目的:观察分化抑制因子2(inhibitor of differentiation 2,Id2)基因转染卵巢癌SKOV3细胞后,对细胞生长和侵袭能力的影响,探讨Id2基因缺失螺旋-环-螺旋(helix-loop-helix,HLH)结构域后对SKOV3细胞的影响。方法:以携带野生型Id2、Id2-DBM和Id2-DBM-δHLH基因的质粒(pcDNA3.1-Id2、pcDNA3.1-Id2-DBM和pcDNA3.1-Id2-DBM-δHLH)经脂质体介导转染SKOV3细胞。采用Western印迹法和RT-PCR法检测转染后SKOV3细胞Id2、Id2-DBM和Id2-DBM-δHLH的表达水平;MTT法检测SKOV3细胞的增殖曲线;划痕试验和Transwell小室检测细胞的迁移能力;Western印迹法检测Id2对MCF-7细胞上皮钙黏附素表达的影响。结果:mRNA及蛋白水平显示质粒成功转入;细胞生长曲线显示各组细胞的增殖无显著差异;与空白对照组相比,转染pcDNA3.1-Id2、pcDNA3.1-Id2-DBM和pcDNA3.1-Id2-DBM-δHLH后,细胞的侵袭能力增强,且pcDNA3.1-Id2-DBM和pcDNA3.1-Id2-DBM-δHLH组细胞明显伴有上皮钙黏附素表达水平的降低。结论:Id2蛋白的过表达可促进卵巢癌SKOV3细胞的侵袭能力,与上皮钙黏附素表达水平的降低有关,且该作用在缺失HLH结构域后依然存在。
Objective：It has been shown that LRP16 is an estrogen-induced gene through its receptor α（ERα）. Although there is evidence demonstrating that inhibition of LRP16 gene expression in MCF-7 human breast cancer cells partially attenuates its estrogen-responsiveness, the underlying molecular mechanism is still unclear. Here, the effect of LRP16 expression on the ERα signaling transduction was investigated. Methods： Cotransfection assays were used to measure the effect of LRP16 on ERα-mediated transcriptional activity. GST-pulldown and immunoprecipitation （ColP） assays were employed to investigate the physical interaction of LRP16 and ERα. The mammalian two-hybrid method was used to map the functional interaction region. Results： the results of cotransfection assays demonstrated that the transcriptional activities of ERα were enhanced in α LRP16 dose-dependent manner in MCF-7 in the presence of estrogen, however, it was abolished in the absence of E2 in MCF-7 cells. The physical interaction of LRP16 and ERα proteins was confirmed by GST-pulldown in vitro and ColP in vivo assays, which was enhanced by E2 but not dependent on its presence. Furthermore, the results of the mammalian two-hybrid assays indicated that the binding region of ERα to LRP16 located at the A/B AF-1 functional domain and E2 stimulated the binding of LRP16 to the full-length ERα molecule but not to the A/B region alone. Conclusion： These results support a role for estrogenically regulated LRP16 as an ERα coactivator, providing a positive feedback regulatory loop for ERα signal transduction. Based on this function of LRP16, we propose that ERα-positive breast cancer patients with high expression of LRP16 might benefit from targeting LRP16 therapy.
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
