miR Let-7a1/f1下调前列腺癌1LNCaP细胞中AMD1蛋白的表达

目的研究let-7a1/f1在前列腺癌LNCaP细胞中对S-腺苷甲硫氨酸脱羧酶(AMD1)表达的影响及作用机制。方法以LNCaP细胞总RNA为模板,RT-PCR扩增let-7a1/f1基因的前体序列,将其克隆到pSilencerTM4.1-CMV neo载体中,构建成let-7a1/f1真核表达载体pSilencer-let7a1/f1。瞬时转染LNCaP细胞后,通过RT-PCR法,检测let-7a1/f1在转录水平的表达,同时通过RT-PCR及Western blot检测AMD1在转录和翻译水平的表达变化。构建AMD1基因3’非翻译区(3’-UTR)荧光素酶报告基因融合质粒(pMIR-AMD1),并与pSilencer-let7a1/f1共转染LNCaP细胞,通过双荧光素酶活性检测,确定let-7a1/f1对AMD1 3’-UTR的作用。结果 pSilencer-let7a1/f1和pMIR-AMD1经酶切电泳及测序鉴定正确,pSilencer-let7a1/f1转染LNCaP细胞后,let-7a1/f1表达明显上升,AMD1在转录水平无变化,在翻译水平明显下降,let-7a1/f1转染后,pMIR-AMD1的荧光素酶活性明显降低。结论 miR Let-7a1/f1可作用于AMD1 3’-UTR靶点,抑制AMD1蛋白的翻译,从而降低LNCap细胞中AMD1蛋白的水平。

miR Let-7a1/f1 down-regulates AMD1 expression in prostate cancer LNCaP cells

Objective To detect the effect and mechanism of miR Let-7a1/f1 on S-adenosylmethionine decarboxylase(AMD1) expression in prostate cancer LNCaP cells.Methods The pre-miR Let-7a1/f1 sequence was amplified by RT-PCR using RNA from LNCaP cells as the template,and then it was inserted into the pSilencerTM4.1-CMV neo vector to generate pSilencer-let7a1/f1.After pSilencer-let7a1/f1 was transiently transfected into LNCaP cells,expression of miR Let-7a1/f1 was verified by RT-PCR.The effect of miR Let-7a1/f1 on expression of the AMD1 gene was detected by RT-PCR and Western blot.The luciferase reporter vector of AMD1 3′UTR(pMIR-AMD1) was constructed and co-transfected into LNCaP cells with pSilencer-let7a1/f1.The luciferase reporter assay was used to detect the effect of miR Let-7a1/f1 on AMD1 3′-UTR.Results The sequences of pSilencer-let7a1/f1 and pMIR-AMD1 proved correct by DNA sequencing technique.After pSilencer-let7a1/f1 was transiently transfected into LNCaP cells,expression of miR Let-7a1/f1 was effectively increased,and expression of AMD1 mRNA was invariant and expression of the AMD1 protein was down-regulated after increased expression of pSilencer-let7a1/f1 in LNCaP cells.The luciferase activity of pMIR-AMD1 was obviously down-regulated by miR Let-7a1/f1.Conclusion miR Let-7a1/f1 inhibits the translation of AMD1 by action on the AMD1 3′-UTR site in LNCaP cells,and therefore decreases expression of the AMD1 protein.