Mechanisms of Progesterone on Proliferation and Migration of Human Ovarian Cancer Cells via HOXA9
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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、100 μmol/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IC50; 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IC50值为2.680 μmol/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。 Abstract: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 100 μmol/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 IC50. 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 IC50 value of H0-8910 cells treated with MPA for 72 h was 2.680 μmol/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-cadherin protein expression decreased (P < 0.001); after knocking down HOXA9, the effect of progesterone on EMT-related protein expression in HO-891 was more obvious (P < 0.05), while after overexpressing HOXA9, the effect of progesterone on EMT-related protein expression in HO-891 was weakened (P < 0.05). Conclusion Progesterone inhibits the development of ovarian cancer by suppressing the expression of HOXA9 gene and therefore suppressing the malignant biological phenotypes such as cell proliferation, migration, invasion and EMT process. -
Key words:
- Ovarian cancer /
- HOXA9 /
- Progesterone /
- Medroxyprogesterone acetate
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图 6 MPA通过降低HOXA9表达抑制人卵巢癌细胞EMT进程
A: Westernblot检测HO-8910细胞株中EMT相关蛋白 Vimentin,N-cadherin,E-cadherin的表达变化;B:HO-8910细胞株中Vimentin的相对蛋白表达;C:HO-8910细胞株中N-cadherin的相对蛋白表达;D:HO-8910细胞株中E-cadherin的相对蛋白表达。*P < 0.05,**P < 0.01,***P < 0.001。
Figure 6. MPA repressed EMT in human ovarian cancer cells by reducing HOXA9 expression
表 1 抗体信息表
Table 1. information of antibodies
抗体(公司) 抗体稀释比例 HOXA9抗体(abcam ab140631) 1∶1 000 Vimentin抗体(CST 5741) 1∶1 000 E-cadherin抗体(CST 14472) 1∶1 000 N-cadherin抗体(CST 13116) 1∶1 000 GAPDH抗体(abcam ab8245) 1∶500 通用二抗(CST 7074) 1∶2 000 -
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