Cell culture

Human gastric cancer cell lines MKN-45 (JCRB0254), AGS (CRL-1739), HGC-27 (C6365), SNU-1 (CRL-5971), normal gastric mucosal GES-1 (C6268) cells, and embryonic kidney HEK293T cells (CRL-11268) were obtained from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan), American Type Culture Collection (Rockville, MD), or Beyotime Biotechnology (Beijing, China). Cell line authentication was performed by short tandem repeat (STR) profiling, and used within 6 months after resuscitation from frozen aliquots. Mycoplasma contamination was verified by using real-time quantitative RT-PCR (qRT-PCR) mycoplasma detection kit (Sigma, St. Louis, MO). The MKN-45, HGC-27, SNU-1, GES-1, and HEK293T cells were cultured in RPMI 1640 media (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Gibco), while AGS was cultured in F12K (Gibco) media supplemented with 10% FBS (Gibco).

RNA and protein isolation

Nuclear and cytoplasmic RNAs or proteins were isolated from cells following the protocol of Ambion® PARIS™ Kit (Thermo Fisher Scientific, Waltham, MA). Briefly, the cells were initially washed with phosphate buffered solution (PBS), then resuspended in 100–500 μl of ice-cold Cell Fractionation Buffer and incubated on ice for 5–10 min. Subsequently, the samples were centrifuged for 1–5 min at 4 °C and 500 × g, after which the cytoplasmic fraction was carefully aspirated from nuclear pellet. The remaining fraction was homogenized in ice-cold Cell Fractionation Buffer to obtain the nuclear lysates. Finally, the sample was applied for RNA isolation or protein analysis. Total RNA was isolated following the manual of TRIzol reagent (Thermo Fisher Scientific) or QIAwave RNA Mini Kit (QIAGEN, Stockach, Germany), while total cellular or tissue proteins were extracted with RIPA lysis buffer (Thermo Fisher Scientific).

RT-PCR and qRT-PCR

The cDNA was synthesized by using a reverse transcription (RT) kit with genomic DNA Eraser (Takara, Dalian, China). Quantitative analysis of mRNA and snoRNA was conducted using SYBR Green PCR kit (Takara), specific primers (Additional file 1: Table S1), and a StepOne real-time PCR system (Applied Biosystems, Carlsbad, CA). The levels of transcripts were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and determined by using the 2−△△Ct method [19, 20].

RNA sequencing (RNA-seq)

Two micrograms of total RNA were utilized for stranded RNA library preparation, while transcriptome sequencing on Novaseq 6000 sequencer (Illumina) with PE150 model was performed at Seqhealth Tech Co. LTD (Wuhan, China). Then, the resulting data were filtered, aligned, and processed to calculate reads per kilobase (kb) of transcript per million mapped reads (RPKM). Alternative splicing events were detected by using rMATS (version 3.2.5) [21] with a false discovery rate (FDR) value less than 0.05. Percent spliced-in (PSI) was applied to quantify alternative splicing events by calculating the expression of individual exons in “exon inclusion” isoforms relative to total expression of all isoforms. Meanwhile, the difference of two PSI values of same gene, termed as delta-PSI (ΔPSI), across samples was used as an indicator of differential splicing. The ∆PSI threshold of 10% was considered to be statistically significant if P-value was less than 0.05 [22]. The sequencing results were submitted to Gene Expression Omnibus (GEO) under the accession number GSE285296 and GSE285402.

Western blot assay

Western blot assay was carried out as previously documented [23,24,25,26,27], with antibodies against CMTR1 (ab70386), MBD2 (ab188474), Flag-tag (ab45766), myelin basic protein (MBP)-tag (ab40390), insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2, ab124930), polypyrimidine tract-binding protein 1 (PTBP1, ab133734, Abcam, Cambridge, MA), ESF1 nucleolar pre-rRNA processing protein homolog (ESF1, 23496–1-AP), serine/arginine-rich splicing factor 1 (SRSF1, 12929–2-AP), nuclear valosin-containing protein-like (NVL, 16970–1-AP, Proteintech, Wuhan, China), hemagglutinin (HA)-tag (3724S), CD44 (3570S), heterogeneous nuclear ribonucleoprotein C (HNRNPC, 91327S), heterogeneous nuclear ribonucleoprotein U (HNRNPU, 34095S), splicing factor 3B subunit 1 (SF3B1, 14434S), U2 small nuclear RNA auxiliary factor 2 (U2AF2, 70471S), GAPDH (2118S, Cell Signaling Technology, Danvers, MA), ELAVL1 (sc-5261), histone H3 (sc-24516), or glutathione S-transferase (GST)-tag (sc-33614, Santa Cruz Biotechnology, Santa Cruz, CA).

