Nutrient Stress-Dysregulated Antisense Lncrna GLS-AS Impairs GLS-Mediated Metabolism and Represses Pancreatic Cancer Progression

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Author Manuscript Published OnlineFirst on December 18, 2018; DOI: 10.1158/0008-5472.CAN-18-0419 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Nutrient stress-dysregulated antisense lncRNA GLS-AS impairs GLS-mediated metabolism and represses pancreatic cancer progression

Shi-jiang Deng 1, 2, Heng-yu Chen 1, 2, Zhu Zeng 1, 2, Shichang Deng 1, Shuai Zhu 1, Zeng Ye 1,

Chi He 1, Ming-liang1 Liu, Kang Huang1, Jian-xin Zhong1, Feng-yu Xu1, Qiang Li1, Yang Liu1,

Chunyou Wang3, Gang Zhao 1,4

1Department of Emergency Surgery, Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan 430022, China.

2These authors contributed equally to this work.

3Deparment of Pancreatic Surgery, Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan 430022, China.

4Corresponding Author: Gang Zhao, Department of Emergency Surgery, Union Hospital,

Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022,

China.

Fax: +86-2785351669

Telephone: +86-2785351621

Email: [email protected]

Running Title: LncRNA-GLS-AS inhibits pancreatic cancer progression.

Key words: Pancreatic cancer, LncRNA, GLS-AS, Myc, Glutaminase

Financial support

This study was supported from the National Science Foundation Committee (NSFC) of China

(Grant number: 81372666 and 81672406 to G. Zhao).

Conflict of Interest Statement The authors declare no conflicts of interest to this work.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 18, 2018; DOI: 10.1158/0008-5472.CAN-18-0419 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract Cancer cells are known to undergo metabolic reprogramming such as glycolysis and glutamine addiction to sustain rapid proliferation and metastasis. It remains undefined whether long non-coding RNAs (lncRNA) coordinate the metabolic switch in pancreatic cancer. Here we identify a nuclear-enriched antisense lncRNA of glutaminase (GLS-AS) as a critical regulator involved in pancreatic cancer metabolism. GLS-AS was downregulated in pancreatic cancer tissues compared with noncancerous peritumor tissues. Depletion of GLS-AS promoted proliferation and invasion of pancreatic cancer cells both in vitro and in xenograft tumors of nude mice. GLS-AS inhibited GLS expression at the post-transcriptional level via formation of double stranded RNA with GLS pre-mRNA through ADAR/Dicer-dependent RNA interference. GLS-AS expression was transcriptionally downregulated by nutrient stress-induced Myc. Conversely, GLS-AS decreased Myc expression by impairing the GLS-mediated stability of Myc protein. These results imply a reciprocal feedback loop wherein Myc and GLS-AS regulate GLS overexpression during nutrient stress. Ectopic overexpression of GLS-AS inhibited proliferation and invasion of pancreatic cancer cells by repressing the Myc/GLS pathway. Moreover, expression of GLS-AS and GLS were inversely correlated in clinical samples of pancreatic cancer, while low expression of GLS-AS was associated with poor clinical outcomes. Collectively, our study implicates a novel lncRNA-mediated Myc/GLS pathway, which may serve as a metabolic target for pancreatic cancer therapy, and advances our understanding of the coupling role of lncRNA in nutrition stress and tumorigenesis.

Significance: Findings show that lncRNA GLS-AS mediates a feedback loop of Myc and GLS, providing a potential therapeutic target for metabolic reprogramming in pancreatic cancer.

Introduction Recent studies had shown that cancer cells exhibit metabolic dependencies to distinguish them from normal tissues. One of these addictions is “Warburg effect” that cancer cells tend to take advantage of glucose via “aerobic glycolysis” pathway, even in the presence of oxygen (1). As an outcome, the pyruvate generated via the aerobic glycolysis is converted to lactic acid, but not acetyl-CoA. In order to compensate for the insufficient citric acid cycle, cancer cells often active glutamine metabolism (2). Therefore, markedly aggravated glucose and glutamine depletion may happen in tumor cells as inadequacies between vascular supply and metabolic requirement (3). Such a situation is especially distinct in pancreatic cancer, of which glucose and glutamine metabolism is reprogrammed by oncogenic Kras to support cancer cell growth (4-6). Therefore, the metabolic characteristics and distinct hypovascular of pancreatic cancer would lead to a dramatic nutrients stress especially caused by glucose and glutamine depletion (7). In fact, such a paradoxical condition affords pathway to rapidly produce the energy and metabolites required for cancer cells proliferation, by which leading to correlatively resistant to metabolism stress including hypoxia and nutrient deprivation (8). Data from Yun suggest that glucose deprivation can drive the acquisition of Kras pathway mutations (9), which is commonly occurred in pancreatic cancer. Results suggested that glucose deprivation increases VEGF mRNA stability, which might facilitate tumor angiogenesis (10). Furthermore, results from Dejure et al. showed glutamine deprivation only halted the proliferation of colon cancer cells, but not killed them (11). Notably, nutrient deprivation has been correlated with poor patient survival, suggesting that instead of killing the tumor, the scarcity of nutrients can make the cancer cell stronger (12). Therefore, it is an emergency to investigate the mechanism that are required to accommodate nutrient stresses for alternative strategy of therapeutic treatment of pancreatic cancer. Long noncoding RNAs (lncRNAs) are a major class of transcripts, longer than 200nt and lack of coding-protein potential. Accumulating evidence suggests that

