Gene expression profile data and the identification of DEGs
The gene expression datasets GSE11399, GSE19826 and GSE27342 were downloaded from GEO database (https://www.ncbi.nlm.nih.gov/geo/). Platforms of GEO datasets is GPL570 (Affymetrix Human Genome U133 Plus 2.0 Array) (Agilent Technologies, Santa Clara, CA, USA). There totally contains 130 cases of GC tumor tissues and 126 cases of non-cancerous gastric mucosa in the datasets for comparison. Dataset with 55 GC tissues and 35 non-cancerous tissues from the Cancer Genome Altas (TCGA) database was intensively explored to further validate the expression profile of the candidate DEGs.
R language, followed by normalization, was conducted by two professional bioinformatics analysts to preprocess the downloaded data, including background correction and transformation from probe level to gene symbol. DEGs differentiated between GC samples and non-cancerous tissues were defined basing on a t-test of linear models for microarray analysis package in R (Version 3.3, http://www.bioconductor.org) (11). A threshold criteria of log2FC≥2.0 and P value＜1.0E-04 was set for DEG selection on gene expression FC calculated in this study.
Funrich Software (Version 3.0, http://funrich.org/index.html) was utilized to analyze the DEGs overlapping characteristic among the three datasets. The online database of Cancer Cell Line Encyclopedia (CCLE: https://portals.broadinstitute.org) was introduced for determining the gene expression status of the candidates among differential tumor cell lines.
Cell culture and surgical specimens
The immortalized gastric epithelium cell line (GES-1) and three GC cell lines (MKN-45, AGS and SNU-16) were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China). RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) was used for cell culture in a humidified cell incubator, under the condition at 37℃ with an atmosphere of 5% CO2, with 100 ug/ml streptomycin and 100U/ml Penicillin in the medium.
Specimens of the real GC patients conducted radical gastrectomy without preoperative therapy were recruited, including 105 paired tumor tissues and the adjacent non-cancerous tissues, at the Department of Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine during 2017-2019. Ethical approval was granted by the research medical ethics committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.
Plasmid construction and transfection
MKN-45 cells in exponential phase were prepared and transfected with shRNA suppressing SPP1 mRNA translation through pGU6/Neo vectors (GenePharma, Shanghai, China) along with the construction of the control ones. Transfected cells were selected using medium mixed with G418 (Santa Cruz Biotechnology, Inc; 400 μg/ml).
Introduction of miR-4262 in MKN-45 cells (MKN-45/miR-4262) was carried out through mimic method similar to the description in our previous publication followed by the Dual-luciferase reporter assay (12, 13). And the control ones were setup simultaneously (NigmiR).
The RT-qPCR assay
Total RNA from cell lines was extracted by using TRIzol reagent (Invitrogen, USA). The first-strand cDNA was synthesized by using High-Capacity cDNA Reverse Transcription Kit (ABI, USA). RT-primers of OPN mRNAs were synthesized as follows: 5’- TCCTAGCCCCACAGACCCTT -3’ (forward) and 5’- CTGTGGAATTCACGGCTGAC -3’ (reverse) by Sangon Biotech Company (Shanghai, China). Real-time quantitative polymerase chain reaction (qRT-PCR) was conducted following the TaqMan Gene Expression Assays protocol (ABI, USA).
Immunohistochemistry assay and the Western blot analysis
Antibodies against SPP1 and GAPDH (Santa Cruz, USA) were prepared, along with horseradish peroxidase-conjugated secondary antibody (Abcam, USA). Immunohistochemistry (IHC) assay was conducted on paired tissues from the patients. SPP1 antibody was utilized as the manufactory instruction described (1:50), IgG antibody was used as control. Samples treated were then exammed by two professional pathologists blindly.
RIPA buffer containing Protease Inhibitor Cocktail (Pierce, USA) was prepared lysing cells. Concentration of the protein was measured \using BCA Protein Assay Kit (Pierce, USA). The extracted proteins were electrophoresed and electrotransfered. SPP1 antibody against (1:1000) and GAPDH (1:5000) were probed afterward. Horseradish peroxidase-conjugated secondary antibody was applied as further probe. GAPDH was regarded as the loading control.
