Human samples
LCa tumor tissues and adjacent normal tissues were taken from forty-six LCa patients who underwent surgery. The inclusion and exclusion criteria of LCa patients were: (1) confirmed pathologic diagnosis of LCa, (2) not receiving preoperative chemotherapy or radiotherapy, and extrahepatic metastases, (3) no previous treatment for LCa, (4) with complete clinical information, (5) randomly selected tissues samples. The excised tissue was immediately frozen in liquid nitrogen and stored at – 80 °C. All experimental procedures were approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University. Each patient received written informed consent.
Cell culture and transfection
The human LCa cells Huh-7 (GDC0134; China Center for Type Culture Collection; CCTCC, Wuhan, China) and HCCLM3 (GDC0289; CCTCC) were cultured in DMEM medium (01-056-1A; Bioind, Israel) and RPMI-1640 medium (01-100-1A; Bioind) at 37 °C with 5% CO2. Human Liver Epithelial-2 cells THLE-2 (T0015001; AddexBio, San Diego, CA, USA) and Human Embryonic Kidney Cells 293 T (T0011002; AddexBio) were maintained in DMEM medium (01-056-1A; Bioind) at 37 °C with 5% CO2. All medium was added with 1 × penicillin/streptomycin (C0222; Beyotime, Shanghai, China) and 10% FBS (04-001-1A; Bioind).
The pLKO.1-puro-sh-circTUBGCP5 vector (sh-circTUBGCP5) containing shRNA targeting circTUBGCP5 junction sites was constructed by Biofeng (Shanghai, China), followed by packaging into the lentivirus by co-transfecting with psPAX2 and pMD2G into 293 T cells, and infection into Huh-7 and HCCLM3 cells. The miR-144-3p mimic (miR-144-3p), miR-144-3p inhibitor (anti-miR-144-3p), the pcDNA-ACSL4 vector containing full-length ACSL4, pmiR-RB-Report™ luciferase vector (circTUBGCP5-wt, circTUBGCP5-mut, ACSL4-3′ UTR-wt, ACSL4-3′ UTR-mut) containing wild type (wt) and mutated (mut) circTUBGCP5 or ACSL4-3′ UTR sequence were obtained from RiboBio (Guangzhou, China), followed by transfecting into Huh-7 and HCCLM3 cells with Lipofectamine 3000 (L3000008; Invitrogen, Carlsbad, CA, USA).
Quantitative real-time polymerase chain reaction (qRT–PCR) analysis
Trizol (R0016; Beyotime) was employed to extract RNA that then was transcribed into cDNA using miRNA First Strand cDNA Synthesis Kit (B532453; Songon, Shanghai, China) and Universal RT-PCR Kit (RP1100; Solarbio, Beijing, China). ChamQ SYBR qPCR Master Mix (Q311-02; Vazyme, Nanjing, China) was implemented to measure the level of circTUBGCP5, miR-1278, miR-1245-5p, miR-144-3p, and ACSL4 mRNA. U6 was used as the internal reference of miR-1278, miR-1245-5p, and miR-144-3p, while β-actin was employed as the internal reference of circTUBGCP5, and ACSL4 mRNA. The relative expression level was calculated via the 2−ΔΔCT method. The primer sequences were synthesized from Songon:
CircTUBGCP5 (Forward 5′-TAACTGTCTCGTCCGTCCTG-3’, Reverse 5′-CAGCCTTGCCATGTCCATTA-3′); ACSL4 (Forward 5′-CATCCCTGGAGCAGATACTCT-3′, Reverse 5′-TCACTTAGGATTTCCCTGGTCC-3′); miR-1278 (Forward 5′-CTCCGAGTAGTACTGTGCATA-3′, Reverse 5′-CTCAACTGGTGTCGTGGAG-3′); miR-144-3p (Forward 5′-GGATTCCGAGTACAGTATAGATGA-3′, Reverse 5′-TCAACTGGTGTCGTGGAGT-3′); miR-145-5p (Forward 5′-ATTCCGAGGTCCAGTTTTCCCA-3′, Reverse 5′-CTCAACTGGTGTCGTGGAGTC-3′); U6 (Forward 5′-CTTCGGCAGCACATATACT-3′,Reverse 5′-AAAATATGGAACGCTTCACG-3′); β-actin (Forward 5′-CTTCGCGGGCGACGAT-3′ Reverse 5′-CCACATAGGAATCCTTCTGACC-3′).
