Cell culture

All HCC cells including Huh7, Hep3B, HepG2, MHCC-97L, SMCC-7721, and immortalized human hepatic cell LO2 were obtained from the Type Culture Collection of the Chinese Academy of Sciences. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose(Gibco, USA), supplement 10% FBS (Gibco, USA) and penicillin–streptomycin (100 U/mL and 100 ug/mL, respectively) and maintained at 37˚C in 5% CO2 humidified incubator.

Oligonuleotide transfection

Small interfering RNA (siRNA), miRNA mimics, miRNA inhibitors and negative control oligos were synthesized by GenePharma (Shanghai, China). The sequences.

are listed in Table 1. Transfection was performed using Lipo8000 (Biyotime, China).

Table 1 The sequences of siRNAs, miRNA mimics, miRNA inhibitors and negative controlsPlasmid construction and stable transfection

The circ_0003570 overexpression plasmids and lentiviruses were synthesized by (Hanheng Biotechnology Co., Ltd., Shanghai, China). The constructs were confirmed by sequencing. Afterwards, HepG2 and MHCC-97L cells were infected with a circ_0003570 overexpression lentivirus or a negative control lentivitus. For stable cell transfection, the transfected cells were selected with puromycin for 2 weeks. Finally, the transfection efficiency was examined via qRT‐PCR analysis.

The isolation of RNA and quantitative real‐time PCR (qRT‐PCR)

Total RNA from cells was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Then, the cDNA was synthesized following the protocol of Evo M-MLV RT Premix for qPCR (Accurate Biotechnology, Hunan, China) and Goldenstar™ RT6 cDNA Synthesis Kit (TsingKe, China). Quantitative real-time PCR was carried out using Master qPCR Mix (TsingKe, China) on a CFX96 Real-Time PCR system (Bio-Rad, CA, USA). The relative gene expression was calculated using the 2−ΔΔCt method. β-actin was served as the internal reference of mRNA/circRNA, and U6 was considered as the internal reference of miRNA. The primers in the study were showed in Table 2.

Table 2 The primers in the studyColony formation assay

After 24 h of transfection, cells were planted into 6‐well plate with 1000 cells per well. After about 1 weeks, when clones formed by single cell possessed at least 50 cells, the clones were fixed with methanol for 10 min and next 0.1% crystal violet were used for staining about 10 min. Finally, the clones were counted for statistical analysis after washed and aired.

MTT assay

Cell viability was assessed by MTT (3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay. Cells were seeded into 96‐well plates at a density of 5000 cells per well. 24 h after seeding, transfection was conducted as the reagent's protocol. After transfection for 24 h, 48 h, 72 h, 96 h, 10 μL of 5 mg/mL MTT was added into the medium in the dark and then cultured for 4 h in incubator. The suspension was then discarded and 150 μL DMSO was added to dissolve the crystal. Using an ELISA reader (Bio-Rad, Hercules, CA, USA), cells viability were measured by the optical density (OD) at 490 nm. Triplicate experiments were performed for each assay.

Transwell assay

After 48 h of cell transfection, 5 × 104 cells were respectively seeded into 8 μm pore size transwell 24‐well chambers (Merck Millipore) coated with Matrigel (BD Biosciences) for invasion assay and uncoated chambers for migration. On the top of chamber is serum-free DMEM medium and lower wells were supplemented 10% FBS. After incubation for further 24 h, chambers were taken out and washed off the non‐invaded and non‐migrated cells gently on the upper membrane. Then the chambers were fixed with 95% ethyl alcohol for 10 min and stained by crystal violet for 10 min, and washed in PBS. Stained cells were observed under optical microscope and calculated.

