IntroductionBreast cancer is the most diagnosed cancer in women and accounts for over 600,000 deaths globally per year1. Treatment regimens are largely based on the expression of three receptors, which define the three clinical breast cancer subtypes2. Hormone receptor positive (HR+) breast cancers express estrogen receptors (ER) and progesterone receptors (PR), and account for 70–75% of cases, and are treated with endocrine therapies, combined with chemotherapy in high-risk cases. Overexpression of the human epidermal growth factor receptor 2 (HER2) is present in 15–25% of breast cancers. Patients with HER2+ breast cancers are treated with HER2-targeting drugs in combination with chemotherapy. Triple-negative breast cancer (TNBC) lacks the three receptors, which prevents their treatment with these receptor-targeting drugs and makes chemotherapy the primary treatment.In addition to having different treatment regimens, the three major clinical breast cancer subtypes have distinct disease progression profiles, with TNBC being the most aggressive and likely to recur3. The subtypes present with distinct gene expression and cell signaling pathways4,5,6 Recent advancements in treatments for TNBCs by targeting genetic factors and molecular pathways associated with TNBC are improving outcomes (e.g., pembrolizumab and olaparib)7. This illustrates how understanding the unique cell signaling and gene expression profiles associated with TNBC can be harnessed into new therapeutic interventions that improve patient outcomes.Key regulators of gene expression and novel proposed targets are the non-coding RNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)8. According to Genecode’s latest release, there are 1879 miRNAs and 34,914 lncRNAs in the human genome and many have now been implicated in cancer progression and treatment responses8,9. MiRNAs are small non-coding RNAs (21–22 bases) that regulate gene expression through their incorporation into the RNA-induced silencing complex (RISC)10. They bind complementary messenger RNAs (mRNAs) causing repression of translation and mRNA degradation. MiRNAs can act in a subtype-specific manner in breast cancer, resulting in distinct gene expression effects in HR+, HER2+, and TNBC subtypes11.LncRNAs also display breast cancer subtype-specific expression12, and this has been especially studied in the context of TNBC. Expression of 300 lncRNAs is elevated in TNBC13. Functional characterization of some of these TNBC-specific lncRNAs has revealed their roles in various cellular processes. For instance, LINP1 is involved in DNA repair regulation13, while another TNBC-enriched lncRNA, non-coding RNA in aldehyde dehydrogenase 1A pathway (NRAD1), previously called LINC00284, promotes breast cancer stem cell properties14. The breast cancer subtype-specific lncRNA expression profiles suggest their potential involvement in the progression of different breast cancer subtypes and highlight their promise as therapeutic targets12. This is especially relevant given that there are increasing examples of lncRNAs affecting cancer-promoting gene expression through multiple mechanism, including through interactions with chromatin and miRNAs15. Review of the literature suggests that competitive endogenous RNA (ceRNA) interactions with miRNA (commonly referred to as “sponging”) is the most often-reported mechanism of lncRNA gene regulation8. The 2019 release of DIANA-LncBase catalogued 240,000 unique tissue and cell-type specific miRNA–lncRNA interactions, including 730 miRNA–lncRNA entries from manual curation of publications16.The subcellular localization of lncRNAs is a determinant factor of their function and impacts potential regulation of miRNAs17,18,19. Cytoplasmic lncRNAs are often reported to be involved in post-transcriptional regulation through miRNA sponging. Some lncRNAs exhibit dynamic localization, shuttling between the nucleus and cytoplasm, which allows them to perform diverse functions. A smaller subset of lncRNAs are mitochondrial non-coding RNAs (mtncRNA). These are typically transcribed from mitochondrial DNA (mtDNA) and play roles in mitochondrial gene expression and function. Recent studies have also identified nuclear-encoded lncRNAs that localize to mitochondria, suggesting a complex interplay between