Patients and samples
A total of 30 HCC tissues and paracancerous tissues were collected from patients who underwent HCC surgery at Affiliated Drum Tower Hospital, Medical School of Nanjing University. All tissues were immediately frozen in liquid nitrogen and stored at a −80°C refrigerator. The written informed consent was ontained from each paticipant. This study was approved by the Ethics Committee of the Affiliated Drum Tower Hospital, Medical School of Nanjing University.
GSE6764 and GSE14520 datasets which contain the PKM2 expression data between normal tissues and HCC tissues, were downloaded from GEO database (https://www.ncbi.nlm.nih.gov/geo/).
Human HCC cell lines 97H and Huh7 were donated by the medical school of Wuhan University. Human embryonic kidney 293T cells were obtained from American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, MA, USA) containing 10% FBS at 37°C in a 5% CO2 incubator.
NEAT1, FOXP3 and PKM2 were silenced by specific small interfering RNAs (siRNAs), termed NEAT1 siRNA1/2 (si-NEAT1 1#, si-NEAT1 2#), FOXP3 siRNA1/2 (si-FOXP3 1#, si-FOXP3 2#), PKM2 siRNA1 (si-PKM2). The full length of NEAT1 or PKM2 was ligated into the pcDNA3.1 vectors to obtain pcDNA3.1/NEAT1 (NEAT1 OE) and pcDNA3.1/PKM2 (PKM2 OE) plasmids. These plasmids above were obtained from GenePharma (Shanghai, China). In addition, the knockdown (KO) vecotor of PKM2 (Psilencer/PKM2) were sythesized by Hanbio (Beijing, China). The plasmids were transfected into 97H and Huh7 cells using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Real time-quantitative PCR (RT-qPCR)
Total RNAs isolated from 97H and Huh7 cells were obtained with the application of the TRIpure Total RNA Extraction Reagent. Next, the EntiLink™ 1st Strand cDNA Synthesis Kit was used to synthesize cDNA. Later on, qPCR was performed using a SYBR Green Mix kit (Beyotime, Shanghai, China) on a FAST7500 Realtime PCR system (American Applied Biosystems, USA). Calculation of gene expressions was conducted on the basis of 2-ΔΔCt method.
The 97H and Huh7 cells were seeded onto 96-well plates at a density of 5×103 cells/well and incubated for 24, 48 and 72 h. After that, cells in each well were added with 10 μL CCK-8 (Dojindo Laboratories, Kumamoto, Japan) solutions and incubated for another 2 h, followed by the measurement of the absorbance value at the wavelength of 450 nm by a microplate reader.
Colony formation assay
The 97H and Huh7 cells were seeded onto 6-well plates and incubated for for 2 weeks. After that, cells were fixed with 4% formaldehyde for 15 min and then stained with 1% crystal violet for 15 min. Later on, cell colonies were observed and photographed by a microscope (Leica, German).
Transfected 97H and Huh7 cells were placed on the upper chamber with 200 μL serum-free RPMI 1640 medium. Meanwile, the RPMI 1640 medium containing 12% FBS was added into the lower chamber. After 24 h of incubation, the cells migrating or invading to the lower membrane surface were fixed with 4% formaldehyde and then stained with 0.2% crystal violet solution for 10 min. Subsequently, the migrated or invaded cells were observed under the microscope. To evaluate cell invasion, the upper chamber was pre-coated with matrigel (BD, USA).
Luciferase reporter assay
The promoter of PKM2 was inserted into the pGL6-miR‐based luciferase reporter vector. 97H or Huh7 cells were co-transfected with the luciferase reporter vector and NEAT1 OE or si-NEAT1 2# plasmids. After 48 h of transfection, the luciferase activity was detected using the Dual Luciferase Reporter Assay System (Beyotime, Shanghai, China).
RNA pull-down assay and mass spectrometry (MS) analysis
The sense and antisense NEAT1 were transcribed in vitro and biotinylated with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche). After that, the cell lysates from 97H or Huh7 cells were incubated with the purified biotinylated transcripts for 1 h at room temperature. Later on, streptavidin agarose beads were used to isolate the biotinylated-labeled RNAs and their binding protein partner. Subsequently, the retrieved proteins were resolved by gel electrophoresis, followed by the MS analysis. Meanwhile, the binding proteins were identified by western blot assay.
RIP assay was performed using the EZ-Magna RIP RNA-binding protein immunoprecipitation Kit (Millipore). 97H or Huh7 cells were lysed in RIP lysis buffer. Next, the cell lysates from 97H or Huh7 cells were mixed with magnetic beads, and FOXP3 antibody or immunoglobulin G (IgG) antibody. After that, the co-precipiated RNAs were examined by RT-qPCR assay.
