目的:为深入研究LRP16基因的生理功能,本研究将小鼠LRP16基因特异打靶载体pBΔ16转染小鼠胚胎干细胞(embryon ic stem cell),并筛选同源重组克隆。方法:通过电转染方法将LRP16插入失活型打靶载体导入ES细胞,经G418筛选,挑取抗性克隆,Southern b lot方法鉴定同源重组ES细胞克隆。结果:打靶载体成功转染ES细胞,Southern b lot筛选100个抗性克隆,结果显示有一个打靶序列同源重组型插入的ES细胞克隆。结论:筛选到同源重组型ES细胞,为下一步建立LRP16缺失型小鼠并为从整体水平研究LRP16基因的生理功能奠定基础。
LRP16 was previously identified as an estrogen-induced gene in breast cancer cells. The responsiveness of LRP16 to estrogen and its functional effects in endometrial cancer （EC） cells are still unclear. Here, we show that the mRNA level and promoter activity of the LRP16 gene were significantly increased by 17β-estradiol （E2） in estrogen receptor ot （ERα）-positive Ishikawa human EC cells. Although the growth rate of Ishikawa cells was not obviously affected by ectopic expression of LRP 16, the results of a Transwell assay showed an approximate one-third increase of the invasive capacity ofLRP 16-overexpressing cells. As a result of molecular screening, we observed that the expression of E-cadherin, an essential adhesion molecule associated with tumor metastasis, was repressed by LRP16. Further promoter analyses demonstrated that LRP 16 inhibited E-cadherin transactivation in a dose-dependent manner. However, the inhibition was abolished by estrogen deprivation, indicating that the downregulation of E-cadherin transcription by LRP16 requires ERα mediation. Chromatin immunoprecipitation analyses revealed that the binding of ERα to the E-cadherin promoter was antagonized by LRP 16, suggesting that LRP 16 could interfere with ERα-mediated transcription. These results suggest that the upregulation of LRP 16 by estrogen could be involved in invasive growth by downregulating E-cadherin in human ECs.
Yuan Guang MengWei Dong HanYa Li ZhaoKe HuangYi Ling SiZhi Qiang WuYi Ming Mu