Gene over-expression and silencing

Human SNORA37 cDNA (129 bp) was obtained by PCR amplification (Additional file 1: Table S2) and validated via Sanger sequencing. The expression vector of human CMTR1 cDNA (2508 bp) was purchased from Genechem (Shanghai, China). Subsequently, SNORA37 cDNA or CMTR1 cDNA was subcloned into CV186 (Genechem), while CMTR1 truncations were obtained via PCR amplification and primer sets (Additional file 1: Table S2), and inserted into pGEX-6P-1 or pCMV-HA (Addgene, Cambridge, MA), respectively. The ELAVL1 vectors were established as previously described [28], while MBP tagged-ELAVL1 truncations were obtained via PCR amplification and primer sets (Additional file 1: Table S2), and inserted into pMAL-c4X (Addgene). Oligonucleotides specific for short hairpin RNAs (shRNAs) targeting SNORA37, CMTR1, or ELAVL1 (Additional file 1: Table S3) were inserted into the GV298 (Genechem). The constructs were transfected using NEOFECT® DNA transfection reagent (NEOFECT, Beijing, China), and stable cancer cell lines were established by selection with puromycin (Invitrogen, Carlsbad, CA).

Rescue of target gene expression

To rescue SNORA37 silencing-altered gene expression, CMTR1 or ELAVL1 construct was transfected into stable cell lines with NEOFECT® DNA transfection reagent (NEOFECT). To restore gene expression induced by SNORA37 over-expression, shRNAs against CMTR1 or ELAVL1 (Additional file 1: Table S3) were transfected into stable cancer cells with NEOFECT® DNA transfection reagent (NEOFECT).

RNA fluorescence in situ hybridization (FISH)

By utilizing a double-stranded DNA template containing a T7 promoter consensus motif (Additional file 1: Table S1) and biotin-16-UTP (Roche, Basel, Switzerland), the probes for SNORNA37, GAPDH, or U1 were synthesized, while antisense probe for SNORA37 was prepared with In vitro Transcription T7 Kit (Takara). Subsequent RNA purification was conducted by using RNeasy Min Elute Cleanup Kit (QIAGEN). Hybridization was performed using FISH kit (RiboBio, Guangzhou, China) following the manufacturer’s instructions, with nuclei counterstained using 4',6-diamidino-2-phenylindole (DAPI). The images were observed and photographed under a Nikon A1Si Laser Scanning Confocal Microscope (Nikon, Japan) [20, 25].

Biotin-labeled RNA pull-down and mass spectrometry

The biotin-labeled SNORA37 sense and antisense probes were synthesized by aforementioned in vitro transcription method, and incubated overnight with streptavidin magnetic beads, cell lysates, and RNAase inhibitors at 4℃. The bound proteins were subsequently retrieved for silver staining detection through Pierce Silver Stain Kit (Thermo Fisher Scientific) [20, 25], while other portion was subjected to mass spectrometry analysis at Wuhan SpecAlly Tech Co. LTD (Wuhan, China).

Cross-linking RNA immunoprecipitation (RIP)

At 254 nm (200 J/cm2), ultraviolet light was used to cross-link cells [20, 25]. RIP assay was conducted according to the protocol provided by Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA). Briefly, cells were lysed in RIP lysis buffer, and incubated with magnetic beads and antibodies specific to CMTR1 (ab70386), HA-tag (3724S), GST-tag (sc-33614), ELAVL1 (sc-5261), HNRNPC (91327S), HNRNPU (34095S), or PTBP1 (ab133734) at 4 °C overnight [20, 25]. Following washing, RNA was purified using RNeasy MinElute Cleanup Kit (QIAGEN), and co-precipitated RNA was detected by RT-PCR or real-time qRT-PCR with specific primers (Additional file 1: Table S1).

In vitro binding assay

The GST-tagged CMTR1 (pGEX-6P-1) or MBP-tagged ELAVL1 (pMAL-c4X) truncation constructs were transformed into E. coli BL21 strain (Thermo Fisher Scientific), while proteins were purified by Pierce GST Spin Purification Kit or Pro-Detect™ Rapid MBP Assay Kit (Thermo Fisher Scientific). The SNORA37 was transcribed by using In vitro Transcription T7 Kit (Takara) and purified with RNeasy Min Elute Cleanup Kit (QIAGEN) [20, 25], followed by co-incubation with GST-tagged CMTR1 or MBP-tagged ELAVL1 protein. The anti-GST beads (Sigma) were used to pull down protein-RNA complex. Protein was then detected through sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and western blot, while RNA was measured through RT-PCR using specific primers (Additional file 1: Table S1).

