Clinical sample collection
The 185 breast cancer tissues, 36 lung cancer, 32 glioma, and 21 gastric cancer tissues and their corresponding normal tissues were obtained from HMUCC. Eligible patients with a histological diagnosis of cancer who had received neither chemotherapy nor radiotherapy before surgical resection were recruited to the present study. All samples were frozen in liquid nitrogen immediately after surgical resection, and only tumors with > 80% tumor cells were selected for RNA extraction. Two independent senior pathologists confirmed the pathological diagnosis and molecular subtype of each cancer tissue. This study conformed to the clinical research guidelines and was approved by the research ethics committee of the Harbin Medical University Cancer Center. We obtained written informed consent from all patients.
Cell line
The human breast cancer cell lines (MCF10A, UACC-812, Hs578T, HCC70, MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-468 and T-47D) , the human gastric carcinoma cell lines (SGC-7901 and MKN-45), the human lung cancer cell lines (NCI-H1975 and NCI-H1650), the human kidney cancer cell line (786-O), the human liver cancer cell line (Hep3B), the human glioma cell line (U251), the human ovarian cancer cell line (SKOV-3) and the human embryonic renal cell line (293T) cells were purchased from the Chinese Academy of Sciences Cell Bank and Cellbio (CAS, China). The cells were grown in basic RPMI-1640 medium (Gibco, USA) or DMEM (Gibco, USA) supplemented with 10% fetal bovine serum. All cells were maintained in a humidified 5%CO2 atmosphere at 37 °C.
Transcriptome sequencing for breast specimens
Transcriptome sequencing of 15 breast cancer tissues, 15 adjacent normal tissues and three breast tissues were obtained from HMUCC. A total of 3 µg of RNA per sample was applied for RNA sample preparation. Ribosomal RNA was first removed using the Ribo-ZeroTM Gold kit (Epicentre, Wisconsin, USA). Next, sequencing libraries were prepared according to the manufacturer’s protocol, and the libraries were then sequenced on an Illumina HiSeq 2500 platform to generate 100-bp paired-end reads. Raw sequencing and processed RNASeq data have been deposited into the NCBI GEO database under accession number GSE71651 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE71651).
BGISEQ-500 RNA-sequencing, ChIP-sequencing and small RNA-sequencing
Total RNA was isolated from the cancer cells using the Promega isolation kit (Promega) according to the manufacturer’s instructions. Raw sequencing and processed RNA-seq data were deposited in the NCBI GEO database under the accession number GSE124961 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE124961). For BGISEQ-500 ChIP-sequencing, six steps used for BGISEQ-500 ChIP-sequencing included sample detection, end repair and adaptor ligation, PCR, circularization, library quality control, and sequencing. Raw sequencing and processed CHIP-seq data were deposited in the NCBI GEO database under the accession number GSE124836 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE124836). For BGISEQ-500 small RNA-sequencing, total RNA was isolated from the cancer cells using the Promega isolation kit (Promega) according to the manufacturer’s instructions. Raw sequencing and processed small RNA-seq data were deposited in the NCBI GEO database under the accession number GSE124961 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc =GSE124961).
Public data access
Genome-wide expression profiles of LINC00036 and EGFR and clinical pathology information were downloaded from TCGA (https://tcga-data.nci.nih.gov/) and CCLE (http://www.broadinstitute.org/ ccle). All transcripts were normalized using log2 transformation. The expression of LINC00036 or EGFR was dichotomized by median expression as the cut-off to define high values (at or above the median) versus low values (below the median). An enrichment peak of c-MYC in the LINC00036 promoter was obtained from the ChIP-seq data of MCF-7 cells in the ENCODE portal (http://www.encodeproject.org). The putative c-MYC-binding site in the LINC00036 promoter was inspected using the JASPAR database (jaspar.genereg.net/). Putative TF-binding site in the transcriptional start of LINC00036 locus are listed in Table S1. The predicted microRNAs binding to the 3’UTR of EGFR TargetScan and RNA22 are listed in Table S2. Venn diagrams were drawn using the VENNY 2.1 online software (https://bioinfogp.cnb.csic.es/tools/venny/).
