MicroRNA-101 suppresses migration and invasion via targeting vascular endothelial growth factor-C in hepatocellular carcinoma cells

Abstract

MicroRNAs (miRNAs) are a class of non-coding RNAs 18-25 nucleotides in length, which play important roles in the regulation of cancer progression through gene silencing. miRNA (miR)-101 has been suggested to be associated with hepatocellular carcinoma (HCC). However, the detailed role of miR-101 in HCC metastasis and the underlying mechanism remain largely unclear. The present study demonstrated that the expression of miR-101 was significantly reduced in HCC tissues compared with that in matched normal adjacent tissues. miR-101 was also found to be downregulated in four HCC cell lines compared with its expression in a normal liver cell line. Vascular endothelial growth factor (VEGF)-C was further identified as a direct target of miR-101, and the protein expression of VEGF-C was downregulated by miR-101 in HepG2 HCC cells. Furthermore, the overexpression of miR-101 and the knockdown of VEGF-C significantly inhibited HepG2 cell migration and invasion, while restoration of VEGF-C reversed the inhibitory effect of miR-101 overexpression on HepG2 cell migration and invasion. Finally, the expression of VEGF-C was notably increased in HCC tissues and cell lines. These findings suggest that miR-101 exerts a suppressive effect on HCC cell migration and invasion, at least in part through the direct inhibition of VEGF-C protein expression. Therefore, the miR-101/VEGF-C axis may serve as a potential therapeutic target for HCC metastasis.

Keywords: hepatocellular carcinoma; invasion; microRNA-101; migration; vascular endothelial growth factor-C.

Figure 1.

Reverse transcription quantitative polymerase chain reaction analysis was performed to examine the determine expression of miR-101 in (A) HCC tissues and matched normal adjacent tissues (**P<0.01 vs. normal); and in (B) human HCC cell lines HepG2, LH86, LMH and PLHC-1, and the normal liver cell line THLE-3 (**P<0.01 vs. THLE-3). HCC, hepatocellular carcinoma; miR, microRNA.

Figure 2.

Seed sequences and luciferase report assay data. (A) Seed sequences of miR-101 in the WT or MUT 3′-UTR of VEGF-C are indicated. (B) Luciferase report assay data demonstrated that co-transfection of HepG2 cells with miR-101 and WT VEGF-C 3′-UTR caused a significant decrease in luciferase activity, whereas co-transfection with MUT VEGF-C 3′-UTR and miR-101 mimics exhibited no difference compared with the control group (cells co-transfected with blank vector and WT VEGF-C 3′-UTR or MUT VEGF-C 3′-UTR). **P<0.01 vs. the control group. WT, wild-type; MUT, mutant; 3′-UTR, 3′-untranslated region; miR, microRNA; VEGF-C, vascular endothelial growth factor-C; NC, negative control.

Figure 3.

RT-qPCR and western blot analysis. (A) RT-qPCR was performed to determine the relative expression of miR-101 and (B) western blot analysis was performed to determine the protein level of VEGF-C in HepG2 cells transfected with NC scrambled miR, miR-101 mimics and a miR-101 inhibitor. GAPDH was used as an internal reference. Control, HepG2 cells without any transfection. **P<0.01 vs. control. RT-qPCR, reverse transcription quantitative polymerase chain reaction; miR, microRNA; VEGF-C, vascular endothelial growth factor-C; NC, negative control.

Figure 4.

The Transwell® assay was performed to determine the migration capacity of HepG2 cells transfected with miR-101 mimics, VEGF-C siRNA, or co-transfected with miR-101 mimics and VEGF-C plasmid. Control, HepG2 cells without any transfection. **P<0.01 vs. control. miR, microRNA; VEGF-C, vascular endothelial growth factor-C; siRNA, small interfering RNA.

Figure 5.

The Transwell® assay was performed to determine the invasion capacity of HepG2 cells transfected with miR-101 mimics, VEGF-C siRNA, or co-transfected with miR-101 mimics and VEGF-C plasmid. Control, HepG2 cells without any transfection. **P<0.01 vs. control. miR, microRNA; VEGF-C, vascular endothelial growth factor-C; siRNA, small interfering RNA.

Figure 6.

VEGF-C mRNA and protein expression. (A) Reverse transcription quantitative polymerase chain reaction analysis was performed to determine the relative mRNA expression and (B) western blot assay was performed to determine the relative protein expression of VEGF-C in hepatocellular carcinoma tissues and matched normal adjacent tissues. GAPDH was used as the internal reference for western blotting; three representative results are shown. *P<0.05 and **P<0.01 vs. normal. VEGF-C, vascular endothelial growth factor-C.