Fluorescence immunocytochemical staining

Cancer cells were grown on confocal dish, and incubated with complete medium until fully adherent. Then, cells were with 4% paraformaldehyde and treated with antibodies specific for CMTR1 (ab70386, Abcam; 1:200 dilution) or ELAVL1 (sc-5261, 1:100 dilution) at 4 °C overnight. Then, coverslips were treated with Cy3-conjugated goat anti-rabbit IgG (1:1000 dilution) or 488-conjugated goat anti-rabbit IgG, and stained by DAPI (300 nmol/L). The images were photographed under a Nikon A1Si Laser Scanning Confocal Microscope (Nikon) [19, 20, 25,26,27].

Co-immunoprecipitation (Co-IP)

Co-IP experiment was conducted as previously reported [19, 20, 25,26,27,28,29], utilizing specific antibodies against CMTR1 (ab70386), ELAVL1 (sc-5261), Flag-tag (ab45766), MBP (ab40390), HA-tag (3724S, Cell Signaling Technology), or GST (sc-33614, Santa Cruz Biotechnology) for immunoprecipitation. Subsequently, proteins bound to Protein A/G Magnetic Beads (MedChemExpress, shanghai, China) were recovered and detected by western blot.

Bimolecular fluorescence complementation (BiFC) assay

Human CMTR1 cDNA (2508 bp) and ELAVL1 cDNA (1818 bp) were respectively inserted into pBiFC-VC155 (22011, Addgene) or pBiFC-VN173 (22010, Addgene; Additional file 1: Table S2). After co-transfection of these constructs by using NEOFECT® DNA transfection reagent (NEOFECT) for 24 h, cells were fixed with 4% paraformaldehyde, stained with DAPI for 10 min, and observed under a Nikon A1Si Laser Scanning Confocal Microscope (Nikon). The excitation and emission wavelengths of confocal microscope were 488 nm and 500 nm, respectively [19, 20, 25,26,27].

In vitro cellular viability, growth, and invasion assays

In vitro viability, growth, and invasive capabilities of cancer cells were detected by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) colorimetric [30], soft agar (Noble agar, Sigma) [19, 20, 25,26,27], and matrigel (BioCoat™ Matrigel®, Corning, NY, USA) invasion assays, respectively [19, 20, 25,26,27].

Xenografts in nude mice

All animal experiments were conducted in accordance with the guidelines established by Experimental Animal Ethics, Huazhong University of Science and Technology and National Institutes of Health’s Guidelines for the Care and Use of Experimental Animals. Subcutaneous xenograft and tail vein metastasis experiments were performed on 4-week-old BALB/c nude mice (National Rodent Seed Center, Shanghai, China), with injection of 1 × 106 cancer cells for subcutaneous xenograft studies, or 1 × 107 cancer cells for tail vein metastasis experiment, respectively [19, 20, 25,26,27]. The condition of nude mice was monitored daily, and their tumor volume, body weight, time of death, and survival were recorded. Subcutaneous xenograft mice were euthanized at 4 weeks after initial injection of cancer cells. Experimental mice with tail vein metastasis were euthanized 7 weeks after initial injection of cancer cells, and their lung or liver tissues were dissected. The in vivo Xtreme II small animal imaging system (Bruker, Billerica, MA, USA) was used for imaging [19, 20, 25,26,27].

Clinical tissues

Human tissue study was approved by the Institutional Review Board of Union Hospital, Tongji Medical College, and conducted in accordance with the guidelines of Declaration of Helsinki. Gastric cancer and adjacent normal epithelial tissues were collected during surgery at Union Hospital, Tongji Medical College, and validated by pathological diagnosis. Written informed consent was obtained from all patients. Fresh tumor tissues were preserved in RNAsafer Stabilizer Reagent (Omega, Guangzhou, China), frozen in liquid nitrogen, and stored at -80 °C.

Immunohistochemistry staining

Immunohistochemical staining and quantitative evaluation were performed as previously described [19, 20, 25,26,27,28], with antibodies specific for CD31 (ab28364, Abcam; 1:100 dilution) or Ki-67 (sc-23900, Santa Cruz Biotechnology; 1:100 dilution). The degree of positivity was measured according to the percentage of positive cancer cells.

Statistical analysis

All data were shown as mean ± standard deviation (SD). Cutoff values were determined by medium gene expression levels. Student′s t-test, Mann–Whitney U test, and analysis of variance (ANOVA) were applied to compare differences in cancer cells or tissues [31]. Statistical significance of overlap analysis was determined by Fisher′s exact test. Log-rank test was used to assess survival differences. All statistical tests were two-sided [31].