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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?