lncRNAs are dysregulated in cancers and involved in development of cancers (13). Specifically, recent results have demonstrated a link between lncRNAs and altered metabolism in cancers. Study reported that a glucose starvation-induced lncRNA-NBR2 reciprocally activates AMPK pathway in response to energy stress (14). LncRNA-UCA1 promotes glycolysis in bladder cancer cells by activating the cascade of mTOR-STAT3/miR143-HK2 (15). Results from Ellis et al. suggested that insulin/IGF signaling-repressed lncRNA-CRNDE promotes aerobic glycolysis of cancer cells (16). LncRNA-ANRIL is upregulated in nasopharyngeal carcinoma and promotes cancer progression via increasing glucose uptake for glycolysis (17). In addition, lncR-UCA1 was found to reduce ROS production, and promoted mitochondrial glutaminolysis in human bladder cancer (18). Nevertheless, the specific lncRNAs which couple nutrient stress and pancreatic cancer have not been elucidated yet. In the present study, we endeavoured to discover a nutrient stress responsive lncRNA which is involved in the pancreatic cancer progression. Glutaminase (GLS) is a phosphate-activated amidohydrolase that catalyzes the hydrolysis of glutamine to glutamate and ammonia to support metabolism homeostasis, bioenergetics, and nitrogen balance (19). Recent studies have revealed GLS is commonly overexpressed in numerous malignant tumors and acts as oncogene to support cancer growth (20, 21). It is noted that GLS is increased in breast cancers compared to surrounding nontumor tissues and positively correlate to the tumor grade (20). Moreover, GLS couples glutaminolysis of the TCA cycle with elevated glucose uptake and consequently the growth of prostate cancer cells (21). Meanwhile, knockdown of GLS significantly blocked the growth and invasive activity of various cancer cells (22). Results from chakrabarti et al. demonstrated that GLS is highly upregulated in pancreatic cancer, thereby targeting glutamine metabolism sensitizes pancreatic cancer cells to PARP-driven metabolic catastrophe (23). In previous study, we discover a cluster of dysregulated lncRNAs in pancreatic cancer (24). Coincidently, one of the significantly downregulated lncRNA, AK123493, is an antisense lncRNA of glutaminase (GLS). Thereby, it draws our attention whether GLS-AS might be

involved in the GLS-mediated metabolism of pancreatic cancer.

Materials and Methods Patients and specimens The clinical tissues were obtained from Pancreatic Disease Institute of Union Hospital from May 2016 to March 2017. We randomly selected 30 pairs of pancreatic cancer and corresponding non-tumor tissues from patients without chemotherapy or radiotherapy before operation. Procedures performed on those patients included pancreatectomy or palliative surgery including I125 seed implantation as well as gastroenterostomy and choledochojejunostomy according to the National Comprehensive Cancer Network (NCCN 2012) guideline for pancreatic cancer. The samples were obtained from surgical resection of patients or biopsy of the palliative surgery patients. The study was conducted in accordance with the Declaration of Helsinki. All samples were collected with the written informed consent of the patients, and the study was approved by the local Research Ethics Committee at the Academic Medical Center of Huazhong University of Science and Technology.

Cell culture BxPC-3 and PANC-1 cells were obtained from American Type Culture Collection (ATCC, USA). They were tested and authenticated for genotypes by DNA

fingerprinting within 6 months. Cells were cultured in 5% CO2 at 37°C and grown in complete medium, which was composed of 90% RPMI-1640(Gibco, USA), 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin and 100 mg/ml streptomycin. In order to build nutrition deprivation model, we incubated cells with complete medium without glutamine (Glutamine (-)) or complete medium with 1mM glucose (Glucose (-)). RPMI-1640 has no glutamine or glucose was purchased from Gibco, USA.

RNA fluorescence in situ hybridization To detect GLS-AS and GLS pre-mRNAs, we purchased a kit from named FISH

Tag™ RNA Multicolor Kit (Invitrogen, USA) to perform fluorescence in situ hybridization (FISH). The probe synthesis, labeling, and purification procedures were following manufacturer’s instructions. While the probe identified GLS pre-mRNA (Probe1) was labeled with green fluorescence, and the GLS-AS probe (Probe2) was labeled with red fluorescence. Cells were fixed in formaldehyde, permeabilized by Triton X-100 and then hybridization was carried out using labeled probes in a moist chamber at 42°C overnight. If necessary, the GLS protein immunofluorescence was conducted after all the FISH procedures were completed.

RNA-binding protein immunoprecipitation (RIP) To detect RNA-protein binding complexes, RIP assays were performed according to the instructions of RNA-Binding Protein Immunoprecipitation Kit (Magna RIP™, Millipore, USA). Firstly, cells were lysed in lysis buffer containing protease inhibitor cocktail and RNase inhibitor. Magnetic beads were pre-incubated with an anti-ADAR1 antibody or anti-Dicer for 30 minutes at room temperature, and lysates were immunoprecipitated with beads bound antibody at 4℃ overnight. Then immobilize magnetic beads bound antibody-protein complexes were obtained, washed off unbound materials, RNA was purified from RNA-protein complexes, and then was analyzed by qPCR.

Northern Blot We purchased a kit, DIG RNA Labeling Kit (Roche, Germany) to perform northern blot analysis for GLS-AS. Firstly, preparing GLS-AS specific DNA template containing T7 promoter sequences from RT-PCR, DNA template was purified. Then DIG-labeled RNA probes were produced according to the kit instructions with the DNA template. DIG-labeled probes were used for hybridization to nylon membrane blotted total RNA. The hybridized probes were detected with anti-digoxigenin-AP, and then were visualized with the chemiluminescence substrate CSPD. The signals were also captured by ChemiDocTm XRS Molecular Imager system (Bio-Rad, USA).

Co-immunoprecipitation (co-IP) For co-IP analysis, anti-ADAR1, anti-Dicer or normal mouse/rabbit IgG were used as the primary antibody, and then the antibody-protein complex was following incubated with Protein A/G PLUS-Agarose (Santa Cruz Biotechnology). The agarose-antibody-protein complex was collected and then analyzed by Western blot.

Chromatin immunoprecipitation (ChIP) The PCR primers are indicated in Supplementary Table S1. We conducted ChIP assays were by using EZ-ChIPTM Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA). All the procedures were performed according to the manufacturer's instructions. Rabbit anti-Myc (Cell Signaling Technology, USA), anti-RNA polymerase II antibodies (Abcam, UK) and corresponding rabbit-IgG (Cell Signaling Technology, USA) was used as controls. The bound DNA fragments were amplified by PCR reactions, and then PCR products were analyzed by gel electrophoresis on 2% agarose gel. The PCR primers used were listed in Supplementary Table S2.

Luciferase activity assay For GLS-AS promoter activity analysis, BxPC-3 cells were transfected with pGL3 vector wild type GLS-AS promoter or mutant GLS-AS promoter with firefly luciferase plasmid, while a plasmid pRL-TK carried Renilla luciferase, was used as internal reference. To confirm the nutrition deprivation impact on GLS-AS promoter activity, cells were cultured under glutamine or glucose deprivation for 24h or 48h. To investigate the relationship between Myc protein and GLS-AS promoter activity, siMyc or the control siNC was co-transfected into the BxPC-3 cells containing luciferase plasmid. The reporter activity was measured by using a luciferase assay kit (Promega, USA) and plotted after normalizing with respect to Renilla luciferase activity. Fireﬂy luciferase activity was normalized to the corresponding Renilla

luciferase activity. The data is represented as mean ±SD of three independent experiments.

