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.
Hepatocellular carcinoma (HCC) is a major health concern and the third leading cause of cancer-related mortality, with an increasing incidence worldwide (
MicroRNAs (miRNAs) are a class of non-coding RNAs 18–25 nucleotides in length, which may lead to mRNA degradation or inhibit protein translation through directly binding to the 3′-untranslated region (UTR) of their target mRNAs. By mediating protein expression of their target genes, miRNAs play key roles in the regulation of cell survival, proliferation, differentiation and motility (
Vascular endothelial growth factor (VEGF)-C is a member of the VEGF family and has been suggested to possess important lymphangiogenic properties (
A total of 20 samples from HCC tissues and their matched adjacent normal tissues were obtained from the Department of Gastroenterology and Hepatology (Jinshan Hospital, Fudan University). All the tissues were immediately snap-frozen in liquid nitrogen following surgical removal and stored at −70°C until use.
The study was approved by the Ethics Committee of Fudan University (Shanghai, China) and all the participants provided written informed consent for the use of their tissue specimens.
The human HCC cell lines HepG2, LH86, LMH and PLHC-1, and the normal liver cell line THLE-3, were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C in a humidified incubator containing 5% CO2.
Total RNA was extracted using TRIzol® reagent (Ambion; Thermo Fisher Scientific, Carlsbad, CA, USA). For miR detection, a TaqMan miRNA Reverse Transcription kit (Thermo Fisher Scientific) was used to convert RNA into cDNA, according to the manufacturer's protocol. qPCR was then performed using an miRNA Q-PCR Detection kit (GeneCopoeia, Rockville, MD, USA) on an ABI 7500 thermocycler (Applied Biosystems; Thermo Fisher Scientific, Foster City, CA, USA). The U6 gene was used as an internal reference. For mRNA detection, a RevertAid First-Strand cDNA Synthesis kit (Fermentas, Carlsbad, CA, USA) was used to convert RNA into cDNA, according to the manufacturer's protocol. qPCR was then performed using iQTM SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) on the ABI 7500 thermocycler. The specific primers were as follows: VEGF-C: forward, 5′-GAG GAG CAG TTA CGG TCT GTG-3′ and reverse, 5′-TCC TTT CCT TAG CTG ACA CTT GT-3′; and GAPDH: forward, 5′-CTG GGC TAC ACT GAG CACC-3′ and reverse, 5′-AAG TGG TCG TTG AGG GCA ATG-3′. The relative mRNA expression of VEGF-C was normalized to GAPDH and analyzed using the 2−ΔΔCq method.
Tissues and cells were solubilized in cold radioimmunoprecipitation assay lysis buffer. The proteins were separated with 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was incubated with phosphate-buffered saline containing 5% milk overnight at 4°C. Subsequently, the PVDF membrane was incubated with mouse monoclonal anti-VEGF-C primary antibody (dilution 1:200; ab191274; Abcam, Cambridge, MA, USA) and mouse monoclonal anti-GAPDH primary antibody (dilution 1:100; ab8245; Abcam) and at room temperature for 3 h, and then with rabbit anti-mouse-IgG (dilution 1:5,000; ab175743; Abcam) at room temperature for 1 h. An enhanced chemiluminescence kit (Pierce Chemical, Rockford, IL, USA) was then used for detection. The relative protein expression was analyzed by Image-Pro plus software 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA) and presented as the density ratio versus GAPDH.
Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific) was used to perform cell transfection according to the manufacturer's protocol. For VEGF-C functional analysis, HepG2 cells were transfected with VEGF-C-specific small interfering (si)RNA or VEGF-C plasmid (Nlunbio, Changsha, China). For miR-101 functional analysis, HepG2 cells were transfected with scrambled miRNA as a negative control (NC), miR-101 mimics or a miR-101 inhibitor (Thermo Fisher Scientific).
A Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) was used to generate a mutant (MUT) 3′-UTR of VEGF-C, according to the manufacturer's protocol. The wild-type (WT) or MUT 3′-UTR of VEGF-C were inserted into the psiCHECK™2 vector (Promega, Madison, WI, USA). For the luciferase reporter assay, HepG2 cells were cultured to ~60% confluence in a 24-well plate, and then transfected with psiCHECK™2-VEGF-C-3′-UTR or psiCHECK™2-mutant VEGF-C-3′-UTR vectors, with or without 100 nM miR-101 mimics. Following incubation for 48 h, a dual-luciferase reporter assay system (Promega) was used to determine the luciferase activity on a LD400 luminometer (Beckman Coulter, Fullerton, CA, USA). Renilla luciferase activity was normalized to firefly luciferase activity.
