靶向 miR-20a 抑制 CFLAR 以增强 TRAIL 诱导的 HepG2 细胞凋亡敏感性。

Targeting of miR-20a against CFLAR to potentiate TRAIL-induced apoptotic sensitivity in HepG2 cells.

机构信息

Infectious Disease Department, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China.

出版信息

Eur Rev Med Pharmacol Sci. 2017 May;21(9):2087-2097.

Abstract

OBJECTIVE

Elevated expression of caspase-8 (CASP8) and Fas-associating protein with a novel death domain (FADD)-like apoptosis regulator (CFLAR) increases sensitivity against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cell apoptosis, but with an unclear mechanism. A previous study showed decreased microRNA-20a (miR-20a) expression in hepatocellular carcinoma (HCC) patient's tumor tissues. Bioinformatics analysis showed potential targeting relationship between the 3-UTR of CFLAR and miR-20a. This study investigated if miR-20a played a role in regulating CFLAR expression and HCC apoptosis.

PATIENTS AND METHODS

Expressions of miR-20a and CFLAR in model rat HCC tissues were compared to normal tissues. HCC patients were also collected for measuring miR-20a and CFLAR expressions between tumor and adjacent tissues. Dual-luciferase reporter gene assay was performed to evaluate the relationship between miR-20a and CFLAR. Cultured HepG2 cells treated with 120 ng/ml TRAIL were mixed with miR-20a mimic and/or si-CFLAR followed by measurement of Caspase-8/3 activity and cell apoptosis by flow cytometry, cell proliferation by MTT assay and protein expression by Western blot.

RESULTS

MiR-20a expression was significantly decreased in rat HCC tissues, while CFLAR was over-expressed. HCC patients had lower miR-20a level and higher CFLAR level in tumor tissues. MiR-20a targeted 3'-UTR of CFLAR to inhibit its expression. TRAIL remarkably up-regulated CFLAR expression, whilst inhibiting miR-20a expression and/or silencing CFLAR significantly potentiated caspase-8 and caspase-3 activity, enhanced sensitivity of HepG2 cells towards TRAIL-induced cell apoptosis, and decreased cell proliferative function.

CONCLUSIONS

HCC had lower miR-20 and higher CFLAR expression. MiR-20a targeted and inhibited CFLAR expression, facilitated activation of caspase-8 and caspase-3, and enhanced sensitivity of HepG2 cells towards TRAIL-induced apoptosis, and subsequently reduced cell proliferation.

摘要

目的

半胱氨酸天冬氨酸蛋白酶-8（CASP8）和 Fas 相关死亡结构域蛋白（FADD）样凋亡调节蛋白（CFLAR）的表达升高可增加对肿瘤坏死因子（TNF）相关凋亡诱导配体（TRAIL）诱导的细胞凋亡的敏感性，但具体机制尚不清楚。先前的研究表明，肝癌（HCC）患者肿瘤组织中 microRNA-20a（miR-20a）的表达降低。生物信息学分析显示 CFLAR 的 3'-UTR 与 miR-20a 之间存在潜在的靶向关系。本研究探讨了 miR-20a 是否在调节 CFLAR 表达和 HCC 细胞凋亡中发挥作用。

方法

比较模型大鼠 HCC 组织与正常组织中 miR-20a 和 CFLAR 的表达。收集 HCC 患者的肿瘤和相邻组织，检测 miR-20a 和 CFLAR 的表达。采用双荧光素酶报告基因检测 miR-20a 与 CFLAR 之间的关系。用 120ng/ml TRAIL 处理 HepG2 细胞，与 miR-20a 模拟物和/或 si-CFLAR 混合，通过流式细胞术检测 Caspase-8/3 活性和细胞凋亡，MTT 法检测细胞增殖，Western blot 法检测蛋白表达。

结果

大鼠 HCC 组织中 miR-20a 表达明显降低，而 CFLAR 表达升高。HCC 患者肿瘤组织中 miR-20a 水平较低，CFLAR 水平较高。miR-20a 靶向 CFLAR 的 3'-UTR 抑制其表达。TRAIL 显著上调 CFLAR 表达，而抑制 miR-20a 表达和/或沉默 CFLAR 可显著增强 caspase-8 和 caspase-3 的活性，增强 HepG2 细胞对 TRAIL 诱导的细胞凋亡的敏感性，并降低细胞增殖功能。