Biotin-RNA pull-down assay The full or partial length of intron-17 of GLS gene sequences was amplified by PCR with SP6/T7-containing primer and then transcribed by MAXIscript™ SP6/T7 Transcription Kit (Thermo Fisher Scientific, MA, USA). The synthetic RNA was Biotin-labeled with Pierce™ RNA 3' End Desthiobiotinylation Kit (Thermo Fisher Scientific, MA, USA). The biotin-labeled RNAs were incubated with cell lysis individually and the target complexes were precipitated by Streptavidin-coupled Dynabeads (Invitrogen, USA). Lastly, whether GLS-AS were pulldown or not was identified by northern blot.

RNA pull-down by MS2-GST We conducted a plasmid expressing GLS-AS tagged with MS2 hairpin loops (GLS-AS-MS2), a plasmid expressing MS2-GST-NSL fusion protein and a plasmid only expressing MS2 (MS2) RNA as control. Pancreatic cancer cells in the test and control groups were transfected with GLS-AS-MS2 and MS2, respectively, along with MS2-GST-NSL fusion protein. After the co-transfection for 48h, cells were harvested and then conducted with RNA pull-down assay as described (25). The purified proteins were analyzed by western blot while RNAs were detected with northern blot.

Xenograft assay Lentivirus containing specific DNA sequences were transfected into BxPC-3 and PANC-1 cells. Five weeks old BALB/c male nude mice were bought from HFK Bio-Technology Co. (Beijing, China). To assess tumor growth in vivo, 100ul RPMI 1640 medium without FBS while containing 4×106 of cells was suspended in and then planted subcutaneously into the of nude mice, each group has 6 mice. Tumor volumes were measured every 4 days according to the formula V= 0.5×L (length) ×W2 (width).

Mice were sacrificed at 3 weeks after cell inoculation. Solid tumor tissues were removed and weighed. To investigate tumor metastasis in vivo, mice were injected with 1×104 tumor cells through the tail vein, tumor visible metastases on liver were counted and then confirmed by H&E stain slides after 3 weeks. Care and handling of the mice were approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology.

Statistical analyses All results were presented as means ± standard deviation (SD). Comparisons between two groups were performed with Student’s t-test. The correlation between GLS-AS and GLS mRNA or GLS and Myc mRNA was revealed by Pearson correlation analysis. And expression of GLS-AS and the clinical characteristics were analyzed by chi-square test, while the log-rank test was conducted to survey pancreatic cancer patient survival. Difference was regarded to be significant at * p<0.05, ** p<0.01.

Results AK123493.1, a nuclear accumulated antisense lncRNA of GLS (GLS-AS), is downregulated in pancreatic cancer The microarray analysis showed AK123493.1 was obviously decreased in the pancreatic cancer (PC) tissues compared to noncancerous peritumoral (NP) tissues (Figure 1A). GLS-AS is an intronic antisense lncRNA embedded within intron-17 of the corresponding sense gene GLS (Figure 1B). In addition, the northern blot validated the expression of GLS-AS in BxPC-3 and PANC-1 cells by using RNA probe (Figure 1C). Moreover, the expression levels of GLS-AS in BxPC-3 and PANC-1 cells were lower than that in the normal human pancreatic duct epithelial cells (HPDE) (Figure 1D). To validate the signal specificity, northern blot analysis was conducted after cells were transfected with siGLS-AS. As shown in Figure 1E, siGLS-AS significantly decreased the expression of GLS-AS. Furthermore, the

northern blot results showed that GLS-AS was obviously lower in PC tissues compared to NP (Figure 1F). Meanwhile, the fluorescence in situ hybridization (FISH) assay showed that GLS-AS is mainly accumulated in the nucleus (Figure 1G), implying GLS-AS may predominantly function in the nucleus. Similarly, separation of nuclear extract and the cytoplasmic fraction showed that GLS-AS retained in the nucleus (Figure 1H). Both Coding-Potential Assessment Tool (26) and Coding Potential Calculator (27) predicted GLS-AS is a non-coding RNA. Furthermore, we blocked new RNA synthesis with RNA polymerase II (Pol II) inhibitor α-amanitin (50μM) in BxPC-3 cells and measured the expression of GLS-AS by qPCR relative to time 0. After treated with α-amanitin, the expression of GLS-AS was significantly decreased, while the 18s mRNA which is transcribed by Pol I was not affected. These results indicated GLS-AS is a Pol II-dependent transcription. (Figure 1I). We further validated the GLS-AS expression level in PC tissues and paired NP tissues by qPCR. Results showed that GLS-AS expression in PC was significantly lower than that in NP (Figure 1J). In addition, the low expression of GLS-AS was associated with large tumor size, lymph node invasion, remote metastasis (Supplementary Table S1) and short overall survival time (Figure 1K).

Low expression of GLS-AS facilitates proliferation and invasion of pancreatic cancer cells To understand the roles of GLS-AS downregulation in pancreatic cancer progression, we depleted GLS-AS expression with siRNA (siGLS-AS) in pancreatic cancer cells (Figure 2A, Supplementary Figure 1A). After downregulation of GLS-AS, the proliferation and colony formation of PANC-1 (Figure 2B, C) and BxPC-3 cells (Supplementary Figure S1B, C) were significantly enforced. Meanwhile, transwell and wound healing assays further revealed an enhanced invasion and migration ability in GLS-AS-depleted PANC-1(Figure 2D, E) and BxPC-3 cells (Supplementary Figure S1D, E). To further confirm whether reduced GLS-AS affects pancreatic cancer progression in vivo, PANC-1 and BxPC-3 cells were stably transfected with lentivirus

containing siGLS-AS (LV-siGLS-AS) or siNC (LV-NC) were transplanted subcutaneously into the mouse respectively. Compared with LV-NC group, the PANC-1 tumor in LV-siGLS-AS group were larger and heavier (Figure 2F), with more visible liver and lung metastases (Figure 2G, H). Similarly, the proliferation and metastasis of BxPC-3 tumor with LV-siGLS-AS were also enhanced (Supplementary Figure S1F-H). These results indicate that the dysregulated GLS-AS expression might contribute to pancreatic cancer development.

GLS is the critical target of GLS-AS to exert function in pancreatic cancer Since GLS-AS is an antisense lncRNA of GLS, we further investigated whether GLS is a functional target of GLS-AS. Coincidently, knockdown of GLS-AS apparently increased the GLS expression both in mRNA and protein levels in both PANC-1 (Figure 3A) and BxPC-3 cells (Supplementary Figure S2A). Coincidently, transfection with a plasmid containing GLS-AS sequence (GLS-AS) obviously decreased GLS expression both in mRNA and protein levels of PANC-1 and BxPC-3 cells (Figure 3B, Supplementary Figure S2B). Subsequently, co-staining fluorescence of GLS-AS transcription and GLS protein further validated GLS was negatively regulated by GLS-AS in PANC-1 and BxPC-3 cells (Figure 3C, Supplementary Figure S2C). In agreement, the co-staining fluorescence assay further showed a decreased GLS-AS accompanied with increased GLS protein expression in PC tissue compared with NP tissue (Figure 3D). Meanwhile, western blot analysis further validated the downregulation of GLS protein in PC tissues compared to NP tissues (Supplementary Figure S2D). Meanwhile, depletion of GLS with siGLS remarkable inhibited proliferation, colony formation, invasion and migration ability of PANC-1 and BxPC-3 cells which was reinforced by siGLS-AS (Supplementary Figure S3A-J). Thus, these data implied that the GLS would be a critical target for dysregulated GLS-AS exert its biological function in pancreatic cancer.

GLS-AS inhibits GLS expression in post-transcription level by ADAR1/Dicer-dependent RNA interference To further identify whether GLS-AS regulates GLS transcription, BxPC-3 or PANC-1 cells were transfected with pGL3 plasmid containing GLS putative promoter region. As shown in Supplementary Figure S4A, B, the luciferase reporter assay showed neither knockdown nor overexpression of GLS-AS did not change the GLS promoter activity, which implied that GLS-AS did not regulate GLS expression in transcription level. The co-RNA FISH assay disclosed both GLS-AS and GLS pre-mRNA hybridized in the same nuclear foci of PANC-1 cells (Figure 4A), which indicating the formation of dsRNA. To further validate the direct interaction between GLS-AS and GLS-pre-mRNA in PANC-1 cells, the RNA-RNA pull-down assay was performed with biotin-labeled full length or partial deletion of intron-17 transcripts (Figure 4B). As GLS-AS is an antisense lncRNA of GLS pre-mRNA, we further presumed GLS-AS might regulate stability of GLS pre-mRNA. To evaluate the stability of GLS pre-mRNA, pancreatic cancer cells were treated with amanitin (50μM). The expression of GLS pre-mRNA was measured by qPCR at the separated time point. Compared to time 0, the stability was dramatically enhanced by siGLS-AS, but impaired by GLS-AS overexpression in PANC-1 cells (Figure 4C, D). Research had demonstrated that adenosine deaminases acting on RNA (ADAR1) is involved in RNA interfering of dsRNA by formation ADAR1/Dicer heterodimer complexes, a member protein of RNA-induced silencing complex (RISC) (28). Meanwhile, the co-IP analysis validated the binding between ADAR1 and Dicer protein in PANC-1 cells (Figure 4E, Supplementary Figure S5A). To confirm whether the ADAR1/Dicer proteins physically bound to GLS-AS/GLS pre-mRNA dsRNA or not, we performed RNA immunoprecipitation (RNA-IP) assays in PANC-1 cells. Compared with the IgG-bound sample, the ADAR1 or Dicer antibody-bound complex showed significant high enrichment of GLS-AS and GLS pre-mRNA in PANC-1 cells (Figure 4F, Supplementary Figure S5B). Thus, we then wonder whether the GLS-AS and GLS

pre-mRNA is regulated by ADAR1/Dicer-mediated RNA silencing. Both siADAR1 and siDicer increased the expression of GLS-AS, GLS mRNA and protein were remarkably increased in PANC-1 cells (Figure 4G, Supplementary Figure S5C), reduced the enrichment of GLS-AS and GLS pre-mRNA in protein (ADAR1/Dicer)-anti-body-bound complex in PANC-1 cells (Figure 4H, Supplementary Figure S5D), as well as strengthened the stability of GLS pre-mRNA in PANC-1 cells (Figure 4I, Supplementary Figure S5E). Meanwhile, the enrichment of GLS pre-mRNA was reduced by siGLS-AS but upregulated by GLS-AS overexpression both by anti-ADAR1 (Figure 4J, K) and anti-Dicer (Supplementary Figure 5F, G) in PANC-1 cells. Additionally, both siADAR1 (Figure 4L) and siDicer (Supplementary Figure S5H) could rescue the expression of GLS mRNA and protein in PANC-1 cells, which was inhibited by GLS-AS overexpression. Furthermore, MS2-tagged RNA affinity purification analysis was performed to further confirm GLS-AS, GLS-pre-mRNA and ADAR1/Dicer can form a complex in PANC-1 cells (Figure 4M). Simultaneously, the experiments described above were also conducted in BxPC-3 cells. Results of BxPC-3 cells also confirmed a interaction between GLS-pre-mRNA and GLS-AS (Supplementary Figure 6A, B) which could regulate the stability of GLS-pre-mRNA (Supplementary Figure 6C and D). Moreover, results further displayed that ADAR1 is required for the regulation of GLS-AS on GLS expression in BxPC-3 cells (Supplementary Figure S6E-M). In addition, results also validated that Dicer is necessary for the ADAR1/Dicer-mediated regulation of GLS-AS on GLS expression in BxPC-3 cells (Supplementary Figure S7A-H). Supplementary Figure S8 is a schematic diagram of MS2-tagged RNA affinity purification analysis.

