2.1 microarray analysis
A comprehensive database of gene expression (GEO, http://www.ncbi.nlm.nih.gov/geo), which is a public repository for archiving and distributing microarrays for free, was screened. GSE97332 and GSE94508 were selected, and the “limma” package was used to analyze differences in gene expression between tumor and non-tumor tissues [14, 15]. circRNAs with adjusted P < 0.05 and |Log FC|>1 were considered as significant dysregulated circRNAs.
A total of 50 HCC patients who underwent surgical treatment in our hospital were enrolled from June 2012 to June 2015. The main screening criteria include: (1) histological diagnosis of primary HCC; (2) receiving surgical resection; (3) not receiving chemotherapy or radiotherapy before surgery; (4) complete preoperative tumor characteristics. In addition, this study Patients with relapsed or secondary HCC and a history of malignant tumors were excluded. The study was approved by the hospital's ethics committee. All patients or their families provided written informed consent.
2.3 Cell lines
human HCC cell lines Hep3B, HepG2, SMMC-7721, and Huh-7, and the human normal liver cell line (LO2) were purchased from the Chinese Academy of Sciences (Shanghai, China) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, California, USA) supplemented with 10% fetal bovine serum (Gibco) and antibiotics (100 U/ml penicillin G and 100 mg/ml streptomycin) at 37 °C in a humidified atmosphere with 5% CO2.
Total RNA was isolated with TRIzol Reagent ((Beyotime, Shanghai, China)) following the manufacturer’s instruction. Then, 1 µg total RNA was reversed into 20 µl complementary DNA (cDNA) with First Strand cDNA Synthesis Kit (Takara, Tokyo, Japan). QRT-PCR was conducted using SYBR Green Master Mix II (Takara) on ABI7900 system (Applied Biosystems, CA, USA) in line with the manufacturer’s procedure. circ_0004913, miR-1290, TEX2 and FOXC1 mRNA expression was determined using the 2−ΔΔCT method. A P-value < 0.05 denotes a statistical significance. GAPDH and U6 were used as internal controls for circRNA and miRNA, respectively. All primers were purchased from GenePharma (Shanghai, China)
2.5 RNase R treatment assay
2 ug RNA and 6 units of RNase R (Geneseed Biotech, Guangzhou, China) were added together to incubate for 20 min at 37 °C. Subsequently, circ_0004913 and TEX2 mRNA expression was detected through qRT-PCR.
2.6 Subcellular localization
The subcellular localization of circ_0004913 was detected using the PARIS Kit (Invitrogen, CA, USA) in accordance with the manufacturer’s instructions.
The lentivirus-pHBLV-CMV-Cicr-MCS-EF1-circ_0004913, lentivirus-microRNA-1290 mimics, and their negative control were purchased from GenePharma. HCC cells were transduced with individual types of lentivirus at a multiplicity of infection (MOI) of 10 in the presence of 5 µg/ml puromycin (Thermo Fisher).
2.8 CCK-8 assay
Transfected SMCC-7721 and Huh-7 cells (1000 cells/well) were seed onto 96-well plates and incubated overnight at 37 °C. Cell proliferation was measured using the Cell Counting Kit-8 (CCK8, Dojindo, Shanghai, China) according to the manufacturer's protocol. OD values were measured at a wavelength of 450 nm using a microplate reader (Bio-Rad, CA, USA).
2.9 5-Ethynyl-2’-deoxyuridine (Edu) Assay
The EdU proliferation assay (RiboBio, Guangzhou, China) was carried out according to the manufacturer’s instructions. Transfected cells were incubated with 50 µM EdU for 2 h. Then an Apollo staining and DAPI staining were performed according to the instructions to detect the EdU positive cells with a fluorescence microscope.
2.10 Flow cytometric analysis
Transfected cells were suspended in 70% cold ethanol overnight after harvest. Then, cells stained with propidium iodide (PI) (Vazyme, Nanjing, China) for 30 minutes were analyzed. The proportion of cells in different cycle phases were calculated and compared.
2.11 Luciferase assay
Dual luciferase reporter system psiCHECKTM (Thermo Fisher) was used for luciferase assay. The wild type (wt) sequences and its mutant type (mut) sequences were cloned into the plasmid psiCHECK2. HEK293T cells (5 × 104 cells/well) were cultured in 24-well plates overnight and transfected with 400 ng of psiCHECK vector (psiCHECK-circ_0004913 wt, psiCHECK-circ_0004913 mut, or psiCHECK-FOXC1 wt, psiCHECK-FOXC1 mut), together with the plasmid for Renilla luciferase expression by lipofectamine 3000. One day later, the luciferase assays were performed after co-transfection with miR-1290 mimics or NC.
2.12 RIP assay
The EZMagna RIP kit (Merck, Darmstadt, Germany) was employed for RIP assay according to the manufacturer's protocol. In brief, RIP lysis buffer was used to HEK293T cells, and the lysate products were incubated at 4 ℃ for 6 h with magnetic beads that were pre-conjugated with anti-Argonaute 2 (AGO2) or anti-IgG antibody. Afterwards, the beads were washed and digested with protease K, so as to remove the proteins. At last, the purified RNA was analyzed by qRT-PCR.
2.13 Western blot
Transfected cells were lysed in ice-cold RIPA buffer (Beyotime) with 10 nM PMSF for 30 minutes and then collected to extract total protein. Total Proteins lysates were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in 5% non-fat milk in TBST for 2 h at room temperature and then immunostained overnight at 4℃ using rabbit anti-FOXC1 and PCNA (1:1000, Cell Signaling Technology, CST, USA). Rabbit anti-GAPDH (CST) was taken as a control. The signals were captured and the intensity of the bands was quantified by using the ChemiDoc XRS + system (Bio-Rad).
2.14 Mice xenograft models
For animal experiment, male BALB/c nude mice (4-6-week-old) were bought from Nanjing Medical University (Nanjing, China) and randomly divided into 2 groups (n = 5 per group). A total of 1 × 106 SMCC-7721 cells transfected with circ_0004913 or con were subcutaneously injected into mice. The tumor volume was calculated every week by the formula: length × width2/2 method. After 5 weeks, the tumors were removed for subsequent experiments. This study weas approved by the Animal Committee of Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University.
2.15 Statistical analysis
Data were shown as Mean ± SD performed at least three independent replicates. SPSS software, 24.0 (SPSS Inc., Chicago, IL, USA) and Graphpad Prism 7.0 (San Diego, CA, USA) were used for one-way ANOVA (multiple groups), a two-tailed Student t-test (2 groups). Kaplan-Meier method test were used for survival analysis. Differences were considered as statistically significant if P < 0.05.