In this study, we used human HCC cell lines, such as Huh7, Bel-7402, QGY-7703, SMMC7721, PLC8024, H2M, and H2P, and the human immortalized normal hepatocellular cell line MIHA, which were gifted by Professor Zhu Xiaofeng’s laboratory, Sun Yat-sen University Cancer Center. The HCC cells were cultured using Dulbecco’s modified Eagles’ medium (DMEM) (Gibco, Big Cabin, UK) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 IU/ml penicillin at 37°C with 5% CO2. For cell transfection, the miRNA mimic, micrONTM mimic negative control (cy3), and siEZH1 were produced by RiboBio (Guangdong, China). The transfection buffer and reagent were also purchased from RiboBio. The miRNA mimic was transfected for 24 h and siEZH1 was transfected for 48 h in six-well plates with 3 × 105 cells per well.
Mirna mimic
Manufactured by RiboBio
Sourced in China
MiRNA mimics are synthetic, double-stranded RNA molecules designed to mimic the function of naturally occurring microRNAs (miRNAs). MiRNAs are small, non-coding RNA molecules that play a crucial role in regulating gene expression. The MiRNA mimics are used as research tools to study the biological functions of specific miRNAs in various cellular and molecular processes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (29 mentions)
Mirna inhibitor, by RiboBio (26 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (15 mentions)
Sirnas, by RiboBio (7 mentions)
Dual luciferase reporter assay system, by Promega (6 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (5 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Inhibitor nc, by RiboBio (3 mentions)
Ribofect cp transfection kit, by RiboBio (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Mimic control, by RiboBio (3 mentions)
Inhibitor control, by RiboBio (3 mentions)
Mirna mimic, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Dapi solution, by Merck Group (2 mentions)
24 well plate, by BD (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
108 protocols using mirna mimic
1
HCC Cell Line Culture and Transfection
2
Transfection of Colorectal Cell Lines
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Cells were seeded into 6-well plates at a density of 2×105 cells/well for siRNA (Genepharma, China) or miRNA mimic (Ribobio, China) transfection. Transfection was performed using Lipofectamine 3000 (Invitrogen, USA), following the manufacturer's instructions. Final concentration of transfections was 100 nM. Sequences of siRNAs were (sense strands are reported as 5′- 3′): si-circ_0000523-1: GAGCAAGAAGAUCUACGGAdTdT; siControl-1: GAGCAAGAAGUAGAUGCCUdTdT; si-circ_0000523-2: GAAGAUCUACGGAAUCCAGAdTdT; siControl-2: CUUCAUCUACGGAAUCCAGAdTdT; si-circ_0000523-3: CAACAGAGCAAGAAGAUCUAdTdT; siControl-3: CAACAGAGCAAGAAGUAGAUdTdT (Table 1 ).
Growth medium used for each cell line.
| Cell line | Growth medium |
|---|---|
| COLO205, COLO320HSR, DLD-1, HCT-15, HCT-8 | RPMI-1640 |
| SW480, SW620, SW1116 | Leibovitz's L-15 |
| HT-29 | McCoy's 5A (Modified) |
| LoVo | F-12K |
| NCM460 | Dulbecco's Modified Eagle's Medium (DMEM) |
| FHC | DMEM/F12a |
| LS 174T | Eagle's Minimum Essential Medium (EMEM)b |
| Caco-2 | EMEMc |
aSupplemented with HEPES (10 mM), cholera toxin (10 ng/mL), insulin (0.005 mg/mL), transferrin (0.005 mg/mL), and hydrocortisone (100 ng/mL); bsupplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL); csupplemented with 20% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL).
3
Cardioprotective Effects of Danlou Tablet
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AC16 cells were bought from American Type Culture Collection (ATCC). Cells were cultured in DMEM medium (DMEM, Corning, NYC, USA, 10-013-CV) containing 10% FBS (Biological Industries, Beit HaEmek, Israel) and 1% P/S (KeyGEN, Nanjing, China, KGY0023) in a 37 °C incubator containing 5% carbon dioxide and saturated humidity. AC16 cells were cultured in a normal glucose medium (5.5 mmol/L), to which mannitol was added to maintain osmolarity balance, and after acclimation, the cells were switched to a high glucose medium (33 mmol/L) and harvested for further assays after 48 h of treatment. Danlou tablet (Dan) was provided by Jilin Connell Pharmaceutical CO. LTD and dissolved in PBS with the stock concentration at 10 mg/mL. AC16 cells were treated with 50 μg/mL of Dan for 48 h followed by high-glucose modeling. Cardiomyocyte transfection was performed with lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The transfection concentration of siRNA, miRNA mimic and inhibitor were 50 nM. The mimic negative control, miR-34a mimic, inhibitor negative control and miR-34a inhibitor were bought from RiboBio. The sequence of shRNA targeting SIRT1 is (5′–3′)：GGAAAUAUAUCCUGGACAATT.
4
Primer Design for Genetic Experiments
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ALL primers, RNA oligoribonucleotides and miRNA mimic used in this study were designed and purchased from RiboBio Co. Ltd (Guangzhou, China).
5
Ovarian Cancer Cell Line Cultivation
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The human ovarian cancer cell lines C13*, OV2008, A2780, and its cisplatin-resistant cell line A2780/DDP were purchased from the American Type Culture Collection (Manassas, VA, USA). HO8910, SKOV3, CaOV3, Hey, COV362, and the immortalized ovarian epithelial cell line (Moody) were conserved in our laboratory. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Auckland, New Zealand) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. MiRNA mimic, inhibitor, and the negative controls were purchased from RiboBio (Guangzhou, China) and transfected into EOC cells with Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.
