RRM
2007 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Tuberous sclerosis complex TSC) is a human genetic syndrome forming benign tumors in the brain, kidney, eyes, heart and skin affecting an estimate of 1 in every 6000 people. TSC is caused mainly by the lost of tuberin (TSC2) and/or Harmatin (TSC1) proteins, two tumor suppressor genes. TSC1 binds and stabilizes TSC2 and prevents its potential ubiquitination and degradation. The TSC1/TSC2 heterodimer has been placed under the phosphatidylinositol 3-OH kinase (PI-3K)-mammalian target of rapamycin (mTOR)/S6K1/4E-BP1 signaling pathway. It functions by inhibiting mTOR activity through a small GTPase RHEB. The TSC2 gene has a very short 3’-UTR (150bp), while TSC1 has a very large 3’-UTR (4887bp). The observation led us to hypothesize that one possible cause of TSC is that microRNAs dysregulates TSC1 gene expression. MicroRNAs (miRNA) are 20-22bp short RNAs that regulate gene expression by binding to their target gene’s mRNA 3’-UTR and either lead to their degradation or repression of translation. Numerous databases have been set up that are used to predict miRNA that might target a given gene. From one such database, www.microma.sanger.ac.uk, we found 31 miRNA that were predicted to target TSC1. We cloned the TSC1 3’-UTR into phRL-TK vector and performed a luciferase reporter assay. Our results indicate that a total of 14 miRNAs (miR- 361, 328, 126, 32, 200a, 326, 19b, 19a, 345, 301, 130b, 214, 523 and let-7e) reduced luciferase gene expression by more than 25%. Western blots analysis using 10 of these miRNA, transfected into both SV7tert and 293T cells, indicated that miR-130b, 200a, 32, and 126 effectively down-regulated the TSC1 gene. Although much work and further confirmatory experiments are needed, the present results suggest that TSC1 is targeted by miRNAs.
Keywords: Tuberous sclerosis complex, microRNA, Luciferase assay
