Patient samples
The 6 cases of GC and its adjacent normal tissues used for LncRNA high-throughput microarray detection were from patients with GC complicated by liver metastasis in general surgery who underwent palliative surgery due to obstruction or gastric bleeding. 120 cases of GC patients used for large sample verification were primary GC patients in the general surgery department of our hospital, all of which were confirmed by surgery and underwent D2 radical resection with R0 resection. All GC patients did not receive chemotherapy, radiotherapy, surgery, and other treatments before surgery. Peripheral blood was collected from 20 patients with GC and 20 healthy controls (not diagnosed with cancer). Preparation of exosomes from peripheral blood. All specimens were collected according to institutional protocol. In addition, the clinicopathological features of patients were collected, including age, gender, tumor location, tumor size, degree of differentiation, Lauren grade, TNM stage, etc.
Microarray analysis
Total RNAs were isolated from the paired tissue samples of six liver metastasis and their adjacent normal tissues and purified using TRIzol reagent (Invitrogen, Carlsbad, CA) and RNeasy mini kit (Qiagen Inc, Valencia, CA) according to the manufacturer's protocol. Following RNA isolation and cDNA synthesis, biotin-labeled cRNA was labeled and hybridized to the 8 × 60 K LncRNA Expression Microarray (ArrayStar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies).
Cell culture
The AGS cell line was purchased from the American Type Culture Bank (ATCC,USA), and the HEK293T, HGC27, MKN45, and GES1 cell lines were purchased from the Shanghai Type Culture Bank of the Chinese Academy of Sciences. HEK293T, HGC27, MKN45, and GES1 cells were cultured in RPMI1640 medium (Gibco, Carlsad, CA, USA). AGS cells were cultured in F12K medium (Wisent, Canada). All cell lines were added with 100 µg/ml streptomycin, 100 U/ml penicillin, and 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37°C.
Isolation and identification of exosomes
Collect exosomes from 20 ml culture medium (1×107 cells). Collect the medium on ice, centrifuge at 800×g for 10 min to pellet the cells, and centrifuge at 12000×g for 30 min to remove cell debris. Centrifuge with an SW32 rotor (Beckman Coulter) at 100,000×g for 2 h to separate exosomes from the supernatant. The exosomal pellets were washed once in a large volume of phosphate buffer, and resuspended in 100 µl of phosphate buffer.
Human serum exosomes were obtained using ExoQuick Exosome precipitate (SBI, CA, USA) according to the instruction manual. In short, the serum was collected and centrifuged at 3000×g for 15 min. The 63 µL Exoquick Exosome precipitate was added to 250 µL supernatant, the serum was refrigerated at 4°C for 30 min, and centrifuged at 1500 ×g for 30 min. Then the Exosome pellets were suspended in 100 µL with 1×PBS.The particle size and shape of exosomes were then identified by transmission electron microscopy (TEM)(Philips Tecnai 20, Netherlands).Western blot method was used to identify exosomal protein markers.Nanoparticle tracking analysis (NTA) was used to measure the total amount of exosomes.
RNA preparation and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells or tissues using TRIzol reagent (Invitgen, MA, USA). The nucleus and cytoplasm were extracted with the Paris™ kit (Thermo Fisher, Massachusetts, USA). The extracted RNA was subjected to quantitative polymerase chain reaction using HiScript Q RT SuperMix (Vazyme, Jiangsu,China). ABI Prism7900 sequence detection system (Applied Biosystems Canada) was used to perform quantitative RTPCR with SYBR Green PCR Master Mix (Vazyme), GAPDH was used as an internal control, and the results of each sample were normalized to GAPDH expression. For RNase R treatment, incubate 2 µg of total RNA at 37°C and add or not add 3 U/µg of RNase R (Epicentre Technologies, WI, USA) in 1× reaction buffer. (The primers are listed in Supplementary Table S1.)
Plasmid and siRNA transfection and lentiviral transduction
The siRNAs of RNF144A-AS1 and HOXA3,and miRNA mimics or inhibitors were designed and synthesized(RiboBio,Guangzhou,China).Plasmids pcDNA3.1-RNF144A-AS1 and pcDNA3.1-PUF60 were designed and synthesized by Jiangsu Genssee Biological Technology Co. Plasmids and miRNA mimics or inhibitors were transfected with liposome 3000 (Invitgen). The cells were transfected with siRNAs with DharmaFECT4 (DharmaCon, IL, USA). An shRNA lentiviral vector (pGLV3/GFP/Puro) targeting RNF144A-AS1 was constructed by GenePharma (Shanghai, China) and transfected into HGC27 cells. A stable cell line was obtained by screening with puromycin. (RNF144A-AS1 and HOXA3 gene siRNA sequences are listed in Supplementary Table S1.)
