Patient tissue samples and cell lines
Human tumor samples were obtained from patients with HCC who had undergone curative resection from 2015 to 2016 at the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. This study was approved by the Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine (No.2021-236) and conformed to the principles of Declaration of Helsinki. Written informed consent was obtained from all patients.
The human HCC cell lines HepG2, Hep3B, HCCLM3, Huh7, and MHCC97H, and the immortalized hepatocyte cell line WRL68 were purchased from Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences. All cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Cat# No. 8,121,393; Gibco) supplemented with 10% fetal bovine serum (FBS; Cat# No. 10,091,148; Gibco) and 1% penicillin/streptomycin (Cat# No.15140-122; Gibco) in an incubator at 37 °C with a humidified atmosphere containing 5% CO2, and passaged following standard cell culture techniques.
Total RNA extraction and quantitative real-time polymerase reaction (qRT-PCR)
Total RNAs of HCC tissues/cells and paired normal tissues/cells were extracted using TRIzol reagent (Cat# No. 15,506,018; Invitrogen, Carlsbad, CA, USA). The concentration of RNA was detected using a Nanodrop 2000 system (Thermo Fisher Scientific). For circRNA and mRNA, reverse transcription was conducted using the HiScript II Q RT SuperMix for qPCR (Cat# No. R223-01; Vazyme Biotech, Nanjing, China) with random primers. For miRNA, reverse transcription was conducted using the HiScript II Q Select RT SuperMix (Cat# No. R233-01; Vazyme Biotech) with specific stem-loop primers. cDNA amplification was performed using ChamQ Universal SYBR qPCR Master Mix (Cat# No. Q711-03; Vazyme Biotech) with an ABI Prism 7500 sequence detection system (Applied Biosystems, CA, USA). Relative RNA expression levels were normalized to those of internal controls (GAPDH and U6) using the 2−ΔΔCT method. All primers were designed by Tsingke Biological Technology (Beijing, China) and are listed in Additional file 1: Table S1.
Nucleic acid electrophoresis
The PCR products were subjected to nucleic acid electrophoresis using 2% agarose gel with 0.5% TAE running buffer at 100 V for 40 min. We used DL500 DNA Marker (50–500 bp) (Cat# No. 3590A; TaKaRa, Osaka, Japan) to observe the bands under ultraviolet irradiation.
HCC cells were seeded and cultured to 50–60% confluence before transfection. Si-circSLC7A11 and the negative control were designed and synthesized by RiboBio (Guangzhou, China). The miR-330-3p mimics, inhibitor, and negative control were synthesized by GenePharma (Cat# No. B03001; Shanghai, China). Lipofectamine 3000 reagent (Cat# No. L3000015; Invitrogen) was used as a transfection medium. All oligonucleotide sequences are listed in Additional file 2: Table S2.
Overexpression vector construction and transfection
The pCDH-ciR vector used in our research was synthesized by RiboBio (Guangzhou, China). To construct the overexpression vector of circSLC7A11, full-length circSLC7A11 cDNA was cloned into the pCDH-ciR vector; empty vector without the circSLC7A11 sequence was used as a negative control.
RNase R treatment
RNAs (10 µg) extracted from HCCLM3 and Hep3B cells were treated with RNase R (3 U/µg, Cat# No. R0301; Geneseed, Guangzhou, China) and incubated for 30 min at 37 °C. The treated RNAs were reverse-transcribed with divergent or convergent primer and detected by qRT-PCR or nucleic acid electrophoresis.
Western blot analysis
Tissues or cells were lysed in radio immunoprecipitation assay (RIPA) lysis buffer (No. P0013B; Beyotime, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF, Cat# No. ST505; Beyotime, Shanghai, China) for 30 min on ice. After centrifugation (15,000 × g, 4 °C, 15 min), the supernatant was collected. A bicinchoninic acid (BCA) protein assay kit (Cat# No. 23,225; Thermo Fisher Scientific) was used to measure protein concentrations and all protein samples were normalized to 2 μg/ul. For electrophoresis, equal amounts of denatured proteins (20ug) were separated on 4–12% NuPAGE Bis-Tris Gel (Cat# No. M00653; GenScript, Piscataway, NJ, USA) and then transferred onto 0.45-µm polyvinylidene fluoride (PVDF) membranes (Cat# No. ipvh00010; Millipore, Burlington, MA, USA). The membranes were blocked in 5% non-fat milk in TBST and incubated with the indicated primary antibodies overnight at 4 °C. Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000) for 1 h at room temperature, followed by detection using an imaging system (Bio-Rad, Hercules, CA, USA). The primary antibodies and dilution of antibodies used in this experiment are listed in Additional file 3: Table S3.
Cell counting Kit-8 proliferation assay
Cell proliferation was evaluated using the Cell Counting Kit-8 (Cat# No. CK04; Dojindo Corp., Japan). A total of 4,000 transfected cells were seeded in each well of a 96-well plate. At the indicated times (0, 24, 48, and 72 h), 10 µL of CCK-8 reagent was added directly to the culture medium. Then, the cells were incubated for 2 h at 37 °C, and the optional density at 450 nm (OD450) was measured using a microplate reader (BioTek Instruments, USA).
Colony formation assay
We seeded 1,000 transfected cells in each well of a 6-well plate for culturing at 37 °C in 5% CO2 for 14 days. After 14 days, cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with 0.1% crystal violet solution for another 20 min before the cell colonies were counted. Colonies containing 30 cells were selected and counted using light microscopy. The average number of colonies was determined from three independent experiments.
