Sample collection and cell lines culture
Sixty-nine GC samples and their paired surgical margin samples were collected from the Affiliated Lihui Li Hospital of Ningbo University between 2016-2017. Samples were stored at liquid nitrogen (-196℃). All individuals provided informed written consents for using their tissues in this experimental study, and the Ethics Committee of Ningbo University approved this study.
The cell line BGC823 was obtained from Beijing Cancer Hospital. HGC27 was obtained from the Medical School of Ningbo University. Both BGC823 and HGC27 were tested by STR authentication (GENEWIZ, China).
Quantitative real-time PCR
According to the manufacturer’s protocol, the total RNA of tissue samples and gastric cancer cell lines was extracted by Trizol reagent (Life Technologies, Carlsbad, USA). cDNA was synthesized by HiFiScript cDNA synthesis Kit (CW2569, CWBIO. China). RNA polymerase (M0276S, NEB, USA) and RT-adaptor were needed for miRNA-specific reverse transcription. The sequences of primers were as follows: RT-adaptor, CGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN; miR-2682-3p PF, CGCCTCTTCAGCGCTGTCTTCC; U6 PF, CGCTTCGGCAGCACATATAC; U6 PR, TTCACGAATTTGCGTGTCAT; MIR137HG PF, CAAGGCATCCAAAGCCTCT; MIR137HG PR, TGTGGTGAGTCAAGATCACGTC; GAPDH PF, GAAGGTGAAGGTCGGAGT; GAPDH PR, GAAGATGGTGATGGGATTTC; Universal miRNA PR, GCGAGCACAGAATTAATACGAC; miR-137 was amplified by All-in-One miRNA qRT-PCR Detection kit according to the manufacturer’s protocol (QP015, GeneCopoeia, USA), using corresponding primer U6 (HmiRQP9001, GeneCopoeia, USA), miR-137 (HmiRQP0175, GeneCopoeia, USA).
Construction of control lentivirus and MIR137HG lentivirus cell strain in GC cell lines
MIR137HG was synthesized and cloned onto pcDNA 3.1b vector by Sangon Biotech Inc. It was then packaged as MIR137HG lentivirus and negative control lentivirus by HANBI Inc. Lentivirus was infected into BGC823 and HGC27 cell lines for 48 h and set up stable strains by puromycin (0.25 μg/ml).
Total proteins were extracted from a cell by RIPA lysis buffer, and the BCA procedure determined protein concentration (C503021, Sangon Biotech. China). The protein (20 μg) was electrophoresed by 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked for 2 h at room temperature with 5% non-fat milk in PBST. The membranes were then incubated overnight at 4℃ with rabbit anti-TLS/FUS (ab23439,1:1000, Abcam, Cambridge, UK), rabbit anti-MET(8198, 1:1000, CST, USA), rabbit anti-RHOC (3430, 1:500, CST, USA), rabbit anti-CTNNB1(8480,1:500, CST, USA), Mouse anti-GAPDH (ab8245, 1:1000, Abcam, Cambridge, UK) and rabbit anti-ACTB (AC026, 1:500, ABclonal, China), followed by incubated with HRP-conjugated Goat Anti-Rabbit IgG (D110058, 1:2000, BBI, Sangon Biotech, Shanghai, China) or HRP-conjugated Goat Anti-Mouse IgG (D110087, 1:2000, BBI, Sangon Biotech, Shanghai, China) at room temperature for 1 h. The targeted proteins were then analyzed by LI-COR model 3600(LI-COR, USA).
Cell migration assays (transwell assay and scratch assay)
About 4 × 104 cells were seeded within 200 μl RPMI1640 onto the upper layer of the chamber. The lower layers were added about 500 μl mixed cultures with 90% RPMI 1640 and 10% FBS. 24 h later, cells were fixed by 2% formaldehyde for 20 min, followed by washing twice by 1 × PBS, and then stained with 1% crystal for 30 min. Cells in the upper well were removed with cotton swabs, and cells on the opposite membranes of the chamber were counted by the microscope system. Each chamber was counted in six fields. BD Matrigel (356234, USA) was diluted by RPMI1640 (1:8) and was seed 80 μl per hole for the invasion assay.
Cells were plated to 70% confluence on 6 well plates and were wounded with 200ul pipette tips. The wound healing status of each group was observed and photographed after scratching 0 and 18 h.
Dual-Luciferase reporter assay
The 3’-UTR segments of the WT and MUT FUS gene were amplified by polymerase chain reaction (PCR) and inserted into PmiR-PB-ReportTM. Co-transfections of WT-FUS-3’-UTR or MUT-FUS-3’-UTR plasmids with miR-2682-3p mimic into the cells were accomplished by using Lipo6000TM. Luciferase activity was measured after 48 h transfection by the Dual-Luciferase Reporter Assay System (E2920, Promega, USA). All assays were performed in triplicated, and each experiment was repeated three times.
