Sample collection
A total of 82 patients with GC admitted to the Cancer Hospital Affiliated to Hainan Medical College and examined in the endoscopy center from January 2017 to December 2018 were recruited in the study after institutional ethics clearance. Patients younger than 80 years of age with complete clinical data, who were undergoing selective GC surgery, who did not receive chemotherapy or other adjuvant treatment before operation, and those without active gastrointestinal bleeding or obstruction were included in the study. Patients with uncontrolled diabetes or hypertension, coronary heart disease, stroke, cardiovascular, and cerebrovascular diseases; with severe basic diseases such as pulmonary, liver, and kidney dysfunction; and those requiring resection of other organs were excluded from the study. Patients admitted to the Cancer Hospital Affiliated to Hainan Medical College and examined in the endoscopy center from January 2017 to December 2018 were recruited to this study following written informed consent and in accordance with the local ethics committee approval. Of the 82 patients, four patients were selected for the circRNA chip screening study. Of these, there were two men (one with T3N1M0, moderately differentiated adenocarcinoma; one with T3N2M0, poorly differentiated adenocarcinoma), and two women (one with T3N1M0, moderately differentiated adenocarcinoma; one with T3N2M0, poorly differentiated adenocarcinoma). The average age, weight, and height of the four patients were 56.7 years, 58.3 kg, and 168 cm, respectively. Another 78 patients with GC (Table 1) were selected to obtain endoscopic biopsy and gastric fluid samples. These patients were included in the validation study of differential circRNA expression. The diagnostic criteria for early GC (EGC) and advanced GC (AGC) were according to the National Comprehensive Cancer Network clinical practice guidelines in oncology (version 3.2016). Additionally, 30 patients with chronic nonatrophic gastritis (CNAG) and 30 patients with chronic atrophic gastritis (CAG) were randomly selected as the the control group. The diagnostic criteria for CNAG and CAG were according to the consensus opinion of the 2012 Chinese Chronic Gastritis of Gastroenterology Branch of the Chinese Medical Association.
The GC specimens were obtained by cutting 0.5 cm3 of the whole layer of the GC tissue, whereas the paracancerous tissue specimens were obtained by cutting 0.5 cm3 of the mucosa at least 5 cm away from the tumor body. The samples were separated from the body, quickly sliced to the required size, and placed into frozen storage tubes stored in liquid nitrogen.
The endoscopic tissue samples and gastric juices were extracted from 78 patients with GC (21 patients with EGC and 57 patients with AGC), 30 patients with CNAG, and 30 patients with CAG. Table 1 illustrates basic characteristics of the patient and control groups. All the specimens were collected and pretreated according to the previously described protocol and preserved at -80°C until RNA extraction [27].
Total RNA extraction and reverse transcription
Total tissue RNA and gastric fluids were extracted using TRIzol reagent (Invitrogen, Life Technologies Inc., Germany). RNA concentration was measured by measuring the absorbance at 260 nm (OD260) using a NanoDrop ND-1000 instrument (Thermo Fisher Scientific, DE, USA). Integrity of the RNA was verified by denatured agarose electrophoresis. Finally, the total RNA was transcribed to the cDNA through the GoScript Reverse Transcription (RT) system (Promega, WI, USA) following the manufacturer’s protocol.
Microarray hybridization of circRNAs
GC tissues and their matched adjacent nontumorous tissues were selected to analyze the circRNA expression profile using Human circRNA Array v2 (Arraystar, MD, USA). Total RNA was digested with RNase R (20 U/μL, Epicentre, Inc., Madison, WI, USA) to remove linear RNAs and enrich circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent cRNA through a random priming method (Super RNA Labelling Kit; Arraystar). Labeled cRNAs were hybridized onto Human circRNA Array v2 (8 × 15 K, Arraystar). Slides were incubated for 17 h at 65°C in a hybridization oven (Agilent, CA, USA). After washing the slides, the arrays were scanned through an Agilent Scanner (G2505C). The scanned images were then imported into the Agilent Feature Extraction software for grid alignment and data extraction. Quantile normalization and subsequent data processing were performed using the R software package. The expression profile of circRNA, identified through volcano plot filtering, between GC and paired adjacent nontumorous tissues was statistically significant [fold change (FC) ≥ 2.0 and P ≤ 0.05]. Hierarchical clustering was performed to exhibit the distinguishable expression pattern of circRNAs among samples. The circRNA/microRNA interaction was predicted using TargetScan [28] & miRanda [29].
Quantitative reverse transcription–polymerase chain reaction
The eight most upregulated and downregulated circRNAs exhibiting the greatest difference in expression between groups were selected for quantitative reverse transcription–polymerase chain reaction (qRT–PCR) verification in the four GC specimens and their adjacent tissues. qRT–PCR was performed using the GoTaq qPCR Master Mix (Promega) on an Mx3005P Real-Time PCR System (Stratagene, CA, USA) in accordance with the manufacturer’s protocols. Divergent primers of the top eight upregulated and downregulated circRNAs and convergent primers of β-actin (H) were designed and synthesized by Aksomics (Shanghai) Biotechnology Co. Ltd. The use of divergent primers could only amplify circRNA and differentiate the contamination from its linear isoforms. Table 2 lists the circRNA primer sequences used for this procedure.
RT-PCR was performed as follows: 40 PCR cycles of 95°C for 10 s and 60°C for 60 s for amplification; followed by annealing at 95°C for 10 s, 60°C for 60 s, and 95°C for 15 s with slow heating from 60°C to 99°C (at 0.05°C/s).
The target and housekeeping genes of each sample were analyzed by RT-PCR. According to the gradient dilution DNA standard curve, the concentration results of the target and the housekeeping gene of each sample were directly generated using an Applied Biosystems ViiA™ 7 Real-Time PCR System (ThermoFisher Scientific, USA). The target gene concentration of each sample divided by its housekeeping gene concentration was considered the corrected relative content of this sample gene.
Statistical analysis
Statistical analyses were performed using the SPSS 22.0 software (SPSS, IL, USA). When comparing the GC and paired nontumorous tissue groups for profile differences, the “FC” (the ratio of the group averages) between the groups for each circRNA was computed. The statistical significance of the difference was estimated by the t test. CircRNAs having FCs ≥ 2.0 were considered as significantly differentially expressed. The analysis outputs were filtered, and the differentially expressed circRNAs were ranked according to characteristics such as the FC value, P value, and chromosome location. The differences in the hsa_circ_000780 levels between the GC and paired adjacent nontumorous tissues were assessed using the t test for paired data and among multiple groups (CNAG, CAG, EGC, and AGC) using one-way analysis of variance with the LSD post-hoc test. The correlations between hsa_circ_000780 levels and clinicopathological factors were further analyzed by the Analyze–Correlate–Bivariate menu of SPSS 22.0. A P value < 0.05 was considered statistically significant.