The Effect of an miRNA Sponge on miR-21 Target Tumor Suppressors (2016)
Undergraduates: Karthika Kandala, Sean Delaney Casey Schmidt and John Noto
Faculty Advisor: Greg Matera
Department: Biology
MicroRNAs (miRNAs), a class of noncoding RNAs, have been examined as potential regulators of cancer cell progression and metastasis pathways. miRNAs
bind to mRNA transcripts and target them for degradation, thereby, serving to control gene expression. miRNA 21 (miR-21), has been identified as a regulator of various tumor suppressor genes in humans. miR-21 is one of the most frequently upregulated miRNAs
in cancer cells. Functional targets of miR-21 include the PTEN, PDCD4, and SMARCA4 mRNAs.
The approach used here is to introduce an exogenous miRNA 'sponge' into HeLa cells that can sequester miR-21. This sponge construct is designed to bind miR-21 and prevent it from negatively regulating
target tumor suppressor mRNAs. In our project, we used tRNAs to generate tRNA intronic circular (tric)RNAs to make an exogenously expressed miRNA sponge. The effectiveness of the sponge has been tested using luciferase assays and RT-PCR. Firefly luciferase
was tagged with a PDCD4 3' UTR, effectively making it a miR-21 target; therefore, firefly luminescence becomes a reporter for miR-21 activity. Preliminary results showed that increased concentrations of the sponge increased expression of luciferase, because
more miRNAs were absorbed by the sponge resulting in increased expression of the miRNA target. RT-PCR was used to show levels of mRNA from the target genes at varying sponge concentrations. Results so far are encouraging, as they show that an increase in sponge
levels results in an increase in PDCD4 expression.