摘要
目的探讨HOXA9在孕激素抑制卵巢癌发展过程中的作用机制。方法(1)按照孕激素醋酸甲羟孕酮(MPA)药物浓度梯度(0.1、1、10、50、100μmol/L)培养人卵巢癌细胞HO-8910,作用24 h、48 h、72 h后,光学显微镜观察细胞形态;CCK-8检测细胞存活率,确定IC_(50);Annexin V-FITC双染法检测细胞凋亡率。(2)将细胞分为H0-8910细胞4组:(1)NC组;(2)孕激素组;(3)孕激素+si-HOXA9组;(4)孕激素+ov-HOXA9组。H0-8910细胞分别转染HOXA9敲降或过表达质粒后使用MPA处理或不转染质粒单独使用使用MPA处理72 h,qPCR和Western blot分别用于检测HOXA9 mRNA和蛋白表达水平以及上皮间质转化(EMT)相关蛋白(Vimentin,N-cadherin,E-cadherin)的表达变化;划痕实验检测细胞迁移能力;Transwell实验用于检测细胞侵袭能力。结果(1)孕激素刺激后,HO-8910细胞株生长状态变差,排列稀疏,细胞增殖,迁移以及侵袭能力受到抑制(P<0.0001),且细胞凋亡率上升(P<0.0001);(2)通过cck8结果计算得MPA处理H0-8910细胞72 h的IC_(50)值为2.680μmol/L;(3)相较于人卵巢上皮细胞CP-H055,HOXA9在人卵巢癌细胞株HO-8910,SKOV3,A2780和OVCAR3中mRNA均高表达(P<0.01),HOXA9蛋白在HO-8910中同样高表达(P<0.001);(4)孕激素可抑制HOXA9的表达(P<0.0001),敲降HOXA9后,孕激素对HO-8910侵袭和迁移能力的抑制更为明显(P<0.0001),而过表达HOXA9后,孕激素对HO-8910侵袭和迁移能力的抑制稍有减弱(P<0.0001);(5)孕激素使E-cadherin蛋白表达上升(P<0.01),而Vimentin,N-cadherin蛋白表达下降(P<0.001);敲降HOXA9后,孕激素对HO-891中EMT相关蛋白表达影响更为明显(P<0.05),而过表达HOXA9后,孕激素对HO-891中EMT相关蛋白表达影响减弱(P<0.05)。结论孕激素通过抑制HOXA9基因的表达,抑制细胞增殖,迁移,侵袭以及EMT进程等恶性生物学表型,从而抑制卵巢癌发展进程。
Objective To investigate the mechanism of HOXA9 in the process of progesterone inhibition of ovarian cancer progression.Methods(1)Human ovarian cancer cell line HO-8910 were cultured according to the gradient of progestin medroxyprogesterone acetate(MPA)concentration(0.1,1,10,50 and 100μmol/L),and the cell morphology was observed after 24 h,48 h and 72 h.CCK-8 was used to detect cell viability and determine IC_(50).The apoptosis rate was detected by Annexin V-FITC double staining.(2)H0-8910 were divided into four groups:1)NC group;2)progestin group;3)progestin+si-HOXA9 group;4)progestin+ov-HOXA9 group.H0-8910 cells were transfected with HOXA9 knockdown or overexpression plasmids and treated with MPA or not transfected with plasmids alone using MPA for 72 h.The qPCR and western blot were used to detect the mRNA and protein expression of HOXA9.as well as the expression of epithelial mesenchymal transition(EMT)-related proteins(Vimentin,N-cadherin,E-cadherin),respectively.Wound scratch assay was used to detect cell migration;Transwell assay was used to detect cell invasion.Results(1)After progesterone stimulation,the growth status of HO-8910 became poor,the arrangement was sparse,cell proliferation,migration and invasion ability were suppressed(P<0.0001),and the apoptosis rate increased(P<0.0001);(2)calculated by cck8 results,the IC_(50) value of H0-8910 cells treated with MPA for 72 h was 2.680μmol/L;(3)compared with human ovarian epithelial cells CP-H055,HOXA9 mRNA was highly expressed in human ovarian cancer cell lines HO-8910,SKOV3,A2780 and OVCAR3(P<0.01),and HOXA9 protein was likewise highly expressed in HO-8910(P<0.001);(4)progesterone could inhibit HOXA9 expression(P<0.0001),and the inhibition of cell invasion and migration ability by progesterone was more obvious after knocking down HOXA9(P<0.0001),while the inhibition of HO-8910 invasion and migration ability was slightly reduced after overexpression of HOXA9(P<0.0001).(5)Progesterone increased E-cadherin protein expression(P<0.01),while Vimentin and N-c
作者
杨霄彦
郝春光
张志坚
YANG Xiaoyan;HAO Chunguang;ZHANG Zhijian(Dept.of Pathology,Inner Mongolia Baogang Group Third Staff Hospital,Baotou Inner Mongolia;Dept.of Anesthesiology,Inner Mongolia Baogang Hospital,Baotou Inner Mongolia 014016,China;Dept.of Pathology,Inner Mongolia Baogang Hospital,Baotou Inner Mongolia 014016,China)
出处
《昆明医科大学学报》
CAS
2022年第10期35-42,共8页
Journal of Kunming Medical University
基金
内蒙古自治区自然科学基金资助项目(2020MS08095)。
关键词
卵巢癌
HOXA9
孕激素
醋酸甲羟孕酮
Ovarian cancer
HOXA9
Progesterone
Medroxyprogesterone acetate