Wound healing assay

After cell transfection, the HCC cells were cultured for 24 h to achieve 90% cell density in six-well plates, and the wells were previously externally marked with a straight black line on the bottom to use as guides for the subsequent photography. Wound (perpendicular to the guide lines) was scratched by a 200 µl sterile tip, and then washed off the floating cells. Under the microscope, the wound size was measured and photographed at 0, 24, and 48 h. All pictures were collected under microscope. Images were aligned using the orientation line to ensure that the identical spots were followed over time.

Dual‐luciferase reporter assay

Bioinformatics tools were used to analyze the miR-182-5p binding sites on circ_0003570 and STARD13 binding sites on miR-182-5p. Cells were seeded into 96‐well plate at approximately 60%‐80% confluence before transfection. Plasmid vector including the wild-type (WT) or mutant (Mut) 3′UTR of circ_0003570 or STARD13 (Hanheng Biotechnology, China) together with miR-182-5p mimics/inhibitor or NC (GenePharma) were co-transfected using the Lipo8000 reagent (Biyotime, China). The basic vector used for the reporter plasmid was psiCheck2 plasmid. The luciferase activity was examined using the Dual Luciferase Assay Kit (Biyotime, China). The Renilla luciferase activity was considered as control.The sequence of synthesized reporter plasmids (wt-circ_0003570-luc, mut-circ_0003570-luc; wt-STARD13-luc, mut-STARD13-luc) was showed in Table 3. The vector maps of wt-circ_0003570-luc, mut-circ_0003570-luc, wt-STARD13-luc, and mut-STARD13-luc used in this study were presented in Fig. S1.

Table 3 The sequence of luciferase reporter plasmidsWestern blot analysis

After transfection for 72 h, all proteins were isolated by RIPA (Pierce). Proteases and phosphatases were added to RIPA in advance to prevent protein degradation. Protein samples were loaded for electrophoresis (5% gel for concentration and 10% for separation), and then the proteins were transferred on a 0.45 μm or 0.22 μm pore size PVDF membrane (Merck Millipore). After blocking with 5% defatted milk gently for 1 h, the membrane was incubated at 4 °C overnight with primary antibodies (STARD13; Proteintech) at 1:2000 dilution. The next day, the membrane was washed extensively and incubated with secondary antibodies (at 1:2500 dilution; Zhuangzhi Biology, China) for 1 h at room temperature. Proteins bands were detected by using ECL immunoblotting kit (Millipore, USA) following the manufacturer’s protocol.

Tumorigenicity assay

Male BALB/c nude mice (4-week-old) were got from the animal laboratory center of Xi’an Jiao Tong University. The mice were divided into two groups randomly, respectively accepted subcutaneously injection for 100μL of stably expressed circ_0003570 or control HepG2 cells in PBS, which contained approximately 5 × 106 cells. After one month or more, all mice involved were sacrificed. 100μL of 2 × 106 lentivirus-transduced HepG2 cells were injected from tail vein for metastasis and after 2 months mice were sacrificed and lung specimens were collected for research next. The study was conducted in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and approved by the medical ethics committee of Xi’an Jiao Tong University.

H&E staining and Immunohistochemistry (IHC)

All tissues were fixed with 10% of formalin for 48 h and embedded with paraffin wax, which were cut for 4 mm thick. After dewaxed by xylene and hydrated using ethyl alcohol of step-rising concentration. Then, H&E staining was preformed and Sections were observed using microscope. For IHC, primary antibody of STARD13 (Proteintech) at dilution rate 1:200 were incubated overnight at 4 °C. The next day, secondary antibodies were incubated at room temperature, and then, slides were stained with DAB and hematoxylin. Images were collected under microscope after dehydration and transparency.

Statistical analysis

All data were showed as means ± SEM and analyzed by SPSS 23.0 (IBM, SPSS, Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software, CA, USA). Student’s t-test or one-way ANOVA was performed to compare the difference between the groups, and Pearson’s correlation coefficient analysis was applied to analyze the correlation between two groups. A two-tailed P < 0.05 was regarded as statistically significant, and P < 0.01 was very significant.