Clinical tissue samples
In this study, thirty NB tissues and paired normal tissues were obtained from patients undergoing surgery at People's Hospital of Rizhao. The patients did not receive any treatment before surgery. Each patient has signed written informed consent. Excised tissues were collected and promptly frozen in liquid nitrogen, and then cryopreserved at -80℃ for subsequent study.
Cell culture and transfection
Human embryonic kidney (HEK293) and NB cell line (SK-N-BE(2)) were purchased from BeNa Culture Collection (Beijing, China). The NB cell line (GI-LI-N) was obtained from Conservation Genetics CAS Kunming Cell Bank (Yunnan, China). These cells were cultured in Dulbecco’s modified eagle medium (DMEM; Invitrogen, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Invitrogen), 100 U/mL penicillin and 100 μg/mL streptomycin in an incubator with 5% CO2 at 37℃.
For this study, small interfering RNA against XIST (si-XIST) and the corresponding control (si-NC), miR-653-5p mimic (miR-653-5p) and the corresponding control (miR-NC), miR-653-5p inhibitor (anti-miR-653-5p) and the corresponding control (anti-miR-NC), XIST or HK2 overexpression plasmid (XIST or HK2) and the corresponding control (pcDNA) were purchased from GenePharma (Shanghai, China). Lentivirus-mediated shRNA interference targeting XIST (sh-XIST) and the corresponding control (sh-NC) were constructed by Genechem (Shanghai, China). GI-LI-N and SK-N-BE(2) cells with 50–60% confluence were transfected with oligonucleotide (50 nM) or plasmid (2 μg) using Lipofectamine 3000 (Invitrogen).
Quantitative real-time polymerase chain reaction (qRT-PCR)
TRIzol reagent (Invitrogen) was applied to isolate total RNA from tissues (NB tissues and normal tissues) and cells (HEK293, GI-LI-N and SK-N-BE(2)). Next, the first strand of complementary DNA (cDNA) was synthesized with a High-Capacity cDNA Reverse Transcription Kit and TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The qRT-PCR was carried out using the SYBR Green PCR kit (Thermo Fisher Scientific) on the ABI 7300 system (Thermo Fisher Scientific). Primers used for qRT-PCR were exhibited as below: XIST (Forward, 5’-CCTCTCCACATACCTCAGT-3’; Reverse, 5’-ACATAATCACACGCATACCA-3’); miR-653-5p (Forward, 5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGTAGAG-3’; Reverse, 5’-ACACTCCAGCTGGGGTGTTGAAACAATCT-3’); HK2 (Forward, 5’-ACAGCCTGGACGAGAGCATC-3’; Reverse, 5’-AGGTCAAACTCCTCTCGCCG-3’); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Forward, 5’-CGCTCTCTGCTCCTCCTGTTC-3’; Reverse, 5’- ATCCGTTGACTCCGACCTTCAC-3’), U6 snRNA (Forward, 5’-CTCGCTTCGGCAGCACATATACT-3’; Reverse, 5’-ACGCTTCACGAATTTGCGTGTC-3’). The expression levels of XIST, HK2 and miR-653-5p were calculated with the 2-ΔΔCt method, followed by normalizing to GAPDH or U6 snRNA, respectively.
Cell viability assay
Methylthiazolyldiphenyl tetrazolium bromide (MTT) was utilized for detecting cell viability. In brief, GI-LI-N and SK-N-BE(2) cells (100 μL) were seeded in 96-well plates at a density of 1 × 104/mL and then transfected with si-XIST, si-XIST + anti-miR-653-5p, miR-653-5p, miR-653-5p + HK2, or matched controls. MTT reagent (5mg/mL, 10 μL, Beyotime, Shanghai, China) was added to each well after transfection for 24, 48 h, or 72 h. After incubation for 4 h, the cultured medium was carefully discarded and dimethyl sulfoxide (DMSO; 150 μL) was added to per well. Lastly, the absorbance was evaluated at 490 nm under a microplate reader (Bio-Rad, Hercules, CA, USA).
Irradiation (IR) and colony formation assay
After transfection for 48 h, GI-LI-N and SK-N-BE(2) cells (150 cells per well) were seeded into six-well plates and the medium was updated every three days. For treatment of IR, cells were exposed to different doses of radiation by a linear accelerator (Varian, Palo Alto, CA, USA) at a dose rate of 3.5 Gy/min. After incubation for 2 weeks, GI-LI-N and SK-N-BE(2) cells were carefully washed with cold phosphate-buffered saline (PBS; pH=7.2) and subsequently fixed with 4% paraformaldehyde for 30 min at 4℃. After that, GI-LI-N and SK-N-BE(2) cells were washed by PBS and then stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). Colonies containing more than 50 cells were counted by a microscope (Olympus, Tokyo, Japan). The survival fraction was calculated as previously described [25].