nuclear and mitochondrial gene regulation15,20.Some lncRNAs are reported to have both chromatin and miRNA interactions, such NRAD1/LINC00284. For example, in breast cancer, NRAD1-mediated gene expression changes were partly explained through chromatin interactions14. However, in other cancers, NRAD1/LINC00284-mediated gene expression changes have been explained through miRNA ‘sponging’ interactions21,22,23,24,25,26. For example, in colorectal cancer, LINC00284 sponged miR-361-5p which led to the increase in transforming growth factor beta (TGF-β) signaling and promotion of colorectal cancer24.Here in we performed a comprehensive analysis of NRAD1 gene expression regulation in TNBC, focusing on characterizing miRNA effects. We find that NRAD1 is at the hub of miRNA–mRNA networks in TNBC cells and mediates the cancer-promoting gene expression changes through altering miRNA levels. We find evidence of ceRNA regulation of some miRNAs that is concentration dependent and NRAD1-transcript-specific. However, the effects of NRAD1 on miRNAs are predominately independent of ceRNA effects. NRAD1 upregulates key miRNA biogenesis protein DICER, alters the sub-cellular distribution of some miRNAs, and affects the biogenesis of mitochondria-localized miRNA miR-4485-3p. Together, this study demonstrates unique modes of interaction between the cancer-promoting lncRNA NRAD1 and miRNAs ultimately leading to changes in the gene expression landscape associated with the aggressive TNBC subtype.Materials and methodsCell cultureMDA-MB-468 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and SUM149 cells which were obtained from BioIVT (previously Asterand, Westbury, NY, USA). MDA-MB-468 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, ThermoFisher Scientific, Burlington, Canada) supplemented with 10% fetal bovine serum (FBS, Life Technologies, ThermoFisher), with the inclusion of antibiotic antimycotic (AA; Life Technologies, ThermoFisher Scientific). SUM149 cells were cultured in Ham’s F-12 Nutrient Mix (F-12; Life Technologies, Life Technologies, ThermoFisher Scientific) supplemented with 5% FBS, AA, HEPES buffer, 1 μg/mL hydrocortisone (Millipore Sigma, Mississauga, Canada), and 5 μg/mL human insulin (Millipore Sigma).NRAD1 knockdownTransient in vitro knockdown of NRAD1 in cancer cells was achieved through previously described protocol14. Briefly, NRAD1 knockdown was achieved by mixing antisense oligonucleotides with that consist of a central DNA “gap” flanked by LNA (Locked Nucleic Acid) regions (LNA GampeRs)27. NRAD1-specific GampeR’s (GapmeR #3 or #4) or negative control GapmeRs (Supplementary Table S1) were purchased from Qiagen (Qiagen, formerly Exiqon, Toronto, Canada) with OptiMEM reduced serum media (Life Technologies, ThermoFisher) and TransIT-BRCA transfection reagent (MJS Biolynk, Brockville, Canada) to a final concentration of 15 nM. Knockdown was confirmed 48 h post transfection with quantitative polymerase chain reaction (qPCR) as described below.Cell treatments with miRNA mimicSUM149 cells were seeded in 6-well plates at 20% confluency and after 18h post seeding, the cells were transfected with miRCURY LNA mimic or negative controls (Qiagen). The mimics were mixed with OptiMEM reduced serum media (Life Technologies, ThermoFisher) and TransIT-BRCA transfection reagent (MJS Biolynk) and added to sub-confluent cells to a final treatment concentration of 25 nM as per the manufacturer’s instructions. Treatment efficacy was confirmed by TaqMan assay at 48 h post transfection as described below.Quantitative polymerase chain reactionFor all transcript expression analyses by qPCR, cells were collected in TRIzol (Life Technologies, ThermoFisher Scientific) and total RNA was purified using a PureLink RNA kit (Life Technologies, ThermoFisher Scientific) as per the manufacturer’s instructions. Equal amounts of harvested total RNA were reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad, Mississauga, Canada) as per the manufacturer’s instructions. QPCR was performed using SsoAdvanced Universal SYBR Super-mix (Bio-Rad) and transcript-specific primers (primer sequences are listed in Supplementary Table S2) as per the manufacturer’s recommended protocol using a CFX96 or CFX384 Touch RealTime PCR Detection System (Bio-Rad). Primer efficiencies, determined