Knockdown of a novel lincRNA AATBC suppresses proliferation and induces apoptosis in bladder cancer.

Long intergenic noncoding RNAs (lincRNAs) play important roles in regulating various biological processes in cancer, including proliferation and apoptosis. However, the roles of lincRNAs in bladder cancer remain elusive. In this study, we identified a novel lincRNA, which we termed AATBC. We found that AATBC was overexpressed in bladder cancer patient tissues and positively correlated with tumor grade and pT stage. We also found that inhibition of AATBC resulted in cell proliferation arrest through G1 cell cycle mediated by cyclin D1, CDK4, p18 and phosphorylated Rb. In addition, inhibition of AATBC induced cell apoptosis through the intrinsic apoptosis signaling pathway, as evidenced by the activation of caspase-9 and caspase-3. The investigation for the signaling pathway revealed that the apoptosis following AATBC knockdown was mediated by activation of phosphorylated JNK and suppression of NRF2. Furthermore, JNK inhibitor SP600125 could attenuate the apoptotic effect achieved by AATBC knockdown, confirming the involvement of JNK signaling in the induced apoptosis. Moreover, mouse xenograft model revealed that knockdown of AATBC led to suppress tumorigenesis in vivo. Taken together, our study indicated that AATBC might play a critical role in pro-proliferation and anti-apoptosis in bladder cancer by regulating cell cycle, intrinsic apoptosis signaling, JNK signaling and NRF2. AATBC could be a potential therapeutic target and molecular biomarker for bladder cancer.


INTRODUCTION
Bladder cancer is a common malignant tumor of genitourinary system [1]. Approximately 70% of the cases are non-muscle invasive bladder cancer (NMIBC) that can be easily resected. However, some of these cases will progress to muscle invasive bladder cancer (MIBC) or metastasis to distant organs and ultimately endanger the lives of patients. Surgery is the main treatment for bladder cancer, but recurrence and metastasis are very common. So far, little is known about the pathogenesis of bladder cancer and no sensitive tumor biomarker has been found [2,3]. Therefore, it is an urgent need to study the carcinogenesis and progression of bladder cancer.
In recent years, accumulating data showed that epigenetic alterations play a significant role in carcinogenesis and progression of malignancies [4,5]. Long noncoding RNAs (lncRNAs, >200nt) are the major noncoding RNAs that regulate gene expression at epigenetic, transcriptional, and post-transcriptional levels [6]. Long intervening noncoding RNAs (lincRNAs), as a subtype of lncRNAs, are transcript units discretely intervening known protein-coding loci. The important functions of lincRNAs have being demonstrated in many publications [7,8]. Studies showed that abnormal expression of lincRNA is in a disease-, tissue-or developmental stage-specific manner [9,10]. Some lincRNAs are involved in pathological processes of cancer, such as proliferation, apoptosis, invasion and metastasis, acting as tumor suppressor genes or oncogenes in various types of cancer [11,12]. Nevertheless, there are thousands of functional lincRNAs yet to be identified. The objective of our study is to elucidate the lincRNAs expression patterns between bladder cancer and normal bladder tissues and try to find a specific lincRNA that may be a potential therapeutic target or biomarker for bladder cancer.
Studies have revealed that some aberrant expression of lincRNAs is closely associated with the progression and prognosis of bladder cancer, such as UCA1 and H19 [13,14]. Our research team previously reported that linc-UBC1 played an important role in tumorigenesis and lymph node metastasis of bladder cancer [15]. In this report, we identified another novel lincRNA, termed as AATBC (Apoptosis-Associated Transcript in Bladder Cancer, also known as LOC284837). We found that AATBC was overexpressed in bladder cancer tissues and cancer cell lines. AATBC expression was positively correlated with tumor grade and pT stage of bladder cancer. AATBC knockdown resulted in inhibition of proliferation and increased apoptosis in vitro and suppressed tumor growth in vivo. The JNK signaling, as an important component of the mitogen-activated protein kinase (MAPK) signal transduction pathway [16], was activated by knockdown of AATBC. In addition, we found that NRF2, which is a critical player in the defense system against diverse stress condition including oxidative stress, was down regulated [17]. Inhibition of JNK by SP600125, ameliorated the cytotoxicity of AATBC silencing and resulted in up regulation of NRF2. These results indicated the key roles of JNK and NRF2 signaling pathway in the occurrence of apoptosis resulted from AATBC knockdown. Taken together, our results demonstrated that AATBC might play a critical role in promoting the tumorigenesis and progression of bladder cancer through the regulation of proliferation and apoptosis.

