Construction of human Bcl-6 3'UTR reporter vector and expression vector and their functional assessment

Objective 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. Methods The 3'UTR and coding region of human bcl-6 gene were amplified by PCR and cloned into pc DNA3.0-Luc and pc DNA3.0-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 Hep G2 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. Results The recombinant plasmids were successfully obtained as confirmed by double digestion and sequence identification. Luciferase assay showed that in293 T and Hep G2 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 G2/M phase and suppressed the growth of Hep G2 cells, and these effects were reversed by Bcl-6 overexpression. Conclusion We successfully cloned wild-type and mutated 3'UTR reporter vectors and expression vector of bcl-6 gene and confirmed their biological functions.