Bioinformatic analysis
The Gene Expression Omnibus (GEO) database was searched for RNA-seq data sets using the term "Gaussian cancer". All data sets were analyzed by the limma method to screen the genes with significant differential expression genes (DEGs) in GC and healthy tissues. DEGs were only considered for further analysis if they passed the following thresholds: P < 0.05 and |logFC| > the mean logFC value. To extract shared DEGs, we finally performed an intersection analysis across datasets (coDEGs). Weighted gene coexpression network analysis (WGCNA) was then used in R version 4.06 to do further screening for coDEGs. Packages "WGCNA" and "reshape2" were loaded to cluster the standardized mRNA expression matrix. The gene matrix with a median greater than 0 was screened, and the missing values were cleared. The samples and genes were clustered hierarchically, and the dynamic hybrid tree-cutting method was used to identify the gene modules related to the characteristics of GC. The DEGs obtained after WGCNA screening were analyzed by the online website Kobas (http://kobas.cbi.pku.edu.cn/kobas3/genelist/) to obtain meaningful Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology (GO), and Reactome (RCTM) pathway. The screening conditions for DEGs function entries is P < 0.05 or FDR < 0.05.
Cerna (Lncrna-mirna-mrna) Network Prediction
The miRNAs that target the 3' untranslated region (UTR) of RAD54 were predicted using the microRNA Data Integration Portal (mirDIP; http://ophid.utoronto.ca/mirDIP/), and the miRNA-lncRNA interaction was predicted using the DIANA tool LncBase predicted v.3 (https://diana.e-ce.uth.gr/lncbasev.3).
Cell Culture
The SGC-7901 human GAC cell line was grown in the lab at 37 ℃ and 5% carbon dioxide (Herocell 180, Radobio, China). RPMI-1640 media was used to cultivate SGC-7901 cells (HyClone, USA). Ten percent fetal bovine serum (F8318, Merck, America) and one percent penicillin and streptomycin were added to all media (C0222, Beyotime, China). The cells were randomly assigned to one of three groups: CON, NC, or SH. A nonfunctional shRNA plasmid (NC) and transfection of an F10-AS1 shRNA plasmid (SH) were performed, respectively.
Design And Preparation Of F10-as1-shrna
Plasmids (shRNA1, shRNA2, shRNA3, and control plasmid sh-NC) for the pLKO.1-EGFP-puro-F10-AS1-shRNA construct were developed and produced by Guangzhou all perfect Biological Technology Co., Ltd. The specific sequences were as follows: shRNA1: AACTCTTTCAACTGATTGCCA, shRNA2: CCACCTTCCCCTTTTCTGGAA, shRNA3: TATGTAACTTCTGTCCCCCTA. Plasmids were transfected into SGC7901 cells separately with Lipofectamine™ Rnaimax (13778150, Invitrogen, America). The interference efficiency was detected by fluorescent quantitative polymerase chain reaction (qPCR). The highest-performing shRNA was chosen for further packaging into lentiviruses with a titer of 1 × 108 TU/mL due to its strong interference effectiveness. SGC7901 cells in the SH group were stably silencing F10-AS1.
Animals Treatment
Eighteen male BALB/C nude mice, aged 5 weeks and with the genotype (foxn1nu) mut/mut, were selected for their high level of sun protection factor (SPF), were purchased from gempharma Co., Ltd., were raised in Guangzhou Jenny Biotech Co., Ltd. The mice were fed with ordinary maintenance feed and drank freely. After one week of adaptive feeding, there was 18 animals total, and they were split into three groups of 6. Suspensions of SGC7901 cells were injected into the left axillary region of nude mice at a concentration of 1.5 × 107 cells per 200 µL. Mice in the CON group were injected with SGC7901 cell suspension, injected with NC-shRNA-SGC7901 cell suspension in the NC group, and injected with F10-AS1-shRNA-SGC7901 cell suspension in the F10-AS1 group. Subcutaneous nodules at the tumor injection point were observed every week, and the diameter of the nodules was measured and recorded. After establishing the tumor-bearing model successfully (subcutaneous nodule was greater than 0.3 mm), shRNA administration intervention was carried out. The tumors' subcutaneous development was evaluated after 28 days when the animals were killed. The National Institutes of Health's Guidelines for the care and use of laboratory animals were strictly adhered to throughout this investigation. The use of animals in research was sanctioned by the Committee of animal research institutes and followed national standards for the care and use of experimental animals. The mice were killed by excessive anesthesia with 4% chloral hydrate. The tumors were excised, measured, photographed, and frozen at -80 ℃ for further study. A vernier caliper was used to measure the tumor's diameter and length weekly during the experiment. Tumor volume was determined using the formula V (cm3) = 0.52 × L × W2, where L and W are the longest and widest dimensions of the tumor block, respectively.
