Abstract: 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 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. Results 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 G2/M phase and suppressed the growth of HepG2 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.