Cell migration and invasion assays were performed using Transwell® chambers (BD Biosciences, Frankin Lakes, NJ, USA). A cell suspension containing 5×105 cells/ml was prepared in serum-free media. For the cell migration assay, 300 µl of cell suspension was added into the upper Transwell® chamber. For the cell invasion assay, 300 µl of cell suspension was added into the upper Transwell® chamber pre-coated with Matrigel™ (BD Biosciences). Subsequently, 500 µl of DMEM with 10% FBS as the chemoattractant was added into the lower Transwell® chamber and the cells were incubated for 24 h. Subsequently, cells that did not migrate or invade through the pores were carefully removed using a cotton-tipped swab. The filters were fixed in 90% alcohol and stained with crystal violet. The cell number was determined in five randomly selected fields under an CX41 inverted microscope (Olympus, Tokyo, Japan) at 400X magnification.
All data are expressed as mean ± standard deviation. SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform the statistical analysis. The differences were analyzed using one-way analysis of variance and P<0.05 was considered to indicate statistically significant differences.
The expression level of miR-101 was determined using RT-qPCR in HCC tissues and matched normal adjacent tissues. As shown in
As described above, bioinformatics analysis suggested that VEGF-C is a potential target of miR-101. To verify this hypothesis, WT and MUT VEGF-C 3′-UTR were generated as shown in
We further investigated the role of miR-101 in the regulation of VEGF-C expression in HepG2 HCC cells. Following transfection of HepG2 cells with scrambled miR, miR-101 mimics or a miR-101 inhibitor, the expression level of miR-101 was first determined in each group, and the transfection efficiency was confirmed to be satisfactory (
The roles of miR-101 and VEGF-C in the regulation of HCC cell migration were further investigated. The findings demonstrated that transfection with miR-101 mimics or VEGF-C siRNA notably suppressed the migration of HepG2 cells. However, the suppressive effect of miR-101 overexpression on HepG2 cell migration was significantly reversed by the upregulation of VEGF-C (
The roles of miR-101 and VEGF-C in the regulation of HCC cell invasion were further investigated. It was demonstrated that transfection with miR-101 mimics or VEGF-C siRNA notably suppressed the invasion of HepG2 cells. However, the suppressive effect of miR-101 overexpression on HepG2 cell invasion was significantly reversed by the upregulation of VEGF-C (
The expression levels of VEGF-C mRNA and protein were determined using RT-qPCR and western blot analysis, respectively, in HCC tissues and matched normal adjacent tissues. As shown in
The present study demonstrated that the expression of miR-101 was markedly reduced in HCC tissues and cell lines. VEGF-C was identified as a novel target gene of miR-101, and the protein expression of VEGF-C was downregulated by miR-101 in HCC cells. The investigation of the underlying molecular mechanism revealed that miR-101 inhibited HCC cell migration and invasion, at least in part via direct inhibition of VEGF-C protein expression. Finally, VEGF-C expression was found to be significantly upregulated in HCC tissues.
miRNAs are frequently deregulated in malignant tumors. The expression of miRNAs such as miR-124 and miR-203 was previously found to be reduced in HCC, and restoration of their expression significantly inhibited HCC cell growth (
In this study, VEGF-C was identified as a novel target of miR-101, and it was indicated that VEGF-C may be involved in the effect of miR-101 on HCC cell migration and invasion. VEGF-C is a member of the VEGF family, which plays an important role in angiogenesis via affecting endothelial cell proliferation and motility and vascular permeability (
In conclusion, miR-101 expression profiling was conducted in human HCC tissues and cells to identify the targets of abnormally expressed miR-101. The findings suggest that miR-101 exerts an inhibitory effect on HCC cell migration and invasion, at least in part by inhibiting the protein expression of its target, VEGF-C. These findings may contribute to the development of molecular-targeted therapies based on miRNAs.
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.
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.
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.
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.
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.
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.