Patient recruitment

In this study, a total of 96 EGC patients (TNM classification: T1A) who were subjected to ESD treatment were recruited and their expression of circFNDC3B was measured for grouping. Subsequently, the median circFNDC3B expression was calculated and utilized as the indicator to divide the patients into a Low expression group (N=48, including all EGC patients whose expression level of circFNDC3B was at or above the median expression of circFNDC3B) and a High expression group (N=48, including all EGC patients whose expression level of circFNDC3B was below the median expression of circFNDC3B). Therefore, for further analysis, since the patients were grouped according to their median, there are same number of patients in each group. The demographic and clinic parameters of both patient groups, including their sex, age, BMI, status of H. pylori infection, history of alcohol abuse and smoking, tumor site, as well as clinical grade of ESD was collected by reviewing their medical records, carrying out breath test for H. pylori infection, serological examinations, as well as bacterial culture, and the demographic and clinic parameters of the two patient groups were compared using Students test. In this study, ESD was defined as a type of adenocarcinoma constrained to the mucosa tissues or submucosa tissues in the stomach. All subjects with a past ESD history or those who received treatment for multiple ESD were not enrolled. An endoscopic forceps biopsy operation was carried out in each patient to collect ESD tissue samples for subsequent Western blot, qPCR and IHC assays. ESD follow-ups were carried out 2, 3, 6, 9, 12, 24, as well as 36months after the initial ESD operation. The level of gastric atrophy was assessed using histological evaluation results based on the endoscopic atrophy border scale developed previously (Kimura et al. 1969; Ito et al. 1996; Satoh et al. 1996). Institutional ethical committee of Chinese PLA NO.254 Hospital has approved the protocol of this study. All methods were performed in accordance with the last vision of the Declaration of Helsinki. Written informed consent was obtained from all patients before the study.

In this study, MKN28 cells, a extensively-studied gastric tubular adenocarcinoma cell line which were established from a 70-year-old female patient, were used to carry out cellular experiments. In brief, MKN28 cells were acquired from American Type Culture Collection (ATCC, Manassas, VA) and cultured according to the recommended conditions provided by the manufacturer, i.e., the cells were cultured in a Roswell Park Memorial Institute 1640 (RPMI 1640) medium (Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented along with 10% of fetal bovine serum as well as 1% of penicillin and 100 U/ml of streptomycin. The culture conditions were 37C, 5% CO2 and saturated humidity. Furthermore, all cells were regularly examined to affirm the absence of Mycoplasma. When the cells reached 70% confluence, they were sub-cultured and divided into different groups. In cell model I, the MKN28 cells were divided into 2 groups, i.e., 1. pGL group (MKN28 cells transfected with an empty plasmid); and 2.pGL-FNDC3B group (MKN28 cells transfected with a pGL3 plasmid inserted with the circFNDC3B fragment). In cell model II, the MKN28 cells were also divided into 2 groups, i.e., 1. NC siRNA group (MKN28 cells transfected with a scramble negative control NC siRNA); and 2.pGL-FNDC3B group (MKN28 cells transfected with circFNDC3B siRNA to silence the expression of circFNDC3B). In cell model III, we utilized the MKN28 cells to establish a H. pylori-infected gastric cancer cell model in comparison with un-infected cell model. The MKN28 cell were established as 2 groups, i.e., 1. Control group (MKN28 cells in unprocessed medium); and 2. rTip- group (MKN28 cells cultured in medium containing 12.5g/mL rTip-).

According to protocols provided by a previous publication22, to obtain rTip-, we transfected Tip- into Escherichia coli for subsequent amplification. And the amplified rTip- was purified for subsequent cell model establishment. All transfections were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the recommended transfection conditions provided by the manufacturer, and the transfected cells were harvested 48h after the start of transfection to analyze the expression of target genes.

The harvested cell as well as tissue samples were treated by utilizing a miRCURY RNA Isolation Kit (Exiqon, Qiagen, Germantown, MD) according to the recommended assay methods provided by the assay kit manufacturer to isolated cellular RNA. Then, the isolated RNA was assayed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Mountain View, CA) in conjunction with an RNA 6000 Pico assay kit (Agilent Technologies, Mountain View, CA) according to the recommended assay methods provided by the assay kit manufacturer to quantify the RNA concentration. In the next step, 1g of isolated total RNA was converted into cDNA by making use of a QuantiTect Reverse Transcription assay kit (Qiagen, Germantown, MD) according to the recommended assay methods provided by the assay kit manufacturer. Finally, real time quantitative polymerase chain reaction (RT-qPCR) was performed on a BX-384 real time PCR apparatus (Bio-Rad laboratories, Hercules, CA) by making use of a QuantiTect SYBR Green PCR assay kit (Qiagen, Germantown, MD) according to the recommended assay methods provided by the assay kit manufacturer to evaluate the relative expression of circFNDC2B, miR-942, miR-510, CD44 mRNA as well as CDH1 mRNA in each sample using the 2Ct approach. The expression of GAPDH in each sample was used as the internal control.

