Cancer Letters 357 (2015) 510–519

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Cancer Letters

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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 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 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 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 , 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 (-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]. The ment (BWA) software; an average of 92.34% of the sequence reads editing sites with significant differences between HCC and non- were clean enough to be used for further analysis and the average HCC samples at the RNA level were defined as RNA over-editing sites coverage depth reached 51.31-fold for each DNA sample. In paral- based on the following criteria: fold change ≥2 and p ≤ 0.01, Q ≤ 0.1 lel, using RNA sequencing, we obtained a total of 371,011,349– by DEGseq. We then identified 3509 RNA editing sites 640,637,068 high-quality reads from the same six samples. We used (Supplemental_data_2.xlsx, ftp://183.62.232.83/) with signifi- BWA software to map these reads to the refseq gene database and cantly different expression levels in the two pairs of HCC tissues human genome (hg19), with an average mapping ratio of 57.81%. (p < 0.01). Of these, 2101 and 1690 RNA editing sites were found Thus, our sequencing coverage was adequate to sensitively and re- in T273/N283 and T311/N321, respectively; there were also 282 sites liably detect various somatic alterations and sequence differences that were RNA edited in both samples. between DNA and RNA at the whole-genome level. Among the 282 shared RNA over-editing sites, we found only 95 And then to explore the RNA editome of human tissues, we first nonsynonymous sites (Appendix: Supplementary Table S5) asso- removed the somatic substitute variations. And then we com- ciated with 84 genes (Appendix: Supplementary Fig. S4a). Seven of pared the genotypes derived from DNA-Seq with those from RNA- these 84 genes with RNA editing events displayed high-frequency Seq using the six specimens listed above (Appendix: Supplementary A→G transcript over-editing in 2 pairs of HCC samples, leading to Table S1) according to strict criteria (Fig. 1). Additionally, to accu- an amino acid substitution. Given the low frequency of transcripts rately obtain the site of RNA editing, we removed any potential RNA with RNA editing events, Sequenom MassARRAY technology and Ion editing sites in the first five and last five positions of the sequenc- Torrent sequencing were used to validate the 7 RNA over-editing ing reads according to previous guidelines published in scientific sites in the DNA and cDNA samples from the two pairs of HCC tissue. journals [15–17]. The final data showed that the RNA editing sites These results showed that 6 genes were confirmed to be RNA over- were randomly located in the 80-bp-length reads (i.e., between the editing genes including BLCAP (Appendix: Supplementary Fig. S4b), 6th and the 85th positions of the sequencing reads) (Appendix: AZIN1 (Appendix: Supplementary Fig. S4c), FDPS (Appendix: 512 X. Hu et al./Cancer Letters 357 (2015) 510–519

Fig. 1. A flowchart for the process of screening and identifying variants and RNA editing sites using high-throughput sequence data. (RE indicates RNA editing.). X. Hu et al./Cancer Letters 357 (2015) 510–519 513

Fig. 2. Characterization of the RNA editome of six HCC specimens. (a) The number of RNA editing sites was identified in six HCC specimens, including known RNA editing sites deposited in the DARNED database and novel RNA editing sites identified in this study. (b) Twelve types of RNA editing were identified in the transcriptome of the six specimens.

Supplementary Fig. S4d), CCNI, PLXNB, and MLL4 (Appendix: nonsynonymous A→I transcript editing, leading to a Tyr→Cys amino Supplementary Table S4). acid substitution in the two pairs of HCC samples. To confirm this result, we investigated the status of BLCAP editing in 179 matched BLCAP RNA over-editing contributes to hepatocarcinogenesis primary HCC and non-HCC samples using a Sequenom MassARRAY. Our results showed that the expression of RNA-edited BLCAP was To study the role of RNA editing in liver carcinogenesis, we se- significantly higher in HCCs than non-HCCs (p < 0.0001, Mann– lected BLCAP gene as a model to observe whether the over-editing Whitney Test, Fig. 3a, Appendix: Supplementary Table S6) and that gene may play roles in hepatocarcinogenesis in some proof of prin- 40.1% of the primary HCC specimens had BLCAP gene over-editing, ciple experiments, Using Sequenom MassARRAY technology, we as defined by a 2-fold increase in RNA-edited BLCAP in HCCs com- found that the BLCAP gene demonstrated a high frequency of pared to non-HCCs (Fig. 3a, Appendix: Supplementary Table S6). The

