Poster abstracts

Poster number 55 submitted by Mohammed Enamul Hoque

piRNA mediated dual gene knockdown via the ping-pong mechanism

Mohammed Enamul Hoque (Chemistry and Biochemistry, Kent State University), Nicole West (Chemistry and Biochemistry, Kent State University), Aiswarya Mukundan Nair (Biological Sciences, Kent State University), Sumirtha Balaratnam (Chemistry and Biochemistry, Kent State University), Helen Piontkivska (Biological Sciences, Kent State University), Soumitra Basu (Chemistry and Biochemistry, Kent State University)

Abstract:
PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that play an essential role in the defense system against the deleterious action of mobile genetic elements (transposons) by cooperating with PIWI proteins. A subset of piRNAs are generated and amplified through the secondary biogenesis of piRNA, or the ping-pong cycle, where one piRNA is utilized for the successive knockdown of at least two target genes. To identify the target genes, we used bioinformatics analysis to search sequences of human protein-coding genes for potential hits that contain 10-nucleotide-long matches at the 5' end. The vast majority of piRNAs contained multiple
hits to the first and second genes in the sequence, up to 142 matches to the first target and up to 283 matches to the second target. Thus, we designed an endogenous-reporter gene system (piR-A → MDM2 → piR-B → luciferase). In this system, piR-A binds at 3’-UTR of MDM2 mRNA and cleaves it by the slicer activity of the PIWI protein which produces the second piRNA (piR-B). piR-B then binds to the complementary target region inserted in the 3’ UTR of the Renilla luciferase gene and represses the expression of the Renilla gene. Quantitative PCR and western blot data indicate the knock-down of both MDM2 and Renilla genes in triple-negative breast
cancer cells with the treatment of piR-A, indirectly suggesting the generation of piR-B. Currently, we are examining longer matches to identify more attainable oncogene targetsto investigate the ping-pong cycle using endogenous genes.

Keywords: piRNA, Ping-Pong Cycle, Bioinformatics