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Gene silencing of BAG-1 modulates apoptotic genes and sensitizes lung cancer cell lines to cisplatin-induced apoptosis 刘红雨 陈 军 天津市肺癌研究所 天津医科大学总医院 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). Chen et al, Oncogene, 2002 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 A549BAG-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. 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. 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. 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 Pathways associated with differential expression genes in A549-BAG-1 gene silencing cells. Pathway Gene Symbol MAPK signaling pathway EGFR、FGF2、GADD45β、HRAS、IL1A、JUN、MAPK1 MAP3K2、MAP3K7、NF-κB2 NR4A1、RPS6KA5、TGFβ2 TGF-beta receptor signaling pathway TGFβ2、E2F5、ID2、MAPK1、KLF11、CDC27、SERPINE1 NF-κB sinaling pathway CARD8、NF-κB2、KAT2B、IL1A VEGF family signaling HRAS、MAPK1、VEGFα、NRP1、NRP2、FN、BRCA1、 EPAS1 Wnt signaling pathway JUN、MAP3K7、FZD1、CLDN1、TCF3 p53 signaling CCNE2、GADD45β、IGFBP3、SERPINE1 B cell receptor signaling pathway HRAS、JUN、LYN、NF-κB2 Cell cycle CCNE2、TGFβ2、GADD45β、HDAC4、EVI5、CUL2、 CDKN2C、NDC80、CDC27、ID2、BCAR1、BTG2、 KAT2B Cell apoptosis CASP7、IL1A、NF-κB2、TNFRSF10D、PDCD6IP 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). Figure 3 (B) The expression level changes of 40 genes shown to be downregulated by microarray (34/40). 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-α. • 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. Figure 4. Western blot analysis of metastasis-related genes in A549 cells transfected with BAG-1 or control siRNA 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. 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. 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”; “973” Plans National Sic-Tech Support Program Tianjin Sci-Tech Support Program Thank You!