Transcript 幻灯片 1

Cloning and functional analysis of FLJ20420: A
novel BAG-1 promoter transcription factor
陈 军
天津市肺癌研究所
天津医科大学总医院肺部肿瘤外科
The structure of human BAG family proteins
Li et al., Chin J Lung Cancer. 2009,
BACKGROUND:
• BAG-1 is a multifunctional protein that interacts with a wide
range of target molecules to regulate apoptosis,
proliferation, transcription, metastasis and mobility (Tang et
al., 2002).
• BAG-1 is widely over-expressed in various human
malignancies and its expression may have clinical utility as
a prognostic marker in early breast cancer (Tang et al.,
2004).
• However, most studies focus on the mechanism by which
BAG-1 interacts with proteins to inhibit apoptosis. The
molecules that regulate BAG-1 over-expression in
malignant cells are currently unknown.
Chen et al, Oncogene, 2002
Chen et al, Oncogene, 2002
Growth factor receptor (HGF, PDGF)
P
Ras
P
Hsp 70
Raf-1
Cell Cycle
Control
Hsp 70
p53
Ubiquitin
Pathways
BAG-1
Bax
Protein
Refolding
Ubiquitin/Proteasome
Complex
Bcl-2
Cytochorome C
Apaf-1
Hormone Receptor (ER/PR)
Protein
Degradation
Apoptosis
Drug Resistance
Nucleotide sequence of human BAG-1 gene 5'- FLanking regions, putative
transcription factor binding sites and CpG island
Yang et al, Oncogene, 1998
Deletion analysis of the 5'-flanking
region of the BAG-1 gene
Yang et al, Oncogene, 1998
DESIGN:
Our lab was the first to isolate the human BAG-1 promoter
and characterize its functions (Yang et al., 1999). Since
BAG-1 is highly over-expressed in human cervical cancer
Hela cell line, we decided to identify transcription
molecules regulating BAG-1 expression using BAG-1
promoter as a probe to screen Human Hela cDNA library
λTripIEx by Southernwestern Blot
Figure 1. Sequence analysis of positive clone cDNA and deduced amino acid
sequence. Numbers indicate the positions of nucleotides.
Figure 2: FLJ 20420 protein expression. (a) GST-FLJ20420 fusion protein (b)
His-FLJ20420 protein.
Figure 3. Gel shift assay. The entire 890 bp of BAG-1 protmoter was separated
finally into several ~30-50 bp DNA fragment by PCR and then used as probe
to perform gel shift assay with purified GST-FLJ20420 fusion protein (+).
Purified GST protein was used as a control (v). (a) EMSA; (b) competition test
of EMSA.
Figure 4. FLJ20420
mRNA expression in
different tissues and cell
lines. Northern blot
analysis of FLJ20420
mRNA expression in
human normal tissues (A)
and various tumor cell
lines (B); (C-D) Real-time
PCR analysis of t
FLJ20420 and BAG-1
mRNA expression in lung
cancer cell lines. (E)
Immunoblot analysis of
BAG-1 protein expression.
Figure 5: (A) Luciferase assay with BAG-1 promoter. (B) The knockdown of FLJ20420
expression in A549 and L9981 cell lines. (C) Microarrays of A549-FLJ-siRNA-1 and
L9981-FLJ-siRNA-1 FLJ20420 gene silencing cells. (D) BAG-1 mRNA expression in the
proper transfected cells. (D) BAG-1 protein expression in the proper transfected cells .
Figure 6: Function of the FLJ20420 gene. The subcellular localization of
FLJ20420 protein. (a) FLJ20420 expression in Hela cells. (b) FLJ20420
expression in NIH/3T3 cells. FLJ20420 fusion protein (Green); Phalloidin (red) ；
DAPI (blue)
Figure 6: Function of the FLJ20420 gene. (B) Cell cycle analysis was evaluated by
FACS after using PI to stain the cellular DNA. (C) Cell viability assay by the MTT
assay. (D) Cells transfected with FLJ20420 siRNA were treated with cisplatin for 24
hours and then analyzed by the MTT assay. (E) Cell apoptosis assay by FACS.
