pIRES2-EGFP-cagC plasmid propagation & purification
Recombinant plasmid pIRES2-EGFP- cagC gene and plasmid pIRES2-EGFP without foreign gene was prepared from the Biotechnology Research Center of the Islamic Azad University of Shahrekord. The pIRES2-EGFP-cagC vector encodes the full-length cagC in the first cistron and the green fluorescent protein in the second cistron. Plasmid amplification was performed at the first step by transferring it to a bacterial host, mainly containing suitable strains of Escherichia coli (TOP10). In this regard, the competent E. coli, TOP10, was transformed by a pIRES2-EGFP-cagC vector according to standard protocol. Briefly, 100 ng of the by pIRES2-EGFP-cagC was added to 1.5×108 of chloride calcium competent cells and kept for 25 min on ice. The mixture was then incubated for 90 sec at 42 °C and moved immediately on ice for 2 min. In the following step, 1000 µl LBBroth edium was added to transformed cells and incubated at 37 °C for 1 hr. The transformed bacteria were selected on an LB-ampicillin (100ng/ml) agar plate. The pIRES2-EGFP-cagC vector was purified from cultured transformed using FavorPrep plasmid (Favorgen Biotech, Tiwan) extraction Mini Kit as per the manufacturer's instructions, and then it was visualized by 1% agarose gel electrophoresis. The PCR technique was done to confirm the plasmid's accuracy extracted from the bacterium, and double digestion by restriction enzymes SalI / SacII was used to confirm the cagC gene's presence in the plasmid pIRES2-EGFP.
AGC cell line expansion
A human gastric epithelial cell line AGS (gastric adenocarcinoma) were obtained from Pasteur institute of Iran.and were grown under standard cell culture conditions (5% CO2, 37°C) in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovin serum (FBS) and standard antibiotics (penicillin-streptomycin 1%). Every three days, the cells had examined by an inverted microscope, and when grown to 70-80% confluence, were trypsinized. Viable cells were counted by trypan blue staining and Neubauer chamber.
Transfection of pIRES2-EGFP-cagC into AGS cells
The lipofectamine 2000 (thermofisher, USA) was used to transfect the cell lines based on the manufacturer's guidance. Briefly, 2 µl of lpofectamine2000 and 2 µg of each plasmid (pIRES2-EGFP-cagC or pIRES2-EGFP) were diluted separately in 100 µl of RPMI reduced serum medium and mixed gently. Then, the Lipofectamine2000 and plasmid were mixed and incubated for 10 min at room temperature to allow the DNA-Lipofectamine2000 complexes to form. The complexes were added to cells grown in RPMI medium without serum and antibiotics. After 4 h., the medium was replaced with RPMI-1640 supplemented with 10% FBS. After 24r, 1%Pen/Strep beside 600 µg/ml neomycin were added to cells grown in serum-containing medium. Finally, the transfected cells were detected.
RNA preparation and real-time RT-PCR
In the present study, the real-time reverse transcription-polymerase chain reaction (RT-PCR) technique was used to evaluate the altered expression of GPR83, CA1, AWP1, and WTAP genes in AGS cells transfected with pIRES2-EGFP-cagC vector. First, Total RNA was extracted from the cells with RNX-Plus solution (Bioidea, Iran), and using quantitative (Nano drop) and qualitative (agarose gel) methods, the quality of extracted RNA was examined. Total RNA was reverse transcribed into cDNA by cDNA synthesis kit (yekta tajhiz, Iran). PCR method was used to confirm the extracted plasmid's accuracy and evaluate the cloned gene (cagC), using specific primers (Table 1). PCR cycling was performed in SYBR Green PCR Master Mix (Yekta tajhiz, Iran) using 25 ng of cDNA. The internal standard GAPDH housekeeping gene was co-amplified with the specific genes. At the end of proliferation, the melting curve analysis was carried out on the PCR product to confirm the propagated original product's specificity and identity. The reaction solution for real-time PCR was prepared by mixing 25ng/ µl of synthesized cDNA solution with 7.5 µl TaqMan Universal PCR Master Mix. Real-time PCR was carried out at 64°C for 25 sec, 95°C for 3 min, followed by 45 cycles at 95°C for 20 s, and at 72°C for 20-sec min and Melting Curve, from 55ºC to 95ºC, read every 0.3ºC, hold 3 sec. Each assay was done in triplicate. The expression of GAPDH was used to normalize that of the target genes. Before using the comparative threshold cycle (Ct) method for the relative quantification, a validation experiment was performed according to the manufacturer’s instructions to verify that the target and GAPDH genes’ efficiencies were approximately equal. The change in the Ct (ΔCt) of the target genes was calculated as ΔCt = (Ct of target genes)–(Ct of GAPDH). We calculated the ratio of the target genes to GAPDH as 2−ΔCt, expressed as 2−ΔCt × 105; this ratio was used to evaluate each target gene’s expression level in each gastric carcinoma cell the unstimulated state. The abundance of the target genes relative to that of GAPDH was calculated as ΔΔCt = (ΔCt of target genes)–(ΔCt of GAPDH). The ratio was calculated as 2−ΔΔCt to evaluate the alteration of the target genes 12 h after co-culture with the gastric cancer cells and H. pylori.
Using SPSS version 22 software and an independent t-test, each gene expression was examined and compared. P-value ≤0.05 was considered statistically significant.