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RNA Over-Editing of BLCAP Contributes To Cancer Letters 357 (2015) 510–519 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet Original Articles RNA over-editing of BLCAP contributes to hepatocarcinogenesis identified by whole-genome and transcriptome sequencing Xueda Hu a,b,1, Shengqing Wan b,1, Ying Ou c,1, Boping Zhou a,d,1, Jialou Zhu b, Xin Yi b, Yanfang Guan b, Wenlong Jia b, Xing Liu c, Qiudao Wang c, Yao Qi c, Qing Yuan c, Wanqiu Huang e, Weijia Liao f, Yun Wang c, Qinghua Zhang c, Huasheng Xiao c, Xinchun Chen a,d, Jian Huang a,c,d,* a Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Third People’s Hospital, Guangdong Medical College, Shenzhen 518112, China b BGI-Shenzhen, Shenzhen 518083, China c Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center and National Engineering Center for Biochip at Shanghai, Shanghai, China d Guangdong Key Laboratory of Diagnosis & Treatment for Emerging Infectious Disease, Shenzhen Third People’s Hospital, Guangdong Medical college, Shenzhen 518112, China e Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China f Hepatology Institute of Guilin Medical University, Guilin, Guangxi Zhuang Autonomous Region, China ARTICLE INFO ABSTRACT Article history: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, although the treat- Received 2 October 2014 ment of this disease has changed little in recent decades because most of the genetic events that initiate Received in revised form 26 November this disease remain unknown. To better understand HCC pathogenesis at the molecular level and to uncover 2014 novel tumor-initiating events, we integrated RNA-seq and DNA-seq data derived from two pairs of HCC Accepted 2 December 2014 tissues. We found that BLCAP is novel editing gene in HCC and has over-editing expression in 40.1% HCCs compared to adjacent liver tissues. We then used RNA interference and gene transfection to assess the Keywords: roles of BLCAP RNA editing in tumor proliferation. Our results showed that compared to the wild-type RNA over-editing BLCAP gene BLCAP gene, the RNA-edited BLCAP gene may stably promote cell proliferation (including cell growth, colony Hepatocarcinogenesis formation in vitro, and tumorigenicity in vivo) by enhancing the phosphorylation of AKT, mTOR, and MDM2 Whole-genome and transcriptome and inhibiting the phosphorylation of TP53. Our current results suggest that the RNA over-editing of BLCAP sequencing gene may serve as a novel potential driver in advanced HCC through activating AKT/mTOR signal pathway. © 2014 Elsevier Ireland Ltd. All rights reserved. Introduction characterizing the genomic profile of HCC [3–7]; these efforts have mainly focused on identifying the somatic DNA variations in tumors Hepatocellular carcinoma (HCC) is a highly malignant tumor with and investigating the relationships between these mutations and a poor clinical outcome and represents the third most common cause various clinical features. of cancer-related deaths worldwide [1]. Many predisposing envi- The transfer of genetic information, as described by the central ronmental factors can contribute to liver cancer, such as infection dogma, has always been considered to be faithful and determinis- with hepatitis B virus or hepatitis C virus, chronic exposure to Af- tic. Therefore, cancer genomic studies have paid the most attention latoxin B1, and alcoholic cirrhosis [2]. In addition, previous studies to DNA sequence variations, the ultimate source of genetic infor- have indicated that the development of HCC is a multistep process mation, as the basis of individual differences in oncogenesis. characterized by the accumulation of genetic and epigenetic al- Moreover, most studies that have examined mRNA and protein levels terations, and many of the genetic abnormalities contributing to HCC, have only analyzed their expression differences rather than the se- such as potential oncogenes and tumor suppressor genes, are well quence differences between individuals. In addition, there are known known. Furthermore, with the rapid development of genomic tech- exceptions to the one-to-one relationship between DNA and mRNA nology, several studies have demonstrated the possibility of sequences. RNA editing is the post- or co-transcriptional modifi- cation of RNA nucleotides (nt) from their complementary DNA sequences [8]. In humans, the most frequent form of RNA editing is the conversion of adenosine to inosine; the transcription ma- chinery subsequently recognizes these inosines as guanosines [9]. * Corresponding author. Tel.: +86 21 51320142; fax: +86 21 51320142. E-mail address: [email protected] (J. Huang). With DNA-seq and RNA-seq as well as next-generation sequenc- 1 These authors contributed equally to this work. ing platforms becoming routine tools in molecular research, several http://dx.doi.org/10.1016/j.canlet.2014.12.006 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved. X. Hu et al./Cancer Letters 357 (2015) 510–519 511 bioinformatic methods have been established to identify RNA editing