The Effect of BRCA1 The Effect of BRCA1 on the Progesterone on the Progesterone

Receptor Receptor

Content Content

1 Background 1 Background

2 Protocol and procedures2 Protocol and procedures

3 Result and Problems3 Result and Problems

4 Discussion and some ideas4 Discussion and some ideas

BackgroundBackground

• 1 BRCA1 gene (breast cancer susceptib1 BRCA1 gene (breast cancer susceptibility gene 1 )ility gene 1 )

wild-type BRCA1 is a anti-oncogene.wild-type BRCA1 is a anti-oncogene. Investigations show that the brca1 Investigations show that the brca1

mutation highly relates themutation highly relates the tumorigenesis of mammary gland.tumorigenesis of mammary gland.

• Data:Data: BRCA1 mutation BRCA1 mutation

rate rate

Hereditary breast cancer family 45% Hereditary breast cancer family 45% Hereditary breast cancer and ovary family Hereditary breast cancer and ovary family 90%90%

In sporadic breast cancer ,brca1 In sporadic breast cancer ,brca1 tumor is also found.tumor is also found.

• BRCA1 function:BRCA1 function:

• cell-cycle, DNA-break repair, cell-cycle, DNA-break repair, apoptosis apoptosis

• an so on.an so on.

• 2 PR (progesterone receptor) 2 PR (progesterone receptor) two isoforms :PRA and PRB two isoforms :PRA and PRB they are two different transcripts frothey are two different transcripts fro

m the same gene with different promm the same gene with different promoters and transcription initiation site.oters and transcription initiation site.

The difference is PRA lacking 164 amino The difference is PRA lacking 164 amino acid in the N-terminalacid in the N-terminal

• ..

•PR is a ligand-dependent trPR is a ligand-dependent transcription factor anscription factor P-PR complex bind with PP-PR complex bind with P

RE (progesterone response RE (progesterone response element) element)

PR expressionPR expression

In the normal mammary gland and cell-In the normal mammary gland and cell-lines , the expression level is the samlines , the expression level is the same . The balance is lost in the breast ce . The balance is lost in the breast cancers. ancers.

PR and breast cancer?PR and breast cancer?

• 3 PR , BRCA1 and breast cancer? 3 PR , BRCA1 and breast cancer? a . estrogen’s (E2) function on breast caner is well –establisha . estrogen’s (E2) function on breast caner is well –establish

ed. ed. b HRT (hormone replace therapy) b HRT (hormone replace therapy) increase the incidence rate of breast cancerincrease the incidence rate of breast cancer

E2 +P (progesterone ) > E2 aloneE2 +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 recbrca1 has effect on glucocorticoid receptor ,ER, androgen rec

eptor eptor

• whether the BRCA1 can whether the BRCA1 can modulate the PR modulate the PR

2 protocol and procedure 2 protocol and procedure

• the whole project include 2 partsthe whole project include 2 parts

A . Whether ?A . Whether ?

B How?B How?

whether BRCA1 can modulatwhether BRCA1 can modulate the expression of PRe the expression of PR

11 Cell linesCell lines

MCF-7 MCF-7 adherent ,adenocarcinomaadherent ,adenocarcinoma epithelial cellepithelial cell

ER(+)ER(+)

PR(+)PR(+)

• T47D T47D Ductal carcinomaDuctal carcinoma

adherent adherent

ER(+) ER(+) PR(+)PR(+)

•

• 2 PROTOCOL2 PROTOCOL

• A) knockdown the BRCA1 in MCF-7 and T47DA) 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-35’-GTC ACG ATA AGA CAA TGA TAT-3

western blotting -protein level changewestern blotting -protein level change

RT-PCR - RNA levelRT-PCR - RNA level changechange

• B) overexpression of wild-type BRCA1 B) overexpression of wild-type BRCA1 • plasmid :pFlag-cmv2-brca1 wtplasmid :pFlag-cmv2-brca1 wt pFlag –cmv2(control)pFlag –cmv2(control) pflag-cmv2-brca1 expression vector inpflag-cmv2-brca1 expression vector in

clude full-length BRCA1 cDNA.clude full-length BRCA1 cDNA.