Transwell assay
Transwell assay was performed using a 24-well plate inserts with 8 μm pores (Corning Incorporated, Corning, NY, USA) to evaluate cell invasion capacity. GI-LI-N and SK-N-BE(2) cells (2×104 cells/well) re-suspended in DMEM medium alone (100 μL) were seeded into the top chamber pre-coated with Matrigel (BD Bioscience, Franklin Lakes, NJ, USA). To induce cells invading through the membrane, the bottom chamber was filled with DMEM containing 10% FBS (600 μL). After incubation for 24 h, non-invaded cells were carefully removed with a cotton bud, and invaded cells were fixed with 95% ethanol and then stained with 0.1% crystal violet. Finally, invaded cells from five random fields were photographed and counted by a microscope.
Measurement of glucose consumption and lactate production
GI-LI-N and SK-N-BE(2) cells (5×104 cells/well) were seeded into six-well plates and transfected with oligonucleotide or/and plasmid. Cell culture media were collected 48 h after the transfection. In accordance with the manufacturer’s instructions, the glucose consumption and lactate production were detected with the glucose assay kit (Sigma-Aldrich) and lactate assay kit (BioVision, Mountain View, CA, USA), respectively. A standard calibration curve was used for determining the data, followed by normalizing to the amount of total protein.
Dual-luciferase reporter assay
The possible binding sites of miR-653-5p and XIST or HK2 were predicted by starBase v2.0. The XIST and HK2 3’UTR fragments containing wild-type (WT) or mutant (MUT) binding sites of miR-653-5p were amplified and inserted into pmirGlO luciferase reporter vector (Promega, Madison, WI, USA) to create the WT plasmids (WT-XIST, HK2 3’UTR-WT) or MUT plasmids (MUT-XIST, HK2 3’UTR-MUT). Afterward, GI-LI-N and SK-N-BE(2) cells were seeded in 24-well plates (2×104 cells/well) and were co-transfected with reporter plasmids and miR-653-5p or miR-NC. After incubating the cells for 48 h, the luciferase activity was estimated via Dual-Luciferase Reporter assay system (Promega).
Western blot assay
GI-LI-N and SK-N-BE(2) cells (1×105 cells/well) were seeded into six-well plates and transfected with oligonucleotide or/and plasmid. After transfection for 48h, cells were lysed using RIPA lysis buffer (Sigma-Aldrich) supplemented with 1mM phenylmethylsulphonyl fluoride (PMSF; Sigma-Aldrich) to extract the total protein, followed by being centrifuged at 12 000 × g at 4℃ for 15 min. After quantification by using bicinchoninic acid protein assay kit (Tanon, Shanghai, China), 30 μg of protein in each group was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then and then transferred to polyvinylidene fluoride membranes (Beyotime). After blocking with 5% non-fat milk in PBS containing 0.05% Tween 20 (PBST), the membranes were immunoblotted by primary antibody against HK2 (1:5000, ab227198, Abcam, Cambridge, UK) or GAPDH (1:2500, ab9485, Abcam) for 12-16 h at 4℃. After washing with PBST, membranes were incubated in horseradish peroxidase-conjugated anti-rabbit IgG (1:4000, D110058, Sangon Biotech, Shanghai, China) for 2 h at room temperature. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL; Tanon) reagent following the manufacturer’s protocol. ImageJ software was utilized to quantify the protein expression. GAPDH was used as an internal control.
In vivo tumor growth assay
BALB/c nude mice (male, 5-week-old) were purchased from Huafukang (Beijing, China). The sh-XIST or sh-NC was transfected into SK-N-BE(2) cells. Subsequently, stably transfected cells (3×106) were subcutaneously injected in BALB/c nude mice. From the 8th day, tumor volume was examined using a caliper every 4 days and calculated with the formula: length × width2/2. After injection for 10 days, sh-XIST group or sh-NC group were randomly divided into two groups. One group was irradiated with 6Gy X-ray once a week, and the other group served as control. All mice were sacrificed after injection for 4 weeks, and tumor samples were weighted and collected for detection of XIST, miR-653-5p and HK2 expression levels.
Statistical analysis
All data in this study were displayed as mean ± standard deviation (SD) and all experiments were repeated at least 3 times. Correlation between miR-653-5p and XIST or HK2 was detected by Spearman rank correlation. The results from different groups were assessed using the two-tailed Student’s t-test (for two groups) and a one-way analysis of variance (ANOVA; for more than two groups). Statistical analyses were performed using Graphpad Prism version 6.0 software (GraphPad Software, San Diego California, USA). P<0.05 indicates a statistically significant result. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).