LincRNAs expression profiles in bladder cancer
To investigate the potential biological functions of lincRNAs in bladder cancer, we firstly examined the expression pattern (fold changes ≥ 2 and ≤0.5, p < 0.05) of lincRNAs and mRNAs in matched sets of muscle invasive bladder cancer tissues and adjacent non tumor tissues obtained from 5 patients. We used a microarray targeting 1238 Entrez protein coding genes and 151 lincRNAs (Agilent), as previously reported [15]. Hierarchical clustering was applied to analyze the systematic variations of lincRNAs expression in these 5 paired tissue specimens. Altogether, 30 lincRNAs were found to be up regulated more than two-fold, while 121 lincRNAs were down regulated more than two-fold (p < 0.05, Fig. 1A-B) in the bladder cancer tissues compared to the adjacent non tumor tissues. The microarray analysis of the expression pattern of lincRNAs clearly implicated that many lincRNAs are linked with bladder cancer. Some of these lincRNAs may have potential functions in the regulation of tumorigenesis and progression of bladder cancer or serve as molecular biomarkers.

AATBC is overexpressed in bladder cancer
Then, we attempted to identify some lincRNAs that are overexpressed in bladder cancer. Firstly, we chose the first ten most overexpressed lincRNAs in the microarray analysis. Secondly we examined the expression levels of these lincRNAs in the bladder cancer tissues of 30 independent cases. Finally, we characterized the most frequently overexpressed lincRNA LOC284837 (we named it AATBC) in bladder cancer tissues compared to the adjacent non tumor tissues (Fig. 1C).
Information from UCSC Browser shows that AATBC is a transcript of 4622bp and localizes in human chromosome 21q22.3, 8465bp downstream of RRP1 gene and 38902bp upstream of CSTB gene (Fig. 1D). TESTCODE tool (http://www.genomicsplace.com/ testcode.html) was employed to predict whether AATBC is likely to code for a protein. A score 0.4502 was obtained, suggesting that AATBC is probably a noncoding RNA.
We further examined its expression levels in bladder cancer tissues of 90 independent cases through quantitative RT-PCR. Compared with the non-tumor counterparts, it was found that AATBC was up-regulated (fold change of ≥1.5) in 54 cases (60%), whereas 36 cases (40%) were down-regulated or without obvious changes (Fig. 1E). To evaluate the significance of AATBC high expression in bladder cancer, we investigated the relationship between AATBC and clinical characteristics of patients (Table. 1). Overall, it was illustrated that the expression of AATBC was higher in MIBC (T2-T4) compared to that in NMIBC (Ta, Tis, T1), and positively correlated with the tumor grade, which indicated that the high expression level of AATBC was associated with tumor development and aggressiveness. However, we found no significant correlation between AATBC expression with gender, age and lymph node status.
We further verified the expression pattern of AATBC in bladder cancer cell lines (T24, EJ, UM-UC-3 and 5637) and an immortalized normal urothelium cell line SV-HUC-1 by quantitative RT-PCR, using β-actin as a reference gene. Similar to the alterations of paired tissue samples, quantitative RT-PCR showed the expression level of AATBC was significantly higher in bladder cancer cell lines than that in the normal urothelium cell (Fig. 1F), which confirmed that the expression of AATBC was correlated with malignancy. We speculated that AATBC could be a potential oncogene in bladder cancer.

