The miR-145 mimic and miR-NC were purchased from Invitrogen. The cells were transfected with miR-145 mimic or miR-NC using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions. In brief, mirVana miRNA mimic hsa-miR-145-5p (Thermo Fisher Scientific Company) or mirVana miRNA mimic NC #1 (Thermo Fisher Scientific Company) was diluted to 30 μM and mixed with diluted Lipofectamine RNAiMAX reagent. After being incubated at room temperature for 5 min, the mixture was then added to the cells accordingly, and the plates were incubated at 37 °C for one day.
Mir 145 mimic
Manufactured by Thermo Fisher Scientific
MiR-145 mimic is a synthetic RNA molecule designed to mimic the function of the naturally occurring microRNA (miRNA) molecule, miR-145. miR-145 is involved in the regulation of various cellular processes. The MiR-145 mimic can be used in research applications to study the biological functions and effects of miR-145 in cell and molecular biology experiments.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Mir 145 inhibitor, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Mir nc, by Thermo Fisher Scientific (1 mentions)
Mir 145 inhibitor, by GenePharma (1 mentions)
Mir 145 mimic, by GenePharma (1 mentions)
Pcdna3.1 vector, by Addgene (1 mentions)
Inhibitor nc, by GenePharma (1 mentions)
Mimic nc, by GenePharma (1 mentions)
Mir 145 mimic inhibitor, by RiboBio (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Control mimic, by Thermo Fisher Scientific (1 mentions)
Control inhibitor, by Thermo Fisher Scientific (1 mentions)
Hk2 overexpression vector, by OriGene (1 mentions)
Control shrna, by OriGene (1 mentions)
Mir 148a inhibitor, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Shrna, by Merck Group (1 mentions)
Mir 497 inhibitor, by Thermo Fisher Scientific (1 mentions)
Mir 148a mimic, by Thermo Fisher Scientific (1 mentions)
6 protocols using mir 145 mimic
1
miR-145 Mimic Transfection Protocol
2
Modulation of MEG3 and miR-145 in LX-2 Cells
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Short hairpin RNAs (shRNAs/sh) targeting MEG3 (forward, 5′-CCGGATAGAGGAGGTGATCAGCAAACTCGAGTTTGCTGATCACCTCCTCTATTTTTTG-3′ and reverse, 5′-AATTCAAAAAATAGAGGAGGTGATCAGCAAACTCGAGTTTGCTGATCACCTCCTCTAT-3′), miR-145 mimic (forward, 5′-GUCCAGUUUUCCCAGGAAUCCCU-3′ and reverse, 5′-AGGGAUUCCUGGGAAAACUGGAC-3′) and negative control (NC)-mimic (forward, 5′-UCACAACCUCCUAGAAAGAGUAGA-3′ and reverse, 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′), miR-145 inhibitor (5′-AGGGAUUCCUGGGAAAACUGGAC-3′) and NC-inhibitor (5′-UCUACUCUUUCUAGGAGGUUGUGA-3′) were obtained from Shanghai GenePharma Co. Ltd. An empty plasmid cloning (pc)DNA3.1(+) vector (Addgene, Inc.) was used as a control for MEG3 and PPARγ overexpression pcDNA3.1(+) constructs. LX-2 cells (5×105 cells/well) were seeded into 6-well plates for 24 h, and then transfected with the aforementioned recombinant vectors (2 µg), shRNA (1 µg), miR-145 mimic (20 nM), miR-145 inhibitor (20 nM) or the controls, at 40–60% confluence using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Transfected cells were harvested 48 h after transfection.
3
Modulation of lncRNA-TUG1 and miR-145 in Cells
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TUG1 and control (shNC) shRNA sequences were purchased from GenePharma (Shanghai, China). MiR-145 mimic, inhibitor, and the respective negative control (NC) were purchased from RiboBio (Guangzhou, China). The lncRNA-TUG1 cDNA was amplified and subcloned into a pcDNA3.1 vector (Invitrogen); the resulting plasmid was named pcDNA3.1-TUG1 WT. The Quik Change Site-Directed Mutagenesis kit (Stratagene) was used to produce mutations in miR-145 response elements and synthesize pcDNA3.1- TUG1 MUT. Sequences and primers are shown in Supplementary Table 2 .
