Four CRC cell lines (including LOVO, SW480, HT-29, and HCT-116) and one normal colon epithelial cell line (FHC) were obtained from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cell lines were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Tianhang, Hangzhou, China) and 1% penicillin-streptomycin in a humidified incubator (37 °C, 5% CO2). Small interfering RNA targeting XIST (si-XIST: 5′-GCCCUUCUCUUCGAACUGUTT-3′) and its matching control (si-NC: 5′-CGTTAATCGCGTATAATACGCGTAT-3′), were obtained from Genepharma (Shanghai, China). A miR-497-5p mimic (miR-497-5p), its inhibitor (anti-miR-497-5p), and their corresponding controls (miR-NC and anti-miR-NC) were obtained from Ribobio (Guangzhou, China). For overexpression vectors, the sequence of FOXK1 was cloned into the pcDNA3.1 vector (vector) according to the manufacturer’s instructions (Life Technologies). Briefly, 100 nM of miR-497-5p mimics or miR-497-5p inhibitor and 1000 ng plasmid were transfected into each 6-well plate for 48 h using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). For animal experiments, cells were stably transfected with an XIST lentiviral vector, constructed by Hanyin Biotechnology Co., Ltd., Shanghai, China. After transfection, DMEM medium was replaced with DMEM supplemented with puromycin (3 µg/mL) as a positive selection for infected cells.
Pcdna3.1 vector vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA3.1 vector is a plasmid used for the expression of recombinant proteins in mammalian cells. It contains a strong CMV promoter and a polyadenylation signal to facilitate high-level protein expression. The vector also includes an ampicillin resistance gene for selection in bacteria and a neomycin resistance gene for selection in mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Si nc, by GenePharma (4 mentions)
Mir nc, by RiboBio (4 mentions)
Anti mir nc, by RiboBio (3 mentions)
Si xist, by GenePharma (3 mentions)
Anti mir 497 5p, by RiboBio (3 mentions)
Small interfering rna targeting, by GenePharma (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Anti nc, by RiboBio (1 mentions)
Sh circ 1, by RiboBio (1 mentions)
Saos 2, by American Type Culture Collection (1 mentions)
Hfob1, by American Type Culture Collection (1 mentions)
Mirna nc, by GenePharma (1 mentions)
Si nc, by RiboBio (1 mentions)
Inhibitor nc, by GenePharma (1 mentions)
Penicillin streptomycin, by Solarbio (1 mentions)
9 protocols using pcdna3.1 vector vector
1
Regulation of XIST and miR-497-5p in CRC
2
XIST and miR-497-5p Regulation in Colorectal Cancer
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Four CRC cell lines (including LOVO, SW480, HT-29, and HCT-116) and one normal colon epithelial cell line (FHC) were obtained from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cell lines were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Tianhang, Hangzhou, China) and 1% penicillin-streptomycin in a humidi ed incubator (37°C, 5% CO 2 ). Small interfering RNA targeting XIST (si-XIST: 5'-GCCCUUCUCUUCGAACUGUTT-3') and its matching control (si-NC: 5'-CGTTAATCGCGTATAATACGCGTAT-3'), were obtained from Genepharma (Shanghai, China). A miR-497-5p mimic (miR-497-5p), its inhibitor (anti-miR-497-5p), and their corresponding controls (miR-NC and anti-miR-NC) were obtained from Ribobio (Guangzhou, China). For overexpression vectors, the sequence of FOXK1 was cloned into the pcDNA3.1 vector (vector) according to the manufacturer's instructions (Life Technologies). Brie y, 100 nM of miR-497-5p mimics or miR-497-5p inhibitor and 1000 ng plasmid were transfected into each 6-well plate for 48 h using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). For animal experiments, cells were stably transfected with an XIST lentiviral vector, constructed by Hanyin Biotechnology Co., Ltd., Shanghai, China. After transfection, DMEM medium was replaced with DMEM supplemented with puromycin (3 µg/mL) as a positive selection for infected cells.