Nutrient stress is responsible for downregulation of GLS-AS in pancreatic cancer We further investigated whether the GLS-AS downregulation in pancreatic cancer is attributed to metabolism stress including hypoxia, acidity, or depletion of glucose and glutamine. Interestingly, GLS-AS was obviously decreased during depletion of

glucose or glutamine, but without significant alteration in BxPC-3 and PANC-1 cells during hypoxia or acidity (Figure 5A). Moreover, qPCR and FISH assays demonstrated a time-dependent GLS-AS downregulation during glutamine or glucose deprivation (Figure 5B-D). Coincident with the GLS-AS downregulation, both GLS mRNA or protein expression was elevated during glutamine or glucose deprivation in a time-dependent manner (Figure 5E). Nevertheless, the expression of ADAR1 and Dicer showed no obvious change during glutamine or glucose deprivation in PANC-1 (Supplementary Figure S9A-C) and BxPC-3 cells (Supplementary Figure S9D-F), which further confirmed the critical function of GLS-AS in the regulation of GLS during nutrient stress. Thus, these results imply dysregulation of GLS-AS/GLS pathway in pancreatic cancer might, at least partially, is attributed to the nutrient stress including glucose or glutamine depletion.

GLS-AS is transcriptionally regulated by Myc under glucose and glutamine deprivation Myc is a multifunctional transcription factor that is deregulated in many human cancers and impacts cell proliferation, metabolism, and stress responses (29). Specifically, DNA sequence analysis showed GLS-AS promoter region contains potential binding sites for Myc (Figure 6A), thereby we presumed GLS-AS might be transcriptionally controlled by Myc. As expected, the chromatin immunoprecipitation (ChIP) assay verified only site 4 locating from -358 to -353bp on the GLS-AS promoter area could bind to Myc, but not site 1 to 3 (Figure 6B). To further confirmed the transcriptional activity of the putative GLS-AS promoter sequence, basic pGL3 plasmid and pGL3 plasmid containing GLS-AS promoter was transfected into BxPC-3 and PANC-1 cells. The luciferase reporter assay showed the luciferase intensity was enhanced in pGL3-GLS-AS promoter transfected cells (Figure 6C), which was further downregulated by siPol II (Figure 6D). In addition, ChIP analysis revealed that Pol-II also could bind to the binding site of Myc on GLS-AS promoter (Figure 6E). Furthermore, GLS-AS promoter sequence containing wild-type (WT) or

mutant site 4 (MUT) was transfected into pancreatic cancer cells. Results showed luciferase activity from the WT was markedly repressed by Myc overexpression, but increased after depletion of Myc (Figure 6F). Coincidently, siMyc substantially increased GLS-AS expression but decreased GLS expression (Figure 6G). These results indicate that GLS-AS is transcriptionally inhibited by Myc, which consequently increases GLS expression. Furthermore, both glucose and glutamine deprivation elevated Myc expression in BxPC-3 and PANC-1 cells (Supplementary Figure S10A). Specifically, the ChIP assay demonstrated the enrichment of GLS-AS promoter by Myc antibody was remarkably increased during glucose and glutamine deprivation (Supplementary Figure S10B). In addition, the decreased activity of GLS-AS promoter was noted during glucose or glutamine deprivation (Supplementary Figure S10C). Moreover, knockdown of Myc could increase GLS-AS expression in glutamine or glucose deprivation stress, coupled with GLS downregulation (Supplementary Figure S10D). Furthermore, Myc induced upregulation of GLS protein levels can be inhibited by GLS-AS overexpression under glutamine or glucose deprivation (Supplementary Figure S10E). Together, these results display the downregulation of GLS-AS in pancreatic cancer might be attributed to energy stress through Myc-depending regulation.

GLS mediates a reciprocal feedback between GLS-AS and Myc Results demonstrated GLS silencing mediates downregulation of Myc protein in glioma cells (30). Moreover, results from Andrew et al. showed that GLS inhibitor, CB-839 markedly reduced the protein levels of Myc in multiple myeloma (MM), acute lymphocytic leukemia, and non-Hodgkin’s lymphoma (31). Therefore, we wonder whether Myc can be regulated by GLS-AS/GLS pathway. Interestingly, GLS knockdown and GLS-AS overexpression significantly inhibited Myc expression at the protein level (Figure 7 A) but not mRNA level (Supplementary Figure S11A, B) in BxPC-3 and PANC-1 cells, which indicating GLS might regulate Myc expression in

post-transcription level. To evaluate whether GLS affects stability of Myc protein, Myc protein was measured in the presence of cycloheximide (CHX), which blocks de novo protein synthesis. The results showed the stability of Myc protein was decreased by GLS knockdown or GLS-AS overexpression in BxPC-3 and PANC-1 cells (Figure 7B, C). Besides, the proteasome inhibitor MG132 could rescue Myc protein level from the depression effect of GLS downregulation or GLS-AS ectopic expression in BxPC-3 and PANC-1 cells (Figure 7D). During the glutamine or glucose deprivation, both GLS knockdown and GLS-AS overexpression obviously inhibited the nutrient stress-induced Myc and GLS expression (Figure 7E). Furthermore, the GLS-AS depletion-induced Myc expression was inhibited by siGLS (Figure 7F) in nutrition deprivation condition. All of these results imply that GLS-AS might regulate Myc expression in protein level in proteasome pathway through GLS-dependent manner.

GLS-AS is conversely correlated with Myc and GLS expression in pancreatic cancer In agreeing to the in vitro and in vivo results, the clinical samples of pancreatic cancer demonstrated an increased expression of GLS mRNA which was conversely correlated with GLS-AS (Supplementary Figure S12A, B). In addition, Myc mRNA was upregulated in pancreatic cancer tissues and associated with GLS mRNA expression (Supplementary Figure S12C, D). Meanwhile, immuno-histochemical analysis validated the overexpression of Myc and GLS in pancreatic cancer tissues (Supplementary Figure 12E). However, analysis of pancreatic cancer database (QCMG and TCGA) by cBioportal revealed that the Pearson correlation value of Myc and GLS mRNA is only -0.007 and -0.049 (32, 33), respectively (Supplementary Figure S12F, G). Similarly, although a positive correlation between Myc and GLS was shown in prostate cancer tissues (34), the similar correlation was not seen in breast tumors and c-Jun was shown to drive GLS expression (35). Therefore, these different results indicate that the Myc-GLS correlation is not universal in human tumors but exist more strongly in a specific sub-group of tumor samples. Also a

possibility, there are multiple mechanisms involved in GLS mRNA regulation causing this complex and diverse complex scenario.