AATBC knockdown inhibits proliferation of bladder cancer cells via cell cycle arrest
We knocked down AATBC in UM-UC-3 and EJ bladder cancer cells using small interfering RNAs (siRNAs). The knockdown efficiency in UM-UC-3 cells was si#1 64.3% ± 3.0% and si#2 81.0% ± 3.6%. In EJ cells, the knockdown efficiency was si#1 61.7% ± 3.5% and si#2 77.7% ± 4.9% ( Fig. 2A). In order to investigate the effect of AATBC on cell growth of bladder cancer cell lines in vitro, CellTiter 96 AQueous One Solution Cell Proliferation assay, panel clonogenic assay, EdU incorporation assay and flow cytometry were employed. These results show that AATBC depletion led to an obvious inhibitory effect on the growth of bladder cancer cells. As shown in Fig. 2B, siRNAs-mediated knockdown of AATBC impaired the proliferation in UM-UC-3 and EJ cells, as revealed by CellTiter 96 AQueous One Solution Cell Proliferation assay. The number of live cells transfected with siRNAs was significantly decreased when compared with that in the negative controls. Consistent with the above results, the ability to form colonies of both cell lines was also suppressed significantly after downregulation of AATBC when compared with that in the negative controls (Fig. 2C).
For the purpose of better understanding the role of AATBC in proliferation, we employed the EdU incorporation assay to examine the effects of AATBC inhibition on the DNA synthesis during cell growth. The result showed that the proportion of S-phase cells (EdUpositive cells) was decreased in siRNA treated groups, suggesting reduced DNA synthetic activity resulted from AATBC depletion (Fig. 2D). Furthermore, we transfected the cancer cells with siRNAs before analyzing the impact on the cell cycle distribution by flow cytometry. Both UM-UC-3 and EJ cells treated with siRNAs show apparent increases in the percentage of cells in G1 phase with concomitant decreases in the percentage of cells in the S phase when compared with negative controls (Fig.  3A), which was consistent with EdU assay. These results proved that AATBC knockdown could lead to a cell cycle arrest in G1 phase, which was responsible for the suppressed proliferation.
To elucidate the possible molecular mechanisms accounting for the anticancer behaviors in proliferation triggered by AATBC depletion, we measured the expression of cell cycle-related proteins by Western blotting. Consistent with the observed phenomena of cell growth, the knockdown of AATBC led to an increased expression of p18 and decreased expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and phosphorylated retinoblastoma (Rb), without any obvious effect on p15 expression (Fig. 3B). In particular, changes of these cell cycle regulatory factors were correlated well with the G1 cell cycle arrest as shown in flow cytometry and EdU incorporation assay. Therefore, these data suggested that AATBC might be capable of promoting cell proliferation in bladder cancer through regulating cell cycle.

AATBC knockdown inhibits tumor growth in NOD/SCID mice
To investigate the biological significance of AATBC knockdown on tumor growth in vivo, we proceeded with the research through the establishment of a subcutaneous xenograft tumor model in NOD/SCID mice. Firstly, Stable AATBC-knockdown in UM-UC-3 cells by lentiviral-mediated RNAi system was successfully established. Quantitative RT-PCR analysis confirmed that the lentivirus-transduced UM-UC-3 cells showed a significant decrease of AATBC in transcript levels. The inhibition efficiency was 79.3% ± 3.1% (Fig. 4A). Equal numbers of UM-UC-3 cells (5×10 6 , per mice) stably expressing AATBC shRNA and negative control were injected subcutaneously into the flanks of NOD/SCID mice (n=4) (Fig. 4B). As expected, suppression of AATBC expression in UM-UC-3 cells treated with shRNA resulted in lower mean tumor mass compared to that of negative control group (0.36 g vs 1.085 g, P<0.05, Fig. 4C-D). The results in vivo indicated that the growth of xenografts was inhibited by AATBC shRNA treatment in UM-UC-3. This response of tumors to AATBC stable depletion revealed that AATBC might have tumor-promoting property in the carcinogenesis and progression of bladder cancer in vivo.