Transient transfections, lentiviral transduction, and plasmid transfection
For transfection, cells were transfected with 40 nM siRNA targeting LINC00036, c-MYC, and PPP1R150, or a control siRNA using LipofectamineTM 2000 (Invitrogen, #11668019) or LipofectamineTM 3000 (Invitrogen, #L3000015) according to the manufacturer’s instructions. Total RNA was isolated after 48 h for real-time PCR analysis. For transduction, lentiviruses were used to infect 5 × 105 cells in a 6-well plate with 4-6 µg/ml polybrene (Catalog number, 107689; Sigma). The infected cells were then subjected to selection with 1 µg/ml puromycin (Catalog Number 540411; Calbiochem, USA). Stable knockdown cell lines were identified using qRT-PCR. For plasmid transfection, cells were transfected with plasmid using LipofectamineTM 2000 or LipofectamineTM 3000 according to manufacturer’s recommendations. The siRNA/shRNA sequences are listed in Additional file 1: Table S1.
Cell proliferation assay
The cell counting kit-8 (Dojindo, Kumamoto, Japan) was used to assess cell growth. Approximately 2 × 103 cells per well were seeded in 96-well plates in a final volume of 100 μL and then transfected with si-LINC00036-1 or si-negative control (NC). The absorbance was detected 0, 12, 24, 48, and 72 h after gene transfection. CCK-8 solution (10 μl) was added to each well and incubated for 2 h at 37 °C. The absorbance levels were measured at a wavelength of 450 nm. All experiments were performed in triplicate. To calculate the half inhibitory concentration (IC50), data were fitted in GraphPad Prism 7.0, and the dose-response curve was plotted using the equation log (inhibitor) vs. response- variable slope. The IC50 was obtained using the following formula: Y=Bottom + (TopBottom)/(1 + 10^((Log IC50-X) *HillSlope)).
Cell apoptosis assay using flow cytometry
The transfected cells were seeded in 6-well plates (5 × 105 cells/well) and treated with 1.0 mg/l adriamycin. The cells were then digested with trypsin (Gibco® trypsin-EDTA, Thermo Fisher Scientific), washed thrice with PBS, suspended in 500 μl binding buffer, and then incubated withFITC Annexin V Apoptosis Detection Kit I (BD Biosciences, 556547) according to the manufacturer’s protocol. The stained cells were detected using the BD FACS Aria II flow cytometer (BD Biosciences, Hercules, CA, USA).
Transwell migration/invasion assays
Transwell migration assays (Corning ® BioCoatTM control insert-No ECM, 8 micron pore size) and invasion assays (Corning BioCoatTM Matrigel invasion chamber) were performed according to the manufacturer’s instructions. Cells which had migrated/invaded to the bottom of the insert were fixed with 70% ethanol and stained with 0.5% crystal violet. The migrated/invaded cells were photographed under an inverted microscope, quantified using ImageJ, and represented as percentage of total area.
Western blot analysis
Briefly, the protein concentrations were determined using a bicinchoninic acid protein assay kit and bovine serum albumin as the standard. Equal amounts of protein were fractionated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The membranes were blocked for 2 h using 5% non-fat milk in Tris-buffered saline with Tween (TBST), and then probed overnight at 4 °C. Following incubation with the primary antibodies, the membranes were incubated with secondary antibody (1: 8000 dilution, Alexa Fluor® 700 goat anti-mouse IgG (H+L) or Alexa Fluor® 800 goat anti-rabbit IgG (H + L); Invitrogen) in PBS at room temperature for 1 h. The protein bands were imaged using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) and quantified using the Odyssey v1.2 software (LI-COR Biosciences, Lincoln, NE, USA) by measuring the band intensity (area × OD) in each group. The band intensities were normalized to that of GAPDH or tubulin as the internal control. The western blot experiments were repeated thrice unless otherwise stated. The antibodies used are listed in Additional file 1: Table S2.
Immunohistochemistry
Paraffin-embedded tissue sections from patients with breast cancer were deparaffinized in xylene, rehydrated in a graded series of ethanol solutions, and then incubated for 20 min in 3% H2O2 to block the endogenous peroxidase activity. Next, the sections were heated in target retrieval solution (Dako) for 15 min in a microwave oven (Oriental Rotor) to retrieve the antigen. Non-specific binding was blocked via incubation with 10% goat serum for 2 h. The slides were then incubated overnight at 4 °C with the anti-EGFR primary antibody. An appropriate secondary antibody was added and incubated for 20 min at 37 °C, and binding was visualized with 3, 39-diaminobenzidine tetrahydrochloride (DAB). After each treatment, the slides were washed thrice with TBST for 5 min. Tissues were fixed in 4% neutral paraformaldehyde for at least 48h and then embedded in paraffin. For safety evaluation of the nanocomplex, consecutive paraffin wax-embedded tissue sections of vital organs (4-5 µm) were dewaxed, rehydrated, and stained with hematoxylin & eosin (H&E). The antibodies used are listed in Additional file 1: Table S1.
