1. The Effect of BRCA1 on the Progesterone Receptor

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

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

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

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

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

7. .

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

9. PR expression 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? PR and breast cancer?

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

11. whether the BRCA1 can modulate the PR whether the BRCA1 can modulate the PR

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

13. whether BRCA1 can modulate the expression of PR whether BRCA1 can modulate the expression of PR 1 Cell lines MCF-7 MCF-7 adherent,adenocarcinoma adherent,adenocarcinoma epithelial cell epithelial cell ER(+) ER(+) PR(+) PR(+)

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

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

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

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

18. 2 How the BRCA1 modulate 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 methylation-specific PCR

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

20. 3 Result and Problems 1 PR expression in two cell lines 1 PR expression in two cell lines Total protein :100ug. Total protein :100ug. Conclusion:PRB expression is higher than PRA Conclusion:PRB expression is higher than PRA PRB 114KD PRA 94KD T47DMCF-7marker b-actin 43KD

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

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

23. 28s 18s

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

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

26. 3 differentiation 3 differentiation reference article: 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 : MDA-MB-231 : PR(-) ER(-) PR(-) ER(-) E-cadherin,a differentiation marker. E-cadherin,a differentiation marker. Brca1 function :cell-cycle,apoptosis Brca1 function :cell-cycle,apoptosis

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

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

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

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