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, where 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 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.
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 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.
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.