Patients and clinical tissue samples
This work was conducted under the approval of the Research Ethics Committee of Zhejiang Provincial People’s Hospital, Hangzhou Medical College (code: 2020QT084). All human researches were based on the Helsinki Declaration. Each participant had provided written informed consent before enrollment.
Forty-one tissue specimens and paired adjacent non-cancerous tissue samples, which obtained from GC patients who underwent radical resection of the primary lesions at Zhejiang Provincial People’s Hospital, Hangzhou Medical College, were collected from May 2017to July 2018 (Table 1). All Patients without any preoperative radiotherapy, chemotherapy, or any other medical interventions were diagnosed as primary GC by postoperative pathological examination. Non-cancerous tissue was obtained 5 cm from the tumor site. Immediately after removal, all samples were frozen in sterile liquid nitrogen test tubes and stored at − 80℃ for further analysis. The tumor stage was accurately defined according to the eighth edition of the tumor–node–metastasis (TNM) classification system of the International Union Against Cancer (UICC, 2009). Two experienced pathologists assessed the histological grade. Detailed clinical and pathological data, including age, sex, diameter, differentiation, lymphatic metastasis, and TNM stage, were available for all patients (Table 1).
Table 1
Clinical characteristics of the patients.
Characteristics | Validation |
Total number | 41 |
Gender (male/female) | 31/10 |
Age (years, mean) | 65.1 ± 11.2 |
Primary GC (yes/no) | 41/0 |
Adenocarcinoma (yes/no) | |
– Tumor size T ≤ 2 cm | 41/0 |
– T > 2 cm | 0/41 |
Number of lymph nodes | |
-0 | 21 |
-1-2 | 6 |
-3-6 | 6 |
-≥7 | 8 |
TNM stage | |
– I | 17 |
– II | 14 |
– III | 9 |
– IV | 1 |
Histologic differentiation | |
Well differentiated | 6 |
Moderately differentiated | 17 |
Poorly differentiated | 18 |
Vascular invasion (yes/no) | 20/21 |
GC: Gastric cancer; TNM: Tumor, node (regional lymph node), metastasis |
Microarray data collection
The microarray data used in this study were obtained from the public GEO (Gene Expression Omnibus) database (https://www.ncbi.nlm.nih.gov/geo/). The circRNA expression profiles of GC were acquired from GSE100170. The raw microarray data were then background corrected, normalized, and log2-transformed. The Bio-conductor Limma package was used to screen for differential expression of circRNAs (DEcircRNAs) between the gastric cancer lymph node metastasis group and the non-lymph node metastasis group in the environment of R-3.6.1, and the cut-off value was set at the adjusted P-value of < 0.05 and |log2 fold change (FC)| >2.0 [13]. The circBase (http://www.circbase.org/) and the circBank (http://www.circbank.cn/index.html) were used to find the original gene associated with the circRNA. The RNA-sequencing (RNA-seq) data were downloaded from the public TCGA (The Cancer Genome Atlas) data portal (https://tcga-data.nci.nih.gov/tcga/).
Cell line, cell culture, and transfection
Normal human gastric epithelium cell (GES-1), highly differentiated cell MGC-803, medium differentiated cell SGC-7901, poorly differentiated cell BGC-823, AGS cell, and HGC-27 cell lines, stored in our laboratory, were purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, where they were tested and authenticated according to American Type Culture Collection standards. These cell lines were cultured in RPMI-1640 or DMEM media (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin mixture (Selleck, China) and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Exponentially growing cells were harvested from the culture flasks using 0.25% Trypsin/EDTA (Beyotime, China), and centrifuged at 1000 rpm for 5 min, re-suspended, and counted for use in subsequent experiments.
Lipofectamine® 2000 reagent (Invitrogen, #11668-027) was used to transfect cells with constructed plasmids (GenePharma, Shanghai) and small interfering RNAs (siRNAs) (RiboBio, Guangzhou), as following the instructions provided by the manufacturer. Human GC cells in logarithmic growth period cultured on the 6-well plate (2.0×105cells/well) were respectively transfected with 2µg circCOL6A3_030 +GFP plasmid, circCOL6A3_030 -ORFmut + GFP plasmid, empty vector (EV), 50 nM negative control (si-NC), and siRNAs of circCOL6A3_030 1#, 2#, and 3 #. circCOL6A3_030+GFP plasmid and circCOL6A3_030-ORFmut+GFP plasmid were synthesized by GenePharma.
The siRNA sequences were as follows:
si-circCOL6A3_030 1#:5′-ACCTGTAATAACCTTCTGCAAUU-3′;
si-circCOL6A3_030 2#:5′-AGACCTTCTGTATCTGACCAAUU-3′;
si-circCOL6A3_030 3#:5′-TAACAATCCTCCTGTACCTAAUU-3′.
Transfection efficiency was verified by real-time quantitative polymerase chain reaction (qRT-PCR) 48h later.
