Gene silencing of BAG-1 modulates apoptotic genes and sensitizes lung cancer cell lines to cisplatin-induced apoptosis

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Gene silencing of BAG-1 modulates apoptotic genes
and sensitizes lung cancer cell lines to
cisplatin-induced apoptosis

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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) and lung cancer (Liu
  et al., Chin J Lung Cancer, 2009).

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Chen et al, Oncogene, 2002
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DESIGN

1. First use BAG-1 siRNA to knockdown the expression
   of BAG-1 in lung cancer cell lines A549 and L9981
   to characterize its function.
2. To analyze the change of gene expression profile
   in A549-BAG-1 silencing cells by Microarray.
   Furthermore, using Real-time PCR and western blot
   to confirm the change.
3. To transfect the changed gene into A549 and L9981
   cells with BAG-1 silencing to reverse its
   function.

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Figure 1. Knockdown of BAG-1 expression in the
A549 and L9981 lung cancer cell lines. (A)
Real-time PCR (B) Western blot for anti-BAG-1.
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Figure 2. Characterization of transfected A549
and L9981 cells. (A) MTT cell viability assay.
(B) Annexin V-FITC flow cytometry assay. (C) Cell
cycle analysis was evaluated with FACS using PI.
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Altered gene expression profile by Bag-1 siRNA
silencing
177 up-regulated genes 145 genes with known
function
412 down-regulated genes 256 genes with known
function
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Pathways associated with differential expression
genes in A549-BAG-1 gene silencing cells.
Pathway Gene Symbol
MAPK signaling pathway EGFR?FGF2?GADD45ß?HRAS?IL1A?JUN?MAPK1 MAP3K2?MAP3K7?NF-?B2 NR4A1?RPS6KA5?TGFß2
TGF-beta receptor signaling pathway TGFß2?E2F5?ID2?MAPK1?KLF11?CDC27?SERPINE1
NF-?B sinaling pathway CARD8?NF-?B2?KAT2B?IL1A
VEGF family signaling HRAS?MAPK1?VEGFa?NRP1?NRP2?FN?BRCA1?EPAS1
Wnt signaling pathway JUN?MAP3K7?FZD1?CLDN1?TCF3
p53 signaling CCNE2?GADD45ß?IGFBP3?SERPINE1
B cell receptor signaling pathway HRAS?JUN?LYN?NF-?B2
Cell cycle CCNE2?TGFß2?GADD45ß?HDAC4?EVI5?CUL2?CDKN2C?NDC80?CDC27?ID2?BCAR1?BTG2?KAT2B
Cell apoptosis CASP7?IL1A?NF-?B2?TNFRSF10D?PDCD6IP
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Figure 3. Real-time PCR analysis of gene
expression in A549 cells transfected with BAG-1
siRNA. (A) The expression level changes of 34
genes shown to be upregulated by microarray
(30/34).
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Figure 3 (B) The expression level changes of 40
genes shown to be downregulated by microarray
(34/40).
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Gene expression chip assay

• Microarray assay the expression of 589 genes was
  altered, including 177 upregulated (145/177) and
  412 downregulated genes (256/412).
• Real-time PCR assay of the changes in gene
  expression indicated a concordance of 86.48
  (64/74) with that observed by microarray.
• The upregulated genes included EGFR, Jun, HSPA1A
  and CASP7, while the downregulated genes included
  Lyn, HRAS, and TGF-a.
• Furthermore, by Western blot, the expression of
  EGFR, Jun, and HSPA1A were significantly
  increased, while expression of Lyn and HRAS were
  dramatically decreased after BAG-1 gene
  silencing.

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Figure 4. Western blot analysis of
metastasis-related genes in A549 cells
transfected with BAG-1 or control siRNA
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Figure 5. Overexpression of Lyn in A549-BAG-1
silencing cells. (A) Real-time PCR analysis of
Lyn expression in A549 cells transfected with
different vectors. (B) MTT cell viability assay.
(C) Annexin V-FITC flow cytometry assay.
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Summary

• After transfection with BAG-1 siRNA, lung cancer
  cell lines A549 and L9981 exhibited significantly
  enhanced sensitivity to cisplatin-induced
  apoptosis.
• Thus, silencing of the BAG-1 gene resulted in
  changes in expression of apoptosis-related genes
  and sensitized A549 and L9981 cells to
  cisplatin-induced apoptosis.

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ACKNOWLEGEMENT
Dr. Shou-Ching Tang Dr. Nian Zhang Dr. Allen
Gao The grants National Natural Science
Foundation of China National 863 973
Plans National Sic-Tech Support Program Tianjin
Sci-Tech Support Program

• 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

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Thank You!