3
XIST and miR-497-5p Regulation in Colorectal Cancer
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Four CRC cell lines (including LOVO, SW480, HT-29, and HCT-116) and one normal colon epithelial cell line (FHC) were obtained from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cell lines were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Tianhang, Hangzhou, China) and 1% penicillin-streptomycin in a humidi ed incubator (37°C, 5% CO 2 ). Small interfering RNA targeting XIST (si-XIST: 5'-GCCCUUCUCUUCGAACUGUTT-3') and its matching control (si-NC: 5'-CGTTAATCGCGTATAATACGCGTAT-3'), were obtained from Genepharma (Shanghai, China). A miR-497-5p mimic (miR-497-5p), its inhibitor (anti-miR-497-5p), and their corresponding controls (miR-NC and anti-miR-NC) were obtained from Ribobio (Guangzhou, China). For overexpression vectors, the sequence of FOXK1 was cloned into the pcDNA3.1 vector (vector) according to the manufacturer's instructions (Life Technologies). Brie y, 100 nM of miR-497-5p mimics or miR-497-5p inhibitor and 1000 ng plasmid were transfected into each 6-well plate for 48 h using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). For animal experiments, cells were stably transfected with an XIST lentiviral vector, constructed by Hanyin Biotechnology Co., Ltd., Shanghai, China. After transfection, DMEM medium was replaced with DMEM supplemented with puromycin (3 µg/mL) as a positive selection for infected cells.
4
Regulation of OS Cell Proliferation
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Normal osteoblast hFOB1.19 and OS cell lines (HOS, U2OS, and SAOS-2) were purchased from the American Type Culture Collection (Manassas, VA, USA). All cells were kept in an incubator with 5% CO2 at 37 °C and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Grand Island, NY, USA) complemented with fetal bovine serum (FBS, 10%, Sigma, Louis, Missouri, MO, USA), streptomycin (100 μg/mL, Sigma), and penicillin (100 U/mL, Sigma).
Short hairpin RNA targeting circ_0005909 (sh-circ#1 and sh-circ#2) and negative control (sh-NC) were bought from RiboBio (Guangzhou, China). MiRNA mimics and inhibitors targeting miR-936 (miR-936 and anti-miR-936) and their negative controls (miR-NC and anti-NC) were procured from RiboBio. The sequence of HMGB1 was cloned into the pcDNA3.1 vector (vector) (Invitrogen, Carlsbad, CA, USA) to construct the overexpression vectors for HMGB1. When the cells were cultured to approximately 80% confluence, OS cells (HOS and U2OS) were transiently transfected with the assigned oligonucleotides or vectors using Lipofectamine 3000 reagent (Invitrogen) and cultured for 48 h.
Short hairpin RNA targeting circ_0005909 (sh-circ#1 and sh-circ#2) and negative control (sh-NC) were bought from RiboBio (Guangzhou, China). MiRNA mimics and inhibitors targeting miR-936 (miR-936 and anti-miR-936) and their negative controls (miR-NC and anti-NC) were procured from RiboBio. The sequence of HMGB1 was cloned into the pcDNA3.1 vector (vector) (Invitrogen, Carlsbad, CA, USA) to construct the overexpression vectors for HMGB1. When the cells were cultured to approximately 80% confluence, OS cells (HOS and U2OS) were transiently transfected with the assigned oligonucleotides or vectors using Lipofectamine 3000 reagent (Invitrogen) and cultured for 48 h.