GLS-AS may be a vital therapeutic target for pancreatic cancer treatment To further validate the function of GLS-AS in pancreatic cancer development, BxPC-3 and PANC-1 cells were transfected with GLS-AS overexpression plasmid (GLS-AS) or empty vector (vector) respectively. GLS-AS overexpression effectively inhibited proliferation, as well as invasion and migration ability of BxPC-3 and PANC-1 cells (Supplementary Figure S13A-D). To further validate the function of GLS-AS in vivo, we transfected PANC-1 and BxPC-3 cells with a lentivirus containing GLS-AS (LV-GLS-AS) or the control (LV-vector). Then the transfected cells were transplanted subcutaneously into the nude mouse to investigate the tumor growth and metastasis. Results showed that PANC-1 tumors of LV-GLS-AS group were smaller and lighter than LV-vector group (Supplementary Figure S14A). Moreover, the number of liver and lung metastases in the LV-GLS-AS group was considerably less than that in the LV-vector group (Supplementary Figure S14B, C). Furthermore, the tumor with LV-GLS-AS displayed higher GLS-AS expression, coupled with lower expression of GLS mRNA (Supplementary Figure S14D, E). Implanted BxPC-3 cells transfected with LV-GLS-AS also demonstrated impaired proliferation and metastasis in nude mice (Supplementary Figure S15A-E). Together, these results suggest GLS-AS may be a novel metabolic target for therapeutic treatment of pancreatic cancer.

Discussion Recently, accumulative researches have revealed that lncRNAs play key roles in modulating various aspects of cancer cellular properties, including proliferation, survival, migration, genomic stability and metabolism (36). Remarkably, aberrant expression of lncRNAs is identified in pancreatic cancer, whether the function of lncRNAs coupling the metabolism and tumorigenesis is far from elucidated (37). In

our present research, we discovered a novel lncRNA GLS-AS was significantly downregulated in pancreatic cancer, and associated with worse clinical outcomes. In addition, the downregulation of GLS-AS dramatically enhanced proliferation and invasion of pancreatic cancer cells both in vitro and in vivo. Therefore, these results intensively indicate that GLS-AS might function as an inhibitor in the progression of pancreatic cancer. Antisense lncRNAs are a cluster of lncRNAs transcribed from the opposite DNA strand compared with sense transcripts (38, 39). Recent findings have shown that antisense lncRNA can regulate the expression of sense gene by acting as epigenetic regulators of gene expression and chromatin remodeling. The antisense transcript for β-secretase-1 (BACE1-AS) is elevated in Alzheimer’s disease, which increasing BACE1 mRNA stability and generating additional amyloid-β through a post-transcriptional feed-forward mechanism (40). Antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level through an embedded SINEB2 repeat (41). In the present study, both GLS mRNA and protein expression was inhibited or increased by GLS-AS overexpression or downregulation. Moreover, GLS knockdown significantly decreased proliferation and invasion of pancreatic cancer cells which was promoted by downregulation of GLS-AS. Furthermore, the clinical samples demonstrated a reversed correlation between GLS-AS and GLS expression. Therefore, our findings indicate GLS is a critical target for GLS-AS exerting inhibition effects on pancreatic cancer. ADAR is a family of enzymes with double stranded RNA (dsRNA) binding domains that converts adenosine residues into inosine (A-to-I RNA editing) specifically in (dsRNA) (28, 42). To date, three ADAR gene family members (ADAR1-3) are discovered in mammals (43). ADAR1 differentiates its functions in RNA editing and RNAi by formation of either ADAR1/ADAR1 homodimer or heterodimer complexes with Dicer (28). Results showed that PCA3, an antisense intronic lncRNA of PRUNE2, forms a dsRNA which undergoes ADAR-dependent RNA editing to downregulate PRUNE2 level (44). However, genome-wide screening

has revealed numerous RNA editing sites within inverted Alu repeats in introns and untranslated regions (43). ADAR1 promotes pre-microRNA (miRNA) cleavage and siRNA process by forming a Dicer/ADAR1 complex (28). Meanwhile, the FISH assay showed a co-localization of GLS-AS and GLS pre-mRNA. Moreover, we found that GLS-AS did not affect transcription of GLS, but impaired stability of GLS pre-mRNA. Moreover, RIP assay further identified both ADAR and Dicer could bind to GLS-AS and GLS pre-mRNA simultaneously. Nevertheless, downregulation of ADAR1 or Dicer increased GLS expression, and also rescued the GLS-AS-induced inhibition of GLS. Therefore, these results intensively imply that GLS-AS inhibits GLS expression in post-transcription level via ADAR1/Dicer-dependent RNA interfering. Recent research showed that a part of lncRNA was dysregulated in cancer due to nutrient stress including glucose deprivation, hypoxia and so on (14, 24, 45, 46). Therefore, we further investigated whether the GLS-AS downregulation is attributed to nutrient stress including deprivation of glucose and glutamine, hypoxia, and acidity. Interestingly, only deprivation of glutamine and glucose dramatically decreased GLS-AS expression, but increased GLS expression. Nevertheless, overexpression of GLS-AS dramatically inhibited the survival and invasion of pancreatic cancer cells in nutrient stress. These results imply the dysregulated GLS-AS/GLS pathway is an adaption to nutrient stress and is required for the pancreatic cancer progression. We further explored the mechanism for downregulation of GLS-AS during nutrient stress. The Myc oncogene is a "master regulator" which controls glucose and glutamine metabolism to maintain growth and proliferation of cancer cells (47). Research demonstrated deprivation of glucose or glutamine dramatically elevated Myc expression and further activated serine biosynthesis pathway (SSP) (48). Results from Wu et al. also showed glucose deprivation upregulates Myc protein in BxPC-3 and PANC-1 cells (49). Meanwhile, study demonstrated Myc-induced mouse liver tumors significantly increase both glucose and glutamine catabolism with GLS upregulation (50). Results indicated Myc is a dual-functional transcription factor

