Human normal liver cell lines (LO2) and HCC cell lines (Huh7, HCCLM3, SMMC-7721, MHCC97H, HepG2) were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Gibco) and antibiotics (1% penicillin/streptomycin, Gibco, USA) in a humidified incubator with 5% CO2 at 37°C.
RNase R treatment
To verify the stability of circITCH molecular structure, RNase R digestion experiment was carried out. 2μg of total RNA extracted from HCC cells were incubated with 3U/μg RNase-R (Geneseed Biotech, Guangzhou, China) at 37 °C for 15 minutes and 70 °C for 10 minutes. Then, the expression levels of linear β-actin and circITCH were determined by qRT-PCR.
RNA extraction and RT-qPCR
Total RNA was extracted from cell lines using TRIzol Reagent. A NanoDrop 2000 microspectrophotometer was used to determine the quality and concentration of total RNA. After spectrophotometric quantification, 1 μg of total RNA in a final volume of 20 μl was used for reverse transcription (RT) with a PrimeScript RT Reagent Kit (Tsingke, China). Then, Fluorescence quantitative PCR was conducted using TB Green Fast qPCR Mix (Tsingke, China). All primers were designed and synthesized by Tsingke (Guangzhou, China). The expression of small nuclear RNA U6 or β-actin genes was used as a control to calibrate the original concentration of miRNA, circRNA respectively. Target gene expression was calculated using the 2-ΔΔCT method. Related primer sequences are shown in Table 1.
Small interfering RNAs (siRNAs) targeting the junction sequence of circITCH (si-circ-1, si-circ-2 and si-circ-3), miRNA-184 mimics, inhibitors and their related negative control oligonucleotides were designed and synthesized by HanBio (Shanghai, China). All transfection operations were performed using EntransterTM -R4000 (Engreen Biosystem, Beijing, China) according to the manufacturer’s instructions. All the sequences used in the present study are listed in Table 2.
Construction of stable cell lines
To establish stable overexpression of circITCH in human HCCLM3 cell lines, we used control lentivirus HBLV-ZsGreen-PURO and purpose lentivirus HBLV-hsa_circ_0001141-Null-ZsGreen-PURO to infect HCCLM3 cells (HanBio, Shanghai, China). In the infected cells after 48 h, by adding and maintaining 1.0 mu g/ml puromycin kill not be infected cells effectively.
Thus under puromycin drugs to maintain stable strains of eventually get circITCH stable expression.
Dual luciferase reporter assay
According to the predictions of Circular RNA Interactome (circinteractome) and Starbase online software, circITCH possessed binding sites for miR-184 in the 3ʹ-UTR region. To experimentally verify the possible miRNA target genes and binding sequences, the wild-type and mutant circITCH 3ʹuntranslated region (3ʹUTR) sequences were synthesized and cloned into dual luciferase reporter plasmids (HanBio, Shanghai, China) containing the psiCheck2 promoter. HCCLM3 cells were inoculated into a 96-well plate and grown to 50%- 70% confluence 24 h at 37°C, 5% CO2. They were transfected in combination with the wild-type or mutant circITCH reporter gene plasmids and miR-184 mimic or miR-NC mimic. After 48 h, the activities of both firefly luciferase (LUC) and Renilla luciferase (RLUC) were measured with a Dual-Luciferase Reporter System Kit (Promega, USA).
RNA fluorescence in situ hybridization (FISH)
FISH kit (RiboBio) was used to detect the location of circITCH in HCC cells following the manufacturer's protocol. Briefly, cells were subjected to pre-hybridization solution for 30 min. Then, cy3-labelled circITCH probe were hybridized at 37°C overnight after pre-hybridization. Next, slides were washed and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). A confocal microscopy was applied to analyze results.
Cell counting kit-8 (CCK8) assay
For the CCK8 assay, transfected HCC cells in logarithmic growth phase were seeded into a 96-well plate at a density of 1×104 cells/well for 6 replicates. The proliferation of cells were monitored every 24 hours for 0, 24, 48,72 h. 10ul of CCK 8 solution (KeyGene, Biotech, China) was added to each well and they were maintained at 37°C for 2h away from light. The absorbance of each well at 450 nm was measured with a microplate reader.
5-Ethynyl-2′-deoxyuridine (EdU) assay
Cell-Light™ EdU Apollo®567 In Vitro Imaging Kit (RiboBio, Guangzhou, China) was used to detect cell proliferation ability according to the manufacturer's instructions. The transfected HCC cells (1 × 104 cells for each group) were seeded in 96-well plates and cultured for 24 h, after, a 50 μM EdU solution was added onto plates and incubated for 2h at 37 °C. Then, the cells were immobilized with 4% paraformaldehyde and stained with Apollo Dye Solution and Hoechst 33342. Finally, the images were acquired using a fluorescence microscope and the ratio of EdU-positive cells was calculated.
To perform the invasion assay, after 24h of transfection treatment, HCC cells (1 × 105 /well) were prepared into the upper chamber (8-μm pore size; Millipore) with 200μL serum-free medium, and 800μL of medium containing 20% fetal bovine serum was added into the bottom transwell chamber. After incubating at 37 °C with 5% CO2 for 24 h, we gently scraped the cells on the upper chamber with cotton swabs, whereas the invasive cells on the bottom chamber were fixed with 100% methanol for 20 min and stained with 0.1% crystal violet for 15 min at normal temperature. Five fields were randomly selected under a microscope (200× magnification) to calculate the average number of invasion cells.
Wound healing assay
Wound healing assays were performed to evaluate the migration ability of HCC cells. Transfected and controlled cells (1 × 106 cells/well) were seeded onto 6-well plates and cultured at 37°C, 5% CO2. After the cells were fully grown, cell monolayers were scratched with a 200-μl pipette tip to draw a vertical line in the middle of the cell and then cultured in DMEM. After routine culture for 24h and 48 h, photographs were taken and the cell migration rate was calculated.
To perform cell apoptosis assay, a FITC Annexin V Apoptosis Assay Kit (BD Biosciences, CA, USA) was used as follows. Cells in logarithmic growth phase were collected, resuspended and evenly spread in a 6-well plate. After 24 h of transfection, a total of 1×106 cells were collected and washed twice with pre-cold PBS. Thereafter, 1× binding buffer (100μL) was used to disperse these cells, 5μL Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and 5μL propidine iodide (PI) were added to the cells for 15 min in a dark at room temperature. Subsequently, the apoptosis rate was analyzed by flow cytometry.
All assays were conducted at least three times, quantitative data are presented as the means ± standard deviation (SD). Differences between two groups or among multiple groups were compared via student’s t test (two-tailed) or ANOVA with GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA) was used to analyze the experimental data. Differences were considered statistically significant with *p < 0.05, **p < 0.01, ***p < 0.001.