Effect of Pokemon gene on the proliferation of colorectal cancer cells and its mechanism

Objective To construct recombinant plasmids containing microRNA targeting the Pokemon gene and investigate the effects of miRNA-mediated downregulation of the Pokemon gene on the proliferation and cell-cycle progression of colorectal cancer LoVo cells.Methods Four miRNAs were designed according to the coding sequence of the Pokemon gene and used to construct recombinant plasmids.The recombinant plasmids were transfected into LoVo cells using Lipofectamine 2000.The expression of Pokemon mRNA was detected by quantitative polymerase chain reaction(QPCR) assay.Cellular proliferation was measured by methyl thiazolyl tetrazolium(MTT) assay.Cell-cycle progression was analyzed by flow cytometry.Results Four recombinant plasmids named mR22-1,mR22-2,mR22-3 and mR22-4 were successfully constructed.The expression of Pokemon mRNA was obviously downregulated in LoVo cells transfected with the recombinant plasmids.The best silencing effect was 50%,which was achieved in cells transfected with the mR22-1 plasmid.MTT assay indicated that mR22-1 transfection could inhibit cellular proliferation.The inhibition rate for 24,48,and 72 hours was(23±2.1)%,(47±3.0)% and(69±2.5)%,and it rose in a time-dependent manner.Flow cytometry assay indicated that mR22-1 transfection led to LoVo cell cycle arrested in G1-phase.Conclusion The recombinant plasmids containing miRNA targeting the Pokemon gene specifically down-regulate Pokemon expression.Pokemon gene can promote proliferation in LoVo cells.