Experimental Verification of Predicted microRNA Within a Frequently Amplified Genomic Region in Lung Carcinoma

MicroRNAs (miRNA) are endogenously processed, single-stranded, small (20-24 nt) non-coding RNAs which post-transcriptionally regulate the expression of most protein-coding genes in human. MicroRNAs control important cellular processes such as proliferation, differentiation, apoptosis, and tumorigenesis. Till now, morethan 2500 miRNAs have been discovered in human, however, it is estimated that the number could reach 50,000.A recent study showed that most of the miRNA genes are located at fragile and cancer related chromosomal regions. We found frequently amplified genome regions in cancers, which are very potential to hold functional miRNAs (oncomirs). Based on the available cytogenetic data, 3q frequently amplified region in lung cancers wassearched for precursor stem-loops potentially containing novel miRNAs. After selecting certain stem-loopstructures for further experimental verification, specific primers were designed in order to amplify and clone the candidate sequences. The clones were then over-expressed in cell-line to verify the generation of mature miRNA from its pre-mir cloned precursors. Following transfection of cell-line, total RNA was extracted from cells andthen polyadenylated in order to synthesize cDNA. The cDNAs were cloned in TA-vector and the sequencing ofthe multiple recombinant vectors confirmed the identity of predicted miRNA. The sequence of the maturemiRNA was submitted to the EBI website and functional assay is underway to find its effect on the cell cycle status and the selected predicted target genes. This novel miRNA, which seems to be functional in lung cancer, has a potential to be used as diagnostic biomarker for early detection of lung cancer.

کلیدواژه ها:

Lung Cancer ، miRNA ، 3q ، genomic amplified regions ، oncomir