Lu Ye, Liao Qin-ping, Chen Chun-ling, Yu Li
Department of Obstetrics & Gynecology, Peking University First Hospital, Beijing 100034,China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2006 Oct 18;38(5):475-9.
To study the functional differences between the two progestrone receptor isoforms(PR-A and PR-B) in human endometrial cancer,using antisense oligodeoxynucleotide(AS-ODN) to downregulate isoform B of progestrone receptor in endometrial carcinoma cell lines, After transfection of the oligodeoxynucleotide, several kinds of hormones were added in the cells to observe the different response, where to study the functional differences between the two isoforms.
The well-differentiated endometrial cancer cell line Ishikawa and moderate-differentiated endometrial cancer cell line Hec-1B were cultured in vitro. The cells were transfected with antisense, sense, and scramble-ODN. After 48 hours, the expressions of two progesterone receptor isoforms were detected by Western blot using specific antibody. Then the cells were planted in 96-well plates, transfected with antisense, sense, scramble-ODN and added in several hormones to search for the response in distinct hormones and oligodeoxynucleotides.
After transfecting antisense-ODN, two cell lines were down-regulated in progesterone receptor isoform B,but progesterone receptor isoform A was not down-regulated,and the progesterone receptor isoform B of cells transfected with sense and scramble-ODN was not changed. When stimulated by 17beta-estradiol(E2)for 72 hours,the growth of Ishikawa cells was significantly higher than that of the control, Hec-1B cells only grew higher than control,but it was to significant in statistics.R5020 inhibited Ishikawa cells significantly after stimulating for 72 hours. There was the same effect in Hec-1B cells after stimulating for 96 hours. On the bases of E2 and R5020, we added mifepristone (RU486) . The cells developed after 96 hours in Ishikawa cells and developed after 48 hours in Hec-1B cells. When PR-B was down-regulated,the stimulating effect of E2 was enhanced, but the inhibitory effect of R5020 was decreased, RU486 antagonized R5020 weaklier than the control.
AS-ODN directed against the human PR-B can inhibit the expression of PR-B effectively,through which the PR-A expresses predominantly. E2 can cause endometrial carcinoma cell growth, PR-B is associated with the stimulating effect of E2 in endometrial carcinoma cells. Progestin (R5020) inhibits the hyperplasia induced by E2,PR-B is involved in the inhibitory effect of R5020. RU486 antagonizes the effect of R5020,inhibiting cell growth, PR-B is involved in the antagonizing effect of RU486.
通过反义寡脱氧核苷酸（AS-ODN）下调子宫内膜癌细胞系中孕激素受体异构体B，研究人子宫内膜癌中两种孕激素受体异构体（PR-A和PR-B）的功能差异。转染寡核苷酸后，向细胞中添加几种激素以观察不同反应，从而研究两种异构体之间的功能差异。
体外培养高分化子宫内膜癌细胞系Ishikawa和中分化子宫内膜癌细胞系Hec-1B。用反义、正义和乱序ODN转染细胞。48小时后，使用特异性抗体通过蛋白质印迹法检测两种孕激素受体异构体的表达。然后将细胞接种于96孔板中，用反义、正义、乱序ODN转染并添加几种激素，以寻找不同激素和寡核苷酸中的反应。
转染反义ODN后，两种细胞系中孕激素受体异构体B表达下调，但孕激素受体异构体A未下调，转染正义和乱序ODN的细胞中孕激素受体异构体B未改变。用17β-雌二醇（E2）刺激72小时后，Ishikawa细胞的生长明显高于对照组，Hec-1B细胞仅生长高于对照组，但统计学上无显著性差异。R5020刺激72小时后对Ishikawa细胞有明显抑制作用。Hec-1B细胞刺激96小时后有相同效果。在E2和R5020基础上，添加米非司酮（RU486）。Ishikawa细胞96小时后生长，Hec-1B细胞48小时后生长。当PR-B下调时，E2的刺激作用增强，但R5020的抑制作用减弱，RU486拮抗R5020的作用比对照组弱。
针对人PR-B的AS-ODN可有效抑制PR-B的表达，从而使PR-A占主导表达。E2可导致子宫内膜癌细胞生长，PR-B与E2对子宫内膜癌细胞的刺激作用有关。孕激素（R5020）抑制E2诱导的增生，PR-B参与R5020的抑制作用。RU486拮抗R5020的作用，抑制细胞生长，PR-B参与RU486的拮抗作用。
