Clinical tissue samples
In this study, thirty neuroblastoma 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 neuroblastoma cell line (SK-N-BE(2)) were bought from BeNa Culture Collection (Beijing, China). The neuroblastoma cell line (GI-LI-N) was obtained from Conservation Genetics CAS Kunming Cell Bank (Yunnan, China). These cells were cultivated 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. The cells were grown in an incubator of CO2 (5%) 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 bought 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 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 (neuroblastoma 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). In this study, the primers were as follows: 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 XIST, HK2 or miR-653-5p expression was evaluated 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 were seeded in 96-well plates and then transfected with si-XIST, si-XIST + anti-miR-653-5p, miR-653-5p, miR-653-5p + HK2, or matched controls. MTT reagent (5 mg/mL, 10 µL, Beyotime, Shanghai, China) was added to each well after transfection. After incubation for 4 h, the cultured medium from per well would be carefully discarded and dimethyl sulfoxide (150 µL) was added. 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 were placed 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 paraformaldehyde (4%) for 30 min at 4℃. After that, GI-LI-N and SK-N-BE(2) cells were washed by PBS and then stained by crystal violet (0.1%, Sigma-Aldrich, St. Louis, MO, USA). Microscope (Olympus, Tokyo, Japan) was applied to count colonies containing more than 50 cells. The survival fraction was calculated as previously described [24].
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 placed 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. After 24 h of incubation, non-invaded cells were carefully removed with cotton bud, and invaded cells were fixed using ethanol (95%) and then stained using crystal violet (0.1%). Finally, a microscope was applied to observe and count invaded cells in five random fields.
Measurement Of Glucose Consumption And Lactate Production
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.
In vivo tumor growth assay
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 (male, 5-week-old, n = 7/group, Huafukang, Beijing, China). From the 8th day, tumor volume was examined using a caliper every 4 days and calculated with the formula: length × width2/2. From the 10th day, mice were irradiated with 6 Gy X-ray once a week. All mice were sacrificed after injection for 4 weeks, and tumor samples were weighted and collected for detection of XIST level.
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 that contained 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 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
Transfected cells were lysed using RIPA lysis buffer (Sigma-Aldrich) supplemented with 1 mM phenylmethylsulphonyi fluoride (PMSF; Sigma-Aldrich) to extract the total protein. After quantification by using bicinchoninic acid protein assay kit (Tanon, Shanghai, China), 30 µg of protein in each group was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After that, polyvinylidene fluoride (Beyotime) membranes were employed to transfer the protein. 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℃. Afterward, membranes were maintained in horseradish peroxidase-conjugated anti-rabbit IgG (1:4000, D110058, Sangon Biotech, Shanghai, China) after washing with PBST. Immunoreactive bands were visualized through 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.
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.