The experiments were reviewed and approved by the Laboratory Animal Ethical Committee at Ningbo University. Five-week-old female Balb/C nude mice (n = 18) were purchased from SLAC.cn (Shanghai, China), and maintained in a specific pathogen-free environment. For tumor formation assay, 1 × 107 GC cells were subcutaneously implanted in nude mice. After 15 days, mice were sacrificed, and tumors were removed for examination. We first injected 1 × 107 GC cells for 7 days for the tumor inhibition assay to form a subcutaneous MIR137HG tumor model and injected the Ago-miR-2682-3p (1 nmol/ time) every 3 days into the tumor model and tested the volume. After 14 days, mice were sacrificed, and tumors were removed for examination. For the metastasis assay, 1 × 107 GC cells were injected into the tails caudal of nude mice. After 42 days, mice were sacrificed, and their lungs were removed for examination.
Paraffin-embedded tissue sections were deparaffinized and rehydrated for IHC with the following procedures: tissue glass slides were immersed in Citrate Buffer (PH 6.0) to repair the antigen. Slides then were successively blocked by 3% hydrogen peroxide and 3% BSA, incubated with primary antibodies overnight at 4℃(FUS: Ab70381, 1:1000, Abcam, Cambridge, UK; KI67: 1:100, A20018, Abclonal, China). Subsequently, the HRP-goat anti-rabbit secondary antibody (1:200, GB23303, Sevicebio, China) was applied and incubated for 1 h at room temperature. The expression of FUS and KI67 was visualized by using DAB (K5007, DAKO, Denmark) and counterstained with hematoxylin (G1004, Servicebio, China).
RNA-FISH and RNA/protein co-location
RNA-FISH kit (R11010.2, RICOBIO.Co., Ltd. Guang Zhou, China) was used to evaluate the sub-location of MIR137HG in cell lines, following the manufacturer’s instructions. For the co-sub-location of MIR137HG and FUS, we added 3 steps into the manufacturer’s instructions: Cells were blocked by DEPC-PBS buffer contains 5% BSA, 0.1% triton after the RNA-FISH prehybridization step for 1 h; The anti-Rabbit FUS antibody (ab70381, 1:300, Abcam, Cambridge, UK) and MIR137HG probe (lnc1100344, RICOBIO.Co., Ltd. Guang Zhou, China) were incubated together overnight at 37 ℃; The Alexa Fluor 488-conjugated Goat anti-rabbit IgG (1:500, Abcam, Ab150077) was incubated 1 h at 37℃, followed by 1×PBS washing for 3 times. The results were tested by a confocal microscope.
The binding of MIR137HG with FUS and DGCR8 was confirmed through the RIP kit according to its manufacturer’s protocol (Millipore/17-701/EZ-Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit, Millipore Corporation, Billerica, MA01821, USA). The enrichment of MIR137HG was assessed via real-time quantitative PCR.
IP and LC–MS/MS
About 2 × 107 BGC823 MIR137HG cell strain was washed with pre-chilled phosphate-buffered saline (PBS) and lysed by RIPA (R0010-20, Solarbio, China). Cell lysates were scratched and centrifuged at 14000 g for 15 min at 4℃ to separate the supernatant. The supernatant was incubated with 100 μl protein A/G beads for 10 min at 4℃ and centrifuged 14000 g for 15 min at 4℃ to get the supernatant. The supernatant was tested the protein concentration by BCA Kit (C503021, Sangon Biotech, China). Protein was diluted by 1×PBS to 2 μg/ul, and separately incubated with rabbit anti-FUS (5 μg, Ab70381,Abcam, UK) and rabbit anti-IgG (PP64B, Millipore, USA) antibody overnight at 4℃. Subsequently, 100ul protein A/G was incubated with the complex overnight at 4℃. Collecting the complex and performed the SDS-PAGE and stained by Coomassie Blue Staining Solution (P1305-1, Solarbio, China). Protein strip around 75KD (location of FUS) was then sent to Sangon Biotech for LC-MS/MS analysis.
The location of MIR137HG and its host miRNAs (miR-137 and miR-2682) were analyzed by UCSC (http://genome.ucsc.edu/). The prediction of candidate target proteins of MIR137HG and miR-2682-3p were separately performed using the online tools: Starbase [23, 24] (http://starbase.sysu.edu.cn/) and TargetScan (http://www.targetscan.org). In addition, the interaction among target proteins was predicted by String (https://string-db.org/).
All statistical analyses were conducted using SPSS 18.0 statistical software. An intergroup comparison was carried out using Mann-Whitney U-test or the Students’ t-test according to data type. A receiver operating characteristics (ROC) curve was generated using the expression level of MIR137HG to separate GC patients with poor differentiation from GC patients with moderate/well differentiation. Differences with a P < 0.05 were considered statistically significant.