1. Differential gene expression in GC
All of the microarray datasetsin the National Center of Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) were searched to identify differentially expressed genes. The original lncRNA, mRNA and miRNA expression profilesof theGSE84787, GSE79973 and GSE93415 datasets, respectively, were obtained. The GSE84787 and GSE79973 datasets had 10 pairs of GC tissue and paracancerous mucosal tissue samples and GSE93415,which included 20 pairs of GC tissue and matched paraneoplastic samples. The robust multi-array average algorithm was used to perform background correction and quartile data normalization of these data[11]. Only the average values of gene symbols with multiple probes were calculated, the others without corresponding symbols were filtered. Student's t-test and fold change (FC) filtering were conducted to screen differentially expressed genes (DEGs) between two groups with the R software limma package[12]. With the threshold of a P-value <0.05 and an absolute FC value > 2, volcano plot filtering was performed using the R software ggplot2 package to identifysignificant DEGs between two groups.
2. Construction of anlncRNA-miRNA-mRNA network
lncRNA gene annotation was performed according to the LNCipedia database[13]. DIANA-LncBase v.2 [14] was used to predict the interactions between miRNAs and lncRNAs with a threshold prediction score >0.8. Differentially expressed lncRNAs and predicted target lncRNAs were intersected to select differentially expressed miRNA-targetedlncRNAs. Then, the interactions between miRNAs and mRNAs were predicted by using mirtarbase[15]. Differentially expressed mRNAs and predicted target mRNAs were intersected to select differentially expressed miRNA-targeted mRNAs. Significantly expressed miRNAs and their significantly expressed targets (mRNAs and lncRNAs) were superimposed onto anlncRNA-miRNA-mRNA network. The network was constructed by Cytoscape (version 3.4.0), and its topology was analyzed with CentiScaPe app[16].A flowchart detailing the construction of the lncRNA-miRNA-mRNA network is shown in Figure. S1.
3.Study population and specimens
This study consisted of 70 GC fresh-frozen tissue specimens and paired adjacent normal tissue specimens (at least 5 cm from the negative resection margin)obtained during surgical resections performed at the Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) betweenFebruary 2017 andFebruary 2018. All participants were self-reported Han Chinese. Both the GC and paracancerous specimens were confirmed by histopathological diagnosis. The tumor clinicopathological data are summarized in accordance with the TNM staging system of the American Joint Committee on Cancer staging manual (8th edition). Histological grades were assessed following the National Comprehensive Cancer Network (NCCN) clinical practice guideline of oncology (V.1.2011). Patients did not receive radiotherapy, chemotherapy or targeted therapy prior to undergoing surgery. The study was authorized by the institutional review boards of the Second Affiliated Hospital of Wenzhou Medical University. Each patient provided written informed consent.
4. Cell culture
Two GC cell lines (AGS and SGC-7901)and the normal gastric mucosal cell line GES1 were purchased from the Shanghai Genechem Co., Ltd (Shanghai, China).AGS and SGC-7901 cells were cultured in DMEM containing 10% FBS in a constant-temperature humidified incubator. Generally, the medium was replaced every 2 days, and the number of cells in the medium became saturated within approximately 5 days; at this time, the cells weresubcultured.
5. Total RNA extraction and reverse transcription
All fresh tissue samples were stored in a freezer at -80°C from the time they were collected to the time of use and fixed with RNA fixing reagent (Baiteke, Beijing, China).TRIzol reagent (Invitrogen, Karlsruhe, Germany) was used to quickly extract total tissue RNA from the GC tissue and adjacent nontumor tissue samples; however, as thisreagent is harmful to the human body, disposable gloves were worn for protection against any spillage. The whole process was performed strictly in accordance with the manufacturer's instructions.Then, total RNA was quantified using a Scandrop100 (Analytikjena, Germany). The A260/A280 ratiowas used to indicate the purity of the isolated total RNA. Most of the sample values werebetween1.8 and 2.0. Then, cDNA was synthesized by reverse transcription (RT) using random primers and the TUREscript 1st Stand cDNA SYNTHESIS Kit (Aidlab, Beijing,China).
