miRNA 3'UTR Clones

Product Details

Creative Biogene offers genome-wide human,mouse,and rat miRNA 3' untranslated region (3'UTR)target clones in mammalian expression vectors.These clones contain the 3'UTR sequence downstream of a reporter gene driven by the SV40 promoter. They are designed to study miRNA-mediated post-transcriptional regulation by quantifying differential luciferase activity. These clones can be used for miRNA target identification, functional verification of predicted targets, and studying the regulatory effects of miRNAs on target genes.

Advantages of Our miRNA 3'UTR Clones

• Genome-wide Coverage:Our miRNA 3'UTR target clones cover the entire human, mouse, and rat genomes, providing comprehensive resources for miRNA target identification and functional studies
• High-Quality Sequences: The 3'UTR sequences used in our clones are obtained from public domain gene sequence databases,ensuring accuracy and reliability in experimental results.
• Mammalian Expression Vector:The clones are constructed in mammalian expression vectors,allowing for efficient expression and functional analysis in mammalian cells.
• Versatile Applications:These clones can be utilized for various applications,including miRNA target identification,functional validation,and investigation of miRNA-mediated gene regulation.
• Quantifiable Results:The differential luciferase activity generated by the interaction between the 3'UTR sequences and miRNAs can be quantified using a colorimetric assay, providing precise and measurable data for analysis.

miRNA 3'UTR Clone List

Application

MicroRNAs(miRNAs)are short regulatory RNAs,approximately 20 to 22 nucleotides in length,found in both plants and animals.In humans,more than 400 miRNAs are encoded by approximately 4%of the genome,and they are estimated to regulate at least 30%of human genes.These miRNAs play crucial roles in various biological processes by binding to complementary sequences in the 3'UTR of target mRNAs. In animals, most miRNAs form imperfect duplexes with sequences located in the 3'UTR region of target mRNAs.In light of these functions,our products offer valuable tools for researchers engaged in understanding miRNA-mediated gene regulation and its implications in various biological contexts.Specifically,our products can facilitate the following research endeavors:

• miRNA Target Validation:These miRNA 3'UTR clones validate gene targets for miRNAs. Researchers transfect or transduce these clones into cells, observing miRNA's regulatory effect by examining its interaction with the target gene's 3'UTR.
• Gene Expression Regulation Studies:These clones investigate the 3'UTR's role in gene expression regulation.Analyzing miRNA-target gene interactions deepens understanding of post-transcriptional regulation mechanisms.
• Signal Transduction Pathway Research:These clones study gene regulation in signal transduction pathways,revealing miRNA's role in modulating cellular signaling.
• Drug Target Screening: Studying these miRNA 3'UTR clones in cells identifies potential drug targets,aiding in developing novel disease therapies.
• Exploration of Disease Mechanisms:Applying these clones in models explores disease mechanisms,and understanding disease progression by investigating miRNA's regulatory effect on target genes.

Case Study

miR-152 inhibits NSCLC cell migration and invasion.

MiR-152 regulates metastases of non-small cell lung cancer cells by targeting neuropilin-1

Paper 2015

International Journal of Clinical and Experimental Pathology | DOI

Background
MicroRNAs (miRNAs) regulate tumorigenesis, but miR-152's role in non-small cell lung cancer (NSCLC) was unclear. This study examined paired NSCLC and adjacent non-tumor tissues from 36 patients and A549 cells. miR-152 was significantly downregulated, while neuropilin-1 was upregulated, with an inverse correlation. Bioinformatics and luciferase reporter assays showed miR-152 targets the 3′-UTR of neuropilin-1 mRNA, inhibiting translation. Overexpressing miR-152 decreased neuropilin-1 protein, migration, and invasion; inhibiting miR-152 increased these. The findings suggest miR-152 suppresses NSCLC metastasis by targeting neuropilin-1 and may be a therapeutic target.
Key Findings
The study found that miR-152 expression was significantly downregulated in NSCLC tissues compared with paired adjacent non-tumor tissues, while neuropilin-1 protein levels were upregulated. Across 36 NSCLC specimens, miR-152 and neuropilin-1 levels showed a strong inverse correlation. Bioinformatics identified a miR-152 binding site in the 3′-UTR of neuropilin-1 mRNA (bases 2135–2142), and luciferase reporter assays confirmed direct targeting. In A549 NSCLC cells, miR-152 overexpression did not alter neuropilin-1 mRNA but decreased neuropilin-1 protein, whereas miR-152 inhibition increased neuropilin-1 protein, indicating translational repression. Functionally, miR-152 overexpression reduced NSCLC cell migration and invasion, while miR-152 depletion enhanced migration and invasion. These results indicate that loss of miR-152 contributes to neuropilin-1-mediated NSCLC metastasis, suggesting miR-152 as a potential therapeutic target.
Contribution
Creative Biogene provided the target sequence clone for the neuropilin-1 miRNA 3’-UTR used to construct luciferase reporters (Methods: Luciferase-reporter activity assay). This reagent enabled the luciferase reporter assay confirming direct miR-152 targeting of neuropilin-1 3’-UTR, contributing to validation of the miR-152–neuropilin-1 regulatory axis.

