Antitumor Effect of Regorafenib on MicroRNA Expression in Hepatocellular Carcinoma Cell Lines

Abstract

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is one of the leading causes of cancer-related deaths worldwide. Regorafenib, a multi-kinase inhibitor, is used as a second-line treatment for advanced HCC. Here, we aimed to investigate the mechanism of the antitumor effect of regorafenib on HCC and evaluate altered microRNA (miRNA) expression. Cell proliferation was examined in six HCC cell lines (HuH-7, HepG2, HLF, PLC/PRF/5, Hep3B, and Li-7) using the Cell Counting Kit-8 assay. Xenografted mouse models were used to assess the effects of regorafenib in vivo. Cell cycle analysis, western blotting analysis, and miRNA expression analysis were performed to identify the antitumor inhibitory potential of regorafenib on HCC cells. Regorafenib suppressed proliferation in HuH-7 cell and induced G0/G1 cell cycle arrest and cyclin D1 downregulation in regorafenib-sensitive cells. During miRNA analysis, miRNA molecules associated with the antitumor effect of regorafenib were found. Regorafenib suppresses cell proliferation and tumor growth in HCC by decreasing cyclin D1 via alterations in intracellular and exosomal miRNAs in HCC.

Keywords: antitumor effect; cell cycle; cell proliferation; cyclin; hepatocellular carcinoma; microRNA; regorafenib.

Figure 1

Cell proliferation assays. Regorafenib suppresses HCC cell proliferation. HuH-7, Hep3B, HepG2, Li-7, PLC/PRF/5, and HLF cells were treated with 0, 1, 3, 5, 10 µM regorafenib for 0, 24, and 48 h. Data points represent the mean cell number in three independent cultures, and error bars represent standard errors. For HuH-7, Hep3B, HepG2, Li-7 cell lines, concentration-dependent inhibition of cell proliferation was observed at 48 h after regorafenib treatment (* p < 0.01).

Figure 2

Regorafenib induces cell-cycle arrest at G0/G1 in HuH-7 cells. (A) Representative results showing the distribution of HuH-7 cells in G0/G1, S, and G2/M phases following treatment with 5 µM regorafenib at 24 and 48 h. (B) Histograms showing the proportion of HuH-7 cells in G0/G1, S, and G2/M phases (* p < 0.05). (C) Western blot showing the expression of cyclin D1, CDK2 and CDK4 in HuH-7 cells at 24 h and 48 h after the addition of 5 µM regorafenib.

Figure 3

Apoptosis-inducing effect of regorafenib. Early apoptotic changes evoked by 5 µM regorafenib at 48 h were assessed by flow cytometry. Annexin-V positive and PI-negative cells were regarded as early apoptotic (enclosed areas in bold squares). The proportion of annexin V-positive and regorafenib-treated cells was slightly higher than that of untreated cells, but this difference was not significant.

Figure 4

Antitumor effect of regorafenib on established HCC in vivo. The tumor volume (mm³) was calculated as follows: (tumor length (mm) × tumor width (mm) × tumor width (mm))/2. All animals were sacrificed on day 9 after treatment. Tumors were significantly smaller in regorafenib-treated mice than in control mice (* p < 0.05).

Figure 5

Hierarchical clustering of (A) HuH-7 cells and (B) HuH-7-derived exosomes cultured with or without 5µM regorafenib. The analyzed samples are presented in the columns, and the miRNAs are presented in the rows. The miRNA clustering color scale presented at the top of the figure indicates the relative miRNA expression levels, with red and blue representing high and low expression levels, respectively.

Figure 6

(A) Relative quantification (RQ) of miRNAs after treatment with or without 5 µM regorafenib for 48 h. miR-3714 expression was significantly upregulated by regorafenib treatment, while miR-494-3p and miR-8073 expression showed no significant changes (* p < 0.05) (n.s. not significant vs. control). (B) Western blot analysis of cyclin D1. The expression of cyclin D1 was attenuated at 48 h post-transfection with an miR-3714 mimic.