For retroviral packaging, 293T cells were co-transfected with transfer plasmids and retroviral packaging vectors. For transduction, cultured cells were incubated with virus-containing supernatant in the presence of 8 mg/ml polybrene. After 48 h, infected cells were selected for 72 h with puromycin (2 mg/ml) or hygromycin (200 mg/ml). MiR-145 mimic, inhibitor, and NC were transfected using Lipofectamine2000 (Invitrogen) or Lipofectamine™ RNAiMAX according to the manufacturer’s instructions.
For retroviral packaging, 293T cells were co-transfected with transfer plasmids and retroviral packaging vectors. For transduction, cultured cells were incubated with virus-containing supernatant in the presence of 8 mg/ml polybrene. After 48 h, infected cells were selected for 72 h with puromycin (2 mg/ml) or hygromycin (200 mg/ml). MiR-145 mimic, inhibitor, and NC were transfected using Lipofectamine2000 (Invitrogen) or Lipofectamine™ RNAiMAX according to the manufacturer’s instructions.
4
Establishing cell lines for HK2 overexpression and knockdown
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Transfection was conducted using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions. The control vector, HK2 overexpression vector, control shRNA, or HK2 shRNA was obtained from OriGene and Santa Cruz Biotechnology, respectively. Control siRNA, HK2 siRNA, control mimic, miR-145 mimic, miR-148a mimic, miR-497 mimic, control inhibitor, miR-145 inhibitor, miR-148a inhibitor, or miR-497 inhibitor, was purchased from Invitrogen. When cells achieved 70% confluence, the above plasmid, shRNA, siRNA, or miRNA mimic (or inhibitor) was transfected into CC cells for 48 h. Finally, stable HK2-overexpressing or HK2 knockdown CC cell lines were established by selecting cells using G418 (Sigma-Aldrich) or Puromycin (Sigma-Aldrich) for 4 weeks.
5
Biotinylated miR-145 mimic pulldown
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MiR-145 mimic (MC11480, Ambion) was biotinylated with the 3′-end Biotinylated Kit (Pierce) and transfected to the PNT1A and PC3 cells with the Lipofectamine RNAiMAX reagent (ThermoFisher). Biotinylated RNA supplied with the kit was used as the control. Biotin pull-down was performed as follows: 11 × 106 cells were centrifuged 48 h after transfection at 1000 rpm for 2 min. Pellets were incubated for 15 min on ice in Pierce lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol) with protease and RNAs inhibitors, then centrifuged (15 min, 13200 rpm). The supernatant was collected and supplemented by an equal volume of 2× TNT buffer (50 mM Tris-Cl, pH 8.0, 2 mM EDTA, and 150 mM NaCl, 1% Triton X-100). Streptavidin agarose beads (30 μl per reaction) were washed 3 times in TENT buffer (2 min, 2000 rpm at 4°C), incubated 30 min at 20 rpm at RT with rotation. Beads were washed 3 times in 500 μl PBS. RNA was purified with TRIAZOL regent and used for qPCR reaction with specific primers:
6
Characterizing miR-145 Regulation of TF 3'UTR
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A partial TF 3′UTR sequence of 434 base pairs containing miR-145 target sites was cloned into psiCHECK-2 Vector (Promega) downstream of Renilla luciferase gene using the XhoI and NotI restriction sites. Mutagenesis of miR-145 target sites was performed by PCR splicing method, and mutated 3′UTR sequences were also cloned into psiCHECK-2 Vector. Primers used for plasmid construction (psiCHECK-2 and TF 3′UTR or mutated TF 3′UTR) are listed in table S4. HEK293 cells were seeded in 24-well plates in DMEM supplemented with 10% FBS and maintained in 5% CO2 incubator at 37 °C. The transfection was done after 24 h using Lipofectamine 3000 (Invitrogen) as per manufacturer's protocol. Cells were transfected with 100 ng of psiCHECK-2 constructs and 40 nM of Pre-miR™ miRNA precursor molecules-negative Control # 1 (non-specific/scr. mimic) and Rno-miR-145 Pre-miR™ miRNA precursor (miR-145 mimic, Ambion). Cells were harvested 24 h post transfection. Luciferase assay was performed by using Dual Luciferase Reporter Assay System (Promega) following the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity and expressed as relative light units (RLU). The relative luciferase activity was reported as the fold change between scr. mimic and miR-145 mimic transfected cells.
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