6. Real-time qRT-PCR detection ofan lncRNA
Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was achieved using 2×SYBR Green Supermix (DBI, Germany) on the qTOWER2.2 real-time PCR System (Analytikjena, Germany) following the manufacturer’s instructions. Primers for LUCAT1 andglyceraldehyde 3-phosphate dehydrogenase (GAPDH) were synthesized by ChenduDangfeng Biotech (Sichuan, China). The primers forLUCAT1were as follows: forward primer, 5'- CCTCCAGAAACCATGTGTCAA-3' and reverse primer, 5'- GTGAGGAAAGGAGCCAGAAGTC-3'. The primers for GAPDH were as follows: forward primer, 5'- CGGAGTCAACGGATTTGGTC-3' and reverse primer, 5'- CGGTGCCATGGAATTTGCCA-3'.Themelting temperature of the LUCAT1 and GAPDH primers was 58°C. The relative expression level for each sample was calculated as the log2ratio with the Pfaffl method, where Ratio=(1+Etarget) ∆ Cttarget (control-sample) / (1+EGAPDH) ∆ CtGAPDH (control-sample)[17, 18].The sample with the lowest expression level in each group was chosen as the control sample. The reaction efficiencies of the target groups and GAPDH group were both 100%.
7. Cell transfection
ShRNAs specifically targeting the lncRNA LUCAT1 anda scrambled negative control shRNA (shNC) were synthesized by GeneChemCo.,Ltd.(Shanghai,China). Following the manufacturer’s instructions, Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to transfect the shRNAs and their controls into the AGS and SGC-7901 cell lines. The cell state and infection efficiency were observed after transfection.
8. Knocking out LUCAT1 inhibits the proliferation and clone formationof gastric cancer cells
Control shRNA- or LUCAT1-specific shRNA-transfected AGS and SGC-7901 cells(2000/well) were allowed to grow in 96-well plates for 24 hrs after shRNAtransfection. After 24h, a CCK8 (Sigma, USA) assay was used to detect cell activity, reflecting cell proliferation. For colony formation, control shRNA- or LUCAT1-specific shRNA-transfected AGS and SGC-7901 cells were plated in a fresh 6-well plate at a density of 1000 cells/well. The inoculated cells were further cultured in an incubator for 14 days, during which time the culture medium was changed every 3days and the cell status was observed. The cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Sangon Biotech, Inc., Shanghai, China). The assay was repeated 3 times, and then the clone formation rate was calculated.
9. Flowcytometric analysis of apoptosis and the cell cycle
Control shRNA- or LUCAT1-specific shRNA-transfected AGS and SGC-7901 cells werecollected and fixed with 75% ethanol for at least 1 hour. The cells were centrifuged at 321g for 5 min and washed with ice-cold PBS 2 times. Then, the cell cycle dyePI (propidium iodide;Sigma, USA) and RNase were added to the cells for 15 min in the dark at 37°C.The cell cycle was then examined by flow cytometry (Millipore, DE).
After centrifugation at 321 g for 5 min, transfected cells were collected and then resuspended in binding buffer. Apoptosis was detected using Annexin V-APC apoptosis detection kits (eBioscience, CA, USA). Cell apoptosis was detected by flow cytometry (Millipore,DE), and the data were analyzed by guava InCyte software.
10.Transwell Assay
To measure the invasive ability of cells, 500 µL serum-free medium was added to the upper and lower chambersofaTranswellsystem (Corning, USA), which was placed in an incubator at 37°C for 2 h. After the basement membrane was hydrated, the medium in the upper chamber was removed, and 500 µL cell suspension was added. In the lower chamber, 750 µL 30% FBS medium was added, and the system was cultured in an incubator at 37°C. Then, a cotton swab was used to removethenoninvasive cells in the chamber, 2-3 drops of a Giemsa (DingguoBiotechnologyCo.,Ltd., Shanghai, China) staining solution was added to the lower surface of the membrane to stain and transfer cells for 3-5 min, and then the chamber was soaked and rinsed several times and air dried. Microscopy (Olympus, Japan) was used to image the attached cells.
11. Wound healing assay
A scratch test was used to assess cell migration.Cells were inoculated into 96-well plates and cultured in an incubator at 37°C and 5% CO2 for 1 night. The next day, the serum medium was changed to low serum medium, the cells cultured in the 96-well plate were aligned with the center of the lower end of a scratch-making device, and the cells were pushed up slightly to form scratches. Then,the cells were rinsed with serum-free medium 3 times. Photographic images along the scrape line were acquired under a microscope at 0, 6 and 24h.
12. Statistical analysis
Statistical analyses and scientific graphing were performed with the Statistical Product and Service Solutions 22.0 softwarepackage (SPSS, Chicago, USA) and GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA), and P<0.05 was considered statistically significant. Associations between the expression levels oflncRNAs and clinicopathologic featureswere evaluated by a t-test or one-way analysis of variance (ANOVA). Receiver operating characteristic (ROC) curves were established to evaluate diagnostic value.