Isolation and clonning of the putative micrornas in the human e-cadherin gene

E-cadherin is a cell adhesion molecule that involved in contact inhibition. Loss of contact inhibition causes cancer cells to metastasis to other organs. Repression of E-cadherin expression in a majority ofepithelial cancers promotes tumor progression and metastasis. icroRNAs (miRNAs) have emerged as post-transcriptional regulators of gene expression and are involved in a wide variety of biological processes such as metastasis. Recent evidences indicated that intragenic miRNAs may regulate the expression of their host genes. In bioinformatic surveys we scanned human E-cadherin gene for findingmiRNA-like structures using different bioinformatics softwares. Out of 27 predicted miRNA-like structure, two which met all criteria of the softwares were chosen for further verification experiments. The present study was conducted to isolation and cloning of these two miRNA precursors. To isolate these precursors, 342 and 386 base pairs fragments were PCR amplified using specific primers andcloned into pTG19-T vector. The presence and orientation of cloned fragment were confirmed by PCR using either primers of insert or vector. EcoRI and HindIII for 342 bp fragment and EcoRI and PstIfor 386 bp fragment were used to clone the mentioned fragments into pEGFP-C1 expression vector. All the recombinant vectors were chosen on the selection media, the recombinant clones were subjected to plasmid purification. Purified plasmid were sequenced to determine the exact sequences of recombinant fragments. In future work, these recombinant vectors will be transfected to the HEK293 to detect the presence of putative mature miRNA. Further studies are needed to investigate the function of obtained microRNAs

Keywords:

Cancer Prevention , Gene and Cancer , Cell and Cancer , Multidisciplinary Cancer Research , Cancer Genetics