核受体Nur77对卵巢癌细胞增殖转移的影响及机制研究

Effect of Nuclear Receptor Nur77 on Proliferation and Metastasis of Ovarian Cancer Cells and its Mechanism

目的研究核受体Nur77对卵巢癌细胞增殖转移能力的影响及其作用机制。方法以人高转移性卵巢癌细胞株HO-8910PM为疾病模型,利用慢病毒载体转染构建HO-8910PM(Nur77-/-)细胞株,下调Nur77的表达。利用Nur77激动剂壳囊孢酮B(Csn-B)处理细胞做比较研究。按不同的实验细胞和处理方式设4个组别进行研究,对照组为转染空载体的HO-8910PM细胞,研究组为HO-8910PM(Nur77-/-)细胞,Csn-B组为HO-8910PM细胞加入Csn-B处理24h,Csn-B(Nur77-/-)组为HO-8910PM(Nur77-/-)细胞加入Csn-B处理24h,流式细胞法检测各组细胞数量在不同细胞周期的分布比例,Western blot法检测磷酸化Nur77(p-Nur77)、磷酸化蛋白激酶B(p-Akt)、周期蛋白-D1(Cyclin D1)、上皮性钙黏附蛋白(E-cadherin)、神经性钙黏附蛋白(N-cadherin)、波形蛋白(Vimentin)的含量,ELISA法检测3种基质金属蛋白酶(MMP-2、MMP-7、MMP-9)细胞培养液中的浓度,Transwell检测细胞的转移、侵袭能力。结果随着p-Nur77的含量增加:研究组<Csn-B(Nur77-/-)组<对照组<Csn-B组,处于S期,G 2和M期细胞数量的比例逐渐增大(P<0.05),p-Akt的含量逐渐上升,Cyclin D1的表达量随之增加(P<0.05),同时E-cadherin的含量逐渐减少,N-cadherin、Vimentin的含量相应递增(P<0.05),细胞的转移侵袭能力随着MMP-2、MMP-7和MMP-9的浓度增加而增强(P<0.05)。结论下调Nur77的表达和磷酸化修饰可抑制卵巢癌细胞的增殖和侵袭。

Objective To study the effect of nuclear receptor Nur77 on proliferation and metastasis of ovarian cancer cells and its mechanism.Methods Using human highly metastatic ovarian cancer cell line HO-8910PM as pathological model,HO-8910PM cells were transfected with lentiviral vector to down-regulate the expression of Nur77,and HO-8910PM(Nur77-/-)cell line was constructed.Cells were treated with Nur77 agonist Cytosporone B(Csn-B)for comparative study.According to different experimental cells and treatment methods,four groups were set up for research:the control group was HO-8910PM cells transfected with empty vectors,the research group was HO-8910PM(Nur77-/-)cells,and the Csn-B group was HO-8910PM cells treated with Csn-B for 24 hours.The Csn-B(Nur77-/-)group consisted of HO-8910PM(Nur77-/-)cells treated with Csn-B for 24h.Flow cytometry was used to detect the distribution ratio of the number of cells in each group in different cell cycles.Western blot method was used to detect the contents of phosphorylated Nur77(p-Nur77),phosphorylated protein kinase B(p-Akt),Cyclin D1,epithelial calcium adhesion protein(E-cadherin),neurogenic calcium adhesion protein(N-cadherin)and Vimentin.ELISA method was used to detect the concentrations of three matrix metalloproteinases(MMP-2,MMP-7 and MMP-9)in cell culture solution,and Transwell was used to detect the metastatic and invasive ability of cells.Results With the increase of the content of p-Nur77,the proportion of the number of cells in S phase,G 2 phase and M phase in the research group<Csn-B(Nur77-/-)group<control group<Csn-B group gradually increased(P<0.05),and the content of p-Akt gradually increased,which thus promoted the expression of Cyclin D1(P<0.05).While the content of E-cadherin gradually decreased.The contents of N-cadherin and Vimentin increased correspondingly(P<0.05),and the metastatic and invasive ability of cells increased with the increasing concentrations of MMP-2,MMP-7 and MMP-9(P<0.05).Conclusion Downregulation of expression and phosphorylation modification of Nur77 could inhibit proliferation and invasion of ovarian cancer cells.