Isolation of mouse MSCs

Mesenchymal Stem Cells Alleviate LPS-Induced Acute Lung Injury in Mice by MiR-142a-5p-Controlled Pulmonary Endothelial Cell Autophagy

Paper 2016

Cellular Physiology and Biochemistry | DOI

Background
Acute lung injury (ALI) damages pulmonary endothelial cells (PECs). This study investigated how mesenchymal stem cells (MSCs) protect against LPS-induced ALI in mice. MSC transplantation reduced lung permeability and histological injury. MSCs enhanced PEC autophagy, increasing LC3-II/LC3-I and Beclin-1 protein without changing Beclin-1 mRNA. Mechanistically, miR-142a-5p directly targeted the Beclin-1 3′-UTR to repress translation. MSCs lowered miR-142a-5p in PECs from LPS-treated mice. Thus, MSCs alleviate LPS-ALI by downregulating miR-142a-5p, relieving Beclin-1 repression and promoting PEC autophagy.
Key Findings
MSC transplantation significantly reduced LPS-induced acute lung injury severity, lowering pulmonary permeability and attenuating histological damage (neutrophil/fibrin deposits, alveolar macrophage prominence, hemorrhage). MSCs promoted pulmonary endothelial cell (PEC) survival by increasing autophagy, shown by elevated LC3-II/LC3-I ratio and Beclin-1 protein; Beclin-1 mRNA was unchanged, suggesting translational regulation. Bioinformatics and luciferase reporter assays identified miR-142a-5p as a direct regulator of Beclin-1 3′-UTR. Modulating miR-142a-5p in human lung endothelial cells altered Beclin-1 3′-UTR luciferase activity, confirming functional binding. In LPS-treated mice, MSC transplantation decreased miR-142a-5p levels in PECs. The findings support a mechanism where MSCs downregulate miR-142a-5p, thereby de-repressing Beclin-1 translation, enhancing PEC autophagy, and mitigating LPS-induced ALI. This highlights a miRNA-based mechanism for MSC therapy in acute lung injury.
Contribution
Creative Biogene (Shirley, NY, USA) supplied the 3′-UTR luciferase reporter clone used to test miR-142a-5p binding to Beclin-1 in vitro. This reagent enabled the functional luciferase assay confirming miRNA-mediated translational repression in pulmonary endothelial cells, directly supporting the study’s mechanistic conclusion.

HFD impairs endothelial cell autophagy in ApoE (-/-) mice.

Endothelial Cell Autophagy in Atherosclerosis is Regulated by miR-30-Mediated Translational Control of ATG6

Paper 2015

Cellular Physiology and Biochemistry | DOI

Background
This study investigates how high-fat diet (HFD) impairs endothelial cell autophagy in atherosclerosis. Using ApoE−/− mice and ox-LDL-treated human aortic endothelial cells, the authors show HFD reduces ATG6 protein without affecting mRNA, and increases miR-30. Bioinformatics and dual-luciferase assays confirm miR-30 binds the ATG6 3′-UTR and suppresses its translation. The findings reveal a miR-30/ATG6 axis that compromises protective endothelial autophagy, suggesting miR-30 as a potential therapeutic target for atherosclerosis.
Key Findings
High-fat diet induced atherosclerosis in ApoE−/− mice within 12 weeks and significantly reduced endothelial cell autophagy, evidenced by decreased LC3-II/I ratio. Among autophagy proteins, ATG6/Beclin-1 protein was specifically downregulated in CD31+ aortic endothelial cells, while ATG6 mRNA remained unchanged, indicating post-transcriptional regulation. miR-30 was elevated in HFD endothelial cells and predicted to target the ATG6 3′-UTR. In miR-30-modified HAECs, miR-30 mimics decreased ATG6 3′-UTR luciferase activity, whereas antisense miR-30 increased it, confirming direct targeting. Ox-LDL treatment of HAECs reproduced these effects, reducing cell viability, LC3-II/I and ATG6 protein, and increasing miR-30 without altering ATG6 mRNA. Together, the data show that HFD/ox-LDL upregulates miR-30, which binds the ATG6 3′-UTR and suppresses ATG6 translation, thereby impairing protective endothelial autophagy and promoting atherosclerosis.
Contribution
Creative Biogene provided the ATG6 3′-UTR reporter plasmid (pRL-ATG6) used in dual-luciferase reporter assays. This reagent enabled the authors to confirm direct binding of miR-30 to the ATG6 3′-UTR, a key mechanistic finding.