Fig. 3. BLCAP over-editing is strongly associated with HCC pathogenesis. (a) BLCAP editing was detected in 179 paired HCC and non-HCC samples using Sequenom MassARRAY. The p values shown were calculated using a Mann–Whitney U test. The upper and lower edges of each box represent the 75th and 25th percentile, respectively; the upper and lower bars indicate the highest and lowest values determined, respectively. (b) Dot Box showing BLCAP editing in HCCs. The p values shown were calculated by Mann– Whitney U test. The HCC specimens were subdivided into six categories according to the tumor size, tumor number and according to the presence or absence of PVTT. T peak indicates wild type BLCAP and C peak indicates RNA editing BLCAP. 514 X. Hu et al./Cancer Letters 357 (2015) 510–519

Table 1 The statistical results of BLCAP overediting status and the clinical characteristics of 179 samples in this study.

Clinical Clinical No. of BLCAP_RE BLCAP_WT characteristics variable patients <2.0 (%) ≥2.0 (%) p Value <2.0 (%) ≥2.0 (%) p Value

Age (years) <55 126 71(56.3) 55(43.7) 0.229 112(88.9) 14(11.1) 0.256 ≥55 53 35(66.0) 18(34.0) 50(94.3) 3(5.7) Gender Female 20 9(45.0) 11(55.0) 0.170 19(95.0) 1(5.0) 0.467 Male 159 97(61.0) 62(39.0) 143(89.9) 16(10.1) Family history No 157 92(58.6) 65(41.4) 0.652 141(89.8) 16(10.2) 0.398 Yes 22 14(63.6) 8(36.4) 21(95.5) 1(4.5) HBsAg Negative 33 18(54.5) 15(45.5) 0.545 30(90.9) 3(9.1) 0.930 Positive 146 88(60.3) 58(39.7) 132(90.4) 14(9.6) AFP (ng/ml) <20 48 30(62.5) 18(37.5) 0.589 45(93.8) 3(6.3) 0.370 ≥20 131 76(58.0) 55(42.0) 117(89.3) 14(10.7) Tumor size (range, cm) <5 44 32(72.7) 12(27.3) 0.036 42(95.5) 2(4.5) 0.240 ≥5 135 74(54.8) 61(45.2) 121(89.6) 14(10.4) Cirrhosis No 21 13(61.9) 8(38.1) 0.790 19(90.5) 2(9.5) 0.996 Yes 158 93(58.9) 65(41.1) 143(90.5) 15(9.5) Tumor number Single 119 62(52.1) 57(47.9) 0.006 102(85.7) 17(14.3) 0.025 Multiple 60 44(73.3) 16(26.7) 58(96.7) 2(3.3) Wine-drinking No 75 44(58.7) 31(41.3) 0.899 69(92.0) 6(8.0) 0.562 Yes 104 62(59.6) 42(40.4) 93(89.4) 11(10.6) TNM stage I–II 66 35(53.0) 31(47.0) 0.198 58(87.9) 8(12.1) 0.360 III–IV 113 71(62.8) 42(37.2) 104(92.0) 9(8.0) PVTT No 133 71(53.4) 62(46.6) 0.030 116(87.2) 17(12.8) 0.039 Yes 46 33(71.7) 13(28.3) 45(97.8) 1(2.2) Distant metastasis No 159 97(61.0) 62(39.0) 0.223 144(90.6) 15(9.4) 0.148 Yes 20 15(75.0) 5(25.0) 16(80.0) 4(20.0) Recurrence No 134 79(59.0) 55(41.0) 0.902 122(91.0) 12(9.0) 0.67 Yes 45 27(60.0) 18(40.0) 40(88.9) 5(11.1)

HBsAg, hepatitis B surface antigen; AFP, alpha-fetoprotein; TNM, tumor-node-metastasis; PVTT, portal vein tumor thrombus. The meaning of p values as defined as p < 0.05.

