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目的检测LNCa P细胞内miR-141-3p对雄激素受体(AR)基因靶向调控作用,明确AR是否为miR-141-3p的靶基因。方法将miR-141-3p mimics转染LNCa P细胞后,利用反转录PCR和Western blot法分别检测LNCa P细胞中AR的mRNA和蛋白表达。采用PCR方法扩增得到含有miR-141-3p结合位点的AR mRNA 3’非翻译区(3’UTR)片段,并插入到pmiR-report载体萤光素酶基因的3’下游区,并将所构建载体命名为pmiR-AR-3’UTR;萤光素酶报告基因检测试剂盒检测miR-141-3p对pmiR-AR-3’UTR靶向抑制效果。结果转染miR-141-3p mimics可降低LNCa P细胞内源AR的mRNA和蛋白水平;萤光素酶活性检测结果显示:与对照组相比,miR-141-3p可明显抑制pmiR-AR-3’UTR萤光素酶活性。结论 AR是miR-141-3p的靶基因。
Objective To detect the role of miR-141-3p in the regulation of androgen receptor (AR) gene expression in LNCa P cells and to determine whether AR is the target gene of miR-141-3p. Methods After transfection of miR-141-3p mimics into LNCa P cells, the mRNA and protein expression of AR in LNCa P cells were detected by reverse transcription PCR and Western blot respectively. A 3 ’untranslated region (3’UTR) fragment of AR mRNA containing miR-141-3p binding site was amplified by PCR and inserted into the 3’ downstream region of the luciferase gene of the pmiR-report vector. The constructed vector was named pmiR-AR-3’UTR. Luciferase reporter assay kit was used to detect the targeted inhibitory effect of miR-141-3p on pmiR-AR-3’UTR. Results Transfection of miR-141-3p mimics decreased the mRNA and protein levels of endogenous AR in LNCa P cells. The results of luciferase assay showed that miR-141-3p significantly inhibited the expression of pmiR-AR- 3’UTR luciferase activity. Conclusion AR is the target gene of miR-141-3p.