INTERNATIONAL JOURNAL OF ONCOLOGY 48: 1617-1627, 2016 Gene expression profiling of microRNAs associated with UCA1 in bladder cancer cells XIAOJUAN XIE1,2, JINGJING PAN1, LIQIANG WEI2, SHOUZHEN WU3, HUILIAN HOU4, XU LI3 and WEI CHEN1 1Department of Clinical Laboratory, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061; 2Shaanxi Center for Clinical Laboratory, Shaanxi Provincial People's Hospital, Xi'an, Shaanxi 710068; 3Center for Translational Medicine, 4Department of Pathology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China Received November 11, 2015; Accepted December 27, 2015 DOI: 10.3892/ijo.2016.3357 Abstract. Emerging evidence indicates that non-coding and positively correlated with miR-196a, whereas UCA1 and RNAs, such as lncRNAs and microRNAs, play important miR-196a were negatively correlated with p27kip1, which was roles in diverse diseases, such as cancer, immune diseases downregulated in bladder cancer patients. Thus, our findings and cardiovascular diseases. Interestingly, lncRNAs could provided valuable information on miRNAs associated with directly or indirectly regulate the expression of miRNAs. UCA1 in bladder cancer, which could be helpful to further However, the expression profiling of miRNAs associated with explore the related genes and molecular networks fundamental UCA1 in bladder cancer remains unknown. Here, we used in bladder cancer progression. Illumina deep sequencing to sequence miRNA libraries from both the UCA1 knockdown and normal high-expression 5637 Introduction cells. We identified 225 and 235 miRNAs expressed in 5637 cells of normal high-expression and knockdown of UCA1, Recently, emerging studies have demonstrated that 70-90% of respectively. Overall, expression of 75 miRNAs showed the human genome is capable of being pervasively tran scribed. significant difference associated with UCA1, of which 38 were Surprisingly, <2% of the total genome is found to have the upregulated and 37 downregulated with UCA1 knockdown. ability to encode proteins, indicating that non-coding RNAs GO analysis of the host target genes revealed that these aber- account for most of the human transcriptome. In fact, recent rantly regulated miRNAs were involved in complex cellular evidence has suggested that the human non-coding RNAs pathways, including biological process, cellular component include approximately 9,000 small RNAs, 10,000-32,000 long and molecular function. We selected 8 candidate miRNAs non-coding RNAs (lncRNAs) and 11,000 pseudogenes (1,2). associated with UCA1 and predicted their targeted mRNAs, Small non-coding RNAs have been well characterized and and found that p27kip1 was a crucial downstream molecule for categorized by transfer RNAs, microRNAs (miRNAs), small- these 8 miRNAs, especially for miR-196a. KEGG pathway interfering RNAs, PIWI-interacting RNAs, small nuclear analysis showed that PI3K-Akt signaling pwathway was RNAs, small nucleolar RNAs and transcription initiation involved in regulating these 8 candidant miRNAs. Among RNAs. these 8 candidant miRNAs, we observed the correlation among lncRNAs can vary in length from 200 nucleotides to UCA1, miR-196a and the host target mRNA, p27kip1, in bladder 100 kilobases. A previous study demonstrated that lncRNAs cancer cells and tissues. UCA1 was upregulated by miR-196a play an important role in a diverse range of biological processes (3-5), such as participating in epigenetics, nuclear import, alternative splicing, RNA decay and translation. More importantly, due to their widespread distribution in nucleus, lncRNAs could regulate the transcription of adjacent genes Correspondence to: Dr Wei Chen, Department of Clinical by binding to the specific transcription factors and polymer- Laboratory, The First Affiliated Hospital, School of Medicine, Xi'an ases (6,7), for example, the expression of microRNAs and their Jiaotong University, Xi'an, Shaanxi 710061, P.R. China bio-function are mainly regulated by lncRNAs. On the other E-mail: [email protected] hand, they can serve as precursors to microRNAs, ceRNA of Abbreviations: lncRNA, long noncoding RNA; UCA1, urothelial microRNAs, and natural microRNA sponges. Therefore, aber- cancer-associated 1 rant lncRNA expression can cause various human diseases and disorders. Key words: urothelial cancer-associated 1, miRNAs, bladder microRNAs, 23-nucleotide-long endogenous RNAs, cancer, expression profiling, deep sequencing can bind with their target RNA transcripts by pairing with microRNA response elements (MREs) and then result in degra- dation or translational repression of the target transcripts (8,9). Recent studies show that microRNAs regulate a number of the 1618 XIE et al: microRNAs Associated with UCA1 in Bladder Cancer transcriptions, including