Chromatin immunoprecipitation (ChIP) assay
EZ-ChIPTM Chromatin immunoprecipitation Kit (Millipore) was used to perform ChIP assay. 97H or Huh7 cells were incubated with formaldehyde for 10 min and then subjected to ultrasonic breaker treatment to break the chromatin. After that, the products were incubated with FOXP3 antibody overnight at 4°C. Next, the protein A-Sepharose magnetic beads were used to precipitate the DNA-protein complex. Later on, cross-linked protein-DNA complexes were reversed at 65°C overnight, and then purified DNA was quantified by RT-qPCR assay.
Western blot assay
Cells were lysed using the RIPA lysis buffer (Sigma-Aldrich), and the total amount of protein was quantified using the BCA method. After that, 40 μg of protein was detached by 10% SDS-PAGE and then transferred to the PVDF membrane. Later on, the PVDF membrane was blocked in 5% skimmed milk and then incubated with the primary antibodies against FOXP3, PKM2, CD63, CD9, HSP90 and GAPDH overnight at 4°C. After incubating with the goat anti-rabbit secondary antibody at room temperature for 2 h, the blots were visualised using the ECL fluorescence detection kit (Beyotime, Shanghai, China) and captured using the Bio-Rad image analysis system. ImageJ software was used to quantify the immunoblots.
Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) assays
Cells were fixed with 4% formaldehyde at room temperature, and then treated with 0.5% Triton X-100. For FISH assay, hybridization was implemented with Cy3-labeled NEAT1 probe overnight. After that, cells were incubated with the secondary antibody at 4°C overnight. For IF assay, 97H or Huh7 cells were incubated with primary antibodies against FOXP3 at 4°C overnight, and then the secondary antibody at 4°C for 1h. The stained cells were observed using the confocal laser microscope (Olympus). Nuclei was counterstained using DAPi.
Exosome extraction and identification
Exosomes were isolated from 97H or Huh7 cells by ultracentrifugation method. Briefly, the supernatant of 97H or Huh7 cells was collected and centrifuged at 300 × g for 10 min, 2,000 × g for 10 min, 10,000 × g for another 30 min. After that, the supernatant was ultracentrifuged at 140,000 × g supernatant for 70 min twice. Later on, the supernatant was removed, the pellet was obtained and resuspended in 50 μL PBS. The number and size of exosomes was analyzed using a Nanoparticle Tracking Analysis (NTA) instrument. In addition, exosomes were also identified by transmission electron microscopy (TEM) western blot and flow cytometry assays.
Xenograft model in nude mice
Female BALB/C nude mice (4-6 week old) were obtained commercially from National Laboratory Animal Center (Beijing, China). Totally, 1×107 HCC cells transfected with pcDNA3.1 (OE NC), pcDNA3.1 NEAT1 (NEAT1 OE), pcDNA3.1 NEAT1 + PKM2 siRNA1 (NEAT1 OE + si-PKM2) plasmids were subcutaneously injected into the left flank of the nude mice. Tumor growth was recorded daily, and tumor volume was measured with a vernier caliper and calculated using the equation: (length × width2)/2. The nude mice were sacrificed at day 23, the tumor tissues were removed completely.
In other experiments, HCC cells transfected with OE NC , NEAT1 OE , NEAT1 OE + si-PKM2 plasmids were injected into the mice via tail vein to observe the distant metastasis. The nude mice were sacrificed at day 23 and the number of lung metastases in these mice was determined. These study were approved by the Ethics Committee of the Affiliated Drum Tower Hospital, Medical School of Nanjing University, and animals were maintained following the guidelines of the Institutional Animal Care and Use Committee.
The tumor tissues were fixed in 10% formaldehyde, embedded in paraffin and then spliced into 6 μm sections. After that, paraffin sections were dewaxed using xylene. Later on, the sections were incubated the primary antibodies against PKM2 and Ki67 at 4°C overnight. Later on, the sections were incubated with biotinylated-labeled second antibody at 37°C for 30 minutes. Next, the sections were visualized with DAB solution and stained with hematoxylin. Pictures were captured under a fluorescence microscope.
All data were repeated in triplicate. Comparisons between two groups were analyzed using the unpaired t test. One-way analysis of variance (ANOVA) and Tukey’s tests were carried out for multiple group comparisons. Values are shown as the mean ± SD. Differences were considered to be statistically significant at *P<0.05.