was performed in T24T and UMUC3 cells transfected with BCUC-1(has_circ_0001361) and compaired vector.

环状RNA（circular RNAs, circRNAs）已被越来越多的研究证实是多种恶性肿瘤发生发展过程中的重要参与因子。我们前期通过高通量测序发现，膀胱癌（bladder cancer, BC）组织中存在数百条环状RNA的异常表达，并已证实下调表达的circHIPK3、BCRC3及circNR3C1可抑制膀胱癌的进展。然而，此类上调表达的环状RNA在膀胱癌中的作用仍有待阐明。本研究对人类组织的环状RNA高通量测序数据进行分析，聚焦上调表达的环状RNA，成功鉴定出一条来源于FNDC3B基因的新型环状RNA hsa_circ_0001361（亦称膀胱癌上调环状RNA-1，BCUC-1），其为全新的候选环状RNA。本研究通过实时定量反转录聚合酶链式反应（qRT-PCR）检测膀胱癌组织及细胞中环状RNA与微小RNA（microRNA, miRNA）的表达水平；采用Transwell迁移实验、Matrigel侵袭实验、细胞计数试剂盒-8（Cell Counting Kit-8, CCK-8）、5-乙炔基-2'-脱氧尿苷（5-ethynyl-2'-deoxyuridine, EdU）、细胞周期实验及体内肿瘤转移实验，评估hsa_circ_0001361对膀胱癌细胞的生物学功能影响；通过转录组测序分析筛选BCUC-1潜在的调控靶基因；采用荧光素酶报告基因实验、RNA pull-down实验及荧光原位杂交（fluorescence in situ hybridization, FISH）技术验证BCUC-1与miR-491-5p的相互作用。为筛选BCUC-1（hsa_circ_0001361）调控的下游靶基因，本研究对转染BCUC-1（hsa_circ_0001361）及对照空载体的T24T与UMUC3细胞进行转录组测序分析。