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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 expression level of LRP16 in primary breast cancer tissues is closely related to the expression of estrogen receptor alpha (ERα) and the number of axillary lymph node invasion.In order to study the effect of LRP16 gene on the invasion and metastasis of breast cancer MCF7 cells and explore the molecular mechanism involved, Matrigel adhesion assay and Transwell method were used to detect the adhesion, invasion and migration of MCF7 cells in vitro.The results showed that inhibiting the expression of LRP16 in MCF7 cells reduced the adhesion, invasion and migration of MCF7 cells in vitro ; The results of experimental metastasis test in FVB mice showed that inhibiting LRP16 significantly reduced the number of lung metastatic nodules in MCF7 cells. To explore the possible molecular mechanism, LRP16 was used to detect the expression of MMP2 , MMP9, CD44 and E cadherin protein expression, the results in LRP16 inhibited MCF7 cells only E cadherin protein expression.Further Northern blot and immunohistochemistry results show that inhibition of LRP16 can up-regulate MCF7 cells in the E Cadherin gene mRNA and protein expression levels; cotransfection and dual-luciferase assay LRP16 pairs of E The results showed that LRP16 inhibits the transcriptional activation of the 5 ’proximal promoter of E-cadherin gene, which is selectively expressed in endogenous ERα-positive cells and is dependent on the expression of estrogen (ChIP) was used to detect the interaction between ERα and E-cadherin promoter. The results showed that ERα antibody precipitated into E-cadherin gene in MCF7 cells with LRP16 deficiency The above results show that inhibiting the expression of LRP16 gene impairs the invasive growth ability of hormone-dependent breast cancer cells and its molecular mechanism involves the regulation of transcriptional activation of E-cadherin gene by LRP16 via ERα.