MiR-20a lowers chemosensitivity of liver cancer Huh-7 cells via regulating NF-кB expression

B.-M. Yang, J.-R. Zhao, T.-T. Huo, M.-L. Zhang, X.-H. Wu

Department of Hepatobiliary Surgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China. xhwu@hbmu.edu.cn

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• Oncology

OBJECTIVE: The aim of this study was to explore the regulatory effect of micro ribonucleic acid (miR)-20a on nuclear factor-κB (NF-кB) in liver cancer Huh-7 cells, and to elucidate its influence on the chemosensitivity of Huh-7 cells.

MATERIALS AND METHODS: Huh-7 cells with overexpression of miR-20a or knockout of miR-20a were first constructed. Quantitative polymerase chain reaction (qPCR) was adopted to detect the expression level of miR-20a in each group of cells. The sensitivity of cells to cisplatin and doxorubicin in each group was measured using methyl thiazolyl tetrazolium (MTT) assay, and the 50% inhibitory concentration (IC50) was calculated. Hoechst 33258 staining was performed to detect the apoptosis of cells in each group. Furthermore, the expression levels of apoptosis-associated proteins and the NF-кB signaling pathway-related proteins in each group of cells were determined via Western blotting.

RESULTS: The expression level of miR-20a in blank control group was considerably higher than that in knockout group (p<0.01). Meanwhile, cells in overexpression group exhibited a notably higher expression level of miR-20a than blank control group (p<0.01). Cells in knockout group had dramatically enhanced sensitivity to doxorubicin and cisplatin (p<0.01), with a prominently decreased IC50 value (p<0.01). However, cells in overexpression group exhibited remarkably weakened sensitivity (p<0.01) and increased IC50 value (p<0.01). After treatment with doxorubicin and cisplatin, the apoptosis level of cells rose substantially in knockout group (p<0.01), whereas declined significantly in overexpression group (p<0.01). Moreover, knockout group exhibited a notably elevated expression level of Caspase-3 (p<0.01), and a considerably decreased ratio of B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein (Bax) (p<0.01). The expression level of Caspase-3 declined remarkably (p<0.01), however, the ratio of Bcl2/Bax increased substantially (p<0.01) in overexpression group. The expression level of NF-кB inhibitor beta (NF-кBIB) was markedly up-regulated (p<0.01), while the expression levels of Livin and Survivin declined remarkably (p<0.01) in knockout group. Furthermore, overexpression group had a considerably decreased expression level of NF-кBIB (p<0.01), but notably increased expression levels of Livin and Survivin (p<0.01).

CONCLUSIONS: MiR-20a up-regulates the expressions of the downstream proteins Livin and Survivin, decreases the expressions of apoptosis-associated proteins, weakens the sensitivity of cells to chemotherapy drugs and lowers the apoptosis level of cells by activating the NF-кB signaling pathway in liver cancer Huh-7 cells.

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