Aim

The regulation of secreted osteopontin (OPN) expression by genistein and its functional sequel in the metastatic cancer cells (MDA-MB-435 and MDA-MB-231) was ascertained.

Main methods

Western blot and Real-Time PCR were used to analyse the proteins and mRNA transcripts, respectively. Possible transcriptional regulation of secreted OPN was analyzed by chromatin immunoprecipitation assay, bioinformatics analysis, transfection and luciferase reporter assay. The specific siRNAs and constitutive p-ERKs were used to evaluate the role of the MAPK pathway. The functional sequel of genistein in these cells was analyzed by colony formation-, migration- and invasion- assay.

Key findings

Secreted OPN expression was inhibited (up to ~0.7-fold) by genistein in these cells. Genistein (50 μM) displayed a reduction in the aggressiveness of these cells concerning colony formation rate, migration, and invasion. The p-ERK½ was increased by ~2.5-fold and ~1.5-fold upon 50 μM genistein and 15 μM resveratrol treatments at 24 h, respectively. Knockdown of ERK½ and PD98059, the inhibitor of MEK, promoted secreted OPN expression in vitro in these cells; while, the transfection of the constitutive active ERK2 (L73P and S151D) decreased the secreted OPN expression. Further, silent mating type information regulation 2 homolog 1 (SIRT1) expression in the cells was increased (~1.6-fold) upon genistein treatment (50 μM) likewise with resveratrol (~1.5-fold), an activator for SIRT1. Knockdown of SIRT1 increased OPN mRNA transcripts expression level and secreted OPN protein level in these cells.

Significance

MAPK pathway and SIRT1 activation are involved in the regulation of secreted OPN by genistein in these cells.

中文翻译：

---

MAPK途径和SIRT1参与金雀异黄素在转移性癌细胞中对骨桥蛋白分泌的下调

目标

确定了染料木黄酮对转移性癌细胞（MDA-MB-435和MDA-MB-231）中分泌的骨桥蛋白（OPN）表达的调节及其功能后遗症。

主要方法

Western blot和实时荧光定量PCR分别用于分析蛋白质和mRNA转录本。通过染色质免疫沉淀测定，生物信息学分析，转染和萤光素酶报告基因测定来分析分泌的OPN可能的转录调控。特定的siRNA和组成型p-ERK用于评估MAPK途径的作用。通过细胞集落形成，迁移和侵袭试验分析了染料木黄酮在这些细胞中的功能性后遗症。

主要发现

金雀异黄素在这些细胞中抑制了分泌的OPN表达（最多约0.7倍）。Genistein（50μM）在集落形成速率，迁移和侵袭方面显示出这些细胞的侵略性降低。在24 h进行50μM金雀异黄素和15μM白藜芦醇处理后，p-ERK½分别增加了约2.5倍和约1.5倍。抑制ERK½和MEK抑制剂PD98059可以促进这些细胞在体外分泌OPN的表达。而组成型活性ERK2（L73P和S151D）的转染降低了分泌的OPN表达。此外，在染料木黄酮处理（50μM）后，同样用白藜芦醇（约1.5倍）（SIRT1的激活剂），细胞中的沉默交配型信息调节2同源物1（SIRT1）表达也增加了（约1.6倍）。

意义

MAPK途径和SIRT1激活参与了染料木黄酮对这些细胞分泌的OPN的调节。