HCC tissue collection and cell culture
Twenty HCC patients who did not receive preoperative radiotherapy or chemotherapy were recruited from May 2015 to July 2018 at Naval Medical University. Tumor and adjacent tissues were collected during surgery. All tissue samples were maintained in liquid nitrogen. All patients signed the informed consent form. All procedures and experiments of this study conformed to the Declaration of Helsinki, and were approved by the Ethics Committee of Naval Medical University.
HCC cell lines (HepG2, Smmc-7721, HLE, Huh7 and Bel-7404) and normal liver cells (LO2) were purchased from the Chinese Academy of Sciences Shanghai Institute of Life Sciences. Cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Vienna, Austria) containing 10% foetal bovine serum. Cells were incubated at 37 °C in 5% CO2 and 95% humidity. The circFAM13B plasmid, circFAM13B siRNA, sh-circFAM13B, E2F5 siRNA, controls, miR-NC, miR-212 mimic, and miR-212 inhibitor were all purchased from Tingzhou Bio (Shanghai, China).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted using TRIzol™ reagent (Invitrogen™, Carlsbad, CA, USA). The cDNA of mRNAs were amplified with oligo (dT) primers and the cDNA of circRNA were amplified with random primers by the PrimeScript™ RT Master Mix reagent kit (TaKaRa, Otsu, Shiga, Japan). The obtained cDNAs were subjected qRT-PCR using SYBR® Premix Ex Taq™ (TaKaRa, Otsu, Shiga, Japan). Gene expression was normalized to GAPDH (for mRNAs) or U6 (for miRNAs) using the comparative cycle time (Ct) method (2−ΔΔCt). Each qRT-PCR was performed in triplicate and the mean values were calculated. Primer sequences are shown in Additional file 1: Table S1.
The targeted binding sites of circFAM13B, E2F5 and miR-212 were generated by bioinformatic methods. CircFAM13B-WT and E2F5-WT luciferase plasmids containing wild-type binding sites and circFAM13B-MUT and E2F5-MUT plasmids containing mutated binding sites were constructed by Tingzhou Bio (Shanghai, China). The luciferase reporter plasmids were transfected with miR-NC or miR-212. The pCMV-AC-GFP-circFAM13B plasmid (Tingzhou Bio, Shanghai, China) and pcDNA3.0-p53 (Tingzhou Bio, Shanghai, China) were transfected with p21-luciferase reporter plasmid (Tingzhou Bio, Shanghai, China) into HepG2 and Smmc-7721 cells. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin, USA). Reporter assays were performed in triplicate.
Cell counting kit-8 (CCK-8)
The proliferation of HCC cells was evaluated by the CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan). Cells grown at logarithmic phase were inoculated into 96-well plates at a density of 5 × 104 cells per well for 48 h at 37 °C with 5% CO2. 10 µl of CCK-8 was added in each well and further incubated for 2 h. OD values were detected under 450 nm by Synergy 4 (BioTek, Winooski, VT, USA). Each test was triplicated.
Cells were counted, diluted in DMEM (Gibco, Vienna, Austria)containing 10% FBS, plated into 6-well plates (500 cell/petri dish), and incubated for 2 weeks. The colony-forming densities were constantly monitored until the colonies were macroscopically observable. Colonies were rinsed with phosphate buffered saline (PBS), fixed in 4% paraformaldehyde (30 min), and stained with crystal-violet solution (2.5% in methanol). The stained cells were subsequently rinsed and dried. Finally, colony counting and imaging were performed.
RNA immunoprecipitation (RIP)
A Magna RIP RNABinding Protein Immunoprecipitation Kit (Millipore, MA, USA) was used to investigate whether the ribonucleoprotein complex contained miRNA and its potential binding circRNA in HCC cells. Ago2 antibody (Millipore, Billerica, USA) and IgG (Millipore, Billerica, USA) were used for immunoprecipitation. The antibodies were added to cell lysates and rotated overnight. After incubating with proteinase K buffer for 30 min the next day, the immunoprecipitated RNAs were isolated and extracted using TRIzol™ reagent (Invitrogen™, Carlsbad, CA, USA). qRT-PCR was performed on the immunoprecipitated RNA. The relevant steps and reagents of qRT-PCR are the same as above.
Fluorescence in situ hybridization (FISH)
To determine the subcellular location of circFAM13B and miR-212 in HepG2 cells, cells were fixed in 10% fixing solution in PBS for 5 min. Glass slides containing cell samples were dipped into fixing solution twice (10 min each), dehydrated in gradual concentrations of iced-cold ethanol solutions (70%, 90 and 100%), and dried. FISH wasperformed in a wet box containing 50% formamide and 50 ml 2× saline sodium citrate buffer at 37℃. The FITC-labelled circFAM13B probe and PE-labelled miR-212 probe were designed by Yansai Co. Ltd. (Shanghai, China). Sequences of the probes are listed in Additional file 1: Table S2.
RNase R treatments
Total RNA of HepG2 cells was incubated for 30 min at 37 °C with 3 U/µg RNase R (Epicentre Technologies, Madison, USA), and subsequently the abundance of linear FAM13B RNA and circFAM13B RNA was analysed by qRT-PCR. The relevant steps and reagents of qRT-PCR are the same as above.
Actinomycin D assay
HepG2 cells were exposed to 100 ng/ml actinomycin D (Sigma-Aldrich, St. Louis, USA) for 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h. Then, the cells were harvested, and total RNA was extracted. The stability of circFAM13B and FAM13B mRNA was analysed using qRT-PCR. The relevant steps and reagents of qRT-PCR are the same as above.
Protein samples were prepared in RIPA lysis buffer (Thermo Scientific, Rockford, IL, USA). Aliquots of 30 µg protein were fractionated by SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Schwalbach, Germany). The membranes were blocked in skim milk, rinsed in PBS and incubated with primary antibodies overnight [E2F5 1:1000 (Abcam, Cambridge, UK, ab59769), PUMA 1:1000 (Cell Signaling Technology, Danvers, MA, USA, 98672), P21 1:1000 (Cell Signaling Technology, Danvers, MA, USA, 2947), GAPDH 1:1000 (Cell Signaling Technology, Danvers, MA, USA, 5174), and β-Actin 1:1000 (Cell Signaling Technology, Danvers, MA, USA, 3700)]. The secondary antibodies, including goat anti-rabbit IgG (1:2000, Proteintech, USA, srbAF488-1) and goat anti-mouse IgG (1:2000, Proteintech, USA, SA00001-1), were then incubated with the membranes. The relative densities of the bands were quantified using densitometry (Quantity One software, BioRad).
In vivo tumor growth
HepG2 cells were stable transfected with sh-circFAM13B and sh-NC lentivirus. Cells (107) were subcutaneously injected under the right arm of each mouse. Ten female BALB/c nude mice were randomly grouped into sh-circFAM13B and sh-NC groups. Tumor growth was evaluated every 3 days after the injection. Mice were sacrificed 30 days after the injection using pentobarbital sodium (150 mg/kg) after the injection. The tumor tissue was collected and analysed.
All statistical analyses were performed using SPSS 20 and GraphPad Prism 5. Data were subjected to independent-sample t-tests for comparisons between 2 groups, and one-way ANOVA for comparison among multiple groups. The significance level was p < 0.05 for all statistical analyses.