Case Study 1

Acute myeloid leukemia (AML) is a highly aggressive cancer that affects individuals of all ages worldwide. The researchers investigated the potential therapeutic effects of Ginsenoside Rh2 (GRh2) on pediatric leukemia. They conducted experiments using mouse models and leukemia cell lines to evaluate the impact of GRh2 on leukemia cell survival and apoptosis. Their findings demonstrated that GRh2 significantly prolonged the survival of mice with pediatric leukemia and decreased the viability of leukemia cells in vitro by inducing apoptosis. Further analysis revealed that GRh2 reduced the levels of the anti-apoptotic protein Bcl-2 in leukemia cells. Interestingly, they discovered that GRh2 may exert its effects by inducing the expression of microRNA-21 (miR-21), which in turn suppressed the translation of Bcl-2 mRNA by binding to its 3'-UTR.

Figure 1.miR-21 significantly reduced the luciferase activity of 3'-UTR reporter for Bcl-2, indicating its role in suppressing Bcl-2 translation. Thus, GRh2-induced apoptosis in leukemia cells may occur through miR-21-mediated suppression of Bcl-2. The Bcl-2 3'-UTR reporter plasmid(pRL-Bcl-2)was purchased from Creative Biogene.(Wang X,et al.,2015)

Case Study 2

Endothelial cell injury and death are crucial in atherosclerosis,with autophagy's protective role unclear; miR-30, an autophagy suppressor, may play a role in atherosclerosis development. Researchers aimed to elucidate the molecular regulation of endothelial cell autophagy in atherosclerosis by investigating the role of miR-30 and its interaction with ATG6 mRNA. Using a mouse model for atherosclerosis, they found that high-fat diet (HFD) led to decreased levels of endothelial cell autophagy and ATG6 protein, but not mRNA. Bioinformatics analyses and luciferase reporter assays confirmed the binding of miR-30 to the 3'-UTR of ATG6 mRNA,suggesting a regulatory mechanism at the post-transcriptional level.In vitroexperiments on oxidized low-density lipoprotein(ox-LDL)-treated human aortic endothelial cells(HAECs)further supported these findings.The upregulation of miR-30 by HFD was associated with impaired endothelial cell autophagy,potentially contributing to the development of atherosclerosis.

Figure 2.miR-30 was identified as targeting the 3'-UTR of ATG6 mRNA, with its levels significantly increased after HFD treatment. By modifying miR-30 levels in HAECs and using an ATG6 3'-UTR reporter plasmid,it was demonstrated that ATG6 translation is suppressed by miR-30,potentially contributing to impaired endothelial cell autophagy in atherosclerosis.The ATG6 3'-UTR reporter plasmid (pRL-ATG6) was purchased from Creative Biogene. (Zhang T, et al., 2015)

Case Study 3

The identification of microRNAs involved in regulating bladder cancer metastases holds promise for uncovering novel therapeutic targets in BC treatment. Researchers aimed to investigate the role of miR-138 in regulating bladder cancer (BC) cell invasion and metastases. They analyzed miR-138 and ZEB2 levels in BC specimens, finding miR-138 significantly decreased while ZEB2 increased compared to normal bladder tissue, with metastatic BC showing lower miR-138 levels. Through bioinformatics analyses and luciferase reporter assays with the ATG6 3'-UTR reporter plasmid,they confirmed miR-138 targeting of ZEB2 mRNA's 3'-UTR to inhibit translation.Additionally,miR-138 overexpression suppressed ZEB2-mediated invasion and metastases,while depletion increased them in BC cells.These findings suggest that miR-138 suppression in BC cells may promote ZEB2-mediated cancer invasion and metastases,highlighting miR-138 as a potential therapeutic target for preventing BC metastases.

Figure 3.Through bioinformatics analyses,researchers identified miR-138 binding sites on the 3'-UTR of ZEB2 mRNA, spanning from the 766th to the 772nd base site. Target plasmids for 3'-UTR of ZEB2 mRNA clone or 3'-UTR of ZEB2 mRNA with a mutant at miR-138 binding sites wre purchased from Creative Biogene. (Sun DK, et al., 2015)

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