6
Transfection of miRNA mimics and inhibitors
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One day before transfection, cells were starved by seeding in medium supplemented with 2% FBS at a density of 5 × 104 cells/ml. On the day of transfection, the medium was changed to 10% FBS medium, and the cells were transfected with miRNA mimic, NC mimic, miRNA inhibitor or NC inhibitor (RiboBio, Guangzhou, China) using a riboFECT™ CP Transfection Kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. The miRNA mimic and inhibitor used are listed in Table 1 .
List of gene
| Gene name | Sequence |
|---|---|
| miR-199a-3p mimics | 5′-AUUGGUUACACGUCUGAUGACA-3′ |
| miR-199a-3 inhibitor | 5′-UAACCAAUGUGCAGACUACUGU-3′ |
| miR-219c-5p mimics | 5′-GGACGUCCAGACGCAACUCUCG-3′ |
| miR-219c-5p inhibitor | 5′-CGAGAGUUGCGUCUGGACGUCC-3′ |
| miR-3572-3p mimics | 5′-UACACUUGUCCUUCUUUCCCCAG-3′ |
| miR-3572-3p inhibitor | 5′-CUGGGGAAAGAAGGACAAGUGUA-3′ |
| mimics NC | 5′-UUUGUACUACACAAAAGUACUG-3′ |
| inhibitor NC | 5′-UUUGUACUACACAAAAGUACUG-3′ |
7
Comprehensive Cell Lysis and RNA Extraction
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RIPA lysis buffer, extraction buffer, and Halt Protease and Phosphatase Inhibitor Cocktail (100×) were purchased from Thermo Scientific and stored at 4°C. BCA protein assay kits used to quantify protein concentration were purchased from Beyotime and stored at room temperature. DMEM (High Glucose), RPMI1640, penicillin-streptomycin, and trypsin-EDTA were purchased from HyClone. Horse serum was purchased from Gibco. Fetal bovine serum (FBS) was derived from Biological Industries. miRNA qPCR primer and miRNA mimic (RiboBio), GW4869 (MCE), PEG8000 (Sigma-Aldrich), PKH67 Green Fluorescent Cell Linker Kit (Sigma), Oligofectamine (Invitrogen), X-tremeGENE 9 (Roche), TRIzol (Vazyme), T4 DNA ligase (Takara), DH5α-competent cells (TIAGEN), SYBR Green (Takara), miRNA mimic (RiboBio), Direct-zol RNA Miniprep (Zymo), and the Dual-Luciferase Reporter Assay System (Promega) were used. Other chemicals, except where specially noted, were purchased from Sigma-Aldrich.
8
Screening of circRNA-miRNA Interactions
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The miRNA binding site of novel_circ_0005319, novel_circ_0005934 and novel_circ_0000134 were screened according to the analysis results obtained with ENCORI software. Cells were seeded in 24-well plates at a density of 1 × 104 cells (HEK-293T) per well 24 h before transfection. Then, the cells were transfected with a mixture of 200 ng of circRNA-WT target or circRNA-MUT target, and 10 μL of miRNA mimic or mimic-NC was utilized with Lipofectamine 2000 (Invitrogen, USA). In this study, the double luciferase vector pmir-GLO was selected (Genewiz, Suzhou, China), and the novel_circ_0005319, novel_circ_0005934 and novel_circ_0000134 sequence were cloned into the reporter gene vector to synthesize the predicted miRNA mimics and control. The miRNA mimic was obtained from RiboBio (Guangzhou, China). After 48 h, luciferase activity was measured with a dual luciferase reporter assay system (Promega, Madison, WI, United States). The expression levels of reporter genes were detected by a multifunctional enzyme labeling instrument, and the assays were performed in triplicate.
9
Immunofluorescence Imaging of Transfected SH-SY5Y Cells
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The SH-SY5Y cells grown on coverslips were transfected with miRNA mimic (Ribo, China) and ATXN3-Q78-GFP.The cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and blocked with 5% fetal bovine serum (FBS) in PBS for 2 h at room temperature. Cells were then incubated with DAPI solution (Sigma, United States) shading at room temperature for 2 min. Finally, cells were mounted and visualized by confocal microscopy.
10
Quantifying Cellular Apoptosis via TUNEL Assay
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The apoptosis rate of cells was measured using the TUNEL Kit (Sigma, United States) according to the manufacturer’s instructions. In this procedure, the SH-SY5Y cells were seeded on the coverslip placed on the 24-well plates (BD Falcon, United States) and then treated with MPP+ after being transfected with miRNA mimic (Ribo, China). The coverslips were washed twice with phosphate buffered saline (PBS), and fixed with 4% paraformaldehyde at 4°C for 30 min. After washed three times with PBS, the slices were covered with 50 μL TUNEL reaction mixture solution with deoxynucleotide fluorescein-12-dUTP and incubated at humidified atmosphere at 37°C for 60 min in the dark. After rinsing the slices with PBS for three times, the cells were treated with DAPI solution (Sigma, United States) shading at room temperature for 2 min. Finally, samples were rinsed twice with PBS, and slides were covered with mounting medium containing glycerin reagent (Sinopharm, China) and cover glasses (Sail Brand, China). Since then, the apoptotic cells were observed and photographed with a fluorescence microscope.
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