RNA extraction, purification, and qRT-PC
Total RNA was extracted from cancerous/noncancerous specimens (stored at -80℃) and cells using Trizol (Invitrogen, USA) as per manufacturer's protocol. The concentration, purity and integrity of RNA were determined by NanoDrop spectrophotometer OD260/280 and 1% formaldehyde-denatured gel electrophoresis. Then, RNA was purified and reverse transcribed into cDNA using the SuperScript™ IVFirst-Strand Synthesis System (Invitrogen, USA). SYBRTM Green PCR Master Mix was used to detect the expression levels of circRNAs and the mRNAs by qRT-PCR in ABI 7500 real-time PCR system. The reaction conditions were according to the manufacturer’s protocol (Thermo Fisher Scientific, USA). We used the human GAPDH reference gene as internal controls. Relative transcription expression value was estimated using a 2−comparative Ct (2−ΔΔCt) method and normalized against the threshold cycle (Ct) of GAPDH. All reactions were conducted in triplicate independently to ensure the reproducibility of all the data. The primer sequence information (RiboBio, China) is presented as follows:
circCOL6A3_030 (Forward, 5ʹ-TGGCTCTCACTGAAACAGAAATG-3ʹ;
Reverse, 5ʹ-GTCGTCAC TGGGTTGGATGTAG-3ʹ),
LineCOL6A3_030 (Forward, 5ʹ-ATGAGGAAACATCGGCACTTG-3ʹ;
Reverse, 5ʹ-GGGCATGAGTTGTAGGAAAGC-3ʹ),
GAPDH (Forward, 5ʹ- AGTCAGCATT TCACAAGACCTC-3ʹ;
Reverse, 5ʹ- CAGGCGAAGATGTTCTGGC-3ʹ)
circRNA confirmatory assay
The divergent primers were synthesized by RiboBio (Guangzhou, China) to verify the back-splicing junction of circCOL6A3_030. Sanger sequencing (GENESEED, Guangzhou, China) of the cDNA PCR product bands (Invitrogen, USA) was performed to further determine the full-length and ensure the accuracy of the circRNA detection.
Fluorescence in situ hybridization (FISH)
RNA fluorescence in situ hybridization (RNA-FISH) was performed with FISH Kits according to the manufacturer’s instructions (RiboBio, China) in gastric cells. RNA FISH probe sequence targeting the back-splicing site of circCOL6A3_030 was designed and synthesized by RiboBio (Guangzhou, China). First, cells were seeded on coverslips, fixed with 4% paraformaldehyde for 10 min, and then permeabilized with 0.5% Triton X-100 PBS for 5 min. Next, hybridization was carried out with a Cy3-labeled FISH probe in a dark moist chamber at 37℃ for 16 h. Afterward, slides were washed in hybridization rinse solution and were stained with 4,6-diamidino-2-phenylindole to label cell nuclei (DAPI; Life Technologies, USA) for 10 min. All images were obtained using a confocal microscopy (Leica, Germany).
Cell counting assay
SGC-7901 and BGC-823 cells were transfected with 50 nM si-circCOL6A3_030 1#, 2#, 3 #, or si-NC in a 6-well plate. Cells were collected 24 h after transfection and placed in a 6-well plate in the form of 2x105 cells/wells, with three duplicate wells of each group. The number of cells was counted after 2 days.
Transwell assay
SGC-7901 and BGC-823 cells were transfected with 2µg circCOL6A3_030 +GFP plasmid, circCOL6A3_030 -ORFmut +GFP plasmid, empty vector (EV), 50 nM negative control (si-NC), and siRNAs of circCOL6A3_030 1#, 2#, and 3 # in a 24-well plate. After transfection for 24h, infected cells were harvested, and then carried out migration experiments. For migration assays, 1×105 infected cells were placed in a Transwell (24-well, Millipore, USA) upper chamber with 200 μl of serum-free RPMI 1640, while 600 μl RPMI 1640 containing 10% FBS was added to the bottom chamber as a chemical attractant. At 24 h of post-incubation, the chamber was fixed with polyformaldehyde for 30 mins and stained with a 0.1% crystal violet solution for 15 mins, and then the cells on the inner surface of the upper chamber were removed by scrubbing with a sterile cotton swab dipped in PBS. After that, five fields were randomly selected under an inverted microscope (Olympus, Tokyo, Japan) to count the average cell number in the lower chamber (200 x). The group under each condition had its duplicate wells.
Scratch wound assays
SGC-7901 and BGC-823 cells were transfected with 2µg circCOL6A3_030 +GFP plasmid, circCOL6A3_030 -ORFmut +GFP plasmid, empty vector (EV), 50 nM negative control (si-NC), and siRNAs of circCOL6A3_030 1#, 2#, and 3 # in a 6-well plate. After 24h infection, transfected cells were collected, and then subjected to a wound-healing assay. For wound-healing assays, the infected cells were plated in 6-well plates (4×105 cells /well). At 24 hours after inoculation, wounds were created by using a 1 ml pipette tip in confluent cells with reaching approximately 80%. The free-floating cells and debris were then washed out with PBS. The cells were incubated at 37°C with RPMI 1640 medium containing 2% FBS. Wound healing was observed at different time points and scrape lines were photographed at the same time. The group under each condition had its duplicate wells, and each experiment was repeated three times.