statistical analysis showed the RNA overediting of BLCAP was not transfected with siRNA-NC (p < 0.05, Fig. 5). These results sug- statistically correlated with gender, age (≥50 or <50), family history, gested that the higher frequency of RNA-edited BLCAP in HCC may hepatitis B surface antigen (HBsAg) expression, history wine- contribute to hepatocarcinogenesis, although we cannot distin- drinking, history of smoking, presence of liver cirrhosis, the level guish edited or wild-type BLCAP using siRNA. of alpha-fetoprotein (AFP), BCLC stage, or presence of distant me- To give these results context in vivo, we then subcutaneously tastasis or lymph node metastasis (p > 0.05, Table 1). However, the injected SMMC7721 cells that were transfected with wild-type, RNA- RNA overediting of BLCAP positively correlated with tumor size (≥5 edited BLCAP as well as parental SMMC7721 cells into 7 athymic or <5 cm, p = 0.036), Tumor number (Single and Multiple, p = 0.006) mice and assessed tumorigenicity. The incidence of tumors derived and the presence of portal vein tumor thrombus (PVTT) (p = 0.03, from RNA-edited BLCAP cells was higher than the incidence of tumors Fig. 3b, Table 1). Our current results suggested that BLCAP editing derived from wild-type BLCAP or parental cells (Fig. 6a). Impor- may be associated with hepatocarcinogenesis. tantly, we also observed that the tumor volumes (mm2) in the group To evaluate the relationship between BLCAP gene functions and of RNA-edited BLCAP-transfected tumors were larger than those of hepatocarcinogenesis, either wild-type or edited BLCAP was first tran- the wild-type BLCAP-transfected and parental cells (p < 0.05, Fig. 6b). siently transfected into SMMC7721 (TP53 wild type, Appendix: In addition, to answer the relationship of BLCAP gene and Supplementary Table S7) and Focus cells (TP53 wild type, Appen- hepatocarcinogenesis is a universal phenomenon for cell lines rather dix: Supplementary Table S7) with pcDNA3.1 plasmid, which both than a specific phenomenon for one type of cell line, we per- express the relatively lower BLCAP based on the previous BLCAP ex- formed a parallel assay by subcutaneously injecting the FOCUS HCC pression pattern of HCC-derived cell lines (Appendix: Supplementary cell line, which had been transfected with wild-type or RNA- Fig. S5a and S5b); an empty pcDNA3.1 vector was used as a control. edited BLCAP, as well as parental FOCUS cells into 7 athymic mice. These results showed that the overexpression of RNA-edited BLCAP We observed the similar results. The incidence of tumors derived significantly promoted the growth and colony formation of these from RNA-edited BLCAP cells was markedly higher and the tumors cells compared to wild-type BLCAP or control cells (p < 0.01; Fig. were markedly larger than those derived from wild-type BLCAP or 4a–d). And then using the same method, we found that RNA- parental cells (Fig. 6c and 6d). These results suggested that RNA edited BLCAP significantly enhanced the anchorage-independent editing of BLCAP may contribute to tumorigenesis in HCC. growth ability of SMMC7721 and MHCC-97H cells compared to wild- type BLCAP and control cells (p < 0.01; Fig. 4e–f). To confirm the effect BLCAP RNA editing in liver cell lines affects the phosphorylation of of BLCAP on cell proliferation in HCC cell lines, we designed and the Akt/mTOR signaling pathway chemically synthesized two small interfering RNAs (siRNAs) to spe- cifically knock down wild-type and RNA-edited BLCAP, including an To explore the molecular mechanisms by which BLCAP RNA siRNA-1 covering nt 888–909 and an siRNA-2 covering nt 1094– editing may contribute to these malignant features, we per- 1115. Huh7 and MHCC-LM3 cells were transiently transfected with formed an antibody protein array to determine whether BLCAP RNA the siRNAs, and a negative control (siRNA-NC) was used as a control. editing affects other or their partners, with a special focus Subsequent analysis by RT-PCR indicated that these two siRNAs sig- on protein phosphorylation. We selected AKT, mTOR, FAK, and nificantly knocked down exogenous BLCAP expression compared to CyclinB1 with more than 2-fold change (Appendix: Supplementa- siRNA-NC. We also found that the silencing of BLCAP significantly ry Table S8) to confirm their phosphorylation level in SMMC7721 inhibited the growth of Huh7 and MHCC-LM3 cells compared to cells cell line (Tp53 wild type, Appendix: Supplementary Table S7) X. Hu et al./Cancer Letters 357 (2015) 510–519 515