Quantitative real-time PCR
Total RNA samples from cells and tissue samples were isolated using the Trizol reagent (Invitrogen, Carlsbad CA, USA) according to manufacturer’s protocols. Total RNA (0.5 μg) was then reverse transcribed using the High-Capacity cDNA reverse transcription kit (Applied Biosystems, MA, USA). The SYBR Green PCR master mix (Applied Biosystems, USA) was used to quantify the RNA levels, and GAPDH or U6 was used as an internal control. The qRT-PCR was performed on a 7500 FAST real-time PCR system (Applied Biosystems, USA). The primers used are listed in Additional file 1: Table S3, 4. LINC00036 and mRNA levels were normalized to those of GAPDH, while microRNAs levels were normalized to those of small nuclear U6. The 2−ΔΔC t method was used to calculate relative expression levels.
Terminal deoxynucleotidyl transferase dUTP nick end labeling assay
Apoptosis-induced DNA fragmentation was determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The cells or tissues under different experimental conditions were fixed with 4% (w/v) paraformaldehyde and processed using a commercial kit (Roche) in accordance with the manufacturer’s instructions. Tumor samples were fixed for 30 min, rinsed with PBS, blocked for 10 min using a solution of 96% methanol mixed with 4% H2O2 at room temperature, and permeabilized with 0.2% Triton X-100 in PBS for 5 min at 4 °C. The TUNEL staining was performed using the in situ cell death detection kit (Roche) and the nuclei were stained with DAPI (Beyotime, China) for 10 min. The number of TUNEL-positive cells and the total number of cells were captured using a fluorescence microscope (IX71 + DP72, Olympus, Japan) and apoptosis was determined using the ImagePro Plus software.
Isolation of nuclear/cytoplasmic fractions
Nuclear/cytoplasmic fractions were isolated using the NE-PERTM nuclear and cytoplasmic extraction reagents (Catalog number 78835; Thermo Fisher) according to the manufacturer's protocol. Cytoplasmic and nuclear fractions were divided for RNA extraction. The GAPDH and U1 enzymes were used as qRT-PCR markers for the cytoplasmic and nuclear RNAs, respectively.
RNA fluorescent in situ hybridization (RNA-FISH)
ViewRNA®Probe (Catalogue Number VA1-3016120, Santa Clara) was purchased to perform Fluorescence in situ hybridization (FISH) assay according to the manufacturer's protocol. LINC00036 hybridization was carried out in a moist chamber. After digestion with a working protease solution, slides were incubated with RNase III (AM2290, Life Technologies, USA) or RNase A (AM2272, Life Technologies) for 2 h. Standard immunofluorescence and imaging were performed by confocal microscopy.
Actinomycin D assay
Cells were treated with 5 μM (final concentration) actinomycin D (Catalog number A1410; Sigma; dissolved in 100% ethanol). Actinomycin D was added to cells 0, 1, 2, 4, 6, 12, or 24 h prior to RNA extraction with the TRIzol reagent. Subsequently, qRT-PCR was used to analyze the changes in the RNA levels.
RNA immunoprecipitation (RIP) and RNA pull down assays
RIP was performed using a Magna RNA-binding protein immunoprecipitation kit (Millipore, Bedford, MA) according to the manufacturer’s instructions. Briefly, cell lysates were incubated with RIP buffer containing magnetic beads conjugated with negative control normal mouse IgG or human anti- PPP1R150 antibody. The samples were then incubated with proteinase K to isolate the immunoprecipitated RNA. Finally, purified RNAs were extracted and analyzed using real-time PCR to confirm the presence of the binding targets. RNA pull-down was performed using a magnetic RNA-protein pull-down kit (Pierce Biotechnology, USA) in accordance with the manufacturer’s instructions.
