摘要
背景与目的:大部分胰腺癌具有高表达诱骗受体-3(decoy receptor 3,Dc R3)的特征,而后者与Fas L凋亡途径相关,可能导致胰腺癌对化疗耐药。该研究旨在探讨RNA干扰沉默Dc R3基因对人胰腺癌细胞化疗药物敏感性的影响及其可能机制。方法:构建带有Dc R3-si RNA序列的稳定表达质粒,通过LipofectamineTM2000转染至人胰腺癌细胞As PC-1细胞株,筛选出转染后稳定低表达Dc R3的胰腺癌细胞,同时设未转染对照组(control组)和转染阴性质粒对照组(mock组)。应用ELISA和蛋白[质]印迹法(Western blot)检测各组As PC-1细胞中Dc R3的蛋白表达;MTT实验检测各组As PC-1细胞对吉西他滨的敏感性;流式细胞术检测各组As PC-1细胞凋亡情况;Western blot和实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测各组As PC-1细胞中Fas L、Caspase-8、Caspase-3蛋白和m RNA的表达。结果:转染Dc R3-si RNA后As PC-1细胞中Dc R3蛋白较其他对照组明显降低;转染Dc R3-si RNA后As PC-1细胞对吉西他滨的敏感性显著增加;沉默Dc R3基因可以上调Fas L、Caspase-8和Caspase-3的表达并促进吉西他滨诱导的细胞凋亡。结论:RNA干扰沉默Dc R3基因可激活Fas L/Caspase凋亡途径,促进肿瘤细胞凋亡,增加人胰腺癌As PC-1细胞对化疗药物的敏感性。
Background and purpose: It has been demonstrated that decoy receptor 3 (DcR3) is overexpressed in pancreatic cancer, and DcR3 correlates with the expression of FasL, which may contribute to chemotherapy resistance in pancreatic cancer. The purpose of this study was to investigate the effect of RNA interference silencing DcR3 gene on chemosensitivity of human pancreatic cancer cells and its possible mechanism. Methods: A stable expression plasmid with DcR3-siRNA sequence was constructed and transfected into human pancreatic cancer cell line AsPC-1 by LipofectamineTM2000. The DcR3-expressing pancreatic cancer cells with stable and low expression were selected. The protein expression of DcR3 in AsPC-1 cells was detected by ELISA and Western blot. MTT assay was used to detect the sensitivity of each group of AsPC-1 cells to gemcitabine. Flow cytometry was used to detect the apoptosis of AsPC-1 cells after treatment with gemcitabine. The expressions of FasL, Caspase-8, Caspase-3 and mRNA in AsPC-1 cells were detected by Western blot and real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR). Results: The expression of DcR3 protein in AsPC-1 cells was significantly lower than those in the other control groups. The sensitivity of AsPC-1 cells to gemcitabine was significantly increased after transfection with DcR3- siRNA compared with control and mock cell lines. Transfection of DcR3-siRNA significantly increased the apoptosis of AsPC-1 cells induced by chemotherapeutic drugs. The expression of FasL, Caspase-8 and Caspase-3 protein and mRNA were up-regulated after transfection with DcR3-siRNA. Conclusion: RNA interference silencing DcR3 gene can activate FasL/Caspase apoptosis pathway, promote tumor cell apoptosis and increase the sensitivity of human pancreatic cancer AsPC-1 cells to gemcitabine.
出处
《中国癌症杂志》
CAS
CSCD
北大核心
2017年第11期873-878,共6页
China Oncology
基金
湖南省自然科学基金(14JJ3136)
郴州市科技局科研基金(CZ2013096)
关键词
诱骗受体-3
RNA干扰
胰腺癌
凋亡
化疗敏感性
Decoy receptor 3
RNA interference
Pancreatic cancer
Apoptosis
Chemosensitivity