which may activate or repress coding or no-coding RNA expression. Hart et al. showed that 534 lncRNAs are either up- or downregulated in response to Myc overexpression in P493-6 human B-cells (51). Zhang et al. showed a Myc-induced lncRNA-MIF inhibits aerobic glycolysis and tumorigenesis (52). On the contrary, Gao et al. reported that Myc transcriptionally represses miR-23a and miR-23b, resulting in greater expression of their target protein, GLS (34). Interestingly, the bioinformatic analysis demonstrated a putative Myc-binding site in the promoter area of GLS-AS gene. Moreover, the ChIP and luciferase reporter assays verified the binding and transcriptional inhibition of Myc on GLS-AS promoter. Coincidently, Myc knockdown significantly increased GLS-AS expression, but inhibited GLS expression. In addition, the deprivation of glucose and glutamine dramatically induced Myc expression and its transcriptional inhibition on GLS-AS. Consistently, our data showed knockdown of Myc dramatically increased GLS-AS expression during nutrient stress. Furthermore, Myc expression was increased and reversely correlated with GLS expression in pancreatic cancer. Therefore, these data indicate that GLS-AS might be transcriptionally inhibited by Myc, leading to GLS upregulation in response to nutrient deprivation. Different from Gao et al had reported a miRNA-mediated GLS regulation (34), our study affords a novel lncRNA-mediated mechanism for the regulation of Myc on GLS transcription, which linking the metabolism reprograms and progression of pancreatic cancer. Interestingly, recent results reminded a potential feedback between Myc and GLS. As elevated GLS activity is under regulatory control of Myc (50, 53) research observed that knockdown of GLS decreased Myc protein expression in glioma cells (30). Recently, Madlen et al demonstrated glutamine depletion with GLS inhibitor is reflected by rapid loss of Myc protein which is dependent on proteasomal activity (54). Similarly, our results showed that downregulation of GLS dramatically inhibited Myc protein expression by impairing its stability. Coincidently, the Myc protein during nutrient stress was also inhibited by GLS-AS overexpression. In addition, siGLS-AS dramatically increased Myc expression, but decreased by siMyc. Therefore,

our data provide further evidence for a reciprocal feedback of Myc and GLS-AS which regulates GLS expression via post-transcriptional level during nutrient deprivation. Given the regulatory mechanism for Myc is complex, the precise mechanism for the regulation of GLS on Myc protein stability needs investigation in the further research. In summary, our study implicates a nutrient stress-repressed lncRNA GLS-AS is involved in progression of pancreatic cancer through mediating reciprocal feedback of Myc and GLS. Furthermore, our findings suggest that the Myc/GLS-AS/GLS axis may be promising molecular targets for the nutrient restrict treatment of pancreatic cancer.

Acknowledgments This study was supported from the National Science Foundation Committee (NSFC) of China (Grant number: 81372666 and 81672406 to G. Zhao).

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Figure legends Figure 1. LncRNA-AK123493.1, an antisense of GLS (GLS-AS), is downregulated in pancreatic cancer. (A) Microarray analysis demonstrated LncRNA-AK123493.1 was distinctly downregulated in 2 pancreatic cancer (PC) samples compared to paired noncancerous pancreatic (NP) tissues. (B) Schematic diagram of GLS-AS and GLS gene location and relationship in genome. Arrows indicate transcript orientation. Probe-1 labeled with red fluorescence was used to detected GLS-pre-mRNA while probe-2 labeled with green fluorescence was for GLS-AS. (C) Northern blot analysis of GLS-AS in pancreatic cancer cells BxPC-3 and PANC-1 with RNA probe. (D) Northern blot analysis of GLS-AS in HPDE cells

compared to BxPC-3 and PANC-1 cells. (E) Northern blot analysis of GLS-AS in BxPC-3 and PANC-1 cells when GLS-AS were knocked down by siRNAs. (F) Northern blot analysis of GLS-AS in five paired cancer and noncancerous pancreatic (NP) tissues. (G) Fluorescence in situ hybridization (FISH) analysis showed the location of GLS-AS in BxPC-3 cells. (H) Histogram showed expression level of GLS-AS in the subcellular fractions of BxPC-3 and PANC-1 cells, analyzed by qPCR. RT-PCR products were run on a 2% agarose gel, U6 and GAPDH were separately used as nuclear and cytoplasmic markers. (I) After blocking new RNA synthesis with α-amanitin (50uM) in BxPC-3 cells, stability of GLS-AS was measured by qPCR compared to time 0. β-actin is transcribed by RNA polymerase II, while 18s RNA is a product of RNA polymerase I. (J) GLS-AS in 30 pancreatic cancer (PC) and corresponding adjacent noncancerous pancreatic tissues (NP) was measured by qPCR. (K) The Kaplan–Meier curves for overall survival analysis of patients with pancreatic cancer by GLS-AS expression. Expression levels of GLS-AS was categorized into “high” and “low” using the median value as the cutoff point. All data were presented as means ± SD of at least three independent experiments. Values are significant at **P< 0.01 as indicated.

Figure 2. Knockdown of GLS-AS facilitates pancreatic cancer proliferation and invasion. (A) qPCR analyzed the knockdown efficiency of GLS-AS by the three siRNAs in PANC-1 cells. (B) After transfected with siGLS-AS #2 or siGLS-AS #3, growth rate of the transfected PANC-1 cells was measured by MTT assays for 5 days. (C) Colony formation assay was performed in transfected PANC-1 cells. Relative colony number (left panel) and representative images (right panel) and are shown. (D) Transwell assay was conducted to observe the invasion ability of the transfected PANC-1 cells. The left histogram represents relative cell number while the representative images are shown on the right. (E) Migration ability of transfected PANC-1 cells was analyzed by wound healing assay. Representative images (left panel) and relative wound size (right panel) and are shown. (F-H) PANC-1 cells

transfected with lentivirus containing sequence of siGLS-AS (LV-siGLS-AS) or empty lentivirus vector (LV-siNC) were transplanted subcutaneously into nude mice to observe tumor growth (5×106 cells per mouse). (F) A photograph of representative nude mice and tumor is presented after 3 weeks when mice were sacrificed (left panel). The tumor volumes were measured every 4 days. Two groups of tumor weights were measured individually. (G) Histogram showed number of visible liver metastases per 5 sections in each nude mouse. Representative images of livers and corresponding H&E staining section vision. (H) Histogram showed number of visible lung metastases per 5 sections in each nude mouse. The representative H&E staining section vision of lungs with metastases. All data were presented as means ± SD of at least three independent experiments. Values are significant at *P < 0.05 and **P< 0.01 as indicated.

