Hsa-miR-107 regulates chemosensitivity and inhibits tumor growth in hepatocellular carcinoma cells

Abstract

Hepatocellular carcinoma is a common type of liver cancer. Resistance to chemotherapeutic agents is a major problem in cancer therapy. MicroRNAs have been reported in cancer development and tumor growth; however, the relationship between chemoresistance and hepatocellular carcinoma needs to be fully investigated. Here, we treated hepatocellular carcinoma cell line (HA22T) with a histone deacetylase inhibitor to establish hepatocellular carcinoma-resistant cells (HDACi-R) and investigated the molecular mechanisms of chemoresistance in HCC cells. Although histone deacetylase inhibitor could not enhance cell death in HDACi-R but upregulation of miR-107 decreased cell viability both in parental cells and resistance cells, decreased the expression of cofilin-1, enhanced ROS-induced cell apoptosis, and dose-dependently sensitized HDACi-R to HDACi. Further, miR-107 upregulation resulted in tumor cell disorganization in both HA22T and HDACi-R in a mice xenograft model. Our findings demonstrated that miR-107 downregulation leads to hepatocellular carcinoma cell resistance in HDACi via a cofilin-1-dependent molecular mechanism and ROS accumulation.

Keywords: chemosensitivity; drug resistance; hepatocellular carcinoma; miR-107.

Figure 1

Expression of microRNA-107 (miR-107) in HA22T and HDACi-R. (A) Detection and comparison of expression of miRNAs between HA22T and HDACi-R (histone deacetylase inhibitor-resistant) cells by microarray assay. (B) Confirmation of decrease in expression of miR-107 in HDACi-R cells by quantitative RT-PCR. (C) Double confirmation of decrease in expression of miR-107 in HDACi-R cells by RT-PCR.

Figure 2

MicroRNA-107 (miR-107) decreased cell survival and induced cell death in hepatocellular carcinoma (HCC) cells. (A) Regulation of miR-107 expression by transfection with mimic and inhibitor in HA22T and HDACi-R (histone deacetylase inhibitor-resistant) cells detected by quantitative RT-PCR. (B) MiR-107 decreased cell viability in HA22T and HDACi-R cells, as assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. (C) MiR-107 promoted apoptosis-related proteins such as cleaved caspase-3 and decreased expression of pro-survival-related proteins. (D) Quantification of Figure 2C. Expression of fold change of cleavage caspase-3, Bcl-2, and p-Akt after normalization using actin. ^*P < 0.05, ^***P < 0.001 vs. HA22T control. ^#P < 0.05, ^###P < 0.001 vs. HDACi-R control.

Figure 3

MicroRNA-107 (miR-107) enhanced chemosensitivity in hepatocellular carcinoma (HCC) cells. (A) Knockdown of expression of miR-107 prevented HDACi (histone deacetylase inhibitor)-induced downregulation of cell viability in HA22T cells, as assessed with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. (B) Upregulation of miR-107 enhanced chemosensitivity in HDACi-R cells, as assessed with MTT assay. Scramble miRNA group treated with 15 μM.

Figure 4

MicroRNA-107 (miR-107) induced reactive oxygen species (ROS) accumulation and cell death by targeting cofilin-1 in hepatocellular carcinoma (HCC) cells. (A) MiR-107 target sequence on cofilin-1 (CFL1) 3' UTR (untranslated region). (B) Luciferase activity assays of luciferase vectors with cofilin-1 3' UTR were performed following transfection with miR-107 or negative control for 24 h. ^**P < 0.01 vs. HDACi-R (histone deacetylase inhibitor-resistant) cells cofilin-1 3' UTR group. (C) MiR-107 induced reactive oxygen species (ROS) accumulation, as detected by MitoSOX staining. (D) H₂O₂-induced HA22T cell death was higher than HDACi-R cells. Transfection with si-cofilin-1 (si-CFL-1) and miR-107 mimic decreased cofilin-1 (CFL-1) expression and enhanced H₂O₂-induced cleavage caspase-3 expression, as detected by western blotting assay. (E) Quantification of Figure 4D. Fold change of cleavage caspase-3 after normalization using actin. ^*P < 0.05, ^***P < 0.001 vs. HA22T control. ^#P < 0.05, ^###P < 0.001 vs. HDACi-R control.

Figure 5

MicroRNA-107 (miR-107) regulates tumor growth and tumor death in hepatocellular carcinoma (HCC) tumors in vivo. (A) The tumoral growth of HCC cell lines xenografted on nude mice. Treatment was administered at day 21 by tumor injection. (B) Hematoxylin and eosin (H&E)-stained sections from the xenograft. Scale bar: 100 μm. (C) TUNEL assay was performed to visualize apoptotic cells (green), and DAPI staining showed the number of nuclei. Scale bar: 100 μm. (D) Overexpression of miR-107 caused a decrease in CFL-1 and p-Akt expression both in two tumors as observed by western blotting assay.