Construction of lenfiviral expression vector for miR-34a and its effect on proliferation of human colon cancer SW480 cells

Objective To construct a lentiviral expression vector for miR-34a and investigate its effect on proliferation of human colon cancer SW480 cells.Methods Partially complementary forward and reverse primers were designed based on has-pre-miR-34a sequence,of which dimmers were formed by annealing and amplified by PCR.The obtained double stranded DNA was used as a template for further amplification by PCR.The PCR product was digested with restriction endonuclease and insered into linearized lentiviral vector pGIPZ.The constructed recombinant plasmid pGIPZ-miR-34a was co-transfected to 293T cells with packaging plasmids Helper 1.0 and Helper 2.0 for packaging.The obtained recombinant lentivirus was determined by gradient dilution.SW480 cells were infected with recombinant lentivirus and determined for transcription level of miR-34a mRNA by qPCR,for proliferative activity by MTT and plate cloning method,and for distribution of cell cycle by flow cytometry.Results Restriction analysis and sequencing proved that recombinant lentiviral expression vector was constructed correctly.The titer of obtained recombinant lentivirus was 6.52 × 108 TU/ml.The recombinant lentivirus up-regulated the transcription level of miR-34a mRNA while inhibited the proliferative activity and increased the percentage at G0-G1 phases of infected SW480 cells(P 0.01).Conclusion The lentiviral expression vector for miR-34a was successfully constructed,which increased the endogenous expression of miR-34a and inhibited the proliferation of SW480 cells.It laid a foundation of further study on function and action mechanism of miR-34a.