IntroductionTriple negative breast cancer (TNBC) is a prevalent form of breast cancer that is often found in women globally1,2,3. It is well characterized based on the molecular typing, which lacks of the hormone receptor and human epidermal growth factor receptor 2 (HER2). TNBC constitutes nearly 20% of breast cancer cases in China and is more often diagnosed in women under the age of 504. Compared to other breast cancer subtypes, TNBC usually associates with a worse prognosis and lower survival rates because of its high invasiveness and tendency to metastasize and relapse. The specific molecular traits of TNBC render it unresponsive to endocrine and molecular targeted therapies, with chemotherapy continuing to be the main treatment. Nevertheless, the outcome of TNBC patients is unfavorable due to the low response rate and drug resistance, which indicates the imperative to investigate new targets and develop novel therapeutic strategies for TNBC.Genome sequencing technology uncovered that up to 98% of human genome lacks protein encoding ability. Among this big proportion, transcripts over 200 nucleotides in length are identified as long non-coding RNAs (lncRNAs). As key regulatory genes, lncRNAs have significant roles in the development and advancement of tumors5,6,7. Increasing evidence has suggested that dysfunction of lncRNAs is closely associated with cancer cell growth, transformation, apoptosis and chemoresistance6,8,9,10. LINC02159 showed increased expression in cases of non-small cell lung cancer (NSCLC)11. Depletion of LINC02159 retarded NSCLC cell proliferation, migration and in vivo tumor growth11. The cancer-promoting function of LINC01259 indicated that LINC02159 could be used as a diagnostic marker and treatment target for NSCLC. Interestingly, our current data revealed that LINC02159 was up-regulated in TNBC, but not the non-cancer tissues. However, the function of LINC02159 in TNBC remains poorly understand. Considering its correlation with tumor progression, the function of LINC02159 in TNBC was explored in this study, which has not been reported.Accelerated glycolysis is a well-characterized metabolic reprogramming in cancer. Unlike normal cell, cancer cell relies on glycolysis rather than mitochondrial oxidative phosphorylation to generate energy, which assists the malignant cells to bona-fine their microenvironment to survive12,13. Due to the unique characteristics of cancer cells, targeting key enzymes involved in glycolysis, such as hexokinase, glucose transporters, pyruvate kinase were reported as promising anti-cancer therapeutic strategy. Glucose-6-phosphate isomerase (G6PI) is a wildly expressed glucose phosphate isomerase, which interconverts D-glucose-6-phosphate and D-fructose-6-phosphate, acting importantly in both glycolysis and gluconeogenesis14,15. Increasing studies have reported that G6PI was specifically secreted by cancer cells14,16. The malfunction of G6PI has been observed in numerous cancers, potentially serving as an independent prognostic marker17. Notably, lncRNAs were reported to regulate glucose metabolism via modulating the expression or activation of predominant glycolysis enzymes, which consequently, participating in cancer progression and therapeutic resistance18. However, the regulation of G6PI function by lncRNAs in cancer is still not well understood.Our study discovered that LINC02159 was abundantly expressed in TNBC and linked to the advanced stages of TNBC patients. The presence of LINC02159 was vital for TNBC cells’ proliferation, migration, and in vivo tumor expansion. Mechanism study revealed that LINCC02159 modulated the function of G6PI in TNBC via sponging miR-1285-3p. These results provided novel understanding about the role of LINC02159 in TNBC.Materials and methodsThis study was reported in accordance with ARRIVE guidelines and approved by the Ethics Committee of Shanxi Province Cancer Hospital. For experiments using human tissues, informed consent was obtained from all participants and performed in accordance with the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations. Materials and Methods are available as below.Tissue samplesBetween October 2019 and June 2020, TNBC tissues and their corresponding normal tissues were gathered at the Shanxi Province Cancer Hospital. The samples were promptly stored in liquid nitrogen before conducting experiments. None of the participants