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

Y. Wang, Y.-R. Zhao, A.-Y. Zhang, J. Ma, Z.-Z. Wang, X. Zhang

Infectious Disease Department, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China. jiexiongzxc@163.com

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• Oncology

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.

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