Cell proliferation assay and cell cycle analysis
MKN-45 cells (1x106) stably transfected were cultured in 96-well microtiter plates in triplicate and incubated at 37˚C with an atmosphere of 5% CO2 for 5 days. Microplate computer software (Bio‑Rad Laboratories, Inc., Hercules, CA, USA) was used for measuring the OD following the Cell Counting Kit‑8 (CCK-8) assay kit protocol (Dojindo, Tokyo, Japan). The cell proliferation curves were plotted.
The aforementioned cells were treated in steps with ethanol fixation, RNase A treatment and propidium iodide staining. Flow cytometry detection by using FACSCalibur (Becton‑Dickinson, Franklin Lakes, NJ, USA) were conducted. Cell populations at the G0/G1, S and G2/M phases were quantified through ModFit software (Becton-Dickinson). Cell debris and fixation artifacts was excluded.
Cell apoptosis analysis
Cell apoptosis rate was calculated by using PE-Annexin V Apoptosis Detection Kit I (BD Pharmingen, USA) according to the product instructions. Stable transfected MKN-45 cells were resuspended in 1ÎBinding Buffer (1Î106 cells/ml). 5μl of FITC and 5μl of PI were added into 100μl of cell suspension, followed by 15 minutes incubation in darkness and 400μlÎBinding Buffer was added. The analysis of apoptosis by flow cytometry (Becton Dickinson, USA) was conducted. Both Annexin V-FITC-positive and PI-negative cells were considered as apoptosis ones.
Verification of the degenerating effect of miR-4246 on SPP1 mRNA
Online database miRDB (http://mirdb.org) and TargetScan, (http://www.targetscan.org) were used for predicting potential miRNAs post-transcriptionally interacting with SPP1 mRNA. By intensively review manuscripts on pubmed (https://www.ncbi.nlm.nih.gov/pubmed/), we noticed miR-4262 is a probable post-transcriptional regulator of SPP1. The expression profile of miR-4262 was further verified among multiple human malignancies by using online tools from the Database of Differentially Expressed miRNAs in Human Cancers (dbDEMC, Version 2.0: http://www.picb.ac.cn/dbDEMC/).
A 202 bp sequence from the 3’-untranslated region (3’-UTR) of SPP1 mRNA covering the potential miR-4262 binding site was cloned into pMIR-REPORT luciferase vectors (Promega, USA), containing Firefly luciferase. The sequence was constructed as follow (Sangon Biotech Co., Shanghai, China): 5’-aauacaauuucucacuuugcauuuagucaaaagaaaaaaugcuuuauagcaaaaugaaagagaacaugaaaugcuucuuucucaguuuauugguugaauguguaucuauuugagucuggaaauaacuaauguguuugauaauuaguuuaguuuguggcuucauggaaacucccuguaaacuaaaagcuucaggguuaugucu-3’. Correspondingly, the mutant sequence was transfected as follow: 5’-auuucuaauacacucauaggaauaacugauauguauauaagguauuuuggauauucauacacaucuucauaagguacauacacugauaaaucgaucauucucuuuguuuaucacugucguauuuaguuaagagauaguuuaauugauaacuaucucgguacuucguaucacgcagaauaguuauaccaugacgcuaaagaca-3’. We used pRL-TK vectors containg Renilla luciferase as control. And MKN-45 cells either over-expressing miR-4262 or the negative control were co-transfected with vectors above. The luciferase activity was measured by Dual-Glo Luciferase assay system (Promega, USA) 48h post-transfection.
Data from GEO, TCGA, CCLE and dbDEMC database was processed as the description above. Survival analysis of SPP1 was conducted by using online tool ((http://www.kmplot.com) and Kaplan-Meier curves, including 876 GC patients with available clinical data. Results derived and generated from specimens and cells were analyzed by using SPSS 18.0. Paired t-test and Fisher’s exact test were considered statistically significant as P values < 0.05.