The 3′UTR segments containing the target sites for miR-15a-5p (TGCTGCT) were amplified by PCR from cDNA and inserted into a pMirTarget vector (#PS100062, OriGene) between the EcoRI and NotI restriction sites for ATG9A (ENST00000361242.9, +3387 to +3648 pb), SMPD1 (ENST00000342245.9, +2108 to +2410 pb) and GABARAPL1 (ENST00000266458.10, +731 to +1611 pb), and between the MluI and NotI restriction sites for ATG14 (ENST00000247178.6, +1842 pb to +4666 pb). All the mutants were generated by the deletion of 5 bp within the site of perfect complementarity using the QuickChange™ XL-II kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s protocol. Mutagenesis primers were synthesized by Eurogentec (Ougrée, Belgium) and are listed in Table 2 . All the constructs were verified by sequencing.
Pmirtarget vector
Manufactured by OriGene
Sourced in United States
The PMirTarget vector is a plasmid-based expression system designed for the study of microRNA (miRNA) function. The vector facilitates the cloning and expression of target miRNA sequences, allowing for the investigation of their regulatory roles in biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Pmirtarget vector, by Promega (2 mentions)
Dual luciferase reporter assay, by Promega (2 mentions)
Dual luciferase assay system, by Promega (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Rapamycin, by Merck Group (1 mentions)
Nvp aew541, by Cayman Chemical (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Akt1 2 inhibitor, by Merck Group (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
U0126, by Merck Group (1 mentions)
Prl renilla luciferase control reporter vector, by Promega (1 mentions)
Opti mem media, by Thermo Fisher Scientific (1 mentions)
Complete rpmi media, by Thermo Fisher Scientific (1 mentions)
Rpmi media, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dharmafect duo, by Horizon Discovery (1 mentions)
Pierce renilla firefly luciferase dual assay kit, by Thermo Fisher Scientific (1 mentions)
N luminometer, by Turner Designs (1 mentions)
16 protocols using pmirtarget vector
1
Cloning and Mutagenesis of miR-15a-5p Targets
2
Investigating IGF-I/II Signaling Pathways
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IGF-I and IGF-II were from Preprotech (Rocky Hill, NJ), lipofectamine 2000 and lipofectamine RNAiMax from Life Technologies Inc. Laboratories (Paisley, UK), scramble (miR-NC), synthetic pre-miR-199a-5p (miR-199a-5p), and mirVana miR-199a-5p inhibitor were purchased from Ambion (Applied Biosystems, CA, USA), fibronectin from Sigma-Aldrich (Saint Louis Missouri, USA).
Constructs encoding either pcDNA.3.1-HA-myr-AKT dominant active construct (AKT) or the empty vector pcDNA3.1 (vector) were kindly provided by prof. P. Tassone (University “Magna Graecia” of Catanzaro).
Construct encoding 3′UTR clone of DDR1 in pMirTarget Vector, the related empty vector (pCMV6) and the human IGF-II cDNAs were from OriGene Technologies (Rockville, USA). The following kinase inhibitors were used: the IGF-IR inhibitor NVP-AEW541 (Cayman Chemical, Ann Arbor, USA); the PI3 kinase inhibitor LY 294002 (Calbiochem, Merck Millipore, Nottingham, UK); the MEK1 inhibitor U0126 (Sigma-Aldrich, Saint Louis Missouri, USA); the TORC1 inhibitor rapamycin (Sigma-Aldrich); the AKT 1,2 inhibitor (Sigma-Aldrich, Saint Louis Missouri, USA). Actinomycin D and cycloheximide were purchased from Sigma-Aldrich, MTT from Amersham Bioscences.
Constructs encoding either pcDNA.3.1-HA-myr-AKT dominant active construct (AKT) or the empty vector pcDNA3.1 (vector) were kindly provided by prof. P. Tassone (University “Magna Graecia” of Catanzaro).
Construct encoding 3′UTR clone of DDR1 in pMirTarget Vector, the related empty vector (pCMV6) and the human IGF-II cDNAs were from OriGene Technologies (Rockville, USA). The following kinase inhibitors were used: the IGF-IR inhibitor NVP-AEW541 (Cayman Chemical, Ann Arbor, USA); the PI3 kinase inhibitor LY 294002 (Calbiochem, Merck Millipore, Nottingham, UK); the MEK1 inhibitor U0126 (Sigma-Aldrich, Saint Louis Missouri, USA); the TORC1 inhibitor rapamycin (Sigma-Aldrich); the AKT 1,2 inhibitor (Sigma-Aldrich, Saint Louis Missouri, USA). Actinomycin D and cycloheximide were purchased from Sigma-Aldrich, MTT from Amersham Bioscences.
