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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。
Objective To investigate the effect of let-7a1 / f1 on the expression of S-adenosylmethionine decarboxylase (AMD1) in human prostate cancer LNCaP cells and its mechanism. Methods The gene sequence of let-7a1 / f1 gene was amplified by RT-PCR using the total RNA of LNCaP cells as a template, and then cloned into pSilencerTM4.1-CMV neo vector and constructed into let-7a1 / f1 eukaryotic expression vector pSilencer -let7a1 / f1. The expression of let-7a1 / f1 at transcriptional level was detected by RT-PCR after transfection of LNCaP cells transiently. The transcriptional and translational changes of AMD1 expression were detected by RT-PCR and Western blot. To construct the 3’-untranslated region (3’-UTR) luciferase reporter gene fusion plasmid (pMIR-AMD1) of AMD1 gene and co-transfected it with pSilencer-let7a1 / f1 into LNCaP cells. Through dual luciferase assay, -7a1 / f1 on AMD1 3’-UTR. Results The expression of let-7a1 / f1 in pSilencer-let7a1 / f1 and pMIR-AMD1 was confirmed by restriction enzyme digestion and sequencing. The expression of let-7a1 / f1 in pSilencer-let7a1 / f1 transfected LNCaP cells was significantly increased. Decreased, luciferase activity of pMIR-AMD1 significantly decreased after let-7a1 / f1 transfection. Conclusion miR Let-7a1 / f1 can target AMD1 3’-UTR and inhibit the translation of AMD1 protein, thereby decreasing the level of AMD1 protein in LNCap cells.