Construction of human Bcl-6 3'UTR reporter vector and expression vector and their functional assessment / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To observe the direct regulation of miR-127 on Bcl-6 and the effect of Bcl-6 in rescuing miR-127-induced cell cycle and cell growth inhibition.</p><p><b>METHODS</b>The 3'UTR and coding region of human bcl-6 gene were amplified by PCR and cloned into pcDNA3.0-Luc and pcDNA3.0-Flag vectors, respectively. Mutations were introduced into the seed sequences of the predicted miR-127 target sites within the Bcl-6 3'UTR using recombinant PCR. Luciferase assay was used to verify the direct targeted regulation of miR-127 on Bcl-6. In HepG2 cell models with overexpression or knockdown of miR-12, the changes of cell cycle and cell growth were investigated after transfection with the constructed vectors.</p><p><b>RESULTS</b>The recombinant plasmids were successfully obtained as confirmed by double digestion and sequence identification. Luciferase assay showed that in 293T and HepG2 cells, miR-127 inhibited the activation of wild-type Bcl-6 3'UTR reporter vector but not mutated Bcl-6 3'UTR vector. Overexpression of miR-127 induced cell cycle arrest at G(2)/M phase and suppressed the growth of HepG2 cells, and these effects were reversed by Bcl-6 overexpression.</p><p><b>CONCLUSION</b>We successfully cloned wild-type and mutated 3'UTR reporter vectors and expression vector of bcl-6 gene and confirmed their biological functions.</p>