RNA pull-down assay and mass spectrometry
According to the manufacturer's protocol, the magnetic RNA protein pull-down kit (TERMO) was used for lncRNA pull-down assay. 107 HGC27 cells were transfected with RNF144A-AS1 overexpressing vector or control vector. After 48 h, the total RNA of the two groups of cells was extracted and incubated with 100 nmol probes at 70°C for 5 min. Then the ribonucleic acid was slowly cooled to room temperature, 50 µl streptavidin magnetic beads were added, and the mixture was incubated at room temperature for 30 min with stirring. The unbound ribonucleic acid was eluted with 20 mMTris, and 100 µl 1×RNA-protein binding buffer with a total protein of 100 µg was added to the test tube containing streptavidin magnetic beads. After the streptavidin magnetic beads were incubated for 1.5 h with rotation at 4°C, they were washed 3 times with washing buffer, and then incubated with 50 µl of elution buffer at 37°C for 15 min with stirring. The supernatant was collected for silver staining and KeyGEN mass spectrometry analysis.
Cell Counting Kit -8(CCK8) analysis
After 48 h of transfection, HGC27 cells (2×103) were seeded in 96-well plates (Corning). Then, add 10 µl of CCK8 (Beyotam, Jiangsu, China) solution to each well at the designated time, and add 10 mmol/l of CCK8 (Beyotam, Jiangsu, China) solution to each well. After incubating at 37°C for 1 h, the absorbance at 450 nm was measured using an automatic microplate reader (Synergy4; BioTek, Winooski, VT, USA).
Wound healing analysis
Cell migration ability was detected by wound healing assay. A total of 2–4 × 105 cells were seeded in 6-well plates, cultured for 12–24 h, and transfected with siRNA or control siRNA and PC-DNA3.1-RNF144A-AS1 or control vector. Once the culture reached an 85% fusion rate, the cell layer was scratched with sterile plastic tips, washed with culture medium, and then cultured for 24 and 48 h. At different time points, images of the steel plate were obtained using a microscope (Olympus Tokyo, Japan), and the relative area of the wound was obtained using ImageJ software to quantify and calculate the significance of the observed event.
Transwell analysis
The Transwell invasion test was performed in a 24-well plate (Corning, Massachusetts, USA). A Transwell cell with a diameter of 6.5 mm and an 8 µm pore polycarbonate membrane insert (Corning) was used. The bottom of the upper cavity is coated with fibronectin (Merck Millipol, Darmstadt, Germany). After 48 h of transfection, HGC27 cells (2×104) were seeded in 50 µl Matrigel (Corning) serum-free medium coated or uncoated in the upper cavity. RPMI1640 containing 10% FBS was added to the lower chamber as a chemical attractant. After incubating for 12 h at 37°C, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and counted under a microscope ×200 times. The determination was repeated three times in duplicate. Take the average of the cell counts in 5 random areas.
Western blot, immunohistochemical (IHC) analysis
RIPA lysis buffer (Thermo Fisher) was used to extract proteins from cells and tissues. Serum protein extraction kit (China Qincheng Biological) to extract serum protein. The immune complexes were detected with ECL Western blotting substrate (Thermo Fisher) and developed with BIO-RAD (BIO-RAD Gel Doc XR+). Western blot analysis was performed using standard procedures. The following antibodies (11000) were used: anti-β-actin (Beyotime, AF0003); anti-α-tubulin (Beyotime, AF0001); anti-GAPDH (Beyotime, AF0006); anti-PUF60 (ab22819, Abcam). Anti-IGF2BP1 (ab184305, Abcam); anti-POU2F2 (ab243153, Abcam); anti-HOXA3(ab28771, Abcam). anti-Calnexin (Abcam, ab92573); anti-CD63 (Abcam, ab134045); anti-TSG101 (Abcam, ab125011); anti-CD81 (Proteintech, 66,866–1-Ig).