Fig. 4. The effect of BLCAP RNA editing on cell proliferation. (a/b) Exogenous RNA-edited BLCAP (RE) was expressed in SMCC7721 (a) and Focus (b) cells transfected with the pcDNA3.1 vector. Parental cells with empty vector and wild-type BLCAP (WT) were used as controls. The growth of these cells was analyzed using the Cell Counting Kit-8 (CCK-8). The experiments were repeated at least 3 times, and the symbols represent the mean values of triplicate tests (mean ± standard deviation (SD)). The western blot analysis shown in the left panel indicates the levels of BLCAP expression in these cell lines. A t-test was used to show significant differences between 2 groups (p < 0.05). (c/d) To observe the effect of BLCAP on colony formation, SMCC7721 (c) and Focus (d) cells were transfected with pcDNA3.1-RNA-edited BLCAP (RE), and the parental cells were transfected with empty vector and wild-type BLCAP (WT) as controls. BLCAP (RE and WT) expression was confirmed by immunoblotting. After transfection for 24 h, the cells were scraped, plated on dishes, and cultured in G418 for 3 weeks. The representative dishes show the inhibitory effect of BLCAP on colony formation. The lower histogram shows that colony formation was significantly promoted by BLCAP (RE), compared to the control (p < 0.05), where the numbers represent the mean values of 3 independent experiments with the SD. (e/f) Quantification of soft agar colonies induced by the indicated stable cell lines. p < 0.05. Scale bar, 100 μm. 516 X. Hu et al./Cancer Letters 357 (2015) 510–519

Fig. 5. The effect of BLCAP silencing on HCC cell growth. (a) RT-PCR confirmation of BLCAP knockdown was performed in Huh7 cells via transient transfection with siRNAs. siRNA-NC was used as the control (left). The growth curves of the Huh7 cells after BLCAP knockdown using siRNAs were plotted based on the CCK-8 assay. siRNA-NC served as a control (right). (b) RT-PCR confirmation of BLCAP knockdown in MHCC-LM3 cells was performed after transient transfection with siRNAs. siRNA-NC was used as the control (left). The growth curves of MHCC-LM3 cells following BLCAP knockdown with siRNAs were plotted based on the CCK-8 assay. siRNA-NC served as a control (right). The experiments were repeated at least three times, and the data points represent the average values of triplicate wells; the SD is included for each mean value.

transfected by BLCAP_RE compared to transfected by BLCAP_WT Then, we focused on the AKT/mTOR signaling pathway, which samples, of which these proteins belongs to signal pathway has been proposed to contribute to cell growth and anchorage- and were reported to play roles in oncogenesis. Using western blot- independent colony formation [20–22]. To systematically assess the ting, we found that the phosphorylation levels of AKT (P-Ser473 and effect of BLCAP RNA editing on the activity of the AKT/mTOR sig- P-Tyr326) and mTOR (P-Thr2446) were clearly increased in BLCAP_RE naling pathway, we used western blotting assays to examine the compared to BLCAP_WT samples (Fig. 7a). phosphorylation level changes of MDM2, p21 and TP53 in BLCAP_RE

Fig. 6. BLCAP editing contributes to augmented tumor-initiating potential and enhanced in vivo tumorigenic ability. (a) An increase in exogenous BLCAP expression pro- moted the growth of xenograft tumors of SMMC7721 cells infected with recombinant pcDNA3.1 carrying BLCAP (WT and RE). These cells were injected subcutaneously into nude mice, and cells carrying an empty vector were used as controls. Tumor growth was monitored for3dbymeasuring the tumor diameters (mean ± SD). Panel A dis- plays tumors derived from BLCAP (WT) and BLCAP (RE) cells 4 weeks after subcutaneous injection (n = 7 mice per group). (b) The growth curves of tumors derived from the indicated cell lines over a period of 4 weeks are shown. The data are presented as the mean ± SD. p < 0.05, as determined with unpaired, 2-tailed Student’s t-tests. (c) In- creased exogenous BLCAP expression promoted xenograft tumor growth of FOCUS cells infected with a recombinant pcDNA3.1 carrying BLCAP (WT and RE). These cells were injected subcutaneously into nude mice, and cells carrying empty vector cells were used as controls. Tumor growth was monitored for 3 d by measuring the tumor diameters (mean ± SD). Panel A displays tumors derived from BLCAP_pcDNA3.1, BLCAP_WT, and BLCAP_RE cells 4 weeks after subcutaneous injection (n = 7 mice per group). (d) Growth curves of tumors derived from the indicated cell lines are shown for a period of 4 weeks. The data are presented as the mean ± SD. p < 0.01, as determined with an unpaired, 2-tailed Student’s t-test. X. Hu et al./Cancer Letters 357 (2015) 510–519 517

