RNA sequencing data Analysis
In order to determine the level of expression of TMEM 219 (IGFBP-3R) and IGFBP-3, four RNA-Seq datasets (SRP135952, SRP133891, SRP012016, SRP073446) from the Sequence Read Archive (SRA) (www.ncbi.nlm.nih.gov/geo) and three datasets (GSE122401, GSE63288, GSE106338) from Gene Expression Omnibus (GEO), all related to patients with GC, were downloaded. The datasets from SRA was analyzed to process raw data by using Ubunto (18.04 LTS) and then was statistically interpreted to determine Differential Expression Genes (DEGs) by using software R (version 3.5.1). According to the low-quality sequence, the sequences were trimmed using Trimmomatic to remove reads with quality less than Q20 and adapter (first 15 bp of reads) (13). High-quality data (All clean reads) were mapped to Homo sapiens genome reference (UCSC version hg38) using HISAT2 (14) with mapping efficiencies around 97%, the counting was performed using HTSeq followed by differential expression analysis on each normal and tumor gastric replicates (15). In the end, normalization of the count was performed using R/Bioconductor limma package. All Geo dataset were already normalized and just the analysis of differential expression was performed using limma to determine DEGs (16).
Clinical Tissue Samples
All of 68 pair gastric cancer samples collected from gastric cancer surgical specimens, between April 2014 until September 2016 from Iran National Tumor Bank (INTB, Tehran, Iran). Each patient was written informed consent form, procedures according with the ethical standards of the institutional and/or national research committee of the 2013 Helsinki declaration and investigation has been approved with ethical committee members of the Medical University of Isfahan (Ethic number: 396386). Specimens without prior radiotherapy, chemotherapy or any treatments were enrolled in the study. All of the samples were evaluated by two independent pathologists blinded to the clinical features. Samples characterized according to the American Cancer Society and tumor-node-metastasis (TNM) classification system guidelines (17). Normal samples were removed from the marginal zone of cancer tissue and used as a control. Immediately, all samples were snap-frozen in liquid nitrogen until the relevant assays were performed. The clinicopathological features of samples were summarized in Table 1.
Table 1
Association of clinicopathological features with IGFBP-3 and IGFB-3R mRNA relative expression in 68 patients with gastric cancer. * (P < 0.05) indicates that the differences have statistical significance. *Pearson chi-square tests.
Parameters | Number of patients | IGFBP-31 mRNA expression | P value 2 | IGFBP-3R3 mRNA expression | P value 2 |
Low | High | Low | High |
Age | | | | 0.250 | | | 0.163 |
< 61 | 28 (41.2%) | 12 | 16 | | 12 | 16 | |
≥ 61 | 40 (58.2%) | 28 | 12 | | 24 | 16 | |
Sex | | | | 0.536 | | | 0.254 |
Male | 54 (79.4%) | 32 | 22 | | 30 | 24 | |
Female | 14 (20.6%) | 8 | 6 | | 6 | 8 | |
Tumor size | | | | 0.146 | | | 0.004 |
< 6 | 34 (50%) | 14 | 20 | | 12 | 22 | |
≥ 6 | 34 (50%) | 26 | 8 | | 24 | 10 | |
Lymph node invasion | | | | < 0.001 | | | < 0.001 |
Positive | 50 (73.5%) | 34 | 16 | | 32 | 18 | |
Negative | 18 (26.5%) | 6 | 12 | | 4 | 14 | |
Differentiation | | | | 0.186 | | | 0.002 |
Poor | 18 (26.5%) | 8 | 10 | | 0 | 18 | |
Moderate | 40 (58.8%) | 26 | 14 | | 30 | 10 | |
High | 10 (14.7%) | 6 | 4 | | 6 | 4 | |
TMN stage | | | | < 0.001 | | | 0.005 |
IB + II | 26 (38.2%) | 10 | 16 | | 10 | 16 | |
IIIA | 18 (26.5%) | 10 | 8 | | 8 | 10 | |
IIIB + IV | 24 (35.5%) | 20 | 4 | | 18 | 6 | |
1 Insulin-like growth factor binding protein-3 |
2 P-value was calculated from one-way ANOVA with Tukey post hoc and independent T-test |
3 Insulin-like growth factor binding protein-3 receptor |
Chemicals, Reagents, And Antibodies
RNA extraction reagent (RNXTM-plus) and DNase I was provided from Cinnagen (Cinnagen, Tehran, Iran). cDNA synthesis kit and the enhanced chemiluminescent detection system (ECL) were purchased from Takara (Tokyo, Japan). All primers and qPCR Master Mixmix SYBR Green (high ROX) obtained from Ampliqun (Herlev, Denmark). Primary sheep polyclonal anti-IGFBP-3R (AF7556-SP) and secondary donkey anti-sheep IgG horseradish peroxidase (HRP)-conjugated antibody (HAF016) were purchased from R&D (Minneapolis, USA). Mouse monoclonal antiβ-actin and anti-IGFBP-3, secondary mouse anti-goat IgG HRP-conjugated was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Electrophoresis reagents and materials provided by Bio-Rad (Hercules, CA, USA). Other chemical and reagent obtained from Sigma Aldrich (St. Louis, MO, USA).
