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(CoIP)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 a 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 CoIP 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.
