Zhang Weijiang, Zhou Jianwei, Zhu Xiaoqing, Yuan Huadi
Department of Gynecology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310000, China.
Department of Day Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310000, China.
Gene. 2017 Sep 5;627:420-427. doi: 10.1016/j.gene.2017.06.055. Epub 2017 Jun 29.
TNF-related apoptosis-inducing ligand (TRAIL) represents one potential and ideal anti-tumor drug, because it kills cancer cells specifically without targeting normal cells. However, acquired drug resistance to TRAIL usually impedes the clinical use of TRAIL on cancer patients. In the present study, we established in vitro TRAIL-resistant cervical cancer cell lines through long-term exposure to TRAIL. Interestingly, we observed significant upregulation of c-FLIP in TRAIL-resistant Hela and SiHa cells (Hela-TR and SiHa-TR) compared to their parental Hela and SiHa cells. Although Hela-TR and SiHa-TR cells exhibited low-sensitivity to TRAIL treatment, knockdown of c-FLIP significantly increased the cytotoxicity of TRAIL to them. In contrast to high protein level of c-FLIP, expression of miR-126 was significantly downregulated in Hela-TR and SiHa-TR cells. Results of western blot analysis, luciferase assays and bioinformatics proved that c-FLIP was the target of miR-126. Furthermore, as c-FLIP is the cellular antagonist to caspase-8, transfection with miR-126 promoted the activation of caspase-8 induced by TRAIL. As a result, miR-126 increased the TRAIL-induced apoptosis in Hela-TR and SiHa-TR cells. In addition, miR-126 was also able to increase the cytotoxicity of TNF-α and FasL (caspase-8 inducers) to Hela-TR and SiHa-TR. We demonstrate that miR-126 impairs drug resistance to TRAIL, TNF-α and FasL through inhibiting the expression of c-FLIP in cervical cancer.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）是一种潜在的理想抗癌药物，因为它能特异性杀死癌细胞而不靶向正常细胞。然而，对TRAIL获得性耐药通常会阻碍TRAIL在癌症患者中的临床应用。在本研究中，我们通过长期暴露于TRAIL建立了体外TRAIL耐药的宫颈癌细胞系。有趣的是，我们观察到与亲本Hela和SiHa细胞相比，TRAIL耐药的Hela和SiHa细胞（Hela-TR和SiHa-TR）中c-FLIP显著上调。虽然Hela-TR和SiHa-TR细胞对TRAIL治疗表现出低敏感性，但敲低c-FLIP显著增加了TRAIL对它们的细胞毒性。与c-FLIP的高蛋白水平相反，miR-126在Hela-TR和SiHa-TR细胞中的表达显著下调。蛋白质印迹分析、荧光素酶测定和生物信息学结果证明c-FLIP是miR-126的靶标。此外，由于c-FLIP是半胱天冬酶-8的细胞拮抗剂，用miR-126转染促进了TRAIL诱导的半胱天冬酶-8的激活。结果，miR-126增加了TRAIL诱导的Hela-TR和SiHa-TR细胞凋亡。此外，miR-126还能够增加TNF-α和FasL（半胱天冬酶-8诱导剂）对Hela-TR和SiHa-TR的细胞毒性。我们证明miR-126通过抑制宫颈癌中c-FLIP的表达来损害对TRAIL、TNF-α和FasL的耐药性。
