LRP16通过调控E-钙黏着蛋白的表达促进MCF-7细胞的侵袭生长

LRP16 Promotes Invasive Growth of MCF-7 Cells by Regulation of E-cadherin Expression

LRP16在原代乳腺癌组织中表达水平与雌激素受体α(ERα)表达状态以及腋窝淋巴结侵袭数目密切相关.为研究LRP16基因对乳腺癌MCF7细胞侵袭生长的影响,并探讨涉及的分子机制,采用基质胶黏附实验与Transwell方法,检测内源性LRP16表达抑制MCF7细胞的体外黏附、侵袭生长与迁移特征.结果表明,抑制LRP16在MCF7细胞中的表达,降低了细胞的体外黏附、侵袭与迁移能力;采用FVB小鼠进行的实验性转移试验结果显示,抑制LRP16显著降低了MCF7细胞的肺转移结节数目;为探索可能的分子机制,采用Western印迹方法,检测了LRP16对乳腺癌转移相关分子MMP2,MMP9,CD44和E钙黏着蛋白表达的影响,结果在LRP16抑制的MCF7细胞中只有E钙黏着蛋白蛋白表达上调.进一步的Northern印迹与免疫组化实验结果表明,抑制LRP16可上调MCF7细胞中E钙黏着蛋白基因的mRNA与蛋白表达水平;共转染与双荧光素酶方法检测LRP16对E钙黏着蛋白基因启动子的表达调控效应,结果显示,LRP16抑制E钙黏着蛋白基因基因5′近端启动子的转录激活,该抑制效应选择性存在于内源性ERα阳性细胞,并且依赖于雌激素的存在;染色质免疫共沉淀方法(ChIP)检测ERα与E钙黏着蛋白基因启动子的相互作用,结果显示,在LRP16基因表达缺陷的MCF7细胞中,ERα抗体沉淀到E钙黏着蛋白基因启动子的DNA序列;上述研究结果表明,抑制LRP16基因表达,削弱了激素依赖型乳腺癌细胞的侵袭生长能力,其分子机制涉及了LRP16通过ERα介导对E钙黏着蛋白基因基因转录激活的调控.

The clinopathyological data indicated that the expression level of LRP16 was positively correlated with the status of estrogen-receptor α（-ERα） and involvement of axillary lymphoid nodes in primary breast cancer samples. Here, the effect of LRP16 on the invasive growth ability of human breast cancer MCF-7 cells and the involved molecular mechanism was investigated. The cellular adhesion, invasion and migration abilities were firstly measured by metrical gel adhesion assay and modified Boyden chamber （Transwell assays） and the results showed that suppression of LRP16 markedly reduced the adhesion, invasive and migration abilities of MCF-7 cells. The results of in vivo metastatic experiments demonstrated that suppression of the endogenous LRP16 significantly reduced the lung metastasis formation of MCF-7 cells. To further screen the expression changes of metastasis associated molecules in LRP16-suppressed MCF：7 cells, the protein levels of MMP-2, MMP-9, CD44 and E-cadherin were analyzed by Western blotting. The results demonstrated that only E- cadherin was up-regulated in LRP16-inhibitory MCF-7 cells. Further results from Northern blot and immunohistochemistry analyses comfirmed that inhibition of LRP16 caused the increases of E-cadherin both at mRNA and protein levels. To further determine the possible regulation effects of LRP16 on E-cadherin transactivation, the luciferase reporter driven by 5′-proximal regulatory region of E-cadherin gene was used to cotransfect with different dose LRP16 expression vector. Results analysis from luciferase assays indicated that the transactivation of E-cadherin promoter （ - 105 bp to ＋ 125 bp） was suppressed by LRP16 in a dosedependent manner in ERa-positive MCF-7 and HT29 cells in the presence of E2, but not in ER a-negative MDA-MB-231 and Caco-2 cells. Finally, the interaction of ERa and E-cadherin regulatory region was investigated by chromatin immunoprecipitation （CHIP） assays and the results suggested that the inhibition of LRP16 gene in MCF-7 cells enhanced the interaction between ERa and the DNA fragment of E-cadherin regulatory region. Taken together, these data demonstrated that inhibition of LRP16 expression attenuated the invasive growth of ERa-positive breast cancer cells, the molecular mechanism of which involved the transcriptional activity of E-cadherin through inhibition of LRP16 via ERa mediation.