Knockdown of AATBC results in the intrinsic apoptosis in bladder cancer cells
We then investigated the involvement of AATBC in cell death of bladder cancer cells through inhibiting AATBC expression with siRNAs. As shown by flow cytometry analysis in Fig. 5A-B, siRNAs treatment led to the increased apoptotic rates in UM-UC-3 and EJ cells compared with that in negative control groups. After cells were induced with cisplatin, the pro-apoptotic effect of AATBC inhibition was augmented significantly when compared to that of no cisplatin-induced groups. Western blotting was used to investigate the alteration of apoptosisrelated proteins induced by AATBC down-regulation. To our knowledge, the cleavages of caspase-9 and caspase-3 are prominent markers of the intrinsic apoptosis. In this present study, the occurrence and intensity of apoptosis resulted from AATBC knockdown were evidenced by the enhanced cleavages of poly (ADP-ribose) polymerase cells was analyzed by flow cytometry. In cisplatin-induced groups, cisplatin was added 24 hours before examination. Data represented as means ± SD. *P < 0.05 (vs. control). B. 48 hours after transfection, apoptotic rate in EJ cells was analyzed by flow cytometry. In cisplatininduced groups, cisplatin was added 24 hours before examination. Data represented as means ± SD. *P < 0.05 (vs. control). C. Western blotting was used to detect the expression of caspase-9, caspase-3 and PARP. β-tubulin served as loading control. www.impactjournals.com/oncotarget (PARP), caspase-9 and caspase-3 in both UM-UC-3 and EJ cell lines (Fig. 5C), indicating that the activation of intrinsic apoptotic pathway was involved in the cell apoptosis due to AATBC down regulation.

AATBC knockdown induces activation of JNK signaling and suppression of NRF2
In view of the evidence that the MAPKs, consisting of ERK1/2, JNK and p38, play critical roles in apoptosis, we examined the activation of MAPK pathway to further uncover the underlying mechanisms for the alterations of apoptosis in UM-UC-3 and EJ cells after transfection with siRNAs. As revealed by Western blotting (Fig. 6A), knockdown of AATBC enhanced the phosphorylation level of JNK in both UM-UC-3 and EJ cells. In contrast, the expression levels of phosphorylated ERK1/2 and p38 were not obviously changed upon the treatment of AATBC knockdown. JNK-mediated apoptosis can occur through multiple pathways, including oxidative stress (OS) pathway [18]. Inspired by this, we detected the factors in OS pathway. In this study, besides the activation of JNK, we found that the expression level of NRF2, a key antioxidant transcription factor in OS pathway, was downregulated (Fig. 6A), indicating the impaired cellular selfprotective response in bladder cancer cells after AATBC depletion. Furthermore, the treatment with the inhibitor of JNK (SP600125) could enhance the expression level of NRF2 (Fig. 6B) and improve the cell adaptive ability to compensate the stress condition, indicating a potential link between JNK activation and NRF2 inhibition. Collectively, these results demonstrated that apoptosis after AATBC knockdown might be mediated by the activation of JNK signaling and suppression of NRF2.

Apoptosis induced by AATBC knockdown is rescued by inhibition of JNK signaling
To verify in depth whether this apoptotic phenomenon was dependent on the activation of JNK signaling pathway, the JNK-specific inhibitor SP600125 was supplemented to block JNK signaling before transfection with siRNAs to see whether the pro-apoptotic effect of AATBC down-regulation could be attenuated. As a result, Western blotting analysis demonstrated that SP600125 reduced the levels of the cleavage fragments of caspase-9, caspase-3 and PARP, and phosphorylated JNK efficiently in UM-UC-3 and EJ cells (Fig. 6B). In addition, it was also found by flow cytometry using Annexin V-FITC/PI staining (Fig. 6C) that SP600125 was able to decrease the percentage of apoptotic cells resulted from siRNA-mediated AATBC down regulation in comparison with the negative controls. These data further illustrated that the apoptosis induced by AATBC depletion in bladder cancer was mediated through the activation of JNK signaling.

