Screen Of HPR Interacted Proteins By Yeast Two-Hybrid Assay And Functional Analysis

hPR(human progesterone receptor),which is the important nucleus transcriptional factor. It has two isomers, hPR-A, hPR-B, which are transcribed from two AUG in a single gene, play the important roles in the development of mammary glands and the proliferation of breast cancer. hPR-B has the more transcriptional activity than hPR-A, they can modulate a lot of key genes such as P21,c-myc,SOX4 and so on. Some research show : the mice lacking of PR do not only ovulate spontaneously, but also limit the development of mammary gland seriously. To find proteins interacting with human progesterone receptor, yeast two-hybrid was applied to screen human breast cDNA library, different domains of hPR-B, as the baits respectively. As the results show: 1. We gained the targeting fragments: AF3, IF, AF1-DBD, AF2, and inserted them into the pGBKT7 vector exactly. 2. We transformed pGBKT7-AF3, pGBKT7-IF, pGBKT7-AF1-DBD, pGBKT7-AF2 into the yeast AH109, then extracted the proteins. By western blotting, the proteins were identified exactly. 3. By testing the self-transcriptional activity, we found that they(pGBKT7-AF3, pGBKT7-IF, pGBKT7-AF1-DBD, pGBKT7-AF2) were all have no self-transcriptional activity except pGBKT7-AF3. 4. By lots of screening, PIAS3, protein inhibitor of activated STAT3, was identified to interact with AF1-DBD domain of hPR-B. 5. We fished the gene PIAS3 from human breast cDNA library, and sequenced exactly. 6. we inserted the gene PIAS3 into the PGEX-KG and PXJ40-HA vector, and expressed the relevant protein successfully. 7. We inserted the gene hPR-B into the eukaryontic expressional vector and express the protein. 8. We established the PR's transcriptional activity system successfully.9. We found that progesterone can affect the cycle of tumour cell by progesterone receptor.