Generation of vectors that coexpress intronic microRNAs and exonic reporters

We have previously identified using a custom microarray a number of microRNAs that differentiate pancreatic endocrine tumors based on clinical characteristics. Using an alternative normalization technique that works well for technical replicates, we have verified that high levels of miR-21 are associated with an enhanced Ki67 proliferation index and metastasis. Intriguingly, miR-21 is overexpressed in all solid tumors that we have investigated including lung, breast, stomach, prostate, colon, and glioblastoma. To ascertain the functional role of microRNAs in cancer, we have generated a novel microRNA expression vector, containing two exons and the intervening intron. The second exon encodes a reporter (hrGFP or LacZ), while the intron encodes either an individual microRNA or microRNA cluster. The utilization of the reporter allows for easy visualization of transfected cells and will prove useful in establishing expression patterns in transgenic mice that overexpress a given microRNA. To date, twelve different microRNAs have been tested that express well. Importantly, the design of the vector allows for efficient replacement of the constitutive promoter (Elongation factor 1 alpha or Rosa 26) for one that is tissue-specific. Utilizing these vectors, we have also explored the possibility that miR-21 regulates the expression of its predicted target and tumor suppressor PDCD4.