DcR3基因对胰腺癌裸鼠移植瘤化疗敏感性的影响

Effect of Decoy receptor 3 on chemosensitivity in nude mice model of pancreatic cancer

摘要:

目的探讨RNA 干扰沉默诱骗受体-3（DcR3）对人胰腺癌细胞裸鼠移植瘤化疗敏感性的影响及其可能机制。方法取对数生长期的AsPC-1 细胞1×106个/ml，接种于5 周龄裸鼠右后肢腹股沟，当皮下肿瘤直径为8 mm 左右，随机分为4 组（ n=10）：对照组、化疗组、阴性质粒+ 化疗组、DcR3 siRNA+ 化疗组。观察DcR3 siRNA联合化疗药物对人胰腺癌AsPC-1细胞裸鼠移植瘤的治疗效果。应用ELISA和Western blot检测DcR3 蛋白的变化；TUNEL 分析肿瘤细胞凋亡；Western blot 和RT-PCR 检测FasL、Caspase-8 和Caspase-3 等凋亡因子的表达。结果化疗组、阴性质粒+ 化疗及DcR3 siRNA+ 化疗组均可抑制移植瘤的生长，与对照组比较，3 组抑瘤率平均值分别为（35.87±4.58）%、（40.68±4.16）%和（90.25±2.53）%，4 组间差异有统计学意义（F =47.736，P =0.000），DcR3 siRNA+ 化疗组抑瘤率较化疗组增加；对照组、化疗组、阴性质粒+ 化疗以及DcR3 siRNA+ 化疗组移植瘤重量平均值分别为（0.95±0.03）、（0.63±0.04）、（0.67±0.02）和（0.17±0.06）g，4 组间差异有统计学意义（F =85.531，P =0.000），DcR3 siRNA+ 化疗组瘤重较化疗组减轻；对照组、化疗组、阴性质粒+化疗组、DcR3 siRNA+化疗组凋亡率平均值分别为（6.3±2.21）%、（14.8±2.65）%、（14.5±3.06）%和（54.6±3.23）%，4 组间差异有统计学意义（F =104.225，P =0.000），DcR3 siRNA+ 化疗组凋亡率较化疗组增加；DcR3siRNA+化疗组的FasL、Caspase-8 和Caspase-3 蛋白表达较其他各组上升（P =0.000）。结论沉默DcR3 可激活FasL/Caspase 凋亡途径，促进细胞凋亡，增加胰腺癌细胞移植瘤对化疗的敏感性。

Abstract:

Objective To investigate the effect of Decoy receptor 3 (DcR3) on chemosensitivity of human pancreatic cancer cells and potential mechanisms. Methods Pancreatic cancer AsPC-1 cells in log-growth phase (1×106 cells) were subcutaneously injected in nude mice. Expression of DcR3 was blocked through small interfering ribonucleic acid (siRNA). Seven days post injection, tumor-bearing mice were then randomly divided into 4 groups (n = 10 group): control group, chemotherapy group, sham siRNA + chemotherapy group and DcR3 siRNA + chemotherapy. Xenograft of human pancreatic cancer AsPC-1 cells was measured among the groups. Expression level of DcR3 was determined by ELISA and Western blot. Apoptosis rate was detected by TUNEL analysis. Expression levels of FasL protein, Caspase-8, and Caspase-3 were measured by Western blot and RT-PCR. Results Tumor weight (gram) in chemotherapy group, sham siRNA + chemotherapy group and DcR3 siRNA + chemotherapy group was significantly decreased when compared with control group (0.63 ±0.04 vs 0.95 ±0.03, P= 0.000, 0.67 ±0.02 vs 0.95 ±0.03, P= 0.000, 0.17 ±0.06 vs 0.95 ±0.03,P =0.000), respectively. Silencing of DcR3 expression dramatically reduced weight of tumor when compared with sham siRNA + chemotherapy group (0.17 ±0.06 vs 0.67 ±0.02 vs 0.95 ±0.03, P= 0.000). Inhibitory rate of tumor growth in chemotherapy group, sham siRNA + chemotherapy group and DcR3 siRNA + chemotherapy group increased when compared with control group [(35.87 ±4.58)% vs (0.00±0.00) %, P=0.000, (40.68 ±4.16)% vs (0.00 ±0.00) %, P=0.000, (90.25 ±2.53)% vs (0.00 ±0.00) %, P=0.000], respectively. Silencing of DcR3 expression dramatically increased inhibitory rate of tumor growth when compared with sham siRNA +chemotherapy group [(90.25 ±2.53) % vs (0.67±0.02) % vs (40.68 ±4.16) %, P= 0.000]. Apoptosis rate in chemotherapy group, sham siRNA + chemotherapy group and DcR3 siRNA + chemotherapy group were significantly increased when compared with control group [(14.8 ±2.65)% vs (6.3 ±2.21) %, = 0.000, (14.5 ±3.06) % vs (6.3 ±2.21) % DcR3 expression dramatically reduced weight of tumor when compared with sham siRNA + chemotherapy group [(54.6 ±3.23)% vs (14.5 ±3.06) %, P= 0.000]. The expression levels of FasL, Caspase-8, and Caspase-3 in DcR3 siRNA + chemotherapy group were downregulated when compared with the remaining 3 groups ( P=0.000). Conclusion Silencing of DcR3 gene can increase the chemosensitivity of human pancreatic cancer by promoting FasL/Caspase-mediated cells apoptosis.