MiR-25 promotes hepatocellular carcinoma cell growth, migration and invasion by inhibiting RhoGDI1

Abstract

MicroRNA (miRNA)-25 is a small non-coding RNA that has been implicated in the tumorigenesis of many cancers, but little is known on the role of miR-25 in HCC metastasis. We hereby found that miR-25 was significantly upregulated in clinical HCC tissues compared with normal liver tissues. We also revealed that miR-25 dramatically stimulates HCC cell growth and activates the epithelial-mesenchymal transition (EMT). MiR-25 is activated by the WNT/β-catenin signaling pathway, and exerts its pro-metastatic function by directly inhibiting the Rho GDP dissociation inhibitor alpha (RhoGDI1). Downregulation of RhoGDI1 enhances expression of Snail, thereby promoting EMT. MiR-25 levels are positively correlated with β-catenin expression, whereas negatively correlated with the level of RhoGDI1 in HCC. Our findings provide new insights into the role of miR-25 in HCC metastasis, and implicate the potential application of miR-25 in HCC therapy.

Keywords: RhoGDI1; hepatocellular carcinoma; metastasis; miR-25.

Figure 1. miR-25 is upregulated in HCC tissues and promotes HCC cell growth in vitro

A. Increased levels of miR-25 were positively correlated with the tumor grades and metastasis of HCC by qRT–PCR. miR-25 expression levels were calculated by the miR-25/U6 expression ratio (2^−ΔΔCT). B–C. The effect of Lv-miR-25 or LEV on the growth of HepG2 and HuH7 cells was examined by EdU incorporation assay (B) and colony formation assay (C). Data are presented as mean ± SD from three independent experiments. **P < 0.01 compared with the control group.

Figure 2. Effect of miR-25 on HCC cell motility, invasion and EMT in vitro

A. Stably upregulating miR-25 increased the migration ability of HepG2 and HuH7 cells in vitro, and knockdown of miR-25 in Lv-miR-25 cells reduced the migration ability. B. Stably upregulating miR-25 induced in vitro invasiveness of HepG2 and HuH7 cells, and specific inhibition of miR-25 in Lv-miR-25 cells reduced the invasion ability. C. Immunoblotting of fibronectin, N-cadherin, and E-cadherin in HepG2 and HuH7 cells. Data are presented as mean ± SD from three independent experiments. **p < 0.01 compared with the control group. Scale bar, 100 μm.

Figure 3. miR-25 directly targets the RhoGDI1 via its 3′-UTR

A. Sequence alignment between miR-25 and the 3′-UTR of human RhoGDI1 mRNA, nt, nucleotides. B. The effect of miR-25 on the activity of firefly luciferase reporter containing either wild type (WT) or mutant type (Mut) 3′-UTR was tested using luciferase reporter gene assays. C. The effect of miR-25 on the endogenous expression levels of RhoGDI1 was examined in HepG2 and HuH7 cells by qRT–PCR. D. Protein levels of RhoGDI1, the activities of Rac1 and Cdc42 in HepG2 and HuH7 cells were determined after transfection with Lv-miR-25 or LEV by Western blot analysis. Data are presented as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01 compared with the control LEV group.

Figure 4. miR-25 overexpression and RhoGDI1 inhibition produce similar changes, which are restored by RhoGDI1 ectopic expression in vitro

A. The expression level of RhoGDI1 was examined by Western blot analyses in HepG2 and HuH7 cells treated with ectopic RhoGDI1 or siRNAs targeting RhoGDI1. EdU incorporation assays B. transwell migration assays C. and Boyden invasion assays D. of HepG2 or HuH7 cells were performed after transfection with NC, Lv-miR-25 and siRNA against RhoGDI1 as indicated. Data are presented as mean ± SD from three independent experiments. **p < 0.01 compared with the control group. Scale bar, 100 μm.

Figure 5. miR-25 inhibits RhoGDI1 and is activated by WNT/β-catenin signaling in HCC cells

A. HepG2 or HuH7 cells were transfected with RhoGDI1 siRNAs, then RhoGDI1, Snail, E-cadherin, N-cadherin and β-catenin protein levels were detected by Western blot analysis. B. HepG2 or HuH7 cells were transfected with negative control (NC), miR-25 mimics, or miR-25 inhibitor, and Snail was then detected by Western blot analysis. C. qRT-PCR analysis of miR-25 in HepG2 and HuH7 cells treated with or without LiCl (20 mmol/L) for 36 hours. D–E. qRT-PCR analysis of miR-25 and Western blot analysis of β-catenin protein levels in HepG2 and HuH7 cells transfected with NC or siRNAs against β-catenin. qRT-PCR data were normalized using U6 RNA. F–G. Transwell migration (F) and Boyden invasion (G) assays were performed in HepG2 and HuH7 cells transfected with NC, Lv-miR-25, and/or siRNAs against β-catenin. Data are presented as mean ± SD from three independent experiments. **p < 0.01 compared with the control group.

Figure 6. The overexpression of miR-25 promotes tumour growth and metastasis in vivo

A–B. Female nude mouse was orthotopically inoculated in the left hepatic lobe with Lv-miR-25-HepG2 or LEV-HepG2 cells. Six weeks later, the body weights (A) and liver weights (B) of the mice were measured. C. Representative images of tumour nodules in primary sites, and metastatic nodules were present at the sixth week after the orthotopic liver inoculation of nude mice. The black arrows indicate the location of primary tumour nodules and metastatic nodules. The number of metastatic nodules in each mouse was counted. D. Representative immunohistochemical staining sections of primary liver tumours formed by Lv-miR-25-HepG2 or LEV-HepG2. For each generated tumour, five fields were randomly selected. The number of Ki67-positive and RhoGDI1-positive cells per 1000 cells was counted by three independent experienced pathologists. Data are presented as mean ± SD. **p < 0.01 compared with the control group. Scale bar, 100 μm.