Human glioma and normal tissues
To detect the endogenous expressions of ZCRB1, circHEATR5B, HEATR5B-881aa and JMJD5, human glioma tissues were collected and divided into two parts: low-grade glioma tissues (LGGTs, WHO Grade I-II, n = 12) and high-grade glioma tissues (HGGTs, WHO Grade III-IV, n = 12) based on the World Health Organization classification guidelines for pathological grades in 2016 . Adjacent non-tumor brain tissues (NBTs, n = 12) served as the negative control.
The human GBM cell lines (U87, U251, U373, and A172) and 293 T cells were purchased from the Shanghai Institutes for Biological Sciences Cell Resource Center and were cultured in DMEM (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (TBD, Tianjin, China) according to standard protocols. Normal human astrocytes (NHAs) were purchased from ScienCell Research Laboratories and were cultured in astrocyte medium (ScienCell, Carlsbad, CA). All cells were cultured at 37 °C in a 5% CO2 humidified incubator.
RNA sequencing (RNA-seq)
Total RNAs were extracted from three GBM and NBTs using TRIzol reagent (Life Invitrogen, Carlsbad, CA) and then treated using the RiboMinus Eukaryote Kit (Qiagen, Valencia, CA) to remove rRNAs before generating RNA-seq library. Next, the RNA-seq library was deep sequenced with the Illumina HiSeq 2000. RNA sequencing reads were aligned to the human reference genome by software STAR and RNA abundance was quantified using software RSEM.
Quantitative reverse transcription PCR assay (qRT–PCR)
Total RNAs were extracted from tissues and cells using TRIzol reagent. RNA concentration and quality were measured using NanoDrop2000 Spectrophotometer. The expressions of circHEATR5B and the mRNAs of ZCRB1, HEATR5B, JMJD5 and β-actin were detected using the One Step TB Green® PrimeScript™ RT-PCR Kit (TaKaRa, Liaoning, China) on the ABI 7500 Fast Real-Time PCR system. The primers are given in Additional file 2: Table S1. The results were calculated using the 2-ΔΔCt relative quantification method and β-actin served as an endogenous control.
Western blot assay
Total proteins were extracted from tissues and cells using RIPA lysis buffer (Beyotime, Shanghai, China). Western blot assay was performed as previously described . Protein samples were added into SDS-PAGE gels followed by electrophoresis and subsequently transferred to PVDF membranes. PVDF membranes were incubated with primary antibodies overnight at 4 °C after blocking for 2 h at room temperature. The next day, after washing by TTBS, PVDF membranes were incubated with the corresponding secondary antibodies at room temperature for 2 h. Proteins were identified using the BeyoECL Star Kit (Beyotime, Shanghai, China) and captured by MicroChemi chemiluminescent imaging system (DNR, Jerusalem, Israel). ImageJ software was used for analyzing the bands and β-actin was used as an endogenous control.
The primary antibodies are as follows: ZCRB1 (Proteintech Cat# 25629–1-AP), JMJD5 (ABclonal Cat# A11606), β-actin (Proteintech Cat# 20536–1-AP), GST tag (Proteintech Cat# 66001–2-Ig), and FLAG tag (Proteintech Cat# 20543–1-AP). HEATR5B-881aa and p-JMJD5-S361 antibodies were prepared by Beijing Huada Protein Innovation. The secondary antibodies are as follows: Goat anti-mouse IgG (Proteintech Cat# SA00001–1), Goat anti-rabbit IgG (Proteintech Cat# SA00001–2).
Immunofluorescence assay (IF)
Cells seeded on glass slides were fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.2% TritonX-100 for 20 min and then blocked with 5% BSA for 2 h at room temperature. Next, the cell slides were incubated with primary antibodies at 4 °C overnight. Then, the cell slides were washed with PBST three times and incubated with fluorescent-conjugated secondary antibodies, Goat anti-rabbit Alexa Fluor 488 or Goat anti-rabbit Alexa Fluor 647 (Beyotime, Shanghai, China), for 2 h at room temperature away from the light. Finally, the nuclei were counterstained with DAPI for 5 min. Fluorescence was visualized under laser confocal microscopy.