by standard curves of diluted cDNA samples, were incorporated into the CFX Manager software (Bio-Rad). Gene expression for all samples was calculated relative to two or three reference genes and relative to the negative control or NRAD1-targeting GapmeR-treated samples.Gene arraysSUM149 cells were treated with NRAD1-targeting antisense oligonucleotides GapmeRs #3 or #4, or negative control GapmeR for 48 h and then collected in TRIzol reagent (n = 3). RNA purification was performed as described above and sent to the Centre for Applied Genomics (TCAG, The Hospital for Sick Kids, Toronto, Canada) for Affymetrix Human Gene 2.0 ST gene chip platform analysis and raw data files are available on the Gene Expression Omnibus (GEO), GSE294997. The data was processed using limma R package v3.54.2 to reveal differential gene expression. MDA-MB-468 cells had been similarly treated with anti-NRAD1 GapmeR #4 or negative control GapmeR and extracted RNA processed for Affymetrix Human Gene 2.0 ST gene chip platform analysis was accesses from GEO (GSE118710)14. The fold-change cutoff of greater than or equal to 1.4 or less than or equal to - 1.4 and had a p-value below 0.05 were used in further analyses (Supplementary File 1).Analysis of patient tumor gene expression dataThe RNA sequencing (RNA‐seq) log2 V2 RSEM expression data for the Breast Invasive Carcinoma (Cell 2015) from The Cancer Geneome Atlas (TCGA were accessed via cBioportal and patients were specifically identified from the clinical data28,29. Gene array expression data from the Breast Cancer Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset was also accessed via cBioportal and TNBC patients specifically identified30.MicroRNA arraysSUM149 and MDA-MB-468 cells were with NRAD1-targeting GapmeRs #3 or #4 or negative control GapmeR for 48 h and then collected using the mirVana™ miRNA Isolation Kit (Life Technologies, ThermoFisher Scientific) as per manufactures instructions for small miRNAs. Total RNA from cell lysates was used to confirm knockdown of NRAD1 using qPCR as described. Small miRNA purification was sent to the Centre for Applied Genomics (TCAG, The Hospital for Sick Kids, Toronto, Canada) for Affymetrix GeneChip miRNA 4.0 array. Data was collected in raw CEL format and differential miRNA expression was visualized using the Affymetrix Transcriptome Analysis Console (TAC). MiRNA names adjusted to the most recent version of miRbase using the miRbaseConverter in R to version V22. Mature miRNAs were identified and those that met the fold-change cut-off of ± 1.4 along with having a p-value less than or equal to 0.05 were used in further analysis (Supplementary File 1). The raw data is deposited on GEO (GSE295075).MicroRNA quantification by TaqMan PCR assaysMiRNA (small and total) was purified from cells using the mirVanna miRNA isolation kit (Life Technoloiges, ThermoFisher Scientific) as per manufactures instructions for small miRNAs. Total miRNA was isolated with modification utilizing miRNA columns and lysis extraction from mirVanna miRNA isolation and wash buffers from PureLink RNA kit (Life Technologies, ThermoFisher Scientific). cDNA was synthesized from the purified small miRNA using the pre-formulated reverse transcription primers and the reagents in the TaqMan microRNA reverse transcription kit (Life Technologies, ThermoFisher). TaqMan Fast Advanced Master Mix (ThermoFisher) was used for the qPCR. MiRNA levels detected using either the TaqMan miRNA assays (miR-4485, miR-484, miR-192-5p, miR-3929, miR-28-3p, miR-769-5p, miR-1303, and miR-452, cat#4427975) were calculated relative to 2 to 3 reference miRNAs (RNU48, miR-25-3p, and miR-93-5p) and relative to the negative control.The miEAA V 2.0 platform was used to perform an over-representation analysis of miRNAs involved in biological processes31. Each cell line was performed separately only utilizing significantly differentiated miRNAs for GapmeR #4 versus negative control GapmeR treatment. The mRNA binding of differentially expressed miRNAs was predicted using MultiMir32. The resulting NRAD1/miRNA/mRNA networks were plotted using Cytoscape V 3.2 software33.Cloning NRAD1-201 and NRAD1-202 transcripts from MDA-MB-468 cellsStandard techniques were used to clone the NRAD1-201 and 202 transcripts from MDA-MB-468 into