DISCUSSION
Because of the unlimited proliferation and apoptosis defect of cancer cells, treatment of cancer is still a huge challenge for human beings. In recent years, increasing studies have revealed that some lincRNAs appear to be correlated with aberrant proliferation and apoptosis of cancer cells, which may in part explain the critical roles of lincRNAs in cancer etiology [19,20]. For example, lncRNA HOXA-AS2 suppresses apoptosis in promyelocytic leukemia. Knockdown of HOXA-AS2 by shRNA resulted in an increase in ATRA-induced apoptosis through the cleavage of caspase-9, caspase-8 and caspase-3 [21]. However, for most of these lincRNAs, the detailed functions, mechanisms and signaling pathways through which lincRNAs perform their biological functions have not been well understood. Studies focused on the regulatory functions and mechanisms of lincRNAs in bladder cancer are yet limited at present. Therefore, we conducted some researches to clarify the possible relationships between bladder cancer and lincRNAs and explore the potential application of lincRNAs in the diagnosis and treatment of bladder cancer.
The expression profiles of lincRNAs detected by microarray analysis provided valuable insight for the elucidation of functional roles and the underlying mechanisms of certain lincRNA in human cancer. In our study, we investigated the aberrant lincRNAs expression profiles in bladder cancer tissues by microarray analysis and identified a new lincRNA AATBC. We found that AATBC was overexpressed in bladder cancer tissues compared with adjacent non tumor tissues and positively correlated with tumor grade and pT stage, implying that AATBC might be an useful diagnostic biomarker or therapeutic target in bladder cancer [22]. This phenomenon was further demonstrated by the relative changes of biological behaviors in bladder cancer cell lines after AATBC knockdown. As mentioned above, down regulation of AATBC resulted in effectively suppressed proliferation in vitro, concomitant with induction of cell cycle arrest and apoptosis, and inhibited tumor growth in NOD/SCID mice in vivo. However, it remains unclear with respect to the relationship between AATBC expression levels and the overall survival of patients. This may be due to the limited number of cases and follow-up time.
An extensive prospective study in larger cohorts may be needed in the future. Cancer progression is often due to disorders in cell cycle that leads to the unlimited proliferation of cancer cells [23,24]. The cell cycle transition from the G1 phase to the S phase is the major regulatory checkpoint in this process. This transition is characterized by the phosphorylation of Rb and the interaction of CDKs, cyclins and CDK inhibitors (CDKI). In our study, the flow cytometry analysis and EdU incorporation assay demonstrated that AATBC down-regulation induced cell cycle arrest at the G1 phase and lower percentage of cancer cells in S phase. We also provided molecular evidences supporting the observed proliferation alterations after AATBC knockdown through evaluating the expression of proteins that play crucial role in G1 phase and the G1/S transition of the cell cycle. We found that knockdown of AATBC inhibited the cyclin D1, CDK4 and p-Rb accompanied by a decrease in total Rb, with an increasing expression of p18. To our knowledge, cyclin D1, CDK4 and Rb play important roles in this process [25]. Cyclin D1 promotes cells to go through the G1 phase via activating CDK4, which leads to increased phosphorylation of Rb (p-Rb) [26]. The Ink4 (Inhibitor of CDK4) family, such as p15 (INK4B) and p18 (INK4C), can specifically inhibit complex of CDK4-cyclin D and further decrease phosphorylation of Rb to regulate cell cycle [27]. Thus, the most likely mechanism underlying the growth arrest involves an increase in p18 expression, which leads to an inhibition of the complex of CDK4-cyclin D1 and phosphorylation of Rb, and then ultimately stimulate cell cycle arrest at the G1 phase. This cell cycle arrest was attributed to, at least in part, the anticancer effect of AATBC knockdown on proliferation. Collectively, these data shed light on a vital role of AATBC in promoting tumorigenesis and progression of bladder cancer. AATBC could be a potential therapeutic target for bladder cancer.
In the process of cell apoptosis, the caspase family is indispensable for the initiation and execution of cell death in response to various kinds of stimuli [28][29][30]. The intrinsic apoptotic signal can recruit and activate initiator caspase-9 and effector caspase (caspase3/6/7), which bring about cellular death ultimately. Our results showed that the knockdown of AATBC by RNAi technology led to the apoptosis of bladder cancer cells via the intrinsic apoptosis signal, as shown by the activation of caspase-9 and caspase-3. Moreover, cisplatin induction could significantly strengthen the pro-apoptotic ability of AATBC knockdown, indicating that apoptosis via AATBC inhibition could enhance the chemosensitivity of bladder cancer cells and AATBC might be an attractive therapeutic target in bladder cancer treatment.
The dysregulation of urothelial epithelial cell apoptosis is one of the causative factors of bladder cancer. Various factors may affect apoptosis of cancer cells through different signaling pathways [31,32]. JNK, as a serine-threonine kinase and a main member of the MAPK pathway, plays an important role in a variety of physiological and pathological processes, including cell death or apoptosis [33,34]. In this study, AATBC downregulation modulated the phosphorylation level of JNK and exerted its influence on apoptosis in bladder cancer cells. The interplay between AATBC and JNK signaling was highlighted by using the JNK inhibitor SP600125, which attenuate the pro-apoptotic ability of AATBC knockdown.
The transcription factor NF-E2-related factor 2 (NRF2) plays a major role in the unfolded protein response (UPR). It has been accepted widely that NRF2 is important in the protective system against oxidative stress, electrophilic stress and the other adverse condition through transcriptional regulation of the genes encoding antioxidant proteins, detoxification enzymes and other stress response regulators [35,36]. Elevated expression of NRF2 can protect cancer cells from harm by chemotherapeutic drugs, including cisplatin, doxorubicin and etoposide [37]. On the contrary, NRF2-deficiency enhances the susceptibility of cancer cells to hostile stimuli [38]. We showed for the first time that AATBC knockdown resulted in decreased level of NRF2, which might partly contribute to the higher apoptotic rates after adding of cisplatin with siRNAs compared to the no cisplatin-induced groups. Although our findings imply a molecular mechanism by which oxidative stress may involve in the apoptosis resulting from AATBC inhibition, further investigation is required to document this in the future.
Therefore, the pro-apoptotic effect of AATBC knockdown in bladder cancer may achieved through activation of JNK signaling and suppression of NRF2. AATBC may be an important regulator of apoptosis in bladder cancer. Its overexpression hiders the apoptosis of tumor cells and increases the number of tumor cells, thereby promoting tumor growth, which partially explain AATBC may serve as a potential oncogene in bladder cancer.
To conclude, the data presented in our study collectively provides convincing evidences that AATBC exhibits potentially pro-proliferative and anti-apoptotic functions in bladder cancer. The most possible mechanisms underlying these functions include disregulation in the cell cycle, activity of the intrinsic apoptosis that involved in JNK and NRF2 signaling pathway. Both in vitro and in vivo results strengthened the positive links between AATBC expression and clinical parameters, including tumor grading and pT stage. Therefore, AATBC could be a promising therapeutic target and molecular biomarker for bladder cancer.