Cells were seeded into 24-well plates and transfected with plasmids using Lipofectamine3000 reagent (Life Technologies, Carlsbad, CA) based on the experimental groupings when cell confluence reached 50–70%. For each well, 0.75 μl Lipofectamine3000 reagent was diluted with 25 μl Opti-MEM Medium (Thermo Fisher, Waltham, MA). 500 ng plasmid DNA was diluted with 25 μl Opti-MEM Medium and 1 μl P3000 reagent. Then, the diluted DNA was incubated with diluted Lipofectamine3000 reagent for 10 min at room temperature. Finally, the mixture was added into the well and incubated with cells for 48 h at 37 °C. The stably transfected cells were screened by neomycin or puromycin and the transfection efficiency was detected by qRT–PCR and western blot assays.
The shRNAs against ZCRB1 (sh-ZCRB1: site #1, 5′-GCACAGTGTATGTATCCAA-3′; site #2, 5′-CTGACAAACAATGACTTGT-3′; site #3, 5′-GGGCAATAAACAACAAACA-3′) and the shRNAs against circHEATR5B (sh-circHEATR5B: site #1, 5′-CTCAACCAGGTTGAAATCGGC-3′; site #2, 5′-AACCAGGTTGAAATCGGCTCG-3′) were synthesized by GenePharma, the shRNAs against JMJD5 (sh-JMJD5: site #1, 5′-CCTGTTCATCCCGGTGAAATA-3′; site #2, 5′-GAGGAGGAAATCACCATCAAT-3′; site #3, 5′-GTCAACGAGTTCATCAGCAAA-3′) were synthesized by GeneChem, and their corresponding empty plasmids (sh-ZCRB1-NC, sh-circHEATR5B-NC, and sh-JMJD5-NC) were constructed as the negative control. The plasmids with ZCRB1 full-length sequence (OV-ZCRB1), circHEATR5B full-length sequence (OV-circHEATR5B) or mutant IRES (circHEATR5B-IRES-Mut), HEATR5B-881aa full-length sequence (OV-HEATR5B-881aa), JMJD5 full-length sequence (OV-JMJD5), JMJD5 with wild-type S361 (JMJD5-WT) or mutant S361 (JMJD5-S361A, JMJD5-S361E), and their corresponding empty plasmids were constructed by GenePharma.
Cell viability assay
Cell viability was detected using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly, cells (1 × 103 in 100 μl) were seeded into 96-well plates and incubated at 37 °C for 48 h. Then, 10 μl of CCK-8 reagent was added into each well and incubated for 1–4 h at 37 °C. Finally, the absorbance at 450 nm was measured by SpectraMax M5 microplate reader.
Glucose and lactate measurement assays
Glucose and lactate concentration were detected using the glucose assay kit and lactic acid assay kit (Nanjing Jiancheng, Jiangsu, China). In brief, cells (1 × 103 per well) were seeded into 96-well plates with 100 μl medium. The supernatants of medium were collected after 24 h and added into glucose or lactate assay kit reagents according to the manufacturer’s protocol. The absorbance at corresponding wavelength was measured by SpectraMax M5 microplate reader. Finally, glucose consumption and lactate production were calculated.
Seahorse XF glycolysis stress assay
Extracellular acidification rate (ECAR) was measured using the Seahorse XF Glycolysis Stress Test Kit (Agilent, Santa Clara, CA) and XF24 Extracellular Flux Analyzer, as previously described . Briefly, cells (5 × 104 per well) were seeded into Seahorse XF24 V7 PS Cell Culture Microplates and cultured overnight. ECAR was measured in XF base medium supplemented with 2 mM glutamine (pH = 7.4) following the sequential injection of glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM). Calculations were as follows: Glycolysis = Maximum rate measurement before oligomycin injection–Last rate measurement before glucose injection, representing basal glycolysis level; Glycolytic Capacity = Maximum rate measurement after oligomycin injection–Last rate measurement before glucose injection, representing maximum glycolysis level.
CircRNA microarray analysis
Total RNAs extracted from ZCRB1-transfected U251 and U373 cells were quantified using the NanoDrop2000 spectrophotometer. The sample preparation and microarray hybridization were performed according to the standard protocols of Arraystar. In brief, circRNAs were enriched from total RNAs by digesting and eliminating linear RNAs with RNase R. Then, the enriched circRNAs were amplified and transcribed into fluorescent cRNAs by utilizing the Arraystar Super RNA Labeling Kit (Arraystar, Rockville, MD). The labeled cRNAs were hybridized onto the human circular RNA array V2.0 (Arraystar, Rockville, MD) and incubated at 65 °C for 17 h. After washing slides, the arrays were scanned by Agilent microarray scanner.