Gene expression profile of FLJ20420silenced A549 cells
Microarray analysis revealed:
655 upregulated genes, including 272 known
genes (272/38,500 = 0.706%).
619 downregulated genes, including 233 known
genes (233/38,500 = 0.605%).
Gene expression profile of FLJ20420-silenced A549 cells
Pathway
Gene Symbol
Apoptosi
AKT1, BCL2L1, CASP3, CASP7, CFLAR, CYCS, IL1RAP, PIK3CB,
TNFRSF10A
Calcium signaling pathway
ADRB2, CAMK2D, ERBB2, MYLK, OXTR, PLCB4, PTK2B, VDAC3
cell cycle
CCNE2, CDC2, CDK6, MAD1L1, TGFB2,
ECM-receptor interaction
COL5A1, ITGA11, ITGA2, SPP1, THBS1, VTN
ErbB signaling pathway
AKT1, CAMK2D, CBLB, ERBB2, GAB1, PIK3CB, RPS6KB1
Insulin signaling pathway
AKT1, CBLB, MKNK2, PDE3A, PFKL, PIK3CB, PPP1R3C, PTPRF,
RAPGEF1, RHEB, RHOQ, RPS6KB1
Jak-STAT signaling pathway
AKT1, BCL2L1, CBLB, CSF2, IFNAR1, IL6, IL6R, IL6ST, JAK3, LEPR,
PIAS1, PIK3CB
MAPK signaling pathway
AKT1, BDNF, CASP3, DUSP3, FGF2, FGFR2, JUND, MAP3K8, MKNK2,
MRAS, PDGFA, PLA2G12A, PPM1A, RASGRP1, STK4, TAOK1, TGFB2,
TGFBR1
p53 signaling pathway
CASP3, CCNE2, CDC2, CDK6, CYCS, IGFBP3, PERP, PTEN, THBS1
TGF-beta signaling pathway
ACVR2A, ID2, INHBC, RPS6KB1, SMAD5, TGFB2, TGFBR1, THBS1
Toll-like receptor signaling
pathway
AKT1, IFNAR1, IL6, IRF5, MAP3K8, PIK3CB, SPP1, TLR1, TRAF3
Wnt signaling pathway
CAMK2D, PLCB4, PPP2R5E, TCF7L1, VANGL1, WNT6
Figure 7: Real-time PCR analysis of gene expression in A549 cells transfected with
FLJ20420 siRNA. (A) The expression level changes of 15 genes shown to be
upregulated (15/17). (B) The expression level changes of 12 genes shown to be
downregulated (12/15) .
Figure 8: FLJ20420 and BAG-1 expression in lung cancer tissues by Microarray
(total 72 paired specimens, which included 29 adenocarcinomas and 43 squamous
cell carcinomas).
Summary
• Identified and characterized a negative BAG-1 regulating
transcription factor FLJ20420.
• There is a high expression level of FLJ20420 in lung cancer
tissues, compared to the paired lung tissues;
• FLJ20420 plays an important role in the apoptosis and
oncogenesis of lung caner.
ACKNOWLEGEMENT
•
•
•
•
•
•
•
•
Dr. Qinghua Zhou
Dr. Hongyu Liu
Dr. Zhihao Wu
Dr. Ke Xu
Dr. Haisu Wan
Dr. Zhigang Li
Wu Heng
Staffs in Dept. of Lung
Surgery
• Staffs in Lung Cancer
Institute
Dr. Shou-Ching Tang
Dr. Nian Zhang
Dr. Allen Gao
The grants:
National Natural Science Foundation
of China ;
National “863” Plans
National Sic-Tech Support Program
Tianjin Sci-Tech Support Program
Thank You!