Microarray and computational analysis
Total RNA from 5 pairs of bladder cancer with matched normal tissue specimens was extracted and purified by RNeasy mini kit (QIAGEN) with RNasefree DNase set (QIAGEN). These total RNAs were then amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Agilent), according to the manufacturer's protocols. Each Slide was hybridized with Cy3-labeled cRNA using Gene Expression Hybridization Kit (Agilent). After hybridization, slides were washed in staining dishes (Thermo Shandon) with Gene Expression Wash Buffer Kit (Agilent), following the manufacturer's instructions. Slides were scanned by Agilent Microarray Scanner (Agilent). The acquired array images were analyzed with Agilent Feature Extraction software (version 11.0.1.1). Quantile normalization and subsequent data processing were performed using the Gene Spring Software 11.0 (Agilent ). The microarray work was performed by KangChen Bio-tech, Shanghai, China.

Patients and tissue samples of bladder cancer
Ninety paired samples of bladder cancer and adjacent non tumor tissues (2 cm away from the tumor) were obtained from patients, who received cystectomy in Sun Yat-sen Memorial Hospital of Sun Yat-sen University. After resection, all samples were immersed immediately in RNAlater solution (Ambion, Austin, Texas) overnight, then stored at -80°C in order to avoid degradation of RNA. All the bladder cancer patients were graded according to the WHO grading system (low-grade, 16 cases; highgrade, 74 cases) and were staged according to the TNM classification (NMIBC Tis, Ta, T1: 41 cases; MIBC >T1: 49 cases). Prior to the use of these clinical materials for research purposes, written consents from all patients and approval of the Hospital Ethic Review Committees were obtained.

Cell culture
The human bladder cancer cell lines (UM-UC-3, T24 and 5637) and an immortalized normal urothelium cell line (SV-HUC-1) were purchased from the American Type Culture Collection (ATCC). Bladder cancer cell line EJ was obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). EJ, T24 and 5637 cell lines were routinely cultivated in RPMI-1640 (Gibco) while UM-UC-3 cell line was grown in DMEM (Gibco), and SV-HUC-1 in F-12K medium (Gibco). All the media contain 10% fetal bovine serum (FBS) (Bioind), 100 U/mL penicillin and 100 μg /mL streptomycin (Gibco). Cells were cultured and maintained under standard conditions of 5% CO 2 and 37°C.