5-Ethynyl-20-deoxyuridine (EdU) incorporation assay
We performed an EdU assay to analyze the proliferative capacity of cells using the Cell-Light EdU DNA Cell Proliferation Kit (Cat# No. C10310; RiboBio, Guangzhou, China) following the manufacturer’s protocol. The cell lines were sequentially stained with Apollo Dye Solution and Hoechst 33,342, and EdU-positive cells were counted under a Leica DMi8 microscope (Leica, Germany).
HCC cells were seeded in a 6-well plate in medium with 10% FBS. After adherence, cells were scratched with a 200-µL pipette tip and replaced with serum-free medium. After washed with PBS, representative images were photographed 0 and 24 h after injury. The diminishing distance across the injury area was measured and normalized to the distance at 0 h. The data are expressed as relative migration rates.
Immunohistochemical (IHC) analysis
Tissue samples were fixed in 4% paraformaldehyde embedded in paraffin and sectioned. The tissue sections were incubated with anti-CDK1, anti-E-cadherin, anti-N-cadherin, and anti-Ki-67 primary antibodies at 4 °C overnight and then incubated with HRP-conjugated secondary antibody. H Score (intensity of staining (4, 3, 2, 1) × % of positive cells staining with that intensity/100%) was used to analyze the quantification of IHC. The dilution of antibodies used in this experiment are listed in Additional file 3: Table S3.
Fluorescence in situ hybridization (FISH)
A FISH assay was performed to determine the subcellular locations of circSLC7A11 and miR-330-3p. Briefly, circSLC7A11 probe and miR-330-3p probe were directly generated by Servicebio (A2061841, Wuhan, China) depending on their inverse complementary sequence, respectively. The corresponding probe sequences are listed in Additional file 2: Table S2. HCCLM3 cells were seeded in a cell slide and grown to 50-60% confluent at the time of fixation. After permeabilized with 0.25% Triton X-100 in PBS for 15 min and pre-hybridized in hybridization buffer (50% formamide, 10 mM Tris-HCl, 200 µg/ml yeast transfer RNA, 1⋅ Denhardt’s solution, 600 mM NaCl, 0.25% SDS, 1 mM EDTA, 10% dextran sulfate) for 1 h at 37 °C, the cell slide was heated to 65 °C for 5 min in hybridization buffer containing 3 nM digoxin-labeled miR-330-3p probe and 300 nM cy3-labeled circSLC7A11 probe and hybridization occurred at 42 °C overnight. The next day, after washed with 2 ⋅ Saline Sodium Citrate Buffer (SSC) for 10 min, 1⋅ SSC for 10 min twice and 0.5⋅ SSC for 10 min at 37 °C, nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min [32, 33]. The images were acquired using a laser scanning confocal microscope (LSM900; Zeiss, Germany).
Biotin-coupled probe RNA pull-down assay
A biotinylated circSLC7A11 probe and negative control probe were synthesized by GenePharma (Shanghai, China). The probes were incubated with M280 streptavidin-coupled Dynabeads (Cat# No. 11205D; Invitrogen) at 25 °C for 2 h to generate probe-coated beads, which were then incubated with the cell lysates at 4 °C overnight. The beads were washed with wash buffer, and the RNA complexes were then purified with TRIzol reagent and subjected to PCR analysis. The probe sequences are listed in Additional file 2: Table S2.
Dual luciferase reporter assay
The sequences of CDK1-3’UTR and their corresponding mutation were synthesized, cloned into luciferase reporter vector PGL3 (Genomeditech, Shanghai, China), respectively. All plasmids were co-transfected with miR-330-3p mimics or controls into HEK-293 T cells. Relative luciferase activity was detected using the Dual Luciferase Assay Kit (Cat# NO. E1910; Promega, Madison, USA).
RNA immunoprecipitation (RIP) assay
RIP assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (No.17-700; Millipore), according to the manufacturer’s instructions. The beads were washed and incubated with proteinase K to remove proteins. Finally, the concentration and quality of the purified RNA were measured and subsequently subjected to PCR analysis. The final expression level was normalized to the fold of the concentration between “IgG” and “Input”, and “AGO2” and “Input”, respectively, in a manner of “%Input”.
All mice were housed under pathogen-free conditions in the animal facility of the Second Affiliated Hospital, Zhejiang University School of Medicine. Animal experiments were performed according to a protocol approved by the Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine (No. 2021-44). Briefly, 6-week-old female BALB/c nude mice were purchased from the Shanghai Experimental Animal Center of the Chinese Academic of Sciences (Shanghai, China). HCCLM3 cells (5 × 106) suspended in 150 µL PBS transfected with sh-circSLC7A11 or negative control were subcutaneously injected into the left flank of each mouse. The tumor volume was measured every 7 days using a caliper and calculated as (length × width2)/2. After 4 weeks, mice were sacrificed and subcutaneous tumors were subjected to qPCR, Western blotting, and IHC staining. Similarly, 5 × 106 sh-circSLC7A11 or negative control HCCLM3 cells were injected into the tail vein of each nude mouse to construct tumor metastasis models. After 8 weeks, the lungs were removed and validated using hematoxylin and eosin (H&E) staining.
Statistical analyses were performed using SPSS (ver. 18.0; SPSS Inc., USA) and GraphPad Prism 6 software (GraphPad Software Inc., USA). Student’s t-test and one-way analysis of variance (ANOVA) were performed to evaluate group differences. Low and high circSLC7A11 expression levels or low and high miR-330-3p expression levels were cut off by median expression values, respectively. Association of circSLC7A11 or miR-330-3p expression level with clinicopathological parameters were evaluated by the chi-squared test or Fisher’s exact test. Pearson’s correlation was used to analyze correlations among circSLC7A11, miR-330-3p, and CDK1. Data are expressed as means ± standard deviation (SD). For all analyses, p < 0.05 was taken to indicate statistical significance.