2 How the BRCA1 modulate2 How the BRCA1 modulate

A ) whether BRCA1 effect PR promoter CpG-methylA ) whether BRCA1 effect PR promoter CpG-methylation ation

As we know ,higher CpG-methylation induce gene iAs we know ,higher CpG-methylation induce gene inactivation nactivation

Konckdown ↘ genomic DNAKonckdown ↘ genomic DNAOver expression ↗ Over expression ↗ ↓↓ methylation-specific PCR methylation-specific PCR

• B) whether BRCA1 can directly interaB) whether BRCA1 can directly interact with PRA or PRB promoterct with PRA or PRB promoter

• EMSA( electrophoretic mobility shift assay)EMSA( electrophoretic mobility shift assay)

3 Result and Problems 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

2 RNAi in T47D2 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 RNAiLane 3 : T47D without RNAi

• No change ?No change ?

3 2 1

Brca1 220kd

PRB 114KD

PRA 94KD

3 RT-PCR 3 RT-PCR

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=0.390OD280=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?

28s

18s

4 discussion and some ideas4 discussion and some ideas

• 1 progesterone receptor function pathway1 progesterone receptor function pathway

• pr is a ligand-dependent transcription factorpr is a ligand-dependent transcription factor• PP• ↓↓ ？？• PR PR

↓ ↓ ？？• PREPRE• ↙ ↘↙ ↘• proliferation differentiation proliferation differentiation

2 PRA and PRB isoforms2 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 PRA inhibit the transcription through inhibiting the

PRB functionPRB function• whether BRCA1 change the Progesterone affinitwhether BRCA1 change the Progesterone affinit

y with PRA or PRB preferentially?y with PRA or PRB preferentially?

• •

3 differentiation 3 differentiation

reference article:reference article:Progesterone receptor induces cellular differentiation in MDA-MB-Progesterone receptor induces cellular differentiation in MDA-MB-

231231Breast cancer cells Transfected with Progesterone receptor CompBreast cancer cells Transfected with Progesterone receptor Comp

lementary DNA. American journal of pathology.vol.162.No.6.julementary DNA. American journal of pathology.vol.162.No.6.june,2003ne,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,apoptosisBrca1 function :cell-cycle,apoptosis

• Plan :Plan :• stable cell-linesstable cell-lines• transfect the MDA-MB-231 with pra/ptransfect the MDA-MB-231 with pra/p

rb expression vector rb expression vector knockdown ↘ e-cadherin changeknockdown ↘ e-cadherin change overexpersssion ↗overexpersssion ↗

• 4 proliferation and apoptosis4 proliferation and apoptosis• two characteristics of tumor :two characteristics of tumor :• malignant proliferation and lower differentiatiomalignant proliferation and lower differentiatio

nn

• Reference article: estrogen-induced loss of progesterReference article: estrogen-induced loss of progesterone receptor expression in normal and malignant ovone receptor expression in normal and malignant ovarian surface epithelial cells.arian surface epithelial cells.

• Oncogene 2005 24,4386-4400Oncogene 2005 24,4386-4400•

• 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 MarkerUsing caspase-3 as a apoptosis Marker

• • Brca1 induced apoptosis through activating caspase-9Brca1 induced apoptosis through activating caspase-9• (reference article?)(reference article?)•

•

• Question 1 :Question 1 :• in apoptosis pathwayin apoptosis pathway , ,• the relationship of caspase-3 the relationship of caspase-3

and caspase-9and caspase-9 ？？• Question 2 Question 2 ： ： • whether there is a interacting whether there is a interacting

effect between BRCA1 and PR in effect between BRCA1 and PR in inducing apoptosis?inducing apoptosis?