Cloning and functional analysis of FLJ20420: A novel BAG-1 promoter transcription factor

陈 军陈 军天津市肺癌研究所天津市肺癌研究所

天津医科大学总医院肺部肿瘤外科 天津医科大学总医院肺部肿瘤外科

The structure of human BAG family proteinsThe structure of human BAG family proteins

Li et al., Chin J Lung Cancer. 2009,

BACKGROUND: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

BAGBAG--11

Growth factor receptor (HGF, PDGF)

Apaf-1

Cytochorome C

Bcl-2Bax

Protein Refolding

Raf-1Ras

P

P

Apoptosis

Drug Resistance

Hormone Receptor (ER/PR)

Ubiquitin/Proteasome Complex

Protein Degradation

Hsp 70

Hsp 70

p53

Cell Cycle Control

Ubiquitin PathwaysBAGBAG--11

Growth factor receptor (HGF, PDGF)

Apaf-1

Cytochorome C

Bcl-2Bax

Protein Refolding

Raf-1Ras

PPP

PPPP

Apoptosis

Drug Resistance

Apoptosis

Drug Resistance

Hormone Receptor (ER/PR)

Ubiquitin/Proteasome Complex

Protein Degradation

Hsp 70

Hsp 70

p53

Cell Cycle Control

Ubiquitin Pathways

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:DESIGN:

Our lab was the first to isolate the human BAG-1 promoter

and characterize its functions (Yang et al., 1999). Since B

AG-1 is highly over-expressed in human cervical cancer H

ela 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 So

uthernwestern 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 FLJ20420-silenGene expression profile of FLJ20420-silen

ced A549 cellsced A549 cells

Microarray analysis revealed:

655 upregulated genes, including 272 known ge

nes (272/38,500 = 0.706%).

619 downregulated genes, including 233 known

genes (233/38,500 = 0.605%).

PathwayPathway Gene SymbolGene Symbol

AApoptosipoptosi AKT1AKT1, , BCL2L1BCL2L1, , CASP3, CASP7, CFLAR, CYCS, IL1RAP, PIK3CB, TNFRSF10A

Calcium signaling pathwayCalcium signaling pathway ADRB2, CAMK2D, ERBB2, MYLK, OXTR, PLCB4, PTK2B, VDAC3

cell cyclecell cycle CCNE2, CDC2, CDK6, MAD1L1, TGFB2,

ECM-receptor interactionECM-receptor interaction COL5A1, , ITGA11, , ITGA2, , SPP1, , THBS1, , VTN

ErbB signaling pathwayErbB signaling pathway AKT1, CAMK2D, CBLB, ERBB2, GAB1, PIK3CB, RPS6KB1

Insulin signaling pathwayInsulin signaling pathway AKT1, CBLB, MKNK2, PDE3A, PFKL, PIK3CB, PPP1R3C, PTPRF, RAPGEF1, RHEB, RHOQ, RPS6KB1

Jak-STAT signaling pathwayJak-STAT signaling pathway AKT1, BCL2L1, CBLB, CSF2, IFNAR1, IL6, IL6R, IL6ST, JAK3, LEPR, PIAS1, PIK3CB

MAPK signaling pathwayMAPK signaling pathway AKT1, BDNF, CASP3, DUSP3, FGF2, FGFR2, JUND, MAP3K8, MKNK2, MRAS, PDGFA, PLA2G12A, PPM1A, RASGRP1, STK4, TAOK1, TGFB2, TGFBR1

p53 signaling pathwayp53 signaling pathway CASP3, CCNE2, CDC2, CDK6, CYCS, IGFBP3, PERP, PTEN, THBS1

TGF-beta signaling pathwayTGF-beta signaling pathway ACVR2A, ID2, INHBC, RPS6KB1, SMAD5, TGFB2, TGFBR1, THBS1

Toll-like receptor signaling Toll-like receptor signaling pathwaypathway

AKT1, IFNAR1, IL6, IRF5, MAP3K8, PIK3CB, SPP1, TLR1, TRAF3

Wnt signaling pathwayWnt signaling pathway CAMK2D, PLCB4, PPP2R5E, TCF7L1, VANGL1, WNT6CAMK2D, PLCB4, PPP2R5E, TCF7L1, VANGL1, WNT6

Gene expression profile of FLJ20420-silenced A549 cellsGene expression profile of FLJ20420-silenced A549 cells

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 Identified and characterized a negative BAG-1 regulating

transcription factor FLJ20420. transcription factor FLJ20420.

• There is a high expression level of FLJ20420 in lung cancer There is a high expression level of FLJ20420 in lung cancer

tissues, compared to the paired lung tissues; tissues, compared to the paired lung tissues;

• FLJ20420 plays an important role in the apoptosis and FLJ20420 plays an important role in the apoptosis and

oncogenesis of lung caner.oncogenesis of lung caner.

ACKNOWLEGEMENTACKNOWLEGEMENT

• 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 S

urgery• Staffs in Lung Cancer Ins

titute

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!Thank You!