RNase R digestion and actinomycin D treatment
RNase R digestion and actinomycin D treatment were both performed to confirm the stability of circHEATR5B in GBM cells. For RNase R digestion, 2 mg total RNAs were incubated with 3 U/mg RNase R (Lucigen, Madison, WI) at 37 °C for 30 min. For actinomycin D treatment, cells were treated with 2 mg/ml actinomycin D (NobleRyder, Beijing, China) and harvested after incubation for 0, 4, 8, 12, 24 h. After treatment with RNase R or actinomycin D, qRT–PCR assays were conducted to determine the expressions of circHEATR5B and HEATR5B mRNA.
Nascent RNA capture assay
Nascent RNAs were prepared using the Click-iT Nascent RNA Capture Kit (Thermo Fisher, Waltham, MA) following the manufacture’s instruction. In brief, ZCRB1-transfected cells were incubated with 0.5 mM 5-ethynyl uridine for 15 min, and then EU-labeled RNAs were isolated using TRIzol reagent and biotinylated by Click reaction. The biotinylated RNAs were captured by streptavidin magnetic beads and reverse-transcribed into cDNA for the qRT–PCR analysis.
RNA Immunoprecipitation assay (RIP)
EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA) was utilized for RIP assay. 100 μl lysates of ZCRB1-upregulated U251 cells (2 × 107) were incubated with 50 μl magnetic beads coupled with 5 μg anti-ZCRB1 overnight at 4 °C. Mouse IgG antibody (Santa Cruz Cat# sc-2025) was used as the negative control. The immunoprecipitated RNA was isolated by proteinase K and analyzed by qRT–PCR assays. The primers are given in Additional file 2: Table S2.
RNA pull-down assay
Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, Waltham, MA) was used for RNA pull-down assays. The flanking sequences and back-splicing junction of circHEATR5B were synthesized segmentally and biotinylated by Pierce RNA 3′ Desthiobiotinylation Kit. Bio1 and Bio3, the probes synthesized by 1 kb flanking sequences upstream and downstream from circHEATR5B exons, respectively; Bio2 and Bio4, the probes synthesized by 1–2 kb flanking sequences upstream and downstream from circHEATR5B exons, respectively; Bio5, 1 kb sequences across circHEATR5B junction. Then, biotinylated probes captured by streptavidin magnetic beads were incubated with the lysates of ZCRB1-upregulated U251 cells for 1 h at 4 °C. Finally, the pull-down proteins were detected by western blot assays after elution for 30 min at 37 °C.
Fluorescence in situ hybridization assay (FISH)
FISH assays were performed using the RNA FISH assay kit (GenePharma, Shanghai, China). U251 and U373 cells were fixed on slides in 100 μl 4% paraformaldehyde at room temperature for 15 min. Then, the slides were treated with 100 μl 0.1% Buffer A (TritonX-100) for 15 min at room temperature. After washed twice with PBS, the slides were treated with 100 μl 2 × Buffer C (Saline-Sodium Citrate buffer) for 30 min at 37 °C and then incubated with 100 μl circHEATR5B probe (5′-Cy3-CGAUUUCAACCUGGUUGAGAAUAUUCCAGG-3′, red-labeled, GenePharma, Shanghai, China) overnight at 37 °C away from the light. The next day, after sequentially washed by 100 μl 0.1% Buffer F (Tween 20), 100 μl 2 × Buffer C, and 100 μl 1 × Buffer C, the slides were stained with 100 μl DAPI for 20 min away from the light. Finally, fluorescence images were captured under laser confocal microscopy.
Nuclear and cytoplasmic extraction
Nuclear and cytoplasmic fractions were isolated using the PARIS™ kit (Thermo Fisher, Waltham, MA). Briefly, U251 and U373 cells were lysed by Cell Fractionation Buffer for 10 min on ice. Then, the supernatants were collected as the cytoplasmic fractions after centrifugation at 4 °C and 500×g for 3 min. Finally, the pellets were lysed by Cell Disruption Buffer to collect the nuclear fractions.
Dual-luciferase reporter assay
The reporter vector constructions were carried out by inserting full-length, truncated, or mutated IRES into dual-luciferase reporter vectors. 293 T cells were transfected with constructed reporter vectors and the relative luciferase activities were detected 48 h after transfection using the Dual-Luciferase® Reporter Assay System Kit (Promega, Madison, WI) following the manufacturer’s protocol.
