Effects of miR-96 on the expression of α-tocopherol associated protein and its effect on the proliferation of prostate cancer cells

Abstract

Objective To screen α-tocopherol associated protein (TAP) -regulating miRNAs and investigate its effect on the proliferation of prostate cancer cells.Methods Bioinformatics analysis and DualLuciferase Reporter Assay were perform to identify TAP-regulating miRNAs.TAP gene (SEC14L2) and its 3' -untranslated region ( 3' -UTR ) sequence were cloned into p3 × FLAG-CMVTM-7.1 expression vector.MiR-96 and TAP expression vector pCMV7.1-TAP,pCMV7.1-TAP-3'-UTR were transfected into prostate cancer cell line DU-145.The expression level of TAP was detected by western blotting and the cell proliferation was valued by methyl thiazol tetrazolium (MTT) assay.Results MiR-96 targets 3' -UTR of TAP.After double restriction enzyme digestion and agarose gel elecrophoresis of pCMV7.1-TAP-3'-UTR,the 1200 bp and 1400 bp purpose bands were sequenced,results was exactly the same with the sequence GeneBank had reported,indicating that the recombinant plasmids pCMV7.1-TAP-3 ' -UTR were successfully constructed.TAP protein expression level mediated by pCMV7.1-TAP-3' -UTR plasmid was down-regulated by miR-96 by 65.1％ (P ＜0.05).MTT assay showed that the cellular absorbance value (A value) of the control group and the empty vector transfection group was 0.972 ±0.023 and 0.967 ± 0.042 each; A value of pCMV7.1-TAP transfection group and pCMV7.1-TAP-3 ' -UTR transfection group was 0.625 ± 0.037 (P ＜ 0.05 ) and 0.731 ±0.043 (P ＜0.05) respectively；A value of pCMV7.1-TAP-3'-UTR and miR-96 co-transfection group was 0.914±0.034 ( P ＞ 0.05 ).Conclusion miR-96 can regulate the expression of TAP by targeting 3' -UTR and attenuates the effect of TAP on the proliferation of prostate cancer cells. Key words: α-tocopherol associated protein; MiR-96; Prostate carcer; Proliferation