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MicroRNAs (miRNAs) have been widely studied in order to elucidate their biological functions. MicroRNA microarrays or miRNA overexpression libraries generated by synthesis and cloning of individual miRNAs have been used to study their different roles. In this work, we have developed a novel methodology to express mature miRNAs and other small RNAs from a double convergent RNA polymerase III promoter. We show that the generated miRNAs function similarly to those processed from primary transcripts or pri-miRNAs. This system allowed us to produce a lentiviral library expressing the whole population of small RNAs present in a metastatic cell line. A functional screening using this library led to the identification of hsa-miR-30b and hsa-miR-30c as negative regulators of cell death induced by loss of attachment (anoikis). Importantly, we demonstrated that the acquisition of anoikis resistance via these miRNAs is achieved through down-regulation of caspase 3 expression. Moreover, overexpression of these miRNAs resulted in a decrease of other types of caspase 3-dependent cell death and enhanced the survival of MCF10A acinar cells in morphogenesis assays, suggesting a putative role as oncomirs. In summary, this novel methodology provides a powerful and effective way for identifying novel small RNAs involved in a particular biological process.

Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance