China Center for Type Culture Collection served as the channel for getting GC cell lines, including AGS, BGC-823, MGC-803, and SGC-7901. Cells were cultivated in 10% FBS in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). And they were cultured with 5% CO₂ at 37°C.

A circPTN template with an artificial flanking sequence was synthesized and inserted into pcDNA 3.1. For transient transfection, siRNA or plasmid was added to Optifectamine 2000 (Invitrogen, Carlsbad, CA, USA) in OptiMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). It is transfected into cells in the form of a complex. The medium was changed after 6 h. And the whole RNA and protein were extracted within two days. In order to stabilize transfection, GenePharma prepared a circPTN lentiviral package (Bornschein et al., 2011). Cells were cultivated in a 24-well plate with 1 × 10⁵ before lentivirus transfection. 24 h later, replaced the original medium with 2 mL fresh RPMI-1640 medium containing 6 g/mL polybrene, and add an appropriate amount of virus suspension at 37°C. After 4 h, 2 mL fresh medium was added to dilute polybrene. Continue to cultivate until the lentivirus contained fluorescent protein was detected in 72 h. After that, the transfected cells were picked out with 5 μg/mL puromycin for a fortnight.

Total RNA in cells was abstracted with Trizol regent (Invitrogen, Carlsbad, CA, USA). The quality and consistence of RNA were tested by utilizing a microplate reader. 2000 ng of RNA was reversed through a reverse transcription kit (Takara, Shiga Prefecture, Japan). RNA amplification was then carried out through SYBR Premix Ex TaqTM II (Takara, Shiga Prefecture, Japan) under the defined procedures. U6 is an internal reference gene. In the end, the expressions were analyzed by utilizing the 2^−ΔΔCt method (Bornschein et al., 2011).

The adoption of cell lysate containing protease inhibitor was to abstract the whole proteins form cells. Protein consistence was tested by BCA (bicinchoninic acid) Protein Assay kit. Western buffer was added to the protein. The protein was denatured by boiling at 100°C for 10 min. 20 μL sample was added to each well of 12% SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel at 100 V for 120 min. The gel was applied to a PVDF (polyvinylidene fluoride) membrane with electrophoresed at 120 V for 90 min. Block with skim milk for 1 h at RT (Reverse Transcription). The primary antibody was cultivated on the membrane at 4°C overnight. After rinsed with PBST (Phosphate Buffered Saline) three times, the secondary antibody was cultivated on the membrane for two hours at RT (Reverse Transcription). After rinsed by PBST (Phosphate Buffered Saline), developing solution was added to the membrane. The outcomes of the experiment were observed through a chemiluminometer.

The cells were seeded into 98-well plates. In the 0–5 days, 10 μL of CKK-8 (Cell Counting Kit-8) solution (Dojindo, Kyushu, Japan) was added per well per day. On the fifth day, the cells were cultivated for two hours at 37°C. In the end, the absorbance of each well was measured at 450 nm by a microplate reader.

Six-well plates were adopted to cultivated cells at 37°C for 10 days. Cells were fixed with 1% paraformaldehyde. The colonies were stained with 0.1% crystal violet dye (RIBOBIO, Guangzhou, Guangdong, China).

Cell migration was analyzed by a Transwell chamber (8.0-μm pore size; 6.5-mm diameter, Corning, NYC, USA). Transfected cells were cultured on the upper chamber with Matrigel-coated of 24-well BioCoat Matrigel Invasion Chambers (Becton Dickinson and Company, Franklin Lakes, NJ, Jiangsu, China) and in serum-free RPMI 1640. After 24 h, cells in the upper chambers were not crystal violet stained until the medium was removed, and the upper chamber cells were scraped.

The adoption of 96-well plates was to cultivate cells. Then 100 μL of medium containing 20 μM EdU (5-Ethynyl-2’-deoxyuridine) was added to each well. The cells were cultivated for two hours at 37°C, 5% CO₂. Following, they were fixed with 4% paraformaldehyde. Wash on a shaker with 0.5% Triton-X-100 PBS for 20 min. Cell staining was conducted utilizing the EdU (5-ethynyl-2’-deoxyuridine) Apollo DNA in vitro kit (RIBOBIO, Nanjing, Jiangsu, China) (Bornschein et al., 2011). In the end, cell proliferation was tested under an immunofluorescence microscope.

All data are expressed as mean ± standard deviation. All data were statistically analyzed by Student’s t-test or one-way analysis of variance. The most significant value is the probability of p < 0.05.

For the sake of disclosing the functions of circPTN in gastric cancer, we devised a divergent primer based on circRNA junction sequences and specifically targeted circPTN to precisely test circPTN expressions through RT-qPCR analysis in line with the circBase database (Glazar et al., 2014). For testing whether circPTN exerted crucial functions in GC, we tested circPTN expression in cells utilizing qRT-PCR. Then, we discovered that circPTN was highly expressed in gastric cancer cells in contrast with normal cells (Fig. 1A). Outcomes indicated that increased circPTN expression was observed in gastric cancer. Thus, we investigated whether circPTN controls the biological behavior of gastric cancer.

The circPTN was proved that is highly expressed in gastric cells. We tried to discover the functions of circPTN in the GC. Though previous studies have reported that circPTN is an oncogene that accelerates cell proliferation, we aimed to prove this effect in gastric cancer cells. Following, we set up the stable circPTN knockdown and overexpression systems by transfecting BGC-823 and AGS cells with lentivirus (Figs. 2A and 2B). By performing a colony formation experiment, we discovered that cell proliferation was restrained after silencing circPTN, while cell proliferation was increased after overexpressed circPTN (Figs. 2C and 2D). It demonstrated that silenced circPTN could suppress cell growth in gastric cancer.

The circPTN was certified that it promoted migration in gastric cells. Similarly, we used stable circPTN knockdown and overexpression systems by transfecting BGC-823 and AGS cells with lentivirus. By performing a Transwell experiment, we discovered that cell migration was reduced after silencing circPTN, while cell migration was increased after overexpressed circPTN (Figs. 3A and 3B). It demonstrated that silenced circPTN could suppress cell migration in gastric cancer.

In order to ulteriorly test the functions of circPTN in gastric cancer cells, we conducted CCK-8 and EdU staining experiments in BGC-823 and AGS cells. We could evidently see that cell viability and EdU positive cells were declined (Figs. 4A and 4B). It indicated that cell proliferation capability was inhibited when circPTN was knocked down. By the large, we concluded that circPTN could induce gastric cancer cell proliferation.

We discovered that previous research had described that circPTN was related to the regulation of stemness or self-renewal (Aponte and Caicedo, 2017). Moreover, countless evidence demonstrated that gastric-stem cell (GSCs), clusters of cancer cells, possess the capacity for self-renewal (Bartfeld and Koo, 2017). Thus, we hypothesized that circPTN might influence self-renewal. We tested stemness markers, which included Nestin, CD133, CD133, SOX2, and SOX9 in BGC-823 and AGS cells. As we thought, the expression of stemness markers determined that these markers were positively associated with the circPTN level (Fig. 5A). Moreover, the outcomes demonstrated that circPTN could exert functions in regulating self-renewal. We examined the protein levels of stemness markers (CD133 and SOX2) in BGC-823 and AGS cells. Consequently, we discovered that the protein levels of these markers were significantly declined in cells, demonstrating that circPTN might exert the crucial roles in regulating stemness (Fig. 5B). Taken together, these outcomes indicated that circPTN may regulate self-renewal.