Prediction of circCOL6A3_030 encodes polypeptide
The circRNADb was used to gather evidence of the circRNA ability to encode proteins (http://reprod.njmu.edu.cn/cgi-bin/circrnadb/circRNADb.php). There were circCOL6A3_030 IRES and ORFs information. Bioinformatic metrics, such as the IRES score and ORF score are used to assess the coding potential of detected IRESs and ORFs (Table 2).
Western blotting
Total protein was extracted from cells using RIPA (Radio-Immunoprecipitation Assay) buffer (Sigma) with a 1% protease inhibitor and 1% PMSF (Beyotime Biotechnology, China). Protein concentrations were measured using the BCA protein assay kit (Beyotime Biotechnology, China). Proteins were denatured with sodium dodecyl sulfate (SDS) buffer at100°C for 10 min, and each sample protein of 30 μg then was segregated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein bands were transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Next, the PVDF membrane was blocked with 5% non-fat milk in TBST buffer for 1 h at room temperature and then were incubated with specific primary antibodies at 4 °C overnight including circCOL6A3_03 0 peptide (1:50; HuaAn Biotechnology Co., Ltd, China) and the internal reference protein antibody GAPDH (1:5000; Cell Signaling Technology, USA). Afterward, the PVDF membrane was washed three times for ten mins in TBST and incubated with Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibodies (1:1500; Cell Signaling Technology, USA) for 2 h at room temperature. After washing in TBST for 3 times, immunoblot bands were visualized by ECL chemiluminescent reagent (Millipore, USA) under a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, USA) and the optical density was analyzed by ImageJ2X software. These experiments were repeated three times.
The peptide antibody was designed and synthesized by HuaAn Biotechnology Co., Ltd (Hangzhou, China). The peptide sequence of circCOL6A3_030 was as follows: 147-161aa CSFSTKKSQPPPPQP; 79-92aa AKLHWERAEPPGPYC.
Xenograft model
The procedures for care and use of animals were approved by the Principles of Laboratory Animal Care of Zhejiang Chinese Medical University. All animal studies were performed following the Guidelines for the Care and Use of Laboratory Animals of the Council of Science and Technology of China.
For the in vivo assay of a gastric cancer mouse model with spleen-to-liver metastasis, thirty-six female (4-week-old) BALB/c nude mice (20±2g) were purchased from the Shanghai SLAC Laboratory Animal Co.Ltd (Shanghai, China) and maintained in SPF condition with a standard 12-h light-dark cycle at the Experimental Animal Center of the Zhejiang Chinese Medical University. On the day of the intrasplenic injection of liver metastases, SGC-7901 cells transfected with si-circCOL6A3_030 3# and si-NC (Ctrl) were harvested by trypsinization and washed twice with serum-free medium and then suspended in PBS (1×106/100 μl in serum-free medium with 50% Matrigel). Cells were injected into the superior pole of the mouse spleen (Fig 1A). The mice were anesthetized with 4 % chloral hydrate (10 ml/kg) by peritoneal injection on day 21 after intrasplenic injection with the tumor cell lines. The livers were removed and the hepatic metastatic nodules were observed. Liver tissues were fixed in 10% buffered formaldehyde solution for hematoxylin and eosin (H&E) staining and immunohistochemical Staining.
Hematoxylin and Eosin Staining and Immunohistochemical Staining
Hematoxylin and Eosin Staining: At room temperature, the specimens were fixed in 4% paraformaldehyde for 24 hours, followed by dehydration and embedding in paraffifin. After dewaxed and rehydrated, the 5-μm-thick tissues were stained with hematoxylin and eosin (H&E). Immunohistochemical Staining: The specimens were blocked with 5% normal goat serum for 30 minutes and then incubated with primary antibody against circCOL6A3_030 (1:50; HuaAn Biotechnology Co., Ltd, China) at 4℃ overnight. Then, sections were processed using the ABC detection kit (Vector Laboratories, Burlingame, CA). All the staining images were obtained using the Olympus microscope (Olympus Co., Tokyo, Japan)
Statistical analysis
Each experiment was repeated in triplicate. Statistical analyses were carried out by SPSS 21.0 software (SPSS, USA). The circRNA expression level was presented as an FC utilizing the 2-ΔΔCT method on qRT-PCR analysis. Differences between individual groups were analyzed using Student’s t-test. All data were expressed as the mean ± standard deviation (SD). A p-value of < 0.05 (*), < 0.01 (**) or < 0.001 (***) was considered significant.