Cell culture

Human gastric cancer cells AGS (CRL-1739), MGC-803 (JCRB0254), MKN-45 (JCRB0254), NCI-N87 (CRL-5822), cervical cancer HeLa cells (CCL-2), prostate cancer PC-3 cells (CRL-1435), embryonic kidney HEK293T cells (CRL-11268), and normal gastric epithelial GES-1 cells (C6268) were obtained from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan), American Type Culture Collection (Rockville, MD), or Beyotime Biotechnology (Beijing, China). Cells were authenticated by short tandem repeat profiling, and used within 6 months after resuscitation of frozen aliquots. Mycoplasma contamination was regularly examined using Lookout Mycoplasma PCR Detection Kit (MP0035, Sigma, St. Louis, MO). Cell lines were cultured in RPMI 1640 medium supplied with 10% fetal bovine serum (Gibco, Carlsbad, CA) in a humidified atmosphere of 5% CO2 at 37 °C, and treated with actinomycin D (ActD, Sigma), benzyl-α-GalNAc (BAG, Sigma), or tunicamycin (Tu, Abcam Inc., Cambridge, MA) as indicated.

RT-PCR and real-time quantitative RT-PCR (qRT-PCR)

Nuclear or cytoplasmic RNA was isolated with RNA Subcellular Isolation Kit (Active Motif, Carlsbad, CA). Genomic DNA (gDNA) and total RNA were extracted with DNA Mini Kit (Qiagen, Valencia, CA) and RNeasy Mini Kit (Qiagen), respectively. For circRNA detection, RNase R digestion (3 U/mg, Epicenter, Madison, WI) was performed at 37 °C for 15 min. Complementary DNA was synthesized with Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN). Quantification of mRNA, circRNA, or gDNA was undertaken with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and primers (Additional file 1: Table S1). To measure mRNA stability, de novo RNA synthesis was blocked with ActD (5 μg/ml), while half-life of mRNA was examined by comparing transcript levels before and after ActD treatment.

Northern blot

The junction-specific probes for circ-hnRNPU (Additional file 1: Table S2) were synthesized and labeled by digoxin (DIG) at TSINGKE (Wuhan, China). Northern blotting was conducted as previously reported [16], and recorded on X-ray films with chemiluminescence substrate CSPD (Roche).

RNA fluorescence in situ hybridization (RNA-FISH)

Biotin-labeled antisense or sense oligonucleotide probe targeting circ-hnRNPU junction (Additional file 1: Table S2) was synthesized by TSINGKE, while probes for GAPDH and U1 were synthesized by in vitro transcription of PCR fragments (Additional file 1: Table S1) with biotin RNA Labeling Mix and T7 RNA polymerase (Roche). Hybridization was undertaken in a humidified chamber at 37 °C for 16 h with or without RNase R (3 U/mg) treatment. The signals were detected by using Fluorescent In Situ Hybridization kit (RiboBio, Guangzhou, China), while nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma).

Western blot

Tissue or cellular protein was extracted by using 1 × cell lysis buffer (Promega, Madison, WI). Cytoplasmic or nuclear fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Inc., Grand Island, NY). Western blot was performed as previously described [17, 18], with antibodies specific for O-GlcNAc (#12,938), maltose binding protein (MBP, #2396, Cell Signaling Technology, Danvers, MA), hnRNPU (ab10297), NONO (ab70335), c-Myc (ab32072), Flag-tag (ab45766), hemagglutinin (HA)-tag (ab9110), glutathione S-transferase (GST)-tag (ab36415), polypeptide N-acetylgalactosaminyl-transferase 6 (GALNT6, ab151329), fibronectin 1 (FN1, sc-59826, Santa Cruz Biotechnology, Santa Cruz, CA), alpha-1,3-mannosyl-glycoprotein 2-beta-N- acetylglucosaminyltransferase (MGAT1, ab180578), N-glycosylated protein glucose transporter 1 (GLUT1, ab115730), polypeptide N-acetylgalacto-saminyltransferase 2 (GALNT2, ab140637), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ab8245), histone H3 (ab1791), or β-actin (ab6276, Abcam Inc.).

Lectin affinity precipitation

Cell lysates containing 600 μg of protein were incubated with N-glycans-recognizing biotinylated phaseolus vulgaris lectin (PHA-E + L, 21510096–1, Glycomatrix, Dublin, OH) conjugated to agarose beads overnight at 4 °C, then with 20 μl of streptavidin-conjugated agarose (Thermo Fisher Scientific, Inc.) for 2 h. Glycoprotein/lectin complexes were collected by brief centrifugation (1400 rpm, 5 min, 4 °C), separated on a 12% denaturing sodium dodecyl sulfate (SDS) gel, and subjected to western blotting assay.

