Clinical samples
Glioma tissues were obtained by surgical excision from glioma patients without any treatment (n=20, 20-50 years old) and age-matched patients with relapsed glioma after therapy (n=12, 20-50 years old during July 2018 to December 2018 at Beijing Tiantan Hospital of Capital Medical University. Our study was approved by the Medical Ethics Committee of Beijing Tiantan Hospital of Capital Medical University. Also, written informed consents were obtained from all patients prior to experiments.
Reagents
Small interference RNAs (siRNAs) targeting RMRP, ZNRF3 and IGF2BP3 and a scramble control siRNA (si-NC) were synthesized by GenePharma Co., Ltd. (Shanghai, China). Sense siRNA sequences were provided in Table 1. The pcDNA3.1-RMRP and β-catenin overexpression plasmid was purchased from Sangon Biotech Co., Ltd (Shanghai, China). TMZ were purchased from MedChemExpress Co., ltd. (Monmouth Junction, NJ, USA). In vitro experiments, TMZ was dissolved in DMSO.
Cell culture and transfection
U251 cells were purchased from Cell bank of Chinese Academy of Sciences (Shanghai, China) and were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific). LN229 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in DMEM medium (Thermo Fisher Scientific) containing 5% FBS (Thermo Fisher Scientific). TMZ-resistant U251 cell line (U251/TMZ) and TMZ-resistant LN229 cell line (LN229/TMZ) were established by continuous exposure to increasing doses of TMZ. All cells were maintained in a 95% air/5%CO2 incubator at 37°C. SiRNAs or plasmids, alone or in combination, were transfected into U251 and LN229 cells using Lipofectamine 3000 reagent (Thermo Scientific) following the protocols of manufacturer.
RT-qPCR assay
Total RNA was extracted from glioma tissues and cells using Trizol reagent (Thermo Fisher Scientific) following the instructions of manufacturer. Next, first strand cDNA was synthesized using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific), random primers and RNA template. Subsequently, quantitative PCR reactions were performed using SYBR™ Green PCR Master Mix (Thermo Scientific), cDNA template and specific PCR primers for RMRP, HOXA-AS3, CASC9, ZNRF3, and GAPDH. GAPDH acted as the house-keeping gene to normalize the expression of other genes. The quantitative PCR primer sequences were presented in Table 2.
Western blot analysis
U251 and LN229 cells were lysed using RIPA lysis buffer (Solarbio Life Sciences, Beijing, China) containing protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). After high-speed centrifugation (12000rpm, 15 min, 4°C), cell supernatants were collected. The concentration of protein in cell supernatants was measured using Bio-Rad Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). Next, protein (30μg/sample) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were sequentially incubated with 5% non-fat milk for 1 h at room temperature, primary antibody for 12 h at 4°C and corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Next, the membranes were exposed to the Pierce™ ECL Western Blotting Substrate (Thermo Scientific) to detect the protein signals.
CCK-8 assay
Cell proliferative ability and survival rate were measured using CCK-8 kit (Beyotime Biotechnology). For the detection of cell proliferative activity, transfected cell suspensions (100µl/well) were seeded into 96-well plates and then co-incubated with CCK-8 solution (10µl/well) at 0, 24, 48, 72 h after plating. After 3 h of co-incubation, the optical density (OD) values were measured at 450 nm using the SpectraMax 190 microplate reader (Molecular Device, San Jose, CA, USA). For the measurement of cell survival rate, transfected cells (100µl/well) were seeded into 96-well plates for 24 h and then treated with different concentrations (8, 16, 32, 64, 128, or 256 µM) of TMZ for another 24 h. Next, 10µl of CCK-8 solution was added into each well. Three hours later, OD values were determined at 450 nm, followed by the determination of 50% inhibitory concentration (IC50). Cell survival rate (%) = (OD (drug treated group)-OD (blank group)/OD (untreated group)-OD (blank group))×100%.
Cell apoptotic rate detection
Cell apoptotic rate was estimated using the Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology) according to manufacturer’s instructions. Briefly, cells were collected at 48 h after transfection and re-suspended in Annexin V-FITC binding solution. Next, cells were co-incubated with Annexin V-FITC and Propidium Iodide (PI) solutions for 15 min at room temperature in a dark place. Next, stained samples were placed in an ice bath and cell apoptotic proportion was determined using a flow cytometry (BD Biosciences, San Diego, CA, USA).
