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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 )
wild-type BRCA1 is a anti-oncogene.
Investigations show that the brca1
mutation highly relates the
tumorigenesis of mammary gland.
• 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.
• BRCA1 function:
• cell-cycle, DNA-break repair, apoptosis
• an so on.
•
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
•
.
• PR is a ligand-dependent
transcription factor
P-PR complex bind with
PRE (progesterone response
element)
PR expression
In the normal mammary gland and celllines , the expression level is the
same . The balance is lost in the
breast cancers.
PR and breast cancer?
• 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
•
whether the BRCA1 can modulate
the PR
2 protocol and procedure
•
the whole project include 2 parts
A .
Whether ?
B
How?
whether BRCA1 can
modulate the expression of PR
1
Cell lines
MCF-7
adherent ,adenocarcinoma
epithelial cell
ER(+)
PR(+)
• T47D
Ductal carcinoma
adherent
ER(+)
PR(+)
•
•
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
western blotting
RT-PCR
-protein level change
- RNA level
change
• B) overexpression of wild-type BRCA1
•
plasmid :pFlag-cmv2-brca1 wt
pFlag –cmv2(control)
pflag-cmv2-brca1 expression vector
include full-length BRCA1 cDNA.
2 How the BRCA1 modulate
A ) whether BRCA1 effect PR promoter CpGmethylation
As we know ,higher CpG-methylation induce gene
inactivation
Konckdown
↘
Over expression ↗
genomic DNA
↓
methylation-specific PCR
• B)
whether BRCA1 can directly
interact with PRA or PRB promoter
•
EMSA( electrophoretic mobility shift assay)
3 Result
•1
and Problems
PR expression in two cell lines
marker
MCF-7 T47D
PRB 114KD
PRA 94KD
b-actin 43KD
• Total protein :100ug.
• Conclusion:PRB expression is higher than PRA
2
RNAi in T47D
Brca1 220kd
PRB 114KD
PRA
3
2
1
94KD
• 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 ?
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?
28s
18s
4 discussion and some ideas
• 1
•
•
•
•
•
•
•
progesterone receptor function pathway
pr is a ligand-dependent transcription factor
P
↓？
PR
↓？
PRE
↙
proliferation
↘
differentiation
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?
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
• Plan :
• stable cell-lines
• transfect the MDA-MB-231 with
pra/prb expression vector
knockdown
↘ e-cadherin change
overexpersssion ↗
•
•
•
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
• 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?)
• 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?