Western blot assay
Mammalian protein extraction kit (C600589; Songon) and BCA Protein Quantification Kit (E112-01; Vazyme) were performed to extract total protein and quantitate protein. SDS-PAGE (P0012A; Beyotime) was used to separate proteins of different molecular weight sizes that then were electrotransferred onto the PVDF membranes (F019531; Songon). Subsequently, the membrane was blocked with 5% Nonfat-Dried Milk (P0216; Beyotime) and incubated with primary antibodies c-myc (Anti-c-Myc; ab32072; 1:1000; Abcam), GLUT1 (Anti-Glucose Transporter GLUT1; ab14683; 1:2000; Abcam), ACSL4 (Anti-FACL4; ab155282; 1:20,000; Abcam), and β-actin (beta Actin Antibody; MA5-15,739; 1:5000; Invitrogen) overnight at 4 °C, followed by incubated with Goat Anti-rabbit IgG/HRP antibody (SE134 1:2000; Solarbio) at room temperature for 2 h. ECL Western Blotting Substrate (PE0010; Solarbio) and Image-Pro Plus software were used for the protein of visualization and quantification.
Immunohistochemistry (IHC) assay
Like the previous study [20], tumor samples were fixed, embedded in paraffin, cut into 5 μm thick sections, and incubated with antibodies c-myc (ab32072; 1:100; Abcam), GLUT1 (ab14683; 1:200; Abcam), ACSL4 (Anti-FACL4; ab155282; 1:200; Abcam), followed by IHC Detection Kit (E-IR-R213; Elabscience, Wuhan, China).
Nuclear/cytoplasmic fractionation
Nuclear and cytoplasmic RNAs in Huh-7 and HCCLM3 cells were isolated using PARIS™ Kit (AM1921; Invitrogen). DNA-free™ DNase (AM1906; Invitrogen) was allowed to remove trace amounts of DNA in the nuclear RNA sample. The level of circTUBGCP5, β-actin, and U6 was estimated by qRT–PCR.
RNase R treatment
Huh-7 and HCCLM3 cells RNA (20 μg) was processed with 4U RNase R (M1228-500; Biovision, Milpitas, CA, USA) at 37 °C for 2 h. The level of circTUBGCP5 and β-actin were explored using qRT–PCR.
Cell proliferation assay
Ethynyl-2′-deoxyuridine (EdU) assay: Cells (2 × 105/well) were inoculated in 24-well plates until the normal growth stage. After being treated with EdU Cell Proliferation Kit (E607204; Songon), cells were fixed with 4% paraformaldehyde and incubated with Hoechst 33,342 to stain cell nuclei. Colony formation assay: Cells (500/well) were cultured in 24-well plates for 14 days. 4% Paraformaldehyde Fix Solution (E672002; Songon), 0.2% crystal violet (A600331; Songon), and Image-Pro Plus software were implemented to fix, stain, and quantify the colony.
Cell apoptosis assay
Cells (2 × 105) were collected and stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) via Annexin V-FITC/PI Detection Kit (E606336; Songon), followed by analysis with flow cytometry.
Caspase-3 and Caspase-9 activity assay
Cells (1 × 106) were collected and processed with Caspase-3 Activity Detection Kit (BC3830; Solarbio) and Caspase-9 Activity Detection Kit (BC3890; Solarbio) to test the hydrolytic activity of Caspase-3 and Caspase-9, which was monitored by microplate reader at wavelength OD405. All values were normalized to cell numbers.
Glucose consumption and lactate production assay
Cells (1 × 106) were collected and dealt with with Glucose Uptake Assay Kit (ab136955; Abcam) and L-Lactate Assay Kit (ab65331; Abcam), followed by measuring output at wavelength OD412 and OD450 with a microplate reader (Invitrogen). All values were normalized to cell numbers.
RNA pull-down assay
The biotin-labeled probe against the circTUBGCP5 junction site (Songon) was incubated with cell lysates, and then Streptavidin immunomagnetic beads (D110557; Songon) were used to pull down biotin-coupled complexes. The circTUBGCP5 bound miR-1278, miR-1245-5p, and miR-144-3p were quantified by qRT–PCR.
Dual-luciferase reporter assay
Luciferase vectors and miR-144-3p were co-transfected into Huh-7 and HCCLM3 cells with Lipofectamine 3000 (L3000008; Invitrogen) for 48 h, followed luciferase activity was assessed via Dual-Lucy Assay Kit (D0010; Solarbio).
Animal experiments
Huh-7 cells (2 × 106) of circTUBGCP5 knockdown were resuspended in 100 μL PBS and then subcutaneously injected into the back of male BALB/cA-nu Mice (HFK, Beijing, China). The length and width of the tumor were assessed with a caliper every 4 days starting on day 3 and the tumor volume was calculated as (length × width2 × 0.5). After 27 days, the mice were sacrificed, followed by qRT–PCR, western blot, and IHC to assess the level of circTUBGCP5, ACSL4, c-myc, and GLUT1 in tumor samples. The animal experiments were permitted by the Animal Care and Use Committee of the First Affiliated Hospital of Anhui Medical University.
Statistical analysis
GraphPad Prism 9 was used to analyze the values that were shown as means ± standard deviations (SD). Student’s t-test or one-way ANOVA with Tukey’s post hoc test respectively were employed for comparisons between two or more groups. P < 0.05 was considered statistically significant.
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