received any treatments previously. The experiment was performed in according with the Declaration of Helsinki. Approval for this study was granted by the Ethics Committee of Shanxi Province Cancer Hospital, with informed consent obtained from all participants.Cell line and transfectionThe TNBC cell lines (MDA-MB-231, BT-549, HCC-1937 and SUM-159) and normal MCF10A cell were sourced from the American Type Culture. Cells were incubated at 37 °C with 5% CO2. MDA-MB-231 cells were cultured in the Leibovitz’s L-15 medium, while BT-549, HCC1937 and SUM-159 cells were cultured in RPMI-1640 medium, which was supplemented with 10% fetal bovine serum (FBS, Gibco). MCF10A cells were cultured in DMEM/F12 (1:1) medium with the addition of 5% horse serum (16050122, ThermoFisher Scientific), 20 ng/ml EGF (P5552, Beyotime), 0.5 mg/ml Hydrocortisone (40109ES08, Yeasen), 100 ng/ml Cholera Toxin (707306ES76, Yeasen) and 10 µg/ml insulin (P3376, Beyotime). Transfection of 25 nM siRNA-control, siRNA-LINC02159, or miR-1285-3p was performed using the Polyethylenimine Linear.RT-qPCR analysisThe extraction of total RNA from tissues or cells was performed using Trizol solution (Invitrogen, Shanghai), and its quantity was determined using the Nanodrop 2000. For cDNA synthesis, 0.5 µg of RNA was reverse transcribed with the PrimeScript RT Kit (Takara, Japan). RT-qPCR analysis was done by the SYBR Green Master Mix on the Light Cycler 480 platform. U6 RNA and GAPDH were selected as internal references for miR-1285-3p and G6PI expression detection, respectively. The primer sequences were: LINC02159-forward: 5’-CCACCCCTTTCCCTGTAAGAG-3’ and reverse: 5’-GGCCTCTGTCATGTCTGCTT-3’; miR-1285-3p forward: 5’-TCTGGGCAACAAAGTGAGACCT-3’ and universal reverse primer; G6PI-forward: 5’-CTTCTTGGCCCAGACAGAGG-3’ and reverse: 5’- TGGTTGGGCGATTTCCTTCA-3’.Cell proliferation assayThe Cell Counting Kit-8 (CCK-8) assay was employed to evaluate the effect of LINC02159 on TNBC the proliferation cells. Cells transfected with siRNA control or siRNA-LINC02159 were seeded into the 96-well plate with 1000 cells per well. After cultured overnight, fresh medium containing 10 µl of CCK-8 solution (Solarbio, Beijing, China) was added for 1 h at the 37 °C incubator and the absorbance of each well at the wavelength at 450 nm was detected.Wound healing assayThe cell migration ability was detected via the scratch assay. TNBC cells (5*105) expressing siRNA-control or siRNA-LINC02159 were seeded in the 6-well dish. When cells achieved a 100% confluent monolayer, the wound was generated using a 200 µl pipette tip, and gently washed to remove the detached cells and debris. Acquire time zero images of the wound with microscope. Cells were then incubated in the fresh complete growth medium at 37 °C, with 5% CO2. After 24 h, the wound monolayer cells were photographed and the wound distance was calculated using ImageJ software (Bethesda, USA).RNA Immunoprecipitation assayTNBC cells were harvested by centrifugating at 3,000 rpm for 5 min at 4 °C. The cells were lysed with the RIPA lysis buffer (P0013K, Beyotime, Shanghai, China) on ice for 30 min with the addition of protease inhibitor and RNase inhibitor. The supernatant was collected and then centrifugated at 12,000 rpm for 15 min at 4 °C. And then, 50 µl of the supernatant was reserved as the input, while the remaining was incubated with 30 µl Protein A + G Magnetic Beads (PB101, Vazyme, Nanjing, China) and 5 µg antibody against Ago2 (04-642, Sigma, Shanghai, China) overnight at 4 °C with constant rotation. IgG was used as the negative control. The beads were collected via centrifugation at 3,000 rpm and the non-specific binding components were washout with washing buffer. The input and immunoprecipitated RNAs were extracted using Trizol reagent (15596026CN, ThermoFisher Scientific, Shanghai, China), followed by reverse-transcription and real-time qPCR analysis.Western blotAfter cells were transfected with indicated expressing vectors, the extraction of total protein from TNBC cells was done using RIPA buffer on ice for 15 min. Proteins were equally separated by 15% SDS-PAGE and transferred onto a PVDF membrane. 