Apoptosis Was Detected By Flow Cytometry
Annexin-V FITC/PI double staining kit (Thermo Fisher Scientific) was used to examine apoptosis. 1× 105 cells per well were suspended in 500 µL of the binding buffer after being digested with trypsin without EDTA (GIBCO trypsin EDTA, Thermo Fisher Scientific), washed with PBS, and suspended at 500 µL in the binding buffer. Next, 5 µL of FITC and 5 µL of PI were added to the cell mixture, and the whole thing was left to incubate in the dark for 15 min at 25°C. Using BD FACS Aria II flow cytometry (BD Biosciences, Hercules, CA, USA), SGC7901 cells were identified.
Dual-luciferase Reporter Assay
Amplified and cloned independently into the pmirGLO Vector were miR-146b-5p sequences from either the wild-type (WT) or mutant (MUT) RAD54L 3'-UTR (HH-LUC-016, Promega, China). Co-transfection of SGC7901 cells with RAD54L-WT or -MUT produced luciferase reporter vectors and miR-146b-5p or miR-NC utilizing Escort™ III Transfection Reagent (L3037-SAMPLE, MERCK, America). The relative luciferase activity was then measured using a kit from MERCK America (SCT152) designed for this purpose (Firefly/Renilla Dual Luciferase Assay).
Rt-qpcr
Trizol reagent (15596026, Invitrogen, America) was used to extract total RNA from cells following the manufacturer's instructions. Use the Hifair® III 1st Strand cDNA Synthesis SuperMix as directed to convert RNA into cDNA (11141ES10, Hifair, China). Using the Hifair® III One Step RT-qPCR SYBR Green Kit, we conducted the RT-qPCR test (11141ES10, Hifair, China). Primers are ordered as follows: Forward primer: 5'-AACCTGGCCTAGTGACTCCT-3'; reverse primer: 5'-TGAGGCCGCAAAACCTTACT-3'; forward and reverse primers for human β-actin: 5'-CCGCGAGTACAACCTTCTTG-3' and 5'-CAGTTGGTGACAATGCCGTG-3', respectively. Primers used to amplify the RAD54L gene in Mus musculus include: 5'-AGTTGGCTAGGAGGAAACCA-3', 5'-CCTTTTCAGGCCCAATGCAC-3', and 5'-GCAGGAGTACGATGAGTCCG-3', and 5'-ACGCAGCTCAGTAACAGTCC-3' for β-actin.
Western Blot
RIPA lysis buffer (P0013B, Beyotime, China) was used to extract proteins, and the BCA kit (P0012S, Beyotime, China) was used to determine their concentration according to the manufacturer's instructions. Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGEs) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). After incubation with RAD54L Rabbit pAb (1:1000, LS-C413241, LifeSpan BioSciences, America), Anti-gamma H2AX (1:1000, ab215967, Abcam, England), and GADPH (1:1000, A19056, ABclonal, China), the membranes were incubated with HRP Goat Anti-Rabbit IgG. (1: 5000, AS014, ABclonal, China). Finally, the ECL technique was used to analyze protein signals.
Statistical analysis
GraphPad Prism 8.0 was utilized in this investigation along with one-way ANOVA. The data were shown as means ± SD. P < 0.05, 0.01, or 0.001 were regarded as statistically significant.