Pyruvate kinase enzymatic activity assay
Pyruvate kinase enzymatic activity was measured using the pyruvate kinase assay kit (Nanjing Jiancheng, Jiangsu, China). Briefly, cell lysates were prepared and the protein concentration was measured. For the colorimetric assay, absorbance was measured at 340 nm at the 30 s after adding reagents to read A1 and measured again at the 15 min 30 s after incubating at 37 °C for 15 min to read A2. Relative pyruvate kinase enzymatic activity was calculated by the ratio of (A1–A2)/(protein concentration).
Co-immunoprecipitation assay (Co-IP)
Co-IP assays were performed using the Pierce Co-immunoprecipitation (Co-IP) Kit (Thermo Fisher, Waltham, MA) following the manufacturer’s instructions. Cell lysates were prepared and incubated with AminoLink Plus Coupling Resin immobilized primary antibody overnight at 4 °C. Then, the samples were washed three times with 200 μl Wash Buffer and eluted with Elution Buffer for 5 min. The eluates were finally analyzed by western blot assays.
GST pull-down assay
Prokaryotic expression plasmids fused with FLAG or GST tag were constructed including FLAG-HEATR5B-881aa, GST-JMJD5(-WT), GST-JMJD5-S361A. These plasmids were transformed into E. coli competent cell BL21 (TaKaRa, Liaoning, China) and protein expression was induced by 0.8 mM IPTG (Solarbio, Beijing, China) at 25 °C and 200 rpm for 6 h. Then, cells were lysed, sonicated, and centrifuged. The proteins were purified using the BeyoMag™ anti-Flag Magnetic Beads and BeyoGold™ GST-tag Purification Resin (Beyotime, Shanghai, China) according to the manufacturer’s procedure. For GST pull-down assay, purified FLAG-HEATR5B-881aa was incubated with purification resin coupled with GST-JMJD5 protein at 4 °C overnight. Then protein complexes were eluted by Elution Buffer and then subjected to SDS-PAGE and analyzed by western blot assays.
In vitro kinase assay
In brief, 10 μg purified GST, GST-JMJD5(-WT), or GST-JMJD5-S361A proteins were incubated with 5 μg recombinant active HEATR5B-881aa in 50 μl kinase buffer (Cell Signaling Technology, Danvers, MA) containing 5 μCi [γ-32P] ATP (PerkinElmer, Waltham, MA) for 30–60 min at 30 °C. The kinase reaction was terminated by adding 12.5 μl 5 × SDS-PAGE loading buffer and boiling for 10 min. Then, the samples were resolved by SDS-PAGE gel electrophoresis. Radioactive signals were detected by autoradiography through a phosphor screen. The gels were subjected to Coomassie brilliant blue staining and destaining to visualize the protein bands.
Mass spectrometry analysis
SDS-PAGE gel electrophoresis separated proteins and the targeted protein bands were excised from the gel. After elution, reduction and alkylation, the proteins were digested at 37 °C overnight. Peptides were collected, desalted and analyzed by timsTOF pro mass spectrometer (Bruker, Bremen, Germany). Sequence and site identification were analyzed using NCBI nonredundant protein database with Mascot Daemon.
Cycloheximide chase assay
Cycloheximide, a protein synthesis inhibitor, was used to determine JMJD5 half-life. U251 cells transfected with JMJD5-WT, JMJD5-S361A, or JMJD5-S361E were treated with 100 mg/ml cycloheximide (NobleRyder, Beijing, China) and collected in 0, 2, 4, 8, 10 h. Then, total proteins were prepared and detected by western blot assays.
Tumor xenografts in nude mice
Four-week-old nude mice (BALB/c) were purchased from Beijing Vital River Laboratory for in vivo study. The experiments were conducted strictly following the protocols approved by the Ethics Committee of China Medical University. The stably transfected U251 and U373 cells were selected and divided into five groups. For subcutaneous xenografts, 3 × 105 cells were subcutaneously injected into the right flank of each nude mouse. Tumor volumes were measured every 4 days and calculated by formula: volume (mm3) = length×width2/2. The mice were sacrificed and then xenograft tumors were separated at the endpoints. For orthotopic xenografts, nude mice were injected with 3 × 105 cells stereoscopically into the right striatum. The surviving numbers of nude mice were recorded, and Kaplan-Meier curves were used for survival analysis.
All experimental data were indicated as mean ± SD. GraphPad Prism 5.01 was used for statistical analysis. Comparison between groups was analyzed by Student’s t-test, one-way ANOVA, or two-way ANOVA. Statistical significance was determined by P-value< 0.05.
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