RNA immunoprecipitation (RIP) assay
RIP assay was carried out in U251 cells using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Temecula, CA, USA) and antibody against IGF2BP3 or IgG following the manufacturer’s protocols. RMRP and ZNRF3 mRNA levels enriched by IGF2BP3 or IgG antibody were measured through RT-qPCR assay.
RNA pull down assay
RNA pull down assay was performed using a Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific) according to the manufacturer’s protocols. Biotinylated ZNRF3 3’ non-coding region (3’UTR) fragment 1 (1-2477 nt), fragment 2 (2445-3539 nt) and fragment 3 (3506-3912 nt) were purchased from Wuhan Genecreate CO., ltd. (Wuhan, China). Briefly, biotin-labeled RNAs were captured by streptavidin-conjugated magnetic beads. Next, the RNA-bead complexes were co-incubated overnight at 4°C with the lysates of U251 cells. Finally, RNA-associated proteins were eluted and the protein level of IGF2BP3 was determined by western blot assay.
mRNA stability analysis
ZNRF3 mRNA stability was measured using actinomycin D (ActD, Sigma-Aldrich) assay. Briefly, 5 µg/ml of ActD as added into cell medium at 24 h post transfection. At 0, 2, 4, 6 h after ActD treatment, RNA was isolated from cells and ZNRF3 mRNA level was tested through RT-qPCR assay.
Subcellular localization analysis of RMRP
Cytoplasmic and nuclear RNA was isolated from U251 and LN229 cells using Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek lnc, Thorold, Canada) following the protocols of manufacturer. Next, the levels of GAPDH, RMRP and U6 snRNA in cytoplasm and nucleus were determined through RT-qPCR assay.
Immunofluorescence (IF) assay
Cells were fixed with 4% paraformaldehyde solution for 15 min and then treated with 0.5% Triton X-100 solution for 20 min. Next, samples on the slides were blocked with normal goat serum (BOSTER Biological Technology Co., ltd., Wuhan, China) for 30 min at room temperature and then incubated overnight with primary antibody against β-catenin at 4°C. On the next day, samples were incubated with FITC-labeled goat anti-rabbit IgG (Ptoteintech Group, lnc., Wuhan, China) for 1 h at 37°C in the dark. Next, cell nuclei were stained with DAPI solution (Beyotime Biotechnology) for 5 min under the dark conditions. After mounted with Fluoromount-G (Birmingham, AL, USA), the slides were imaged using the Olympus BX53 microscope (Olympus Opticol Co. ltd., Tokyo, Japan).
In vivo experiments
Lentiviruses carrying interference fragment targeting RMRP (sh-RMRP) and lentiviruses bearing scramble control fragment (sh-NC) were obtained from Novobio Biotech Co., ltd. (Shanghai, China). U251 cells were infected with RMRP-targeting or non-targeting lentiviruses for 3 days and then screened with puromycin (1 μg/ml) for 1 week to establish cell lines with or without RMRP stable knockdown.
Mouse experiments were conducted with the approval of the Animal Care and Use Committee of Beijing Tiantan Hospital of Capital Medical University and performed with the standard experimental procedures. The BALB/C nude mice (male, 6 weeks old) were obtained from Laboratory Animal Center of of Beijing Tiantan Hospital of Capital Medical University and acclimatized to the new surroundings for 1 week. Mice were randomly divided into control, sh-NC and sh-RMRP groups (n=6/group). U251/TMZ cells (1×107 cells/mouse), U251/TMZ cells infected with sh-NC (1×107 cells/mouse), U251/TMZ cells infected with sh-RMRP (1×107 cells/mouse) were injected subcutaneously into the right back of mice in the control, sh-NC and sh-RMRP groups. TMZ (25 mg/kg body weight) were intraperitoneally injected into mice every other day for 14 days when tumor volumes reached to approximately 100 mm3. TMZ was dissolved in 70% normal saline and 30% DMSO. Tumor volume was monitored using a vernier caliper every 2 days and calculated using the following formula: tumor volume (mm3) = (L×W2)/2, where L and W respectively represents tumor’s length and width. Tumors were resected, photographed and weighed at the end of experiments.
Statistical analysis
Data were analyzed using GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA) with results presenting as means ± standard deviation. Differential analysis between clinical samples was performed using paired t-tests. Differences between cell line data were compared using unpaired t-tests. Differences among groups were compared using one-way ANOVA or two-way ANOVA along with Bonferroni post hoc test. Differences were regarded as statistically significant when P value was less than 0.05.