3
miR-21-5p Regulation of SMAD7 3'UTR
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The complete sequence of SMAD7 3′ untranslated region (UTR) was constructed into the pMirTarget vector (Origene, Rockville, MD, USA). The plasmid containing the putative binding sequence of miR-21-5p “ATAAGCTA” is named as “SMAD7 3′UTR”. For luciferase assay, 50 nm of control miRNA or miR-21-5p mimics, 0.8 µg of pMirTarget vector containing SMAD7 3′ UTR, and 0.08 µg of pRL renilla luciferase control reporter vector (Promega, Madison, WI, USA) were co-transfected into HEK-293 cells in 96-well plates for 48 h. Cell were harvested and analyzed using the dual-luciferase reporter assay (Promega) according to the manufacturer’s instruction.
4
Transfection of LOVO and SW480 cells
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One day before transfection, LOVO (8 × 105) and SW480 (5 × 105) cells were seeded in 6-well plates in RPMI media (Life Technologies) overnight. The following day, media were removed and cells were transfected with 50 ng of the pMirTarget vector (Origene), and 100 nM of the rellevant pre-miRNA or pre-miR-Negative Control#1 (Life Technologies) using Lipofectamine 2000 in Opti-mem media (Life Technologies) according to the manufacturer’s protocol. After 6 h transfection, the transfection reagent was replaced with complete RPMI media (Life Technologies). Cells were harvested for other experiments at 24 h after transfection. Transfection efficiency was monitored by qPCR.
5
miR-30e Regulates Caspase-3 and p21 Expression
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To determine the influence of miR-30e on caspase-3 expression, cells were transfected using DharmaFECT Duo (Dharmacon, Lafayette, CO, USA) with the pMiR-Target vector containing the firefly luciferase cDNA with or without the 3′-UTR of the caspase-3-mRNA (Origene Technologies, Rockville, MD, USA) together with miR-30e or a non-targeting control miR. To determine the influence of miR-30e on p21 expression, cells were transfected with a firefly luciferase reporter plasmid under the control of either the CMV or the p21 promoter together with miR-30e or the control miR. In both setups, a renilla luciferase construct (pRL-TK) was co-transfected for normalization purposes. Cells were either left untreated or irradiated 24 h post transfection, and firefly and renilla luciferase activities were determined another 24 h later with the dual-luciferase reporter assay (Promega, Fitchburg, WI, USA) and a Centro LB960 luminometer (Berthold Technologie, Bad Wildbad, Germany).
6
miR-148a Binding Validation on Target mRNA
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To verify miR-148a-directed binding on targeted mRNA, two 3′-UTR segments including one wild-type and the other mutant type were cloned and integrated into the pMirTarget Vector (OriGene Technologies, Inc., Rockville, MD, USA), respectively. Both the wild-type and mutant 3′-UTR segments of c-Met or ROCK1 were constructed, because c-Met and ROCK1 were predicted to be the potential target genes of miR-148a. The cells (1 × 104) were seeded in a 96-well plate for 24 h and co-transfected with two plasmids—wild-type or mutant 3′-UTR construct and pTK-Green Renilla Luc Vector (Thermo Fisher Scientific)—by using Lipofectamine 3000 (Thermo Fisher Scientific). After transfection for 48 h, a luciferase reporter assay was performed using the Pierce™ Renilla-Firefly Luciferase Dual Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For the normalization of firefly activity, the Renilla luciferase activity was used as the internal control.
7
Luciferase Assay for MET and EGFR 3'UTR
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The 2,300 bp 3’-UTR of the human c-MET or 1,700 bp 3’-UTR of the human EGFR was cloned into the pMirTarget vector (Origene, Rockville, MD) to produce the 3-UTR-luciferase plasmids. The 3’-UTR-c-MET, 3’UTR-EGFR or the empty pMirTarget vector was co-transfected with miR-206 mimic (100 nM) in SKOV3 and A498 cells, and luciferase activities were measured in cell lysates using the Dual Luciferase Assay System (Promega Corporation, Madison, WI) with a 20/20n luminometer (Turner Designs, Sunnyvale, CA) [57 ].
8
TFAM 3′-UTR Luciferase Assay
9
Luciferase Assay for miRNA-mRNA Interaction
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The luciferase assay was performed in HEK293 cells using the dual luciferase reporter assay system (Promega) and analyzed as described
[3 (link)]. cDNA fragments of HPV16 transcript and E6AP 3′UTR were subcloned into a pMiR-Target vector between EcoRI and NotI restriction enzyme sites downstream of Firefly luciferase (FL) coding sequence (OriGene Technologies, Rockville, MD). Four nucleotides in the seed binding sites were mutated to disrupt the miRNA-mRNA interaction as described previously
[3 (link)]. Renilla luciferase reporter was used to normalize the data. All primers used for cloning luciferase construction and mutagenesis are listed in Additional file
1 : Table S1.
[3 (link)]. cDNA fragments of HPV16 transcript and E6AP 3′UTR were subcloned into a pMiR-Target vector between EcoRI and NotI restriction enzyme sites downstream of Firefly luciferase (FL) coding sequence (OriGene Technologies, Rockville, MD). Four nucleotides in the seed binding sites were mutated to disrupt the miRNA-mRNA interaction as described previously
[3 (link)]. Renilla luciferase reporter was used to normalize the data. All primers used for cloning luciferase construction and mutagenesis are listed in Additional file
10
Analyzing miRNA Binding to ATP6 Gene
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