MicroRNAs in control of cell proliferation

ED02-03

We are using three approaches to discover microRNAs that regulate cell proliferation. In the first approach, we focused on miRNAs induced during differentiation of C2C12 myoblasts into myotubes in vitro. One of the microRNAs induced during differentiation, miR-206, suppresses cell proliferation. By surveying mRNAs that are downregulated by miR-206 for genes predicted to be targets of the microRNA, we discovered the mRNA of DNA polymerase alpha p180 is a direct target that is destabilized upon miR-206 transfection. We now report that additional microRNAs induced during muscle differentiation target other cell-cycle regulators.
 After noting that targeted mRNAs are often destabilized by microRNAs, in the second approach, we tested whether global downregulation of microRNAs by knockdown of Dicer or Drosha can help identify microRNA-repressed mRNAs. The principle was proved by the discovery that HMGA2 oncogene is repressed by the growth-suppressive let-7 microRNA. Indeed, chromosomal translocations in lipomas and leiomyomas de-repress HMGA2 by deleting the 3' UTR that is normally repressed by let-7. Conversely, downregulation of let-7 has been noted in lung cancers and large leiomyomas and is correlated with over-expression of the HMGA2 oncogene.
 In the third approach we have cloned short RNAs from androgen-dependent prostate cancer cells grown in the presence or absence of androgens and subjected them to ultra-high-throughput sequencing. The frequency of a number of clones present in the libraries is changed by androgens and we have identified several microRNAs that change upon androgen-depletion. Over 30-40% of the short RNAs cloned, however, do not correspond to known microRNAs and are produced by cleavage of known mRNAs and noncoding RNAs. The diversity and abundance of the non-micro-short RNAs (nmsRNAs) contrast with how little is known of their function and suggest that much remains to be done before we understand the biological functions of many short RNAs present in the cell. In an extension of the project we have identified several microRNAs that are reproducibly altered in expression level between the androgen-dependent LnCAP cells and androgen-independent derivative C4-2 cells. Interestingly, the microRNAs decreased in C4-2 cells were also reported by others to be decreased as prostate cancers advance in Gleason score or metastasize. This raises the possibility that a microRNA signature may be established for following the molecular stage of prostate cancer and for predicting its predilection to recur in an androgen-independent form.