Fig. 7. BLCAP RNA editing modulates the phosphorylation of the AKT/mTOR signaling pathway. (a) The overexpression of RNA-edited BLCAP enhanced the phosphorylation of AKT (P-Ser473 and P-Tyr326), mTOR (P-Thr2446), and MDM2 (P-Ser166) and inhibited the phosphorylation of TP53 (P-Ser6 and P-Ser15) in SMMC7721 cell line (TP53 wild type). (b) The overexpression of RNA-edited BLCAP did not affect the phosphorylation of AKT (P-Ser473 and P-Tyr326), mTOR (P-Thr2446), MDM2 (P-Ser166) and TP53 (P-Ser6 and P-Ser15) in Hep3B cell line (TP53 null). β-actin was used as a loading control. Quantification of phosphorylation and non-phosphorylation levels, as indicated by the numbers above the corresponding panels, was performed by normalizing the phosphorylation and non-phosphorylation concentrations to the β-actin loading control. compared to BLCAP_WT samples with more than 2-fold change experiment. We did not observe the same phenomenon in Hep3B in protein chip assay. Surprisingly, the phosphorylation of TP53 (P- cells (Fig. 7b). These results demonstrate that BLCAP RNA editing Ser6 and P-Ser15) were markedly suppressed in SMMC7721 cells imparts a malignant phenotype to liver cancer cells through mod- transfected with RNA-edited BLCAP compared to SMMC7721 cells ulation of the AKT/mTOR signaling pathway [23,24]. transfected with wild-type BLCAP, whereas the phosphorylation of MDM2 (P-Ser166) was markedly enhanced (Fig. 7a). The current Discussion results of the western blotting suggest a potential p53-dependent mechanism. To further validate the potential p53-dependent Recurrently, Mingyao Li compared RNA sequences to the corre- mechanism by BLCAP editing type, we also selected Hep3B (TP53 sponding DNA sequences from the same individuals and uncovered null, Appendix: Supplementary Table S7) to repeat the above more than 10,000 exonic sites where the RNA sequences did not 518 X. Hu et al./Cancer Letters 357 (2015) 510–519 match the DNA sequences [13]. Zhiyu Peng also performed whole- the expression of ADAR1, ADAR2 (ADARB1), and ADAR3 (ADARB2) genome and transcriptome sequencing and identified 22,688 RNA in 20 pairs of HCC samples; we observed that ADAR1 was mark- editing events in a normal human cell line [10]. Thus, scientists have edly upregulated in 85% (17/20) of these HCC samples as compared begun to pay close attention to RNA editing events in the to non-HCC samples. However, we did not detect differential ex- transcriptome of human cells, with the hypothesis that RNA editing pression of ADAR2 between the tumors and adjacent liver tissue, events play important roles in driving disease pathogenesis, par- and we could not detect ADAR3 expression in any of the samples ticularly oncogenesis. In the current study, we evaluated whether (Fig. S7a). To validate whether BLCAP RNA editing was regulated by there are widespread RNA and DNA sequence differences in the ADAR1, liver cancer cell lines were transfected with ADAR1 in the human transcriptome, whether there is differential expression of pcDNA3.1 plasmid, and the results demonstrated an increased level genes demonstrating RNA editing events in HCC compared to non- of BLCAP RNA editing (Fig. S7b). Furthermore, we found that using HCC samples, and whether any of the genes demonstrating RNA over- RNA interference against ADAR1 effectively reduced the level of editing events play roles in hepatocarcinogenesis. BLCAP RNA editing (Fig. S7c). In summary, these studies of the Using high throughput sequencing data, we identified seven genes overexpression and knockdown of ADAR1 support Chan’s conclu- with nonsynonymous loci demonstrated RNA over-editing in both sion. To observe the molecular events (overediting of BLCAP) also paired HCC samples. Of these seven genes, BLCAP, AZIN1, and FDPS happening on other kinds of cancers, we added the detection of were confirmed by Sequenom MassARRAY assay, while CCNI, MLL4, BLCAP-editing level in colorectal cancer (CC), gastric cancer (GC), and PLXNB1 were validated by Ion Torrent PGM platform. Differ- esophageal cancer (EC) and hepatocellular carcinoma (HCC) com- ent technologies possess different levels of sensitivity and pared with the related non-tumor tissues using Ion Torrent PGM quantitative characteristics; therefore, we did not expect to vali- platform. We found that the over-editing event also reasonably date all sites using any individual platform, especially those with happen on colorectal cancer (Appendix: Supplementary Fig. S8). low levels of RNA editing sites. Interestingly, we detected pep- Except that the BLCAP-editing level was significantly elevated in tides translated from the edited RNA sequences by analyzing the HCCs, interestingly, we also found BLCAP-overediting phenome- proteome of N321 and T311 cells using mass spectrometry, and we non in CCs which is different from Galeano’s result [33]. The potential found wild-type and RNA-edited peptides of the CCNI and FDPS gene reason will be explored in further research. products (Appendix: Supplementary Table S9). These results suggest In addition, we analyzed the expression of ADAR family genes that RNA-edited transcripts are available to be translated. at RNA levels in CCs, GCs, ECs and HCCs compared with the related In addition, to explore the biological functions of RNA editing non-tumor tissues using RT-PCR technology. We found that ADAR1 events, we selected three genes (BLCAP, AZIN1, and CCNI) that were gene only up-regulated in HCC compared with non-tumor liver easy to clone for further research. We found that RNA-edited AZIN1 tissues and ADAR2 (ADARB1) gene up-regulated in CCs compared promoted the growth of QGY7703 HCC cells compared to wild- with non-tumor intestine tissues (Appendix: Supplementary Fig. S8). type AZIN1 (Appendix: Supplementary Fig. S6a and S6b), and it was Overexpression of ADAR1 and ADAR2 genes may be the potential also previously reported that AZIN1 contains an RNA over-editing reason of BLCAP-overediting in tumor tissues. event in HCC that contributes to hepatocarcinogenesis [25].Inad- Taken together, our study provides a view of the RNA over- dition, we also found that RNA-edited CCNI promoted the growth editing events that may serve as a potential driver in advanced HCC. of QGY7703 HCC cells as compared to wild-type CCNI (Appendix: In the future, RNA-overedited BLCAP in HCC may become a novel Supplementary Fig. S6a and S6c). However, this phenomenon was biomarker and also represent a target of molecular therapies to block not observed in additional HCC cell lines, and we did not research or reverse heptacarcinoma development. the biological functions of these genes in depth. Instead, we se- lected BLCAP for further research. Acknowledgements This study was the first to report that the RNA editing of BLCAP shows a relatively higher frequency in HCC specimens and that there We gratefully acknowledge support from the National is an accumulation of the substitution of A283G (NM_001167820) High Technology Research and Development Program of China in these specimens. BLCAP is located on 20q11.23, (863 Program, 2012AA02A205), the Chinese National Key which is referred to as RP11-425M5.2 or BC10. BLCAP was origi- Program on Basic Research (973 Program, 2014CB965002 and nally found to be downregulated in human transitional cell 2010CB529206), the National Natural Science Foundation of China carcinomas (TCCs) by mRNA differential display and therefore rep- (81272306 and 81472639), the Shanghai Commission for Science resents a novel human bladder cancer-associated protein with a and Technology (11JC1408800), Program of Shanghai Subject Chief conserved genomic structure [26,27]. Therefore, BLCAP may have Scientist (12XD1421400), Program of Shenzhen Science Technol- physiological importance in the context of bladder tissue. In addi- ogy and Innovation Committee (JCYJ20130329171031740, tion, other researchers have reported that BLCAP may act as a tumor CXZZ20130515163643 and JCYJ20120831144704366). suppressor gene in cervical carcinoma and tongue carcinoma and may have some prognostic value in breast cancer [28–30]. Conflict of interest Levanon reported that BLCAP was an ADAR-mediated A-to-I RNA- edited gene [31] and should therefore play a critical role during early The authors declare no competing financial interests. human development [32]. In 2010, Galeano and his colleagues re- ported that they observed a general decrease in BLCAP-editing level Appendix: Supplementary material in astrocytomas, bladder cancer and colorectal cancer when com- pared with the related normal tissues. They thought that the newly Supplementary data to this article can be found online at identified editing events could be useful for future studies as a di- doi:10.1016/j.canlet.2014.12.006. agnostic tool to distinguish malignancies or epigenetic changes in different tumors [33]. In this study, our results was opposite with Galeano’s result. Hence, it is important to understand whether the References A-to-I RNA overediting of BLCAP plays a role in hepatocarcinogenesis. 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