5
Silencing circ_0008450 Regulates Hypoxia Response
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Small interference RNA targeting circ_0008450 (si-circ_0008450, 5′-ACAAGACAGAAGGGTCTCTCA-3′) and negative controls (si-NC, 5′-GCGCGATAGCGCGAATATA-3′) were purchased from Guangzhou RiboBio Co., Ltd. miR-431 mimics (3′-ACGUACUGCCGGACGUUCUGU-5′), inhibitors (3′-ACAGAACGTCCGGCAGTACGT-5′) and their negative controls (miRNA NC and inhibitor NC) were obtained from Shanghai GenePharma, Co., Ltd. The full-length sequences of circ_0008450 and AKAP1 were cloned into the pcDNA3.1 vector (vector; Invitrogen; Thermo Fisher Scientific, Inc.) or pCD5-ciR vector (pc-NC; Greenseed Biotech, Co.) to obtain the overexpression vectors for circ_0008450 and AKAP1, respectively. Oligonucleotides or vectors were transiently transfected into SNU-387 and Huh7 cells using the Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The transfection concentration of oligonucleotides was as follows: si-NC (40 nM), si-circ_0008450 (40 nM), miR-431 Mimics (50 nM), miRNA NC (50 nM), miR-431 inhibitors (50 nM), inhibitor NC (50 nM). After transfection, cells were treated under hypoxia conditions for 48 h.
6
Modulating NSCLC Cell Proliferation
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A549 cells (CL‐0016, Procell) and H1975 cells (CL‐0298, Procell), as well as human bronchial epithelial cells BEAS‐2B (CL‐0496, Procell), were cultured in Roswell Park Memorial Institute‐1640 medium (Sigma) supplemented with fetal bovine serum (10%, Solarbio) and streptomycin/penicillin (1%, Solarbio).
Transfection of NSCLC cells was executed by using Lipofectamine 3000 reagent (Invitrogen). siRNA targeting circ_0008037 (si‐circ_0008037) and matched negative control (si‐NC), miR‐433‐3p inhibitor and mimic (anti‐miR‐433‐3p and miR‐433‐3p), as well as corresponding controls (anti‐miR‐NC and miR‐NC), were be synthesized by Genechem. The pcDNA3.1‐NUCKS1 (NUCKS1) vectors were constructed using the empty pcDNA3.1 vector (vector) (Invitrogen). Sequences for siRNAs are shown in TableS1 .
Transfection of NSCLC cells was executed by using Lipofectamine 3000 reagent (Invitrogen). siRNA targeting circ_0008037 (si‐circ_0008037) and matched negative control (si‐NC), miR‐433‐3p inhibitor and mimic (anti‐miR‐433‐3p and miR‐433‐3p), as well as corresponding controls (anti‐miR‐NC and miR‐NC), were be synthesized by Genechem. The pcDNA3.1‐NUCKS1 (NUCKS1) vectors were constructed using the empty pcDNA3.1 vector (vector) (Invitrogen). Sequences for siRNAs are shown in Table
7
Circular RNA Regulates NSCLC Cell Lines
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NSCLC cell lines (A549, H1299, and H1975) were procured from American Tissue Culture Collection (Manassas, VA, USA). Human bronchial epithelial cells 16HBE were obtained from Bena Culture Collection (Suzhou, Jiangsu). The Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) was employed to maintain the above cell lines. All cells were kept in an incubator with 5% CO2 at 37°C. Additionally, fetal bovine serum (10%, FBS, Gibco) and streptomycin/penicillin (1%, HyClone, Logan, UT, USA) were added to DMEM in order to maintain cell growth.
Small interference RNA targeting circ_0016760 (si-circ_0016760) and negative control (si-NC) were procured from GenePharma (Shanghai, China). MiR-577 mimics and inhibitors (miR-577 and anti-miR-577) and their negative controls (miR-NC and anti-NC) were obtained from Ambion Inc (Austin, TX, USA). The ZBTB7A overexpression vectors (ZBTB7A) were constructed using the pcDNA3.1 vector (vector; Invitrogen, Carlsbad, CA, USA). Lipofectamine 3000 reagent (Invitrogen) was utilized to transfect oligonucleotides or vectors into A549 and H1299 cells.
