刘红雨 陈 军 天津市肺癌研究所 天津医科大学总医院

1. 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

2. 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).

3. Chen et al, Oncogene, 2002

4. DESIGN: • First use BAG-1 siRNA to knockdown the expression of BAG-1 in lung cancer cell lines A549 and L9981 to characterize its function. • 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. • To transfect the changed gene into A549 and L9981 cells with BAG-1 silencing to reverse its function.

5. 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.

6. 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.

7. 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

8. Pathways associated with differential expression genes in A549-BAG-1 gene silencing cells.

9. 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).

10. Figure 3 (B) The expression level changes of 40 genes shown to be downregulated by microarray (34/40).

11. 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.

12. Figure 4. Western blot analysis of metastasis-related genes in A549 cells transfected with BAG-1 or control siRNA

13. 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.

14. 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.

16. 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

17. Thank You!