Perform IHC using standard procedures. Anti-IGF2BP1, POU2F2 and HOXA3 antibodies were used for IHC(Abcam,1: 200). Images were scanned by Pannorama Scan(3DHistech, Hungary). IP was performed with anti-PUF60 antibody (AbCAM, 1:150) and appropriate control IgG(Merck micropore). Immunoprecipitates were collected by centrifugation and analyzed by SDS-PAGE.
RNA fluorescence in situ hybridization (FISH)
A CY3-labeled RNF144A-AS1 specific probe was designed and synthesized using RIBOBIO, and the signal was detected using FISH kit (Ribobio) according to the manufacturing instructions.The cells grew to the exponential stage, and fusion reached 40%~50% during fixation.After cells were permeated (1×PBS/0.5%Triton X-100), they were hybridized with RNF144A-AS1, U6 and 18S specific probes overnight in a 37°C hybrid buffer. The hybridization buffer was gradually eluted with 4×SSC (containing 0.1% Tween-20), 2×SSC and 1×SSC at 42°C, and the nuclei were counter-stained with 4,6-diamino-2-phenylindole (DAPI)(RiboBio). Confocal images were captured using the Zeiss AIM and Zeiss LSM 700 confocal microscopy systems (Carl Zeiss Jena, Oberkochen, Germany).
RNA-protein immunoprecipitation (RIP) analysis
The MagnaRIP RNA Binding Protein Immunoprecipitation Kit (Merck Microwells) was used according to the manufacturer's instructions. The cell lysate was incubated with microspheres coated with 5 µg anti-Argonaute-2 antibody (AGO2) (Abcam, MA, USA) and anti-PUF60 (Abcam), and the control IgG was rotated overnight at 4°C. Then total RNA was extracted, and the expression of RNF144A-AS1 was detected by qRT-PCR.
Dual-luciferase reporter analysis
HEK-293T cells were seeded in a 24-well plate at a density of 6×104 cells per well, 24 h before transfection. The luciferase reporter vector (PmirGLO) containing RNF144A-AS1-miR-361-3p/miR-619-5p binding sequence or mutation sequence was co-transfected with miRNA mimic (20 Nm) to detect the binding ability of miRNA. After 24 h, the luciferase activity was measured using the dual-luciferase reporter analysis system (Promega, Madison, WI, USA) according to the manufacturer's protocol.
RNA sequencing(RNA-seq) analysis
Total RNA was extracted with Trizol reagent, and the amount and purity of RNA were verified by Nanodrop2000 (Thermo Fisher). RNA integrity and gDNAs contamination were detected by denatured agarose gel electrophoresis.Before the RNA-seq library was constructed, the RNA of each sample was removed by the Ribominus Eukaryotic Cell Kit (QIGEN) for ribosomal RNA.The sequencing library was determined by the Agilent 2100 bioanalyzer using the Agilent DNA 1000 chip kit (Agilent, CA, USA).The library was adjusted to 10 NM before clustering. The cDNA was then sequenced using the HiSeq2000 system (Illumina, Sandiego, CA, USA) and a 100 bp paired-end run.
Animal research
All animal experiments were conducted following the procedures approved by the Institutional Animal Care and Utilization Committee of Jiangsu University. HGC27 cells of RNF144A-AS1 stable silence LV3-sh-RNF144A-AS1 and control group LV3-sh-NC cells were collected and suspended in frozen PBS. Ten 5-week-old male BALB/cnu/nu mice were randomly divided into 2 groups, and liver metastasis models (1.5 × 106 cells in 150 µl of PBS) were prepared by infrasplenic injection. After 3 weeks, bioluminescence imaging was performed to check liver metastases every week for 4 consecutive weeks. Prepare D-luciferin sodium salt stock solution with 15 mg/ml PBS. In order to produce bioluminescence, mice received intraperitoneal injection of fluorescein stock solution (150mg/kg). All mice were anesthetized with 2% isoflurane immediately, and imaging was performed 10 minutes later. These images were captured using the IVIS Spectrum Xenogen imaging system (Caliper Life Sciences). The mice were sacrificed 7 weeks later, and the liver samples were taken for H&E staining, Western blot, and IHC detection. Blood samples were collected for exosome analysis.
Statistical Analysis
All statistical analyses were performed using SPSS software version 20.0 (SPSS Inc., USA) and GraphPad Prism version 7.00 (GraphPad Software, USA). The differences between the two groups were assessed using Student’s t-test. Overall survival curves were estimated by the Kaplan–Meier method, and the difference in survival was evaluated using the log-rank test.p Values < 0.05 were considered statistically significant.