mir-34a targets cell cycle genes CCND1 (cyclin D1) and MYCN, while mir-21 does not target PTEN and PDCD4 in neuroblastoma
ForfatterUtnes, Peter Andree
Neuroblastoma is an embryonal cancer of the post-sympathetic nervous system and is the most frequent extra-cranial solid tumor in childhood. Two important microRNAs, the tumor suppressor mir-34a and the oncogenic mir-21, have been found to increase during MYCN-knockdown induced differentiation. However their distinct roles are not clear. This thesis employs a method to identify the mRNA targets of mir-21 and mir-34a by the capture of biotin-labeled microRNA-mRNA complexes. The function of the biotin-labeled microRNA duplexes mir-21 and mir-34a were proven to be biologically active by targeting of the antisense mir-21 and MYCN 3’ UTR cloned into the firefly luciferase, respectively. Further, these luciferase vectors were used to prove the applicability of the pull-down approach where mir-21 and mir-34a enriched for their respective targets. A biotinylated, negative control did not enrich for the luciferase transcript nor did the biotinylated mimics enrich for HPRT1 (a housekeeping gene). Moreover, the method was used to search for endogenous targets in the MYCN-amplified neuroblastoma cell line SK-N-BE(2)c. The pull-down results of mir-34a yielded high enrichment of MYCN and CCND1 (cyclin D1). Additionally, SIRT1 was a probable target of mir-34a. Two mRNAs that previously were shown to be targeted by mir-21 in human cancers, PTEN and PDCD4, were not enriched from neuroblastoma cell extracts. Previous results from our lab have also shown that pre-mir-21 over-expression do not affect PTEN and PDCD4 protein levels. Over-expression of the biotinylated mir-21 duplex did neither affect mRNA levels of PTEN nor PDCD4. mir-21 was as such concluded to not target PTEN and PDCD4 in the human neuroblastoma cell line SK-N-BE(2)c. Moreover, mir-34a over-expression in BE(2)c showed a 4-fold reduction of MYCN and a 20 % increase in CCND1. Mechanisms of mir-34a were therefore believed to repress and perhaps direct CCND1 to P-bodies. mir-34a over-expression resulted in the respective 1.75 and 7.8 fold increase of the two neuronal differentiation markers NPY and GAP43. This was conclusive with the tumor suppressing role of mir-34a. Additionally, the role of mir-34a in neuroblastoma is discussed in detail. Concluding, this thesis demonstrates how biotinylated microRNA duplexes can be used to identify miRNA-mRNA targets.
ForlagUniversitetet i Tromsø
University of Tromsø
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