Novel microRNAs located in the human E-cadherin gene. Novel microRNAs finding

This study examined the presence of putative microRNAs located in the human E-cadherin gene. To predict miRNA-like structures in this gene, bioinformatics programs were used. In order to facilitate the experimental identification of these miRNAs, over-expression of them was performed in the HEK293 cell line. At first, genomic regions of the putative miRNA precursors were amplified by PCR and cloned into the pEGFP-C1 expression vector. The recombinant vectors were transfected into the HEK293 cell line. RNA was extracted and polyadenylated from transfected cells and expression of putative miRNAs were investigated by RT-PCR by forward primers designed for probable mature miRNA sequences. Validation of these predicted miRNAs were confirmed by sequencing of RT-PCR products.