the pcDNA3 plasmid. First-strand cDNA was synthesized from MDA-MB-468 cell RNA, using SuperScript III, and oligoDT12-18 (Life Technologies, Thermo Fisher Scientific), as per manufacturer’s instructions. The NRAD1-201 and 202 sequence were retrieved and amplified using specific primers (Supplementary Table S3) with flanking 5′-HindIII (forward) and 3′-BamHI (reverse) sites, in the presence of Platinum SuperFi enzyme (Life Technologies, Thermo Fisher Scientific). The amplified NRAD1 cDNA with the primer-introduced restriction sites were digested with BamHI and HindIII and ligated into double-digested (BamHI and HindIII) pcDNA3 plasmid using T4 ligase (New England Bioscience, Whitby, Ontario, Canada). Competent TOP10 E. coli cells were transformed and incubated overnight at 37 °C. The plasmids were purified with a plasmid purification kit, and the resulting plasmids. The clones were verified to have the correct insert by sequencing (Plasmidsaurus, Supplementary File 2).Predicted miRNA binding to NRAD1 transcriptsTo identify miRNAs predicted to bind NRAD1 transcripts, we utilized the LncBook 2.0 database, which integrates comprehensive lncRNA–miRNA interaction predictions34. We queried the database for all available NRAD1 transcripts that had transcript level support in Ensembl (Release 113). The LncBook tool employs three computational prediction tools (miRanda, TargetScan, and RNAhybrid) and we focused on interactions supported by at least two tools35,36,37. By filtering for high-confidence predictions, we generated a list of miRNAs with potential binding sites on the target NRAD1 lncRNA transcripts (Supplementary File 3).Western blottingCollected cells were lysed in RIPA buffer and sonicated for 5 min (15S ON, 45S OFF, 40% amplitude, 4 °C) and quantified with Pierce BCA Protein Assay Kit (ThermoFisher scientific). Laemmli buffer was added to the lysates and the samples were boiled 10 min and 40 μg of MDA-MB-468 lysate samples and 20 μg of SUM149 lysate samples was run per lane in Mini-PROTEAN TGX Stain-Free Precast Gel (Bio-Rad) and run for 1 h at 100 V in Tris–Glycine-SDS buffer. The lysates were transferred onto PVDF membranes in transfer system (Bio-Rad), in transfer buffer (3 g TRIS, 144 g Glycine, 200 μL MetOH, 800 μL H2O), for 19 h 30 V at 4 °C. The membranes were blocked in 5% skim milk in TBST for 8 h at 4 °C. The membranes were incubated with 1:1000 anti-DICER (Dicer (D38E7) Rabit mAb #53625, Cell Signaling Technology, Danvers, Massachusetts, USA) diluted in 5% bovine serum albumin, overnight at 4 °C followed by peroxidase conjugated affiniPure goat anti-rabbit IgG (H + L, #111-035-144, Jackson Immunoresearch, West Grove, PA, USA) antibody (1:1000 in 5% milk TBST) for 1 h at room temperature. The chemiluminescence was imaged with the ChemiDoc imaging system (Bio-Rad) and the band intensities were calculated and plotted using Imagelab software (Bio-Rad) versus total protein.Luciferase assayOligos specific to the wildtype (WT) NRAD1-miR-4485-3p predicted binding region and the mutated version of the sequence (MUT) are listed in Supplementary Table S4. To make double stranded sequences for cloning, the oligos were admixed into oligo annealing buffer and heated to 90 °C for 3 min, followed by cooling to 37 °C for 15 min. The WT and MUT annealed oligos (Life Technologies, ThermoFisher Scientific) were cloned into the multiple cloning sites of the pmirGLO Dual-Luciferase miRNA Target Expression Vector (ThermoFisher Scientific, using SacI and XhoI restriction enzymes (New England Biolabs, Ipswich, MA, USA). The confirmed vectors were co-transfected into MDA-MB-468 cells with the pRLTK vector (Promega, ThermoFisher Scientific), using TransIT-BRCA transfection reagent. 