1 / 31

The Effect of BRCA1 on the Progesterone Receptor

The Effect of BRCA1 on the Progesterone Receptor. Content. 1 Background 2 Protocol and procedures 3 Result and Problems 4 Discussion and some ideas. Background. 1 BRCA1 gene (breast cancer susceptibility gene 1 )

riona
Télécharger la présentation

The Effect of BRCA1 on the Progesterone Receptor

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. The Effect of BRCA1 on the Progesterone Receptor

  2. Content 1 Background 2 Protocol and procedures 3 Result and Problems 4 Discussion and some ideas

  3. Background • 1 BRCA1 gene (breast cancer susceptibility gene 1 ) wild-type BRCA1 is a anti-oncogene. Investigations show that the brca1 mutation highly relates the tumorigenesis of mammary gland.

  4. Data: BRCA1 mutation rate Hereditary breast cancer family 45% Hereditary breast cancer and ovary family 90% In sporadic breast cancer ,brca1 tumor is also found.

  5. BRCA1 function: • cell-cycle, DNA-break repair, apoptosis • an so on.

  6. 2 PR (progesterone receptor) two isoforms :PRA and PRB they are two different transcripts from the same gene with different promoters and transcription initiation site. The difference is PRA lacking 164 amino acid in the N-terminal

  7. .

  8. PR is a ligand-dependent transcription factor P-PR complex bind with PRE (progesterone response element)

  9. PR expression In the normal mammary gland and cell-lines , the expression level is the same . The balance is lost in the breast cancers. PR and breast cancer?

  10. 3 PR , BRCA1 and breast cancer? a . estrogen’s (E2) function on breast caner is well –established. b HRT (hormone replace therapy) increase the incidence rate of breast cancer E2 +P (progesterone ) > E2 alone c steroid hormone : glucocorticoid E2 ,androgen and P brca1 has effect on glucocorticoid receptor ,ER, androgen receptor

  11. whether the BRCA1 can modulate the PR

  12. 2 protocol and procedure • the whole project include 2 parts A . Whether ? B How?

  13. whether BRCA1 can modulate the expression of PR • Cell lines MCF-7 adherent ,adenocarcinoma epithelial cell ER(+) PR(+)

  14. T47D Ductal carcinoma adherent ER(+) PR(+)

  15. 2 PROTOCOL • A) knockdown the BRCA1 in MCF-7 and T47D BRCA1-specific SiRNA sequence: 5’-AAT GCC AAA GTA GCT AAT GA-3’ scrambled SiRNA (as a negative control): 5’-GTC ACG ATA AGA CAA TGA TAT-3

  16. western blotting -protein level change RT-PCR - RNA levelchange

  17. B) overexpression of wild-type BRCA1 • plasmid :pFlag-cmv2-brca1 wt pFlag –cmv2(control) pflag-cmv2-brca1 expression vector include full-length BRCA1 cDNA.

  18. 2 How the BRCA1 modulate A ) whether BRCA1 effect PR promoter CpG-methylation As we know ,higher CpG-methylation induce gene inactivation Konckdown ↘ genomic DNA Over expression ↗ ↓ methylation-specific PCR

  19. B) whether BRCA1 can directly interact with PRA or PRB promoter • EMSA( electrophoretic mobility shift assay)

  20. MCF-7 T47D marker PRB 114KD PRA 94KD b-actin 43KD 3 Result and Problems • 1 PR expression in two cell lines • Total protein :100ug. • Conclusion:PRB expression is higher than PRA

  21. Brca1 220kd PRB 114KD PRA 94KD 3 2 1 2 RNAi in T47D • Lane 1 :T47D transfected with brca1-SiRNA(80nmol/L) • Lane 2 :T47D transfected with scramled-SiRNA (negative control) • Lane 3 : T47D without RNAi • No change ?

  22. 3 RT-PCR 3 RT-PCR problem met in RT-PCR: RNA isolation OD260=0.717 OD280=0.390 OD260/OD280=1.84 (1.80-2.0) NO band in PCR product?

  23. 28s 18s

  24. 4 discussion and some ideas • 1 progesterone receptor function pathway • pr is a ligand-dependent transcription factor • P • ↓？ • PR ↓ ？ • PRE • ↙ ↘ • proliferation differentiation

  25. 2 PRA and PRB isoforms They have different function on transcription PRB enhance the transcription PRA inhibit the transcription through inhibiting the PRB function • whether BRCA1 change the Progesterone affinity with PRA or PRB preferentially?

  26. 3 differentiation reference article: Progesterone receptor induces cellular differentiation in MDA-MB-231 Breast cancer cells Transfected with Progesterone receptor Complementary DNA. American journal of pathology.vol.162.No.6.june,2003 • MDA-MB-231 : PR(-) ER(-) E-cadherin ,a differentiation marker. Brca1 function :cell-cycle,apoptosis

  27. Plan : • stable cell-lines • transfect the MDA-MB-231 with pra/prb expression vector knockdown ↘ e-cadherin change overexpersssion ↗

  28. 4 proliferation and apoptosis • two characteristics of tumor : • malignant proliferation and lower differentiation • Reference article: estrogen-induced loss of progesterone receptor expression in normal and malignant ovarian surface epithelial cells. • Oncogene 2005 24,4386-4400

  29. it refer : • P induce ovarian cancer cell lines apoptosis Antiprogestin induced apoptosis in MCF-7 • Using caspase-3 as a apoptosis Marker • Brca1 induced apoptosis through activating caspase-9 • (reference article?)

  30. Question 1 : • in apoptosis pathway , • the relationship of caspase-3 and caspase-9？ • Question 2 ： • whether there is a interacting effect between BRCA1 and PR in inducing apoptosis?

  31. that's all ,thank you! that's all,thank you!

More Related