MicroRNA let-7a negatively regulates the expression of NIRF in A549 lung cancer cells
AIM: To explore the regulatory effect of let-7a on expression of NIRF, one of the predicted let-7a target, in A549 lung cancer cells. METHODS: Computational approaches were usedfor let-7 a target prediction. Among all the predicted candidate targets, NIRF was chosen for further investigation. A lucifer-ase gene expression vector containing full-length 3' untranslated region (3' UTR) of NIRF gene was constructed. The resulting luciferase expression plasmids were named pRL-TK-NIRF 3' UTR. The Firefly luciferase expression plasmid pGI3-control and the Renilla luciferase construct pRL-TK or pRL-TK-NIRF 3' UTR with either let-7a mimics or control mimics were cotransfected into A549 cells. The Firefly and Renilla luciferase activity were detected by using the Dual Glo Luciferase Assay System. After transfection of let-7a mimics or control mimics in AS49 cells, expression of NIRF protein was determined by Western Blot. RESULTS: By bioinformatics analysis, we found NIRF 3' UTR contains a phylogenetically conserved binding site for let-7a. The recombinant vector pRL-TK-NIRF 3' UTR was verified by sequencing. Luciferase activity was significantly reduced by approximately 50% in A549 cells transfected with pRL-TK-NIRF 3' UTR and pGL3-control in the presence of let-7a mimics, compared with cells transfected with pRL-TK and pGL3-control (P<0. 01). Western Blot detection demonstrated a significant decrease of NIRF protein level in cells transfected with let-7a mimics (P < 0.01) , but not in cells transfected with mimics control (P>0.05) , when compared to A549 cells. CONCLUSION: These data indicated that let-7a can negatively regulate the expression of NIRF by binding to NIRF 3' UTR in A549 lung cancer cells.
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