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  • Tunicamycin br Table br Association of circVAPA level


    Table 1
    Association of circVAPA level with clinicopathologic features in CRC patients.
    Clinicopathologic features Total (n = 60) CircVAPA expressiona p value
    Age (years)
    Tumor site
    Tumor size (cm)
    Lymphovascular invasion
    Depth of invasion
    Lymph node metastasis
    Distant metastasis
    TNM stage
    2.5. Actinomycin D and RNase R treatment
    To block transcription, 2 mg/ml Actinomycin D or dimethylsulph-oxide (Sigma-Aldrich, St. Louis, MO, USA) as a negative control was added into the cell culture medium. For RNase R treatment, total RNA (2 μg) was incubated for 30 min at 37 °C with or without 3 U/μg of RNase R (Epicentre Technologies, Madison, WI, USA). After treatment with Actinomycin D and RNase R, qRT-PCR was performed to de-termine the expression levels of circVAPA and VAPA mRNA.
    2.6. Isolating RNAs from nucleus and cytoplasmic fractions
    The nuclear and cytoplasmic fractions were isolated using PARIS™ Kit (Invitrogen, Carlsbad, CA, USA) following the manufacture’s pro-tocol. Briefly, Tunicamycin were collected and lysed with cell fractionation buffer, followed by centrifugation to separate the nuclear and cyto-plasmic fractions. The supernatant containing the cytoplasmic fraction was collected, and transferred to a fresh RNase-free tube. The nuclear pellet was lysed with Cell Disruption Buffer. The cytoplasmic fraction and nuclear lysate were mixed with 2X Lysis/Binding Solution and then added with 100% ethanol. The sample mixture was drawn through a Filter Cartridge, followed by washing with Wash Solution. The RNAs of nuclear and cytoplasmic fractions were eluted with Elution Solution. U6 snRNA and 18S rRNA were employed as positive control for nuclear and cytoplasmic fractions, respectively.
    2.7. Transfection
    Small interfering RNAs (siRNAs) targeting the back-splice junction 
    sequences of circVAPA (Fig. 1), miR-101 mimics, and their respective negative control oligonucleotides were synthesized by GenePharma (Shanghai, China). The oligonucleotides were transfected into CRC cells at a final concentration of 50 nM using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instruc-tions. The sequences of siRNAs and negative control oligonucleotide were as follows: si-circVAPA#1: 5’-ATGATAAATTGGCCCCTTC-3’; si-circVAPA#2: 5’-TAAATTGGCCCCTTCACAG-3’; negative control: 5’-TTCTCCGAACGTGTCACGT-3’.
    2.8. Construction of stable circVAPA-overexpressing cells
    CircVAPA sequences were synthesized and inserted into pHBLV-cir vector (Hanbio, Shanghai, China). Then the constructed vectors were packaged into lentivirus. CRC cells were transfected with lentivirus following the manufacturer’s instructions. Then the cells were selected by puromycin for 1–2 weeks. The surviving cells were regarded as stable circVAPA-overexpressing cells and the circVAPA level was measured using qRT-PCR.
    2.9. Luciferase reporter assay
    CircVAPA sequences containing wild-type or mutant miR-101 binding site (predicted by miRanda v3.3a software) were respectively synthesized and inserted into psiCHECK-2 vector (Promega, Madison, WI, USA). Then the wild-type or mutated vector was co-transfected with miR-101 mimics or mimics control into HEK-293T cell using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). 48 h after transfection, cells were harvested and luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA), with Renilla luciferase activity as internal control.
    2.10. Colony formation assay
    Twenty-four hours after transfection, cells were seeded into 6-well plates and then cultured for about two weeks. Then the colonies were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 15 min at room temperature. The colonies were pho-tographed and analyzed.
    Cell proliferation assay was performed using CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) according Tunicamycin to the manufacturer’s in-structions. Approximately 1 × 103 cells were seeded into 96-well plates and treated with the CCK-8 reagent (10 μl) at 0, 24, 48, 72 and 96 h. After incubation for 2 h, the optical density (OD) at 450 nm was read using a Varioskan Flash Spectral Scanning Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA). Cells in each group were tested for 5 replicates.
    2.12. Flow cytometry for cell apoptosis analysis
    Cell apoptosis assay was performed using the Annexin V-FITC Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China) following the manufacturer’s instructions. 48 h after transfection, cells were collected and stained with Annexin V-FITC and propidium iodide, followed by flow cytometry in a FACS Canto II (BD Biosciences). The data were analyzed using BD FCSDiva Software and FCS Express 5 software (De Novo Software, Los Angeles, CA).