Small interference RNA targeting circ_0016760 (si-circ_0016760) and negative control (si-NC) were procured from GenePharma (Shanghai, China). MiR-577 mimics and inhibitors (miR-577 and anti-miR-577) and their negative controls (miR-NC and anti-NC) were obtained from Ambion Inc (Austin, TX, USA). The ZBTB7A overexpression vectors (ZBTB7A) were constructed using the pcDNA3.1 vector (vector; Invitrogen, Carlsbad, CA, USA). Lipofectamine 3000 reagent (Invitrogen) was utilized to transfect oligonucleotides or vectors into A549 and H1299 cells.
8
Culturing Hepatic Cell Lines and Transfection
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Hepatic epithelial cells THLE-2 and HCC cell lines (HCCLM3, Huh7, and MHCC97-H) were purchased from BeNa Culture Collection (Suzhou, China). All cells were kept in an incubator with 5% CO2 at 37 °C and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma, Louis, Missouri, MO, USA) complemented with fetal bovine serum (FBS, 10%, HyClone, Logan, UT, USA), streptomycin (100 μg/mL, Sigma), and penicillin (100 U/mL, Sigma).
Small interference RNA targeting hsa_circ_0000517 (si-hsa_circ_0000517#1 and si-hsa_circ_0000517#2) and negative control (si-NC) were obtained from GenePharma (Shanghai, China). MiR-326 mimics and inhibitors (miR-326 and anti-miR-326) and their negative controls (NC and anti-NC) were procured from GenePharma. The sequence of hsa_circ_0000517 or SMAD6 was cloned into the pCD5-ciR vector (circ-NC) (Greenseed Biotech, Guangzhou, China) or pcDNA3.1 vector (vector) (Invitrogen, Carlsbad, CA, USA) to construct the overexpression vectors for hsa_circ_0000517 and SMAD6, respectively. When the confluence reached 80%, HCC cells were transiently transfected with the designated plasmids or oligonucleotides using Lipofectamine 3000 reagent (Life Technologies, Grand Island, NY, USA).
Small interference RNA targeting hsa_circ_0000517 (si-hsa_circ_0000517#1 and si-hsa_circ_0000517#2) and negative control (si-NC) were obtained from GenePharma (Shanghai, China). MiR-326 mimics and inhibitors (miR-326 and anti-miR-326) and their negative controls (NC and anti-NC) were procured from GenePharma. The sequence of hsa_circ_0000517 or SMAD6 was cloned into the pCD5-ciR vector (circ-NC) (Greenseed Biotech, Guangzhou, China) or pcDNA3.1 vector (vector) (Invitrogen, Carlsbad, CA, USA) to construct the overexpression vectors for hsa_circ_0000517 and SMAD6, respectively. When the confluence reached 80%, HCC cells were transiently transfected with the designated plasmids or oligonucleotides using Lipofectamine 3000 reagent (Life Technologies, Grand Island, NY, USA).
9
Characterization of GBC Cell Lines
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Human GBC cell lines (SGC-996, GBC-SD, and NOZ) and human intrahepatic bile duct epithelial cells (HIBEpiC) were used in the study. SGC-996, GBC-SD, and HIBEpiC cells were obtained from Jingkang Biological Engineering Co., Ltd. (Shanghai, China). NOZ cells were obtained from Bena culture collection (Suzhou, China). Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scienti c, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scienti c) and 100 U/mL penicillin/streptomycin (Corning Inc., Corning, NY, USA) was used to culture all cell lines. All cell lines were incubated in an atmosphere with 5% CO 2 at 37˚C. Short hairpin RNA (sh-RNA) targeting UCA1 (sh-UCA1) and negative control (sh-NC) were synthesized by Genepharma (Shanghai, China). The sequences of UCA1 and SPOCK1 were cloned into the pcDNA3.1 vector (vector) (Invitrogen, Carlsbad, CA, USA) to construct the overexpression vectors for UCA1 and SPOCK1, respectively. MiR-613 mimic and inhibitor (miR-613 and anti-miR-613) and their corresponding negative controls (miR-NC and anti-miR-NC) were purchased from Genepharma. Oligonucleotides or plasmids were transfected into NOZ and GBC-SD cells using the lipofectamine 2000 reagent (Thermo Fisher Scienti c).
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