GC patients and tissue acquisition
A total of 63 patients (38 males and 25 females) diagnosed as GC through histopathological exam were enrolled at People’s Hospital of Xinjiang Uygur Autonomous Region. The 63 patients included 30 cases at AJCC stage I or II, and 33 cases at AJCC stage III or IV. All cases were adenocarcinoma. All patients signed informed consent. This hospital Ethics Committee approved this study. Age range of patients was 48 to 68 years, with a median of 58 years. Initiated therapy and other severe clinical disorders, such as metabolic disorders, severe infections, other malignancies, were excluded from this study. All patients were diagnosed for the first time, and no recurrent cases were included. Prior to therapy, fine needle aspiration was carried out to collect GC and paired adjacent (with 3 cm around tumors) non-tumor tissues from the patients. Tissue samples were kept in liquid nitrogen storage prior to the subsequent assays.
GC cells and transfections
The cell model of GC was AGS (adenocarcinoma) cell line purchased from ATCC (USA). RPMI-1640 medium was mixed with FBS (10%) to serve as the culture medium of AGS cells. Cells were cultivated in an incubator at 5% CO2, 95% humidity and 37 °C.
Expression vector of cRAPGEF5 was constructed using pcDNA3.1(+) CircRNA Mini Vector (Addgene). MiR-22 mimic and negative control (NC) miRNA, as well as miR-22 inhibitor and inhibitor NC were purchased from Sigma-Aldrich. AGS cells were cultivated to reach about 80% confluence, followed by transfecting expression vector of cRAPGEF5 (1μg), miR-22 mimic (40 nM) or miR-22 inhibitor (40 nM) into 108 cells. To perform NC experiments, NC miRNA-, NC inhibitor or empty vector-transfection was performed. Untransfected cells cultivated in fresh medium were also included to serve as a control (C) experiment. Prior to the subsequent experiments, AGS cells were cultivated in fresh medium for further 48h in fresh medium.
RNA isolation and quality analysis
Tissue samples and AGS cells were subjected to total RNA isolations, followed by digesting genomic DNA for 2h at 37 °C using DNase I (Invitrogen). Electrophoresis performed using a 5% urea-PAGE gel was performed to analyze RNA integrity. Analysis of the purity of RNA samples was performed by measuring the OD 260/280 ratios. RNA samples with an OD 260/280 ratio around 2.0 were pure RNA samples.
A Reverse Transcription System (A5003, Promega Corporation) was used to synthesize cDNA samples through reverse transcriptions (RTs) with RNA samples as template. With GAPDH as an internal control, qPCRs were performed using a QuantiTect SYBR Green PCR Kit (Qiagen) to analyze the expression of cRAPGEF5. Expression of mature miR-22 and miR-22 precursor was analyzed by RT-qPCR. To measure the expression levels of mature miR-22, poly (A) was added, followed by using poly (T) as reverse primer to perform both RTs and qPCRs. To analyze miR-22 precursor expression, sequence-specific primers were used in RTs and qPCRs U6 was used as the internal control. Three technical replicates were performed for each experiment, and 2-ΔΔCT method was used to normalize Ct values of target gene to corresponding internal controls.
Cell proliferation assay
AGS cells with transfections were subjected to proliferation analysis using a CCK-8 kit from Sigma-Aldrich (USA). Cells were cultivated at 37 °C in a 96-well cell culture plate (3000 cells in 0.1ml fresh medium per well, three replicate wells were set for each experiment), and cell proliferation was analyzed by measuring the OD values at 450 nm every 24h for a total of 96h. At 2h prior to the determination of OD values, CCK-8 solution was added to reach 10%.
Expression of cRAPGEF5, mature miR-22 and miR-22 precursor in paired tissues was expressed as heatmaps plotted using Heml 1.0 software. Correlations between cRAPGEF5 and mature miR-22 or miR-22 precursor were analyzed by Pearson’s correlation coefficient. Mean+/-SD values were calculated to express data of multiple cell transfection groups and data comparisons were analyzed by ANOVA and Tukey’s test. P<0.05 was deemed statistically significant.