We utilized online bioinformatic tools including TargetScan (http://www.targetscan.org/vert_80/) and miRDB (http://mirdb.org/) to compare the sequences of circFNDC2B, miR-942, miR-510, CD44 mRNA and CDH1 mRNA. Accordingly, we detected a putative binding site of miR-942 on circFNDC3B, while a putative binding site of miR-942 was detected on the 3UTR of CD44 mRNA. Similarly, we detected a putative binding site of miR-510 on circFNDC3B, while a putative binding site of miR-510 was detected on the 3UTR of CDH1 mRNA. In the next step, we performed luciferase assays in MKN28 cells to confirm the regulatory relationship of circFNDC2B/miR-942, circFNDC2B/miR-510, miR-942/CD44 mRNA, and miR-510/CDH1 mRNA. In brief, the wild type sequences of circFNDC2B, CD44 mRNA, and CDH1 mRNA containing the corresponding miRNA binding sites were cloned into pGL plasmid vectors to generate wild type plasmids of circFNDC2B, CD44 mRNA, and CDH1 mRNA. At the same time, the sequences of circFNDC2B, CD44 mRNA, and CDH1 mRNA containing the corresponding miRNA binding sites were subject to site-directed mutagenesis to generate mutant type sequences of circFNDC2B, CD44 mRNA, and CDH1 mRNA containing the corresponding miRNA binding sites, which were also cloned into pGL plasmid vectors to generate mutant type plasmids of circFNDC2B, CD44 mRNA, and CDH1 mRNA. In the next step, MKN28 cells were co-transfected with the plasmids carrying wild type or mutant type circFNDC2B, CD44 mRNA, and CDH1 mRNA along with miR-510 and miR-942. At 48h post transfection, the luciferase activity of transfected cells was assayed by utilizing a GloMax Multi Detection assay kit (Promega, Madison, WI) according to the recommended assay methods provided by the assay kit manufacturer.

The recurrence-free rate of the patients was analyzed by using R statistical software (version 3.0). The recurrence-free rate in each group was calculated at various follow-up time points, and the recurrence-free rates of the two groups were compared at the level of statistical significance of 0.05. The KaplanMeier survival curves were generated for high and low groups of circFNDC3B expression.

Cells as well as tissue samples were first lysed in a phosphatase- and protease-inhibitor containing 1X RIPA buffer (Thermo Fisher Scientific, Waltham, MA) according to the recommended assay methods provided by the assay kit manufacturer. The collected lysates were then centrifuged to collect proteins in the supernatant, whose concentration of total proteins was examined by using a BCA protein assay kit (Pierce, Thermo Fisher Scientific, Waltham, MA) according to the recommended assay methods provided by the assay kit manufacturer. In the next step, the protein in each sample was resolved by 10% SDS-PAGE and blotted onto nitrocellulose membranes (Hybond, GE Medical Care, Pittsburgh, PA), which was blocked with TBSS containing 5% of skim milk and subsequently incubated with primary anti-CD44 and anti-CDH1 antibodies as well as HRP-conjugated secondary antibodies in sequence according to the recommended antibody incubation conditions provided by the assay kit manufacturer (Abcam, Cambridge, CA). Finally, after the protein blots (all original protein blots are shown in Supplementary file) were developed by using an enhanced chemiluminescence Western blot substrate (Pierce, Rockford, IL) according to the recommended assay methods provided by the assay kit manufacturer, the relative protein expression of CD44 and CDH1 in each sample was calculated.

Collected tissue samples were paraffin embedded, sliced into 4 um sections, deparaffinized, gradient alcohol hydrated, and incubated with primary anti-CD44 antibodies and biotin-labeled secondary antibodies in sequence according to the recommended antibody incubation conditions provided by the assay kit manufacturer (Abcam, Cambridge, CA) to determine the positive protein expression of CD44 in each sample under a Zeiss Axioskop microscope.

Unless otherwise specified, all results are presented as meanS.E.M of 4 independent tests. Statistical evaluations were done by making use of the Student's t test in SPSS 21.0 software (IBM, Chicago, IL) and Prism 8.0 program (GraphPad, San Diego, CA), and P<0.05 was deemed as statistically significant.

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