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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 )

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The Effect of BRCA1 on the Progesterone Receptor

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  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!

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