5% non-fat milk was added to block the membrane at RT for 1 h with followed by an overnight incubation with primary antibodies against G6PI (#57893, Cell Signaling Technology), GAPDH (ab8245, Abcam) or Flag antibody (#2368, Cell Signaling Technology) at 4 °C. After extensive PBS washing, the membrane was incubated for 1 h with the secondary antibody at RT. Enhanced chemiluminescence substrate (GE Healthcare) was employed to visualize the protein bands.Target prediction and luciferase reporter assayTo detect the potential binding of LINC02159 with miR-1285-3p, the transfection of TNBC cells was carried out with a reporter vector containing wild-type (WT) or mutated (Mut) LINC02159 and miR-1285-3p or a miRNA mimic control, using Lipofectamine 2000. Additionally, the interaction between the 3’-UTR of G6PI and miR-1285-3p was determined by co-transfecting reporter plasmid expressing the WT or Mut 3’-UTR of G6PI and miR-1285-3p. The Dual-Luciferase Reporter Assay System from Promega was employed to detect the luciferase activity 48 h post-transfection, adhering to the manufacturer’s instructions.In vivo tumor growth assay.Female nude mice aged 6–8 weeks were purchase from the Vital River Co., Ltd (Beijing, China). To create the xenograft mouse model, mice received subcutaneous injections of MDA-MB-231 cells that were transfected with lentiviral vectors expressing either shRNA-control or shRNA-LINC02159. Tumor volume was assessed twice weekly and calculated using the formula: 1/2 x length2 x width (mm3). At day 30, mice were euthanized via carbon dioxide (CO2) inhalation using a gradual-fill method with the displacement rate from 10% to 30% of the chamber volume/min. When the mouse fainted and lost the ability to exercise, the gas flow rate was increased, and the maximum flow rate was not exceeded 0.5KPa. After confirming that the mouse was motionless and not breathing, turned off CO2 after pupil dilation, and then observed for two minutes to confirm that the mouse was dead. And then the tumors were isolated, weighted and collected for further analysis. This research received approval from the Ethics Committee of Shanxi Provincial Cancer Hospital and performed according to the ARRIVE guidelines19.Glucose consumption and lactate production analysisTo assess glucose consumption in TNBC cells, the Glucose Uptake Assay kit (ab136955, Abcam, UK) was employed as per the manufacturer’s guidelines. Cells expressing either shRNA-control or shRNA-LINC02159 were briefly seeded into a 96-well plate at the density of 1,500-2,000 cells/well and cultured one day prior to use. After being washed twice with PBS, the cells were kept in a serum-free medium overnight. Subsequently, the starved cells were stimulated with insulin for 20 min, followed by the addition of 2-DG and cultured for another 20 min. Exogenous 2-DG was removed by washing with PBS. The glucose uptake was measured at the wavelength at OD412 nm on a microplate reader at 37 °C protected from light. Similarly, lactate level was determined using the Lactate Assay Kit (MAK329, Sigma) according to the guidelines and quantified by detecting the absorbance at 450 nm.ATP detectionThe cellular ATP levels of TNBC cells expressing siRNA-control or siRNA-LINC02159 were detected using the ATP detection kit (S0027, Beyotime, Shanghai, China) according to the manufacturer’s protocol. Cells were lysed using the provided lysis buffer and the supernatant was collected via centrifugated at 12,000 rpm for 10 min at 4 °C. The ATP level was measured by determining the luminescence with the microplate reader (BioTek). The protein concentration was also detected using the BCA kit (PP102-02, Beyotime) to normalize the ATP level between different samples.Extracellular acidification rate (ECAR) analysisSeahorse XF24 FluxPak from the Agilent Technologies (Santa Clara, CA) was used to detect the ECAR of TNBC cells. Cells expressing either siRNA-control or siRNA-LINC02159 were seeded into a 24-well plate at a density of 20,000 cells per well and cultured in complete growth medium overnight. Afterwards, cells were washed with the XF assay medium and exposed sequentially to glucose, oligomycin, and 2-DG at the indicated time points. The ECAR was analyzed using the Seahorse software and normalized to the cell counts.Statistical analysisResults are expressed as mean ± standard deviation, with statistical analysis done by the Student’s t-test or one-way ANOVA followed by the Tukey test. Kaplan-Meier method was used to determine the survival curves and analyzed using the log-rank test with the Graphpad Prism (version 6.0). The difference was considered as significant when p value