PHD1通过下调NF-κB介导的cyclinD1的表达抑制肺癌细胞的生长和增殖

PHD1 Inhibits NF-κB Mediated CyclinD1 Expression and Proliferation of Lung Cancer Cells

目的:探讨PHD1在肺癌中的功能,并进一步研究其分子机制,为肺癌的治疗寻找新的靶点。方法:选取人肺癌细胞A549,以脂质体为载体一方面过表达PHD1,另一方面合成设计靶向PHD1的siRNA沉默PHD1,利用荧光素酶检测NF-κB的活性,分别用western blot和real-time PCR检测cyclinD1的表达水平。在A549细胞中过量稳定表达带GFP标记的PHD1,流式细胞仪检测细胞周期变化,测量细胞的生长曲线,并将细胞注射到裸鼠皮下观察其成瘤情况。结果:过表达PHD1可明显抑制NF-κB的活性和IκBα的降解,降低cyclin D1的mRNA和蛋白表达水平;而干扰PHD1的表达可显著增加NF-κB的活性,并上调cyclin D1的mRNA和蛋白表达水平,而不影响cyclinE1。过表达IκBαSR可以阻止干扰PHD1引起的cyclinD1 mRNA水平的上调。过表达PHD1可引起细胞周期的停滞,显著抑制细胞的增殖和移植瘤的生长。结论:PHD1可能通过下调NF-κB介导的cyclinD1的表达抑制肺癌细胞的生长和增殖。

Objective： To find new target for lung cancer treatment through investigating the function and the mechanism of PHD1 in lung cancer cells. Methods： PHD1 was over expressed using the lipidosome or silenced using siRNA in A549 cells, then NF-κB activity was detected by luciferase assay and the expression level of cyclinDl was measured by real-time PCR and western blot. The A549 stable cell line which expresses GFP-PHD1 was established and the cell cycle and cell growth were analyzed by flow cytometry. Finally, the growth of tumor was observed after injecting cells subcutaneously to the nude mice. Results： Overexpression of PHD1 significantly inhibited the NF-κB activity and degradation of IκBα, and decreased the mRNA and protein expression of cyclinD1; Conversely, knocking down PHD1 significantly increased the NF-κB activity and up regulated the mRNA and protein expression of cyclinD1, but it had no effect on the expression of cyclinE1. Over expression of IκBαSR inhibited the up regulation of cyclinD1 mRNA level induced by knocking down of PHD1. Over expression of PHD1 caused cell cycle arrest and significantly inhibited the cell proliferation and tumor growth. Conclusion： Over expression of PHD1 might inhibit the growth and proliferation of lung cancer cells through down-regulating the cyclinD 1 expression, which was mediated by NF-κB.