孕激素受体B亚型在子宫内膜癌细胞中的功能

Functional study of progesterone receptor isoforms in endometrial cancer cell lines

目的:通过反义寡核苷酸(antisense oligodeoxynucleotide,AS-ODN)技术特异性下调子宫内膜癌细胞系中孕激素受体亚型B(progestrone receptor isoform B,PR-B),在此基础上观察激素对转染前后细胞作用的改变,了解子宫内膜癌细胞中孕激素受体亚型功能的不同。方法:体外培养高分化子宫内膜癌细胞Ishikawa和中分化子宫内膜癌细胞Hec-1B,转染PR-B的反义寡核苷酸和正义寡核苷酸及错义寡核苷酸后,提取细胞蛋白,采用Western blot方法测定各个条件下内膜癌细胞中两种孕激素受体亚型的表达;并在转染PR-B的反义寡核苷酸和正义寡核苷酸及错义寡核苷酸后,用MTT法测定转染前后各种激素对细胞生长作用的改变。结果:转染孕激素受体亚型PR-B的反义寡核苷酸后,两种细胞系PR-B的表达较非转染组显著下调,孕激素受体亚型A(progesterone receptor isoformA,PR-A)的表达无明显变化。雌激素(17β-estradiol,E2)作用后72h Ishikawa细胞生长显著高于对照组,Hec-1B细胞较对照组有升高趋势,但差异无统计学意义。孕激素(R5020)作用72h后对Ishikawa细胞有显著抑制作用,96h后对Hec-1B细胞有显著抑制作用。在雌、孕激素作用基础上加入米非司酮(mifepristone,RU486),96h后Ishikawa细胞明显增生,而仅48h后HEC-1B细胞明显增生。转染PR-B的反义寡核苷酸后,雌激素对内膜癌细胞生长的刺激作用较转染前增强,孕激素对细胞的生长抑制作用减弱甚至消失,米非司酮拮抗孕激素的作用较转染前显著降低。结论:(1)向子宫内膜癌细胞系中转染PR-B的反义寡核苷酸可以下调PR-B的表达,使细胞优势表达PR-A;(2)雌激素可以刺激内膜癌细胞的生长,PR-B可能参与下调雌激素对内膜癌细胞的促生长作用;(3)孕激素在雌激素基础上抑制内膜癌细胞的生长,主要可能通过PR-B起抗内膜癌细胞增生作用;(4)米非司酮有拮抗孕激素抑制子宫内膜癌细胞生长的作用。PR-B可能参与介导了米非司酮拮抗孕激素的作用。

Objective：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, whereh to study the functional differences between the two isoforms. Methods： The well-differentiated endometrial cancer cell line Ishikawa and moderate-differentiated endometrial cancer cell line Hec- 1B were cuhrued 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. Results： 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 17β-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. Conclusion： 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.