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目的:探讨LRP16基因促进MCF-7细胞增殖的分子生物学机制。方法:(1)将ERα模式启动子调控的荧光素酶报告子(3×ERE-Luc)与ER,αLRP16真核表达载体共转染MCF-7细胞,测定3×ERE-Luc的相对荧光素酶活性;(2)将3×ERE-Luc,ERα真核表达载体、针对LRP16的小干扰RNA(LRP16-siRNA374,LRP16-siRNA668)或对照小干扰RNA(control-siRNA)共转染MCF-7细胞,测定3×ERE-Luc的相对荧光素酶活性;(3)用Northern印迹与W estern印迹方法检测抑制LRP16基因后MCF-7细胞中ERα下游靶基因对雌激素刺激的反应性。结果:(1)LRP16增强3×ERE-Luc的相对荧光素酶活性,并呈现剂量依赖性,该效应依赖于雌激素对ERα的激活;(2)North-ern印迹结果显示,抑制LRP16表达限制了雌激素对ERα下游靶基因表达水平的刺激上调,包括E2F1、视黄酸受体(RARα)、c-fos和MTA3,但没有影响组织蛋白酶D(cathepsin D)的反应性;W estern印迹结果显示,抑制LRP16限制了雌激素对细胞周期蛋白D1(cyc lin D1)的刺激。结论:(1)雌激素反应性靶基因LRP16反馈增强ERα介导的转录激活活性;(2)LRP16基因通过增强ERα介导的转录激活活性调节其下游靶基因的表达,主要通过改变cyc linD1的水平促进MCF-7细胞的增殖。
Objective: To investigate the molecular mechanism of LRP16 gene in promoting the proliferation of MCF-7 cells. Methods: (1) The luciferase reporter (3 × ERE-Luc) regulated by ERα mode promoter and ER, αLRP16 eukaryotic expression vector were co-transfected into MCF-7 cells and the relative fluorescence intensity of 3 × ERE-Luc (2) MCF-7 was co-transfected with 3 × ERE-Luc and ERα eukaryotic expression vector, LRP16 siRNA (LRP16-siRNA374, LRP16-siRNA668) or control siRNA (3) Northern blotting and Western blotting were used to detect the responsiveness of ERα downstream target genes to estrogen stimulation in MCF-7 cells after inhibition of LRP16 gene. RESULTS: (1) LRP16 enhanced the relative luciferase activity of 3 × ERE-Luc in a dose-dependent manner, which was dependent on the activation of ERα by estrogen. (2) North-ern blotting showed that the inhibition of LRP16 expression was limited Estrogen up-regulated the expression of downstream ERα target genes, including E2F1, RARα, c-fos and MTA3, but did not affect the reactivity of cathepsin D; Western blot results It has been shown that inhibition of LRP16 limits the stimulation of cyclin D1 by estrogen. CONCLUSION: (1) LRP16, an estrogen-responsive target gene, enhances ERα-mediated transcriptional activation. (2) LRP16 regulates the expression of its downstream target genes by enhancing ERα-mediated transcriptional activation, The level promotes the proliferation of MCF-7 cells.