Antibodies and reagents
Antibodies, including anti-cyclin D1, CDK4, p15, p18, Rb, p-Rb, caspase-3, caspase-9, PARP, JNK, p-JNK, p-P38, p-ERK1/2, β-Tubulin and HRP-linked secondary antibody were obtained from Cell Signaling Technology. The anti-NRF2 antibody was purchased from Bioworld Technology. All the antibodies were diluted 1:1000. SP600125, as a JNK inhibitor, was purchased from Sigma. SP600125 was dissolved in DMSO as a 20 mmol/L stock solution and stored at -20°C. To avoid precipitation of SP600125, DMSO was added to a final concentration of 0.1% in the media. UM-UC-3 and EJ cells were treated with 5 μmol/L SP600125 1 hour before transfection with siRNAs. Cisplatin was obtained from the institutional pharmacy (Qilu Pharmaceutical Co., China). In the groups of cisplatin-induced apoptosis of tumor cells, cisplatin was added at concentration of 1.5 μg/ml to UM-UC-3 ells and μg/ml to EJ 3 cells, respectively.

RNA extraction and quantitative RT-PCR
According to the manufacturer's instructions, total RNA from cell lines and tissue samples was extracted using Trizol reagent (Invitrogen). First-strand cDNA was synthesized with PrimeScript RT Master Mix (TAKARA, Dalian, China). After reverse transcription of the total RNA, quantitative RT-PCR was conducted to examine the expression of AATBC using SYBR-Green PCR Master mix (Roche) on LightCycler 480 Real-Time PCR instrument (Roche). β-actin was used as an internal reference gene to normalize RNA levels between different samples for an exact comparison of transcription level. The sequences of primers were as follows (in the 5' to 3' orientation): β-actin forward, ACTGGAACGGTGAAGGTGAC, β-actin reverse, AGAGAAGTGGGGTGGCTTTT; AATBC forward, AAGGCCGGTTATCAACGT, AATBC reverse, GCCAGTCCCTCACTGCTCT. Relative quantitative expression of AATBC was determined using the ΔΔCT method and presented as relative units.
In the animal experiment, for stable knockdown of AATBC in UM-UC-3 cells, we constructed lentiviruses containing AATBC shRNA, which was synthesized by Shanghai GeneChem Co. Ltd (Shanghai, China). The short hairpin RNAs (shRNAs) were cloned into the GV248-GFP lentiviral vector. The sh-AATBC targeting sequence was CAGATACACTGACTACGAT. A nontargeting scrambled shRNA GV248-GFP vector was generated as a negative control. These modified plasmids were further cotransfected into HEK 293T cells with lentiviral packaging plasmids to generate an AATBC shRNA-expressing lentivirus or a control shRNA-expressing lentivirus. For cell infection, UM-UC-3 cells were cultured in 6-well plates at a density of 3 × 10 5 cells per well and infected with the constructed lentiviruses expressing AATBC shRNA and scrambled shRNA, respectively. Forty-eight hours after infection, 1 mg/ml puromycin (Invitrogen) was added into the media to select puromycin-resistant clones. The inhibitory efficiency of AATBC was detected by quantitative RT-PCR.

5-ethynyl-2'-deoxyuridine (EdU) incorporation assay
UM-UC-3 and EJ cells (1.0 × 10 4 cells/well) were seeded into 24-well culture plates, followed by transfection with siRNAs to knockdown AATBC expression. Fortyeight hours after transfection, cell proportion in S-phase was analyzed by EdU assay (Cell-Light™ EdU DNA Cell Proliferation Kit, Ribobio, Guangzhou, China) according to the manufacturer's instructions. The procedures were as follows: cells were incubated in complete medium supplemented with 50 μM EdU for 2 hours at 37 °C, washed with phosphate-buffered saline (PBS) and then cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature. The excess paraformaldehyde was neutralized with 2 mg/ml glycine. After being washed with PBS and 0.5% Triton X-100 in PBS, cells were washed with PBS again and dyed with 1× Apollo® reaction cocktail in dark for 30 minutes. Cells were washed twice with 0.5% Triton X-100 in PBS again, then incubated with 1× Hoechst 33342 solution for 30 minutes in dark at room temperature. At the end, after washing with PBS for three times, cellular immunostaining was observed with fluorescence microscopy and photographed. Digital images were acquired and analyzed with Image J software.