Gene over-expression or knockdown

The circ-hnRNPU (594 bp) with circularization framework sites (Additional file 1: Table S3) was synthesized by TSINGKE and subcloned into pLCDH-ciR (Geenseed Biotech Co., Guangzhou, China). To generate corresponding construct of linear circ-hnRNPU (lin-hnRNPU), the reverse circularization framework sites were removed by QuikChange II Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA) and primers (Additional file 1: Table S3). Human NONO cDNA (1416 bp) and c-Myc cDNA (1365 bp) were provided by Drs. Jean-Yves Masson [19] and William P. Tansey [20], respectively. Their truncations were amplified with PCR primers (Additional file 1: Table S3) and subcloned into pCMV-3Tag-1A, pCMV-HA, pMAL-c4X, or pGEX-6P-1 (Addgene, Cambridge, MA), respectively. Mutation of nuclear localization signal (NLS) was undertaken with QuikChange II Site-Directed Mutagenesis Kit (Agilent) and primer sets (Additional file 1: Table S3). Human hnRNPU, GALNT6 and MGAT1 vectors were obtained from GeneChem Co., Ltd (Shanghai, China). Oligonucleotides specific for short hairpin RNAs (shRNAs) targeting circ-hnRNPU, NONO, or c-Myc (Additional file 1: Table S2) were synthesized by TSINGKE and inserted into GV298 (GeneChem Co., Ltd). Lentiviral vectors were co-transfected with packaging plasmids psPAX2 and pMD2G (Addgene) into HEK293T cells. Stable cell lines were obtained by selection for neomycin or puromycin (Invitrogen, Carlsbad, CA) resistance.

Rescue of target gene expression

To rescue circ-hnRNPU over-expression-altered gene expression, NONO or c-Myc construct was transfected into stable cell lines. To restore gene expression induced by circ-hnRNPU silencing, shRNAs against NONO or c-Myc (Additional file 1: Table S2) were transfected into cancer cells with Genesilencer Transfection Reagent (Genlantis, San Diego, CA).

RNA sequencing (RNA-seq)

Total RNA of cancer cells (1 × 106) was extracted with TRIzol® reagent (Life Technologies, Inc.). Library preparation and transcriptome sequencing on an Illumina HiSeq X Ten platform were carried out at Wuhan SeqHealth Technology Co., Ltd. (Wuhan, China). Gene set enrichment analysis (GSEA) was undertaken as previously reported [17], with application of indicated gene sets.

Biotin-labeled RNA pull-down and mass spectrometry

Biotin-labeled oligonucleotide probes targeting junction sites of circRNAs were synthesized (Invitrogen, Additional file 1: Table S2). The lysates of 2 × 107 cancer cells were incubated with 3 μg of biotin-labeled probe for 2 h, and incubated with 35 μl of Streptavidin C1 magnetic beads (Invitrogen) for 1 h. Retrieved proteins were detected through western blot or mass spectrometry at SpecAlly Life Technology Co., Ltd (Wuhan, China).

Cross-linking RNA immunoprecipitation (RIP)

Cells were cross-linked by ultraviolet light (200 J/cm2, 254 nm). RIP assay was undertaken with Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Temecula, CA), using antibodies specific for NONO (ab70335) or Flag-tag (ab45766, Abcam Inc.) and protein A-Sepharose beads (Santa Cruz Biotechnology) at 4 °C for 4 h. Co-precipitated RNAs were detected by RT-PCR or real-time qRT-PCR with primers (Additional file 1: Table S1).

RNA electrophoretic mobility shift assay (EMSA)

The 5'-monophosphorylated linear circ-hnRNPU was in vitro transcribed by using Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase, and circularized with guide oligonucleotide targeting circular junction (Additional file 1: Table S2) and T4 RNA ligase I (Qiagen) [21]. RNA EMSA assay using recombinant NONO protein and biotin-labeled circular probe of circ-hnRNPU was performed with LightShift Chemiluminescent RNA EMSA Kit (Thermo Fisher Scientific, Inc.).

In vitro binding assay

By using the FLAG® M Purification Kit (Sigma), Flag-tagged NONO protein was prepared from HEK293 cells transfected by corresponding constructs. The GST-tagged NONO proteins were produced from E. coli as previously described [21]. The Flag-tagged NONO protein was purified by using FLAG® M Purification Kit (Millipore). The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma). Circ-hnRNPU was validated by RT-PCR using divergent primers (Additional file 1: Table S1), while protein was detected by western blot.