24 h later the mirVana miRNA negative control mimic or mimic-hsa-miR-4485-3p (Life Technologies ThermoFisher Scientific) was transfected into the cells using TransIT-BRCA. The resulting firefly and renilla luciferase activity in the cells were measured 24 h later using the Dual-Glo® Luciferase Assay System with a SpectraMax® M3 Multi-Mode Microplate Reader (Molecular Probes, San Jose CA, USA). Binding of the mimic sequence to the luciferase reporter vector would inhibit production of luminescence.Nucleus, cytoplasm and mitochondria fractionationNuclear and cytoplasmic fractionation followed by RNA extraction was performed using the PARIS™ kit (Life Technologies, ThermoFisher Scientific) as per manufactures instructions. Confirmation of fractionation was determined through the comparison of qPCR levels of cytoplasmic lncRNA DANCR or for miRNAs miR-25-3p and nuclear lncRNA NEAT1 or for miRNAs RNU48 (Supplementary Table S2, for NEAT1 and DANCR). Mitochondria separation from nuclear-cleared cytoplasm was performed using the Mitochondria Isolation Kit for Cultured Cells (Life Technologies ThermoFisher Scientific) following the using the reagent-based methods with modifications. Briefly, cells were harvested by trypsin and pelleted by centrifugation at 500×g for 5 min. Cells were counted and resuspended in PBS and centrifuged in a 2.0 mL RNA/DNA free microfuge tube at 850×g for 2 min to remove nuclei. Supernatants were removed and cell pellets were resuspended in 800 µL Mitochondrial Isolation Reagent A with RNA inhibitor (SUPERase·In™ RNase Inhibitor (20 U/μL), cat# AM2696, Life Technologies ThermoFisher Scientific) and using a 25G × 1 ½” TW (0.5 mm × 40 mm, BD PrecisionGlide Needle) five times aspirations followed by five times with a 30G × ½” (0.3 mm × 13 mm, BD PrecisionGlide Needle). The remainder of the procedure was followed as suggested by manufacture, all buffers were supplemented with RNA inhibitor and mitochondria and cytoplasm fractions were isolated for RNA analysis. QPCR confirmed separation of mitochondria from cytoplasm using primers against mitochondria-specific COX-2 and MT-12S compared to cytoplasmic-localize DANCR (Supplementary Table S2). For miRNA analysis, the mirVanna miRNA isolation kit (Life Technologies, ThermoFisher Scientific) was applied to fractions, following the manufacturer’s instructions for small miRNA isolation.ASncmtRNA2Antisense non-coding mitochondrial RNA 2 (ASncmtRNA2) was quantified adapting a protocol by Fitzpatrick et al., with modifications38. Total RNA was isolated from TRIzol as described above. Briefly, 100 ng of total RNA was mixed with 1 μL of 50 μM Random Hexamers (Life Technologies ThermoFisher Scientific) and 1 μL of 10 mM dNTP (Applied Biosystems, ThermoFisher Scientific) to make a total volume of 14 μL contents were mixed and heated for 66 °C for 5 min, placed on ice for 1 min. This was then combined with 1 μL of 100 mM DTT (Life Technologies, ThermoFisher Scientific), 1 μL of Super Script IV Reverse Transcriptase (200 U/μL) (Life Technologies, ThermoFisher Scientific) and 4 μL 5 × Superscript IV RT buffer, mixed and heated at 23 °C for 10 min, 55 °C for 10 min, 80 oC for 10 min. The resulting cDNA was diluted 1 in 10 and PCR was performed using 4 μL of Diluted cDNA, 5 μL SYBR green (Bio-Rad) and 1 μL of primer mix 4 μM ASncmtRNA-2 forward and reverse, primers are listed in Supplemental Table S2. QPCR setting 95 °C for 30 s, 95 °C for 10 s, 60 °C for 30 s × 45 cycles. QPCR was compared to reference primes (18S rRNA and 16 s rRNA, Supplementary Table S2). Confirmation of product was done using 2% agarose DNA gel, 20 μL of qPCR product and 4 μL of TrackIt cyan/orange loading buffer (catalog #10482028, Life Technologies, ThermoFisher Scientific), TrackIt ladder (catalog# 10488085, Life Technologies, ThermoFisher Scientific). QPCR products were purified and sequenced by Sanger Sequencing to verify the amplification of ASncmtRNA2 (Supplementary File S4).StatisticsAll data points represent biological replicates, not technical replicates. For qPCR assays, multiple technical replicates were averaged into a single biological replicate data point. All statistical analyses were performed in the GraphPad Prism software (GraphPad Software, San Diego, CA, USA). In all cases where three or more groups are compared, a one-way or two-way ANOVA was performed (with Dunnett’s or Tukey’s multiple comparisons post-test as indicated in the figure legend). Comparisons between two groups were done using a two-tailed student’s t-test. For co-expression analyses, p values were determined by the cor.test() function with the method argument set to “spearman” in Rv4.2. LncBook data analysis was performed in R studio, Wilcoxon test, Cliff’s delta and adjusted p-value by false discovery rate was performed on each group. Significant p values are indicated as follows in the figures: p