Clinical samples

Tissue samples were collected from 32 patients diagnosed with gastric cancer in The Affiliated Huaian No. 1 People’s Hospital of Nanjing Medical University, and gastric cancer tissues and adjacent normal tissues were collected through surgical resection. All patients signed informed consents and had no other treatment before surgery. This study obtained the approval of the Ethics Committee of The Affiliated Huaian No. 1 People’s Hospital of Nanjing Medical University.

Cell culture

Human normal gastric mucosal epithelial cell line (GES-1) and human gastric cancer cell lines (MKN-74, HGC-27 and AGS) were bought from BeNa Culture Collection (Beijing, China). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), and maintain the conditions of 37 °C and 5% CO2.

RNA extraction and RNAse R treatment

Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer instructions. Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek Corp, Ontario, Canada) was used to extract cytoplasmic and nuclear RNA. For RNAse R treatment, total RNA (5 μg) was incubated with 20 U RNAse R (Epicentre Biotechnologies, Madison, WI, USA) for 1 h at 37 °C.

Quantitative real-time polymerase chain reaction (qRT-PCR)

RNA was reversely transcribed into cDNA by PrimeScript RT Reagent Kit (Takara, Dalian, China). And then, qRT-PCR was performed with SYBR Green Master Mix (Takara) in accordance with the instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were used as internal controls, and the expression level was calculated by 2−∆∆Ct method. The primers were listed in Table 1.

Table 1 The primer sequences used for qRT-PCRVector construction and cell transfection

For the overexpression of circCNIH4, human circCNIH4 cDNA was cloned and inserted into the pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), and the empty vector was severed as control (NC). Small interfering RNA (siRNA) against circCNIH4, DKK2 and FRZB (si-circCNIH4, si-DKK2 and si-FRZB) and the control (si-NC) were obtained from GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for transfection of vector or siRNA. For stable transfection, circCNIH4 cDNA was inserted into the PLCDH-ciR lentivirus expression vector (Geneseed, Guangzhou, China) and infected into MKN-74 cells. The cells were selected with 0.8 μg mL−1 puromycin.

Western blot analysis

A protein extraction kit (Beyotime, Shanghai, China) was used to extract total protein. Then protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). 5% nonfat milk was used to block the membrane, and then the membrane was incubated with the primary antibodies against β-catenin (1:5000; ab32572, Abcam, Cambridge, UK), Ki67 (1:5000; ab92742, Abcam, Cambridge, UK), DKK2 (1:1000; ab95274, Abcam, Cambridge, UK), FRZB (1:1000; ab205284, Abcam, Cambridge, UK) or GAPDH (1:5000; ab181602, Abcam, Cambridge, UK) overnight at 4 °C. Subsequently, the membrane was incubated with the secondary antibody (1:5000; ab205718, Abcam, Cambridge, UK) at room temperature for 1.5 h. The protein signals were showed by enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA).

Cell proliferation assay

Cell proliferation was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cells were cultured in a 96-well plate for 24 h after transfection. Then each well was added with 20 µL of MTT (5 mg mL−1) to incubate the cells for 4 h. Subsequently, cells were collected and added with 150 µL of dimethyl sulfoxide for the dissolution of formazan crystals. The absorbance at 490 nm wavelength was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Flow cytometry assay

Apoptosis was determined according to annexin V-fluorescein isothiocyanate (V-FITC)/propidium iodide (PI) apoptosis kit (BD Bioscience, San Diego, CA, USA) instructions. Cells were collected after transfection for 48 h and then added annexin V-FITC and PI. After incubation for 20 min in a dark condition, cell apoptosis was tested by flow cytometry (BD Bioscience, San Diego, CA, USA).

Transwell assay

Cell migration and invasion were detected with transwell chambers (Corning, Tewksbury, MA, USA) coated without and with Matrigel (BD Biosciences, San Diego, CA, USA), respectively. Cells with serum-free medium were added into the upper chamber, and the basolateral chamber was added cell medium with 10% serum. After cultured for 24 h, the migrated or invaded cells adhering to the lower surface of the membrane were fixed by 4% paraformaldehyde and dyed with 0.5% crystal violet solution. The stained cells were counted under a microscope (Thermo Fisher Scientific, Waltham, MA, USA).

In vivo tumor formation assay

The animal experiment was approved by the Animal Care Committee of The Affiliated Huaian No. 1 People’s Hospital of Nanjing Medical University. 5-week-old nude mice were randomly divided into two groups (circCNIH4 overexpression group and negative control (NC, transfected with empty vectors) group. MKN-74 cells stably transfected with circCNIH4 overexpression vector or empty vector were subcutaneously injected into nude mice. After 10 days for injection, tumor length and width were gauged every 4 days. After 34 days, the tumor tissues were resected and weighed. Tumor volume was computed using the formula length × width2/2.

Statistical analysis

Data were presented as mean ± standard deviation and analyzed by SPSS 22.0 (IBM Corp., Armonk, NY, USA). Each experiment was completed with at least three repeats. Differences between two groups or multiple groups were compared by Student’s t-test or One-Way Analysis of Variance (ANOVA). p < 0.05 was considered statistically significant.