Co-immunoprecipitation (co-IP) assay

Co-IP was performed as previously described [17, 18, 21], with antibodies (1:200 dilutions) specific for NONO (ab70335), c-Myc (ab32072), Flag-tag (ab45766), HA-tag (ab9110), GST-tag (ab36415, Abcam Inc.), or MBP (#2396, Cell Signaling Technology). Bead-bound proteins were released and analyzed by western blot.

Fluorescence immunocytochemical staining

Cancer cells were grown on coverslips, incubated with antibody specific for NONO (ab70335, Abcam lnc.; 1:100 dilution) or c-Myc (ab32072, Abcam lnc.; 1:100 dilution) at 4 °C overnight, and treated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:1000 dilution) and DAPI (300 nM) staining.

Bimolecular fluorescence complementation (BiFC) assay

Human NONO cDNA (1416 bp) and c-Myc cDNA (1365 bp) were respectively subcloned into BiFC vectors pBiFC-VN173 or pBiFC-VC155 (Addgene), and co-transfected into cancer cells for 24 h. The fluorescence emission was observed under a confocal microscope, with excitation and emission wavelengths of 488 and 500 nm, respectively [17, 18].

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was undertaken by using EZ-ChIP kit (Upstate Biotechnology, Temacula, CA), with antibodies (1:100 dilution) specific for c-Myc (#18,583, Cell Signaling Technology, Inc.) and primers targeting gene promoters (Additional file 1: Table S1).

Dual-luciferase reporter assay

Complementary oligonucleotides containing three or four canonical binding sites of transcription factors, and promoter fragments of GALNT6 (-460/ + 50) or MGAT1 (-1595/-1196) amplified from genomic DNA (Additional file 1: Table S3) were subcloned into pGL3-Basic (Promega). Human NONO activity reporter was established by inserting complementary oligonucleotides containing four canonicial NONO binding sites (Additional file 1: Table S3) into 3'-UTR of Renilla luciferase within psiCHECK2 (Promega). Dual-luciferase assay was performed as previously reported [18, 22].

In vitro cell viability, growth, migration, and invasion assays

The 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) colorimetric, soft agar, and matrigel invasion assays were conducted to measure the viability, growth, and invasive capabilities of cancer cells in vitro [17, 21, 22].

In vivo growth, metastasis, and therapeutic assays

All animal experiments were approved by the Experimental Animal Ethics, Huazhong University of Science and Technology, and undertaken according to NIH Guidelines for the Care and Use of Laboratory Animals. For in vivo tumor growth and experimental metastasis studies, cancer cells (1 × 106 or 0.4 × 106) were injected into dorsal flanks or tail vein of blindly randomized four-week-old male BALB/c nude mice (National Rodent Seeds Center, Shanghai, China) [17, 18, 21]. For in vivo therapeutic studies, cancer cells (1 × 106 or 0.4 × 106) were injected into dorsal flanks or tail vein of nude mice, respectively. One week later, mice were blindly randomized and treated by tail vein injection of lentivirus (1 × 107 plaque-forming units) as indicated. The in vivo Optical Imaging System (In-Vivo FX PRO, Bruker Corporation, Billerica, MA) was applied to acquire fluorescent images of xenograft tumors in nude mice.

Patient tissue samples

Human tissue study was approved by the Institutional Review Board of Union Hospital, Tongji Medical College. All procedures were conformed to principles set forth by Declaration of Helsinki. Written informed consent was obtained from all patients without preoperative chemotherapy or other treatment. Fresh cancer tissues were collected at surgery, validated by pathological diagnosis, and stored at -80 °C.

Immunohistochemistry

Immunohistochemical staining and quantitative evaluation were performed as previously described [17, 18], with antibodies specific for Ki-67 (1:500, sc-23900, Santa Cruz Biotechnology), CD31 (1:500, ARG52748, Arigo, Hsinchu City, Taiwan), or NONO (1:500, ab70335, Abcam Inc.).

Statistical analysis

All data were shown as mean ± standard error of the mean (s.e.m.). Cutoff values for gene expression were defined by medium or average levels. Student’s t test, analysis of variance (ANOVA), Mann–Whitney U test, and χ2 analysis were used to evaluate differences. Statistical significance of overlap between two gene lists was determined by Fisher’s exact test. Pearson’s correlation coefficient assay was used to analyze expression correlation. Log-rank test was used to assess survival difference. All statistical tests were two-sided and considered statistically significant when P values were less than 0.05.