Figure 3. GLS is the critical target of GLS-AS in pancreatic cancer. (A) PANC-1 cells were transfected with GLS-AS siRNA (siGLS-AS) or siRNA negative control (siNC). The mRNA and protein level of GLS were analyzed by qPCR and Western blot, respectively. (B) PANC-1 cells were transfected with GLS-AS overexpression vector (GLS-AS) or empty vector as negative control (Vector). The mRNA and protein level of GLS were analyzed by qPCR and Western blot, respectively. (C) Combined immunofluorescence of GLS protein (red) and RNA-FISH analysis of GLS-AS (green) in PANC-1 cells transfected with siGLS-AS or ectopic GLS-AS were compared to the negative control cells individually. (D) Combined immunofluorescence of GLS protein (red) and RNA-FISH analysis of GLS-AS (green) in pancreatic cancer (PC) and corresponding noncancerous pancreatic (NP) tissues. All data were presented as means ± SD of at least three independent experiments. Values are significant at *P < 0.05 and **P< 0.01 as indicated.

Figure 4. GLS-AS inhibits GLS expression via ADAR1/Dicer-dependent RNA silencing in PANC-1 cells. (A) Co-RNA-FISH analysis of GLS-AS and

GLS-pre-mRNA transcripts was performed with specific probe which is against GLS-pre-mRNA (probe1, red) or against GLS-AS (probe2, green) in PANC-1 cells. (B) Biotin-labeled RNAs containing full or part length of intron-17 were subjected to RNA-RNA pulldown assay and the pulldown GLS-AS was analysed by northern blot. (C) and (D) After treated with α-amanitin (50uM), stability of GLS-pre-mRNA was measured by qPCR compared to time 0 in PANC-1 cells transfected with siGLS-AS or GLS-AS plasmid. (E) The representative Western blot of the co-IP analysis with anti-ADAR1 or IgG antibody validated the binding between ADAR1and Dicer protein. (F) RIP assay detected the relative quantification of GLS-AS and GLS pre-mRNA in RIP with ADAR1 or IgG antibodies from cell lysis, measured by qPCR assays. (G) After transfected with siADAR1, the expression of GLS-AS and GLS transcription was detected by qPCR in PANC-1 cells, Western blot assay showed the expression of GLS and ADAR1 protein of treated PANC-1 cells. (H) After knockdown of ADAR1 and Dicer, RIP assay was performed with ADAR1 antibody, relative enrichment of GLS-AS and GLS pre-mRNA were measured by qPCR. (I) After transfected with siADAR1, cells were treated with α-amanitin (50uM), stability of GLS-pre-mRNA was measured by qPCR compared to time 0. (J) and (K) After knockdown or overexpression of GLS-AS, RIP assay was performed with ADAR1 antibody, relative enrichment of GLS-AS and GLS pre-mRNA were measured by qPCR. (L) After co-transfection with GLS-AS or siADAR1, the expression of GLS and GLS-AS were examined by qPCR or Western blot, respectively. (M) Western blot analysis of Dicer and ADAR1 protein levels in the pull-down complex. And the GLS-AS and GLS-pre-mRNA measured by northern blot. All data were presented as means ± SD of at least three independent experiments. Values are significant at *P < 0.05 and **P< 0.01 as indicated.

Figure 5. Nutrient stress is responsible for downregulation of GLS-AS in pancreatic cancer cells. (A) BxPC-3 and PANC-1 cells were exposed to stressors including hypoxia, acidosis, glucose or glutamine starvation. The GLS-AS level was

measured by qPCR and normalized to GAPDH as an endogenous control. (B) qPCR analysis detected the GLS-AS expression during glutamine or glucose deprivation in gradient time. (C) and (D) Representative images of RNA-FISH analysis displayed the GLS-AS expression during glutamine or glucose deprivation in gradient time. (E) Expression of GLS mRNA and protein were measured during glutamine or glucose deprivation conditions, respectively. All data were presented as means ± SD of at least three independent experiments. Values are significant at *P < 0.05 as indicated.

Figure 6. GLS-AS is transcriptionally inhibited by Myc under glucose and glutamine deprivation. (A) Schematic illustration showed the GLS-AS promoter region and the 4 sites of potential Myc binding sites. (B) ChIP assay with anti-Myc antibody or IgG was conducted to explore the binding capacity between Myc and GLS-AS promoter in BxPC-3 and PANC-1 cells. (C) Luciferase activity assays were performed in BxPC-3 and PANC-1 cells transfected with pGL3 reporter vector containing GLS-AS promoter or the pGL3 basic vector as control. The luciferase density was measured when cells were transfected for 48h. (D Luciferase activity assays of GLS-AS promoter and Western blot of Pol II were performed in BxPC-3 and PANC-1 cells after knockdown of Pol II. (E) ChIP analysis with anti-Pol II antibody or IgG was conducted to reveal the binding capacity between Pol II and site-4 sequences on GLS-AS promoter. (F) After overexpression or knockdown of Myc in BxPC-3 and PANC-1 cells, the luciferase activity of BxPC-3 and PANC-1 cells transfected with reporter containing wild-type GLS-AS promoter (WT) or mutant type (MUT) was measured. The site-4 potential binding sequences were mutated as indicated. (G) After knockdown of Myc with siMyc, expression of GLS-AS, GLS mRNA and protein in BxPC-3 and PANC-1 cells were measured by qPCR or Western blot, respectively.

Figure 7. GLS mediates a reciprocal feedback between GLS-AS and Myc in PANC-1 cells. (A) Western blot analysis of Myc protein in BxPC-3 and PANC-1 cells

upon GLS knockdown or GLS-AS overexpression. (B) and (C) After treated with siGLS or GLS-AS overexpression, the stability of Myc protein in BxPC-3 and PANC-1 cells were compared to time 0 for periods of time with treatment of cycloheximide (CHX, 50ug/ml). (D) Expression of Myc protein in BxPC-3 and PANC-1 cells, which were treated with the proteasome inhibitor MG132 (20uM) and simultaneously transfected with siGLS or GLS-AS. (E) The expression of Myc and GLS protein were analyzed in BxPC-3 and PANC-1 cells treated with siGLS or GLS-AS overexpression during glucose or glutamine deprivation, respectively. (F) BxPC-3 and PANC-1 cells were co-transfected with siGLS-AS and siGLS or the paired NC, and then cultured in glutamine or glucose deprivation medium for 48

hours. Western blot analysis of GLS and Myc in those cells were conducted.

Nutrient stress-dysregulated antisense lncRNA GLS-AS impairs GLS-mediated metabolism and represses pancreatic cancer progression

Shi-jiang Deng, Heng-yu Chen, Zhu Zeng, et al.

Cancer Res Published OnlineFirst December 18, 2018.

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