Quantitative RT-PCR
Before RNA extraction, tissues were washed with ice-cold phosphate buffer saline (PBS). Total RNA was extracted from all tissues by using RNXTM-plus according to the manufacturer’s protocol. Frozen tissues (20–30 mg) homogenized by the bead-milling method in 1 ml of RNXTM-plus reagent as described previously(18). Subsequently, for the elimination of genomic DNA, the suspension was treated with DNase I. RNA concentration was then determined by ultraviolet spectrophotometer (BioTek,Winooski, VT, USA) using A260/A280 ratio and then the RNA samples were stored at -80 °C.
Total RNA (2 µg) was used for cDNA synthesize according to the kit’s protocol as following: reaction mixture up to 10 µl and without a hot start, 1 cycle at 37 °C for 15 min for reverse transcription and 94 °C for 30 s for enzyme inactivation. Real-time PCR was performed utilizing an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). The sequences of primers were used as follow: IGFBP-3 forward: 5′-GGTGTCTGATCCCAAGTTCC-3′, IGFBP-3 reverse: 5′-ACCATATTCTGTCTCCCGCT-3′; IGFBP-3R forward: 5′-TGACCACCTTGAACTTCG-3′, IGFBP-3R reverse: 5′-GCAGAAGATCCTTTCAATC-3′; GAPDH forward: 5′-CAGCCTCAAGATCATCAGC-3′, GAPDH reverse: 5′-GGCAGTGATGGCATGGACT-3′.
The final volume of the reaction mixture (10 µl) contained 1 ng of cDNA template, 200 nM each of sense and antisense primers and 5 µl of 2X SYBR Green PCR. The reaction conditions were as follows: after an initial hot start (95 °C) for 10 min, amplification was performed for 40cycles containing denaturation for 10 s at 94 °C, annealing for 30 s at 50 °C, and extension for 40 s at 72 C. The amplification kinetics was recorded as sigmoid progress curves for which fluorescence was interoperated against the number of amplification cycles. The threshold cycle number was used to define the initial amount of each template. Fluorescence readings were carried out in every amplification cycle, using StepOnePlus (Applied Biosystems, Foster City, CA, USA). All measurements were performed in triplicate. The sizes of the amplified fragments were confirmed by agarose gel 2% electrophoresis.
The relative expression was normalized with the GAPDH as an internal control. Then fold change calculated according to the 2−ΔΔCtmethod: ΔΔCt = [Ct gene of interest -Ct GAPDH] (cancerous tissues) - [Ct gene of interest -Ct GAPDH] (normal-adjacent tissue) similar with our previous study
Western Blotting
IGFBP-3 and IGFBP-3R protein expression were evaluated with western blotting as described previously(12). Summary, 100 milligrams of the fresh frozen tissues washed three times with ice-cold PBS. The tissues were homogenized by the bead-milling method in 1 ml ice-cold RIPA buffer (50 mM Tris-Cl [pH 7.4], 150 mM NaCl, 15 mM Na4P2O7, 20 mM NaF, 1 mM EDTA,1 mM PMSF, 6 mM EGTA, 100 mM glycerol 3-phosphate, 1% NP-40 [NP-4] and 1% Sodium Deoxycholic acid supplemented with 0.5%freshly protease and phosphatase inhibitors cocktail (Melford). The lysates were harvested with centrifugation (14,000 rpm) at 4ºC for 25 min and the supernatant stored in -80ºC.
The protein concentrations were measured with the Bradford protein assay. All protein samples were incubated with Laemmli buffer at 100 °C for 5 min, an equal amount (40 µg) of total proteins were separated by electrophoresis in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Ready Gel, Bio-Rad). Following, transferred onto a polyvinylidene fluoride membrane (PVDF, Amersham Pharmacia Biotech), membranes were blocked with Tris-buffered saline, containing 5% non-fat dried milk and 0.1%Tween (TBS-T) for 2 h in room temperature. After 3 time wash with TBS-T, membranes incubated overnight with primary antibodies at 4 °C (1:1000 in TBS-T, 0.1% Tween 20 and 0.1% BSA). After washing three times with TBS-T for 5 min, the membranes were incubated in secondary antibodies (1:2500 in TBS-T, 0.1% Tween 20 and 0.1% BSA) horseradish peroxidase (HRP). After three times washing with TBS-T protein bands were detected with ECL reagent. All bands were normalized by β-actin as the internal control. The relative intensity of all bands was quantified by densitometry, using the Image J software (NIH, Bethesda, MA, USA).
Statistical analysis
Datasets from SRA database was analyzed to process raw data using Ubuntu (18.04 LTS) and then, to statistical calculation and interpretation of Differential Expression Genes (DEGs) used Statistical software R (version 3.5.1, https://www.r-project.org/).The comparison of RNA and protein relative expression levels between the normal and the tumor tissues assessed with paired Student’s t-test. The One-way ANOVA (with Tukey posthoc) and independent sample T-test were used to evaluate the relationship between clinicopathological parameters and IGFBP-3/IGFBP-3R expression. The overall survival rates were calculated by the Kaplan–Meier method and differences in survival rates for between subgroup patients (high and low expression) were analyzed with the log-rank test. All experiments performed as triplicate and data were presented as mean ± SEM. Statistical significance was determined at the level of P < 0.05. All data were analyzed with SPSS 22 (SPSS, Chicago, IL, USA).