Supplementary Fig. S1a and S1b). In total, 52,792 potential RNA sites on a genome-wide basis via high-throughput sequencing data editing sites (Supplemental_data_1.xlsx, ftp://183.62.232.83/) (21,886 [8,10–12]. Additionally, some genome-wide studies of human RNA in T311, 18,048 in N321, 23,723 in T273, 11,442 in N283, 8235 in editing have been reported [13,14], although these studies were LM6, and 8124 in M97L) were identified (Fig. 2a). Of these, only 900 mostly performed at the population genetics level and examined RNA editing sites were detected among all six specimens. An average healthy individuals, and very few studies have examined cancers. of 5379 RNA editing sites (range from 2001–10,492) were found spe- To analyze the RNA editome in HCC and further understand its po- cifically in the individual samples (Appendix: Supplementary Fig. tential role in hepatocarcinogenesis, we integrated DNA-Seq and S2). Furthermore, we found that approximately 40% of the RNA RNA-Seq analysis of two pairs of HCC tissues, their matched non- editing sites were located in the DARNED database [18], and 60% tumor tissues, and 2 HCC-related cell lines. Our analysis focused on of the RNA editing sites were first reported in this study (Fig. 2a). the RNA editing of BLCAP (bladder cancer-associated protein and These data also showed that 12 types of differences were found regulated by ADAR1), which was subjected to further study in in each of the 6 samples (Fig. 2b). A total of 82.15% of the RNA editing large sample cohorts and functional analyses, and the results sites were A-to-G changes, which may have been the result of deami- demonstrated that BLCAP RNA over-editing may contribute to nation by adenosine deaminases acting on RNA (ADAR). An additional hepatocarcinogenesis. 12.09%, 1.70%, and 0.98% of the RNA editing sites involved T-to-C, G-to-A, and C-to-T alterations, respectively; the other eight types Materials and methods accounted for only 3.07% of the changes (Fig. 2b and Appendix: Sup- plementary Table S3). The relative proportion of each type across HCC tissue specimens individuals was similar to previous findings [10]. All HCC tissue specimens were obtained from patients who underwent surgi- To experimentally validate our calls, we randomly sequenced 123 cal resection of their tumors and provided informed consent prior to liver surgery. potential RNA editing sites in T273, N283, T311, and N321, using The primary tumor specimens were immediately frozen at −80°C until DNA/RNA ex- an Ion Torrent sequencer. These results validated 66.0%, 81.0%, 87.0%, 3 traction. Specimens (approximately 1 cm ) of both tumor and adjacent liver tissue and 90.0% of the editing sites in T273, N283, T311, and N321, re- were taken from each patient, and the diagnosis of HCC was confirmed by patho- spectively (Appendix: Supplementary Table S4), which suggested logical examination. The HCC specimens presented in this study were grouped according to differentiation grades II–III following the Edmondson–Steiner grading that these identified RNA editing sites have high credibility and that system. The clinical characteristics of the patients and tumors are summarized in these data could be used for further analysis. Appendix: Supplementary Table S1. This project and its protocols involving human and animal tissues were approved by the ethics committee of the Chinese Nation- Over-editome analysis of HCCs al Human Genome Center. Laboratory methods Interestingly, we found that the number of RNA editing sites was significantly different in the six samples and that the number of RNA See the Supporting Materials and Methods section for detailed experimental editing sites was greater in HCC tissue than adjacent liver tissue (non- procedures. HCC) (p < 0.05, Appendix: Supplementary Fig. S3). No differences between the LM6 and 97L cell lines were observed (p > 0.05, Ap- Results pendix: Supplementary Fig. S3), although these two cell lines were also derived from the same genetic background (one male patient Editome analysis of six samples using whole-genome and suffers from HCC) [6]. In addition, we found that hepatoma cell lines transcriptome sequencing data display less RNA editing sites compared to non tumoral liver. The potential reasons may be related with that cell line homogenized By carrying out next-generation DNA sequencing, we obtained under subculture, whilst hepatoma cells have frequent heteroge- a total of 1,810,665,526–1,910,437,344 high-quality reads (Appendix: neity in tumor tissue. Supplementary Table S2) from six samples including two pairs of To identify how many genes are involved in RNA over-editing HCC tissues and 2 HCC cell lines used in this study (Appendix: Sup- events, we first statistically analyzed the differential expression of plementary Table S1). These reads were aligned to the human genes with RNA editing events in HCC samples compared to non- genome reference sequence (hg19) using Burrows–Wheeler Align- HCC samples according to a previously described criterion [19].
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