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed using the EZ ChIP kit (Millipore, Bedford, MA, USA) according to the manufacturer's protocol. In total, 1 × 106 cells were fixed in 1% formaldehyde at room temperature for 10 min, and the nuclei were isolated with nuclear lysis buffer supplemented with a protease inhibitor. The chromatin DNA was sonicated and sheared to 100-200 bp fragments. The sheared chromatin was immunoprecipitated at 4 °C overnight using an anti-c-MYC antibody (Cell Signaling Technology). Normal mouse IgG was used as the negative control and an anti-RNA pol II antibody (Millipore) was used as the positive control.
Luciferase reporter assay
Luciferase reporter vector-harboring lentiviral particles were constructed with the wild type or mutant 3¢ UTR of EGFR or LINC00036 and packaged by Shanghai GeneChem, Co., Ltd. The H293T cells were transfected with wild type or mutant reporter plasmid vector using Lipofectamine 2000 and were then cotransfected with microRNA mimics or the negative control. After 48 h, luciferase assays were performed using the dual luciferase assay system (Promega, catalogue E1910). Firefly luciferase activity was normalized to the Renilla luciferase activity for each sample.
In vivo xenograft model
All experimental procedures involving animals were performed in accordance with the animal protocols approved by the Animal Care and Use Committee of Harbin Medical University. Briefly, 4-5-week-old female BALB/c nude mice were obtained from the Shanghai Laboratory Animal Center (CAS), housed in a pathogen-free and temperature-controlled environment. Stably transfected cancer cells (1 × 107 UACC-812 cells, 5 × 106 SGC-7901 cells and 5 × 106 NCI-H1975 cells)in 100μl PBS together with anequal volume of Matrigel basement membrane matrixwere injected subcutaneously into the right abdomento establish breast cancer xenograft model, lung cancer xenograft model and gastric cancer xenograft model respectively. One week after the tumor cell inoculation, the mice were treated with gefitinib at 20 mg/kg/day by asingle intraperitoneal injection (i.p). Tumor volumes were measured every 4 days after being apparently observed and calculated with the following formula: Volume = (length × width2)/2. After 30 days, all mice were sacrificed under anesthesia.
LINC00036 targeting nanoparticles preparation and biodistribution
The LINC00036 targeting ASO was designed and synthesized by Integrated DNA Technologies (Skokie, Illinois, USA). The ASO and DOTAP liposomal transfection reagent were dissolved in methylene dichloride and allowed to stand for 20 min at room temperature according previous studies [43, 44]. Next, the mixture was rehydrated in normal saline for future use. The mice of the control group were administrated with 0.9% normal saline via tail vein injection. The treatment groups were administrated with 20μl ASO or 20μl LINC00036 targeting nanoparticles (TTN) via tail vein injection (t.v.i). Three groups (four mice per group) of female BALB/c nude mice were used to evaluate the biodistribution of the TTN in vivo. Cyanine 5.5 was used to label TTN and ASO. The mice of the control group were administrated 200μl 0.9% normal saline and the treatment groups were administrated 150μl cyanine 5.5/ASO or 150μl cyanine 5.5/TTN via tail vein injection at time intervals of 1, 3, and 24 h. Imaging of cyanine 5.5-labeled ASO DNA oligo was monitored over time by detecting near infrared (NIR) fluorescence in the whole animal according to the instructions of the IVIS Lumina Imaging System 100 series (Xenogen, Alameda, CA, USA).
Statistical analysis
All statistical analyses were performed using the R 4.1.2 (R Foundation for Statistical Computing, Vienna, Austria) software program or GraphPad Prism 7 software (San Diego, CA). All immunohistochemistry, immunoprecipitation, western blot, and in vitro assays were conducted for more than three times to ensure reproducibility. The number of mice in each group has been indicated for specific in vivo experiments. Correlations between the LINC00036 and EGFR expression were assessed using Pearson correlation coefficients. Unpaired Student’s t-tests were used to detect significant differences among tumors or between tumor and normal samples. The OS was calculated as the time from surgery until the occurrence of death. The DSS indicated the duration of disease-specific survival. Survival curves were constructed using the Kaplan-Meier method and were compared between subgroups with the log-rank test. All P values were two-tailed, and P < 0.05 was considered statistically significant. All data are presented as the mean ± standard deviation (SD) from at least three independent replicates.