the coding and noncoding transcrip- Huada BGI-Tech (Shanghai, China) for Illumina sequencing tome (10). Therefore, a better understanding of microRNA of the small RNAs. profiling associated with lncRNA in cancer is imperative for the development of novel therapeutic strategies. However, Analysis of sequencing data. The small RNA sequencing reads many details remain unknown regarding microRNA profiling obtained were then subjected to the following: i) reads were regulated by lncRNA. selected by removing the low-quality reads with the Solexa In this study, we used Illumina deep sequencing to Chastity quality filter; ii) the adapter sequences were trimmed sequence miRNA libraries prepared from the 5637 cells of and the reads longer than 15 were retained; and iii) low two-copy normal high-expression and knockdown of UCA1, which are sequences were discarded. The filtered short reads were then specifically upregulated in bladder cancer. Overall, expression mapped to the Repbase database (available online: http://www. of 75 miRNAs showed significant difference associated with girinst.org/repbase/), Rfam database, NCBI database (available UCA1: 38 were upregulated and 37 downregulated. GO anal- online: http://www.ncbi.nlm.nih.gov/) (available online: http:// ysis of the host target genes revealed that these dysregulated rfam.janelia.org/). The unmappable sequences not mapped to miRNAs are involved in complex cellular pathways, including miRBase 19.0 were used to predict potentially novel candidate biological process, cellular component and molecular func- miRNAs with the Mfold RNA folding prediction web server tion. After confirming with RT-qPCR, 8 miRNAs associated (available online: http://mfold.rna.albany.edu/). with UCA1 were selected to draw the TF-miRNA-mRNA network and enrich KEGG pathway analysis. miR-196a was Identification of differentially-expressed miRNAs.To compare one of these 8 candidate microRNAs and its targeted gene the differential miRNA expression in 5637 cells of normal p27kip1 participated in PI3K-Akt signaling pathway. Then the high-expression and knockdown of UCA1, the number of raw correlation among UCA1, miR-196a and p27kip1 in bladder tags in each library was normalized to tags per million of the cancer cells and patients was analyzed. total miRNA reads. Differentially-expressed miRNAs were determined according to the criterion of a fold change of ≥2 in Material and methods the sequence counts between libraries. Tissue specimen collection. In this study, 35 bladder cancer Real-time PCR. The total RNA was extracted from cells or tissues and 16 bladder non-tumor tissues were obtained from bladder cancer patient tissues using TRIzol reagent (Invitrogen) August 2012 to September 2013 at the Department of Clinical as described above. RNA was subjected to reverse transcription Laboratory, the First Affiliated Hospital of Xi'an Jiaotong reactions using the PrimeScript RT reagent kit (Invitrogen). University, Xi'an, China. All samples were identified by two Selected miRNAs were subjected to RT-qPCR using SYBR independent pathologists. Informed consent was provided Premis Ex Tag Ⅱ (Takara, Dalian, China) and monitored with by each patient, and this study was approved by the Ethics the CFX96 Touch Real-time PCR detection system (Bio- Committee of the First Affiliated Hospital of Xi'an Jiaotong Rad, Hercules, CA, USA). The PCR reaction mixture (20 µl) University and accordance with the Declaration of Helsinki. consisted of 2 µl of cDNA, 10 µl of 2X SYBR-Green PCR master mix, 0.8 µl each of the microRNA-specific forward Cell culture and treatment. Human bladder cancer cell lines primer, a universal reverse primer (10 µM) and 6.4 µl of (5637 cell line and UMUC2 cell line) obtained from the nuclease-free water. The cycling parameters were 94˚C for American Type Culture Collection (ATCC) were cultured in 5 min followed by 40 cycles of 94˚C for 30 sec and 60˚C for RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented 30 sec. U6 was used as the endogenous control for normaliza- with 10% heat inactivated bovine calf serum and 150 µg/ml tion. The relative expression levels were calculated with the -ΔCt G418 at 37˚C in a humidified atmosphere with 5% CO2. Stable 2 method. All the miRNA primers were obtained from cell lines with UCA1 knockdown in 5637 cells and UCA1 over- Ribobio (Guangzhou, China). Three independent biological expression in UMUC2 cells were a kind gift of our colleague replicates were performed. Zhengkun Li, the Center for Translational Medicine of the First Affiliated Hospital, School of Medicine, Xi'an Jiaotong miRNA target prediction. The potential target genes of the University (Xi'an, China). differentially-expressed miRNAs were predicted with four miRNA