A Functional Assay for MicroRNA Target Identification and Validation

Abstract 3874

MicroRNAs are a class of small RNA molecules that regulate numerous critical cellular processes including proliferation, differentiation and apoptosis. Several microRNAs play important roles in normal haematopoiesis such as mir-181 which inhibits differentiation, mir-223 induces myeloid differentiation and mir-150 and mir-155 that are involved in T and B cell differentiation.

Increased levels of mir-155 and mir-181 have been documented in diffuse large B-cell lymphomas and acute myeloid leukaemia respectively and mir-15a and mir-16 are frequently deleted in chronic lymphocytic leukaemia. More recently, down-regulation of mir-145 and mir-146a, located to the critical deleted region of 5q in MDS 5q- syndrome has been implicated in this disease.

To obtain insight into the function of miRNAs, much effort has gone into different computational algorithms that identify miRNA targets. However, a major drawback of these prediction models is the substantial false positive rate and an inevitable bias due to reliance on the few known miRNA:target gene interactions. The lack of sensitivity and specificity of the developed computational algorithms is clearly shown by the fact that for the 940 human miRNAs identified (miRecords release 10 April 2010), only 152 miRNAs have experimentally validated targets. There is thus, a clear need to develop methodologies for the identification and validation of the functional targets of specific miRNAs.

To enable the identification of biologically relevant microRNA targets we have developed a novel functional assay for the isolation of microRNA target sequences by selection, relying directly on downregulation, by a miRNA, of a selectable marker expressed in frame with a library of 3′ RNA sequences. The library was derived from human brain tissue cDNA, brain being the tissue expressing the largest number of individual genes (∼11,000).

Cells with low or absent levels of the miRNA of interest are transfected with this 3′ UTR library inserted downstream of a TKzeo fusion gene in plasmid p3′TKzeo. Zeocin selection results in a population of cells that are expressing the TKzeo fusion protein and are resistant to zeocin and sensitive to Ganciclovir (GCV). The zeocin resistant cells are next transfected with the miRNA of interest cloned into the pbabepuro expression vector and selected in puromycin to isolate microRNA transduced cells. GCV treatment then selects for cells that have downregulated the TKzeo fusion protein expression either by inhibition of translation or mRNA cleavage. The 3′UTR sequences present downstream of the TKzeo fusion from GCV resistant cells are PCR amplified and sequenced.

As proof of concept we identified targets for mir-130a which is involved in megakaryopoeisis and for which validated targets have been identified. This microRNA is not expressed in MCF7 cells, therefore the library was introduced into MCF7 cells and selected in 500μg/ml zeocin. Introduction of mir-130a, GCV selection, PCR amplification, cloning and sequencing of the introduced 3′ UTR sequences resulted in the identification of musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB), a known validated target for mir-130a. In addition, we identified tumour protein translationally-controlled 1 (TPT1), proline rich 14 (PRR14), kinesin-associated protein 3 (KIFAP3), microtubule interacting and transport, domain containing 1 (MITD1) and cytochrome P450, family 27, subfamily A, polypeptide 1 (CYP27A1). All targets were validated by western blot analysis of cell extract from cells over-expressing mir-130a or cells treated with hairpin inhibitors directed against mir-130a. Deep sequencing of the total PCR product identified 107 putative targets for mir-130a which are being verified by western blot analysis of the genes for which antibodies are available.

This strategy makes no assumptions based on previously identified sequences, relying directly on downregulation, by a miRNA, of a selectable marker expressed in frame with a library of 3′ RNA sequences.

This strategy will lead to identification of functional targets for all the majority of microRNAs. For example the microRNAs present on the critical deleted chromosomal region of a number of haematological malignancies, including those on chromosomes 5 and 7 such as mir-143, mir-145, mir-146a, mir-378 etc.