- 3'UTRs and miRNAs
- Empty Vector Suite
- Lightswitch Assay Reagents
- Transfection Reagents
miRNA Target Validation Grant Winners
All of our grantees will be using the LightSwitch Luciferase Assay to validate the targets of their miRNAs of interest:
- 3’UTR GoClone reporters
- Empty control construct
- Synthetic constructs
- Mimics or inhibitors and a non-targeting control
- LightSwitch Luciferase Assay Reagent
Who: Bo Kong
Postion: Senior Scientist
Institution: University of Kansas Medical Center
FGF19 (mouse ortholog FGF15) is a target gene of nuclear receptor FXR. FGF19 has been shown to play a role in liver proliferation and regeneration, and may be involved in liver hepatocellular carcinoma (HCC). miR-21 is overexpressed in most human cancers and overexpession can induce cancer in mice. Dr. Kong’s preliminary data showed that miR-21 in mice liver increased three-fold 1 hour after FGF15 protein injection through the tail vein, clearly indicating that FGF15 increased miR-21 expression. Kong’s hypothesis is that FGF15/19 in intestine can promote liver regeneration through microRNA miR-21. He will use several 3’UTR GoClones and the LightSwitch Luciferase Assay System to validate the gene targets of miR-21 which inhibit liver regeneration in 2/3 partial hepatectomy (2/3 PH) model (ref: Song G et al, Hepatology. 2010, 51(5):1735-43; Rebecca T et al, Am J Physiol Gastrointest Liver Physiol. 2010, 298(4): G535–G541).
Who: George Yousef
Postion: Principal Investigator
Institution: St Michael’s Hospital, Toronto, On , Canada
Dr. Yousef’s lab reported miRNA dysregulation in kidney cancer (RCC) and provided evidence for miRNA involvement in RCC pathogenesis. Dysregulated miRNAs are predicted to effect hypoxia–related pathways and epithelial-mesenchymal transition (EMT). Hypoxia is central to RCC pathogenesis, and EMT drives metastasis. Yousef’s lab will be validating the targets of two miRNAs that are biologically active regulators of RCC pathogenesis, and which simultaneously modulate hypoxia pathway and EMT. They will integrate the miRNAs into the gene regulatory network of RCC pathogenesis.
Who: Kimberly Cordes
Postion: Postdoctoral Fellow
Institution: Gladstone Institute of Cardiovascular Disease
My research focuses on understanding the mechanisms that regulate vascular smooth muscle proliferation, which is a hallmark of atherosclerosis. Recently, I identified miR-29 to be enriched in the adventitial layer of the artery, made up primarily of fibroblasts, which is associated with extra-cellular matrix (ECM) breakdown in the formation of aortic aneurysms. Interestingly, I found it is up-regulated during proliferation in some arterial disease models, such as Elastin haploinsufficiency. I will use the 3′UTR GoClone reporters for the targets of this miRNA and the LightSwitch Luciferase assay reagent to collect the results. If repression of luciferase is seen by miR-29, I will further mutate the miR-29 binding sites in the target 3′UTRs to verify miR-29′s binding in the predicted regions. In addition, I plan to verify target expression in human smooth muscle cell lines from normal and Elastin haploinsufficient patients. I will further test whether inhibition of miR-29 can rescue the disease phenotype of hyper- proliferation seen in Elastin-mutant cells.
Who: David Mu
Institution: Penn State University College of Medicine
I am interested in understanding the roles of the microRNA network in the biology of the lung. We used an essential lung developmental gene called thyroid transcription factor 1 (TTF-1) as a starting point and discovered the first microRNA (miR-365) that directly regulates TTF-1 expression (Qi et al. Cell Cycle, in press 2012). While characterizing miR-365, we uncovered a series of putative targets to the little-known miR-365* species. We wish to further characterize miR-365*.
Who: Seth Masters
Postion: Lab Head
Institution: Trinity College Dublin
Our lab is focused on inflammation, and in particular, innate immune pathways that are triggered during infection or chronic disease, to make inflammatory cytokines. Recently we have identified a miRNA (miR-223), which is highly expressed in monocytes and targets the Nlrp3 inflammasome, and reduces production of the cytokine IL-1b. We now wish to find which inflammatory cytokines are directly targeted by miR-223 and other miRNA expressed from inflammatory cells like monocytes.
We have selected 5 putative 3’UTR targets which are all cytokines produced during infection and chronic inflammatory disease. It is particularly interesting that several gamma herpes viruses encode their own miRNA, for example CMV. The cytokines we selected are also involved in anti-viral defence, so could be targets of CMV miRNA, which we plan to study in the future.
Who: Stephanie Murphy
Postion: Associate Professor, Anesthesiology and Perioperative Medicine
Institution: Oregon Health and Science University
Stroke occurs more frequently in men than women, and ischemic sensitivity of rodent brain is sex-specific, with males being more sensitive than females. We hypothesize that sexually dimorphic ischemic responses are due to sex differences in the brain’s epigenome mediated by DNA methyltransferases (DNMTs) whose expression is regulated by miRNAs. Our quantitative PCR arrays identified several miRNAs which target DNMTs in brain and show sex differences in expression. Thus we have focused validation studies on these miRNAs.