CellTiter 96 Aqueous One Solution Cell Proliferation assay
The effect of AATBC knockdown on cell growth of UM-UC-3 and EJ was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega). One day after treatment of siRNAs, cells were trypsinized and collected. 1000 cells per well were seeded in 96-well plates and allowed to attach overnight. From the second day, CellTiter 96 Aqueous One Solution was added in at least five replicate wells at one-fifth of the total volume and incubated for 4 hours at 37°C. Absorbance was measured with the multifunctional microplate reader SpectraMax M5 (Molecular Devices) at 490 nm. The measurement of cell proliferation was conducted every 24 hours, and lasted 5 days. Cell growth curve was constructed with absorbance as ordinate and time as abscissa.

Colony formation assay
UM-UC-3 and EJ cells, treated by siRNAs and negative control for 24 hours, were routinely trypsinized and seeded in 6-well plates (1000 cells/well). The medium was changed every three days. After two weeks, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes, and then stained with crystal violet for 30 minutes for visualization and counting.

Flow cytometry assay
Flow cytometry analysis was performed to determine whether AATBC regulates the growth phase and apoptosis of bladder cancer cells. UM-UC-3 and EJ cells were seeded into 6-well plates. After tightly attaching to sidewall of plates, cells were transfected with www.impactjournals.com/oncotarget siRNAs. Forty-eight hours after transfection, the cells were harvested. In cisplatin-induced groups, cisplatin was added 24 hours before harvest. When evaluating the effect of JNK pathway blockage, the cells were pre-incubated with 5 μmol/L SP600125 for 1 hour, and then treated with siRNAs for 36 hours. The cells were stained with annexin V-FITC and propidium iodide (PI), according to the manufacturer's instructions. Cellular apoptotic rate was evaluated by FACSVerse TM flow cytometer (Becton Dickinson, CA, USA). Cells for growth phase analysis were resuspended in 200 μl PBS, fixed with 70% ice-cold ethanol overnight, and stained with PI. The cell cycle was detected by FACSVerse TM flow cytometer.

Protein extraction and Western blotting
Cells were rinsed twice with cold PBS and lysed by RIPA buffer (Thermo) containing protease inhibitor cocktail (Roche). Total protein was extracted according to the manufacturer's instruction. The concentration of protein was quantitated by bicinchoninic acid method. For Western blotting analysis, fifty μg protein of each sample was separated by electrophoresis in 10%~12% sodium dodecylsulfate-polyacrylamide gel and was then transferred to polyvinylidene fluoride membranes (Millipore). Blotted membranes were blocked in 5% skimmed milk in TBST, followed by incubation with appropriate primary antibodies overnight at 4°C. Then, the membranes were washed 5 minutes for three times with TBST, and subsequently incubated 1 hour with HRPlinked secondary antibody at room temperature. β-Tubulin was used as an internal control. The blots were detected by enhanced chemiluminescence kit (Millipore) and autoradiography using X-ray film.

Xenograft model in NOD/SCID mice
Male NOD/SCID mices (4 weeks, 14-16 g body weight) were raised under specific pathogen free (SPF) conditions. All the experimental procedures were performed according to the institutional ethical guidelines for animal experiment. For establishing bladder cancer xenograft model, UM-UC-3 cell line was used. Eight NOD/SCID mice were randomly divided into two groups: the lentiviral-mediated AATBC stable knockdown group (n=4) and the negative control group (n=4). Eight NOD/ SCID mice were subcutaneously injected into the flank with UM-UC-3 cells at the dose of 5 ×10 6 cells suspended in 200 μl PBS. Animals of both groups were sacrificed and tumor weight was measured after 4 weeks.

Statistic analysis
All the experiments were performed at least three times, and then mean values and standard deviation were calculated. Differences between two groups were analyzed by Student's t-test. The relationship between AATBC and the other clinical characteristics was determined using SPSS 22.0 Pearson Chi-square test. A value of P < 0.05 was considered to be statistically significant.