Role of microRNA-199a-5p and discoidin domain receptor 1 in human hepatocellular carcinoma invasion

Abstract

Background: Micro-ribonucleic acid (miRNA)-199a-5p has been reported to be decreased in hepatocellular carcinoma (HCC) compared to normal tissue. Discoidin domain receptor-1 (DDR1) tyrosine kinase, involved in cell invasion-related signaling pathway, was predicted to be a potential target of miR-199a-5p by the use of miRNA target prediction algorithms. The aim of this study was to investigate the role of miR-199a-5p and DDR1 in HCC invasion.

Methods: Mature miR-199a-5p and DDR1 expression were evaluated in tumor and adjacent non-tumor liver tissues from 23 patients with HCC undergoing liver resection and five hepatoma cell lines by the use of real-time quantitative RT-PCR (qRT-PCR) analysis. The effect of aberrant miR-199a-5p expression on cell invasion was assessed in vitro using HepG2 and SNU-182 hepatoma cell lines. Luciferase reporter assay was employed to validate DDR1 as a putative miR-199a-5p target gene. Regulation of DDR1 expression by miR-199a-5p was assessed by the use qRT-PCR and western blotting analysis.

Results: A significant down-regulation of miR-199a-5p was observed in 65.2% of HCC tissues and in four of five cell lines. In contrast, DDR1 expression was significantly increased in 52.2% of HCC samples and in two of five cell lines. Increased DDR1 expression in HCC was associated with advanced tumor stage. DDR1 was shown to be a direct target of miR-199a-5p by luciferase reporter assay. Transfection of miR-199a-5p inhibited invasion of HepG2 but not SNU-182 hepatoma cells.

Conclusions: Decreased expression of miR-199a-5p contributes to increased cell invasion by functional deregulation of DDR1 activity in HCC. However, the effect of miR-199a-5p on DDR1 varies among individuals and hepatoma cell lines. These findings may have significant translational relevance for development of new targeted therapies as well as prognostic prediction for patients with HCC.

Figure 1

Expression of miR-199a-5p and DDR1 in HCC specimens. qRT-PCR analysis was performed using 23 paired surgical specimens of HCC tissues and adjacent normal tissues for miR-199a-5p (A) and DDR1 (B). Expression levels normalized to expression of reference genes, RNU44 and HMBS (A) and HMBS and GAPDH (B), are shown. Quantitative values representing the mean and SD from experiments performed in triplicate are presented.

Figure 2

Expression of miR-199a-5p and DDR1 in human HCC cell lines. Expression levels of miR-199a-5p (A) and DDR1 (B) were determined by qRT-PCR analysis for 5 HCC cell lines (Hep3B, HepG2, HuH7, SK-Hep-1 and SNU-182) and primary human hepatocytes (HH). The expression levels were normalized to reference genes, RNU44 and HMBS (A) and HMBS and GAPDH (B). Quantitative values representing the mean and SD from experiments performed in triplicate are presented. *p < 0.001 vs. primary human hepatocytes.

Figure 3

Box plots of DDR1 expression in AJCC/UICC stages of HCC. The relative expression levels of DDR1 in 23 human HCC tissues categorized according to AJCC/UICC stage I-II and AJCC/UICC stage III-IV are presented. The boxes enclose the interquartile ranges (IQRs), with the median values denoted by the horizontal lines. Circles (ο) represent outliers (values >1.5 × IQR). * p < 0.01 vs. AJCC/UICC stage I-II.

Figure 4

DDR1 is a target gene of miR-199a-5p. (A) Cells were cotransfected with 200 ng of each vector (pMIR-DDR1-3'-UTR construct or empty pMIR-REPORT firefly luciferase reporter vector, together with beta-galactosidase (β-gal) expression construct pMIR-REPORT β-gal and miR-199a-5p precursor or control miRNA precursor using lipofectamine 2000. Luciferase assays and β-gal enzyme assays were performed 24 hours after transfection in triplicate. Firefly luciferase activity was normalized to β-gal expression for each sample. Data are shown as mean ± SD from 3 independent experiments. *p < 0.05 vs. respective control. (B) HepG2 or (C) SNU-182 cells were transfected with 10 nM miR-199a-5p precursor or 100 nM DDR1-siRNA. DDR1 mRNA expression was assessed by qRT-PCR and normalized to the expression of HPRT1 and SDHA. Cells transfected with control miRNA precursor or control siRNA were used as controls. Data are shown from 3 independent experiments each performed in triplicate as mean ± SD. *p < 0.05 vs. respective controls.

Figure 5

MiR-199a-5p differentially regulates the expression of DDR1. (A) HepG2 or (B) SNU-182 cells were transfected with 10 nM miR-199a-5p precursor or 100 nM DDR1-siRNA. Cell lysates were obtained 48 hours after transfection for immunoblot analysis of DDR1 protein expression. GAPDH protein levels were determined as a loading control. Cells transfected with control miRNA precursor or control siRNA were used as controls. Lane M indicates the molecular weight marker and lane numbers 1 to 4 represent control miRNA precursor, miR-199a-5p precursor, control siRNA, and DDR1-siRNA respectively. Arrow, DDR1 protein; stars, protein bands resulting from nonspecific binding to DDR1-antibody. (C) For mRNA stability assays, HepG2 cells were plated in 24-well plates and transfected with 10 nM of miR-199a-5p, miR-control or 100 nM si-DDR1 as a positive control. After transfection for 6 hours, cells were incubated with or without 4 μg/ml of actinomycin D for additional 36 hours. Total RNA was extracted from cells and DDR1 mRNA was quantified by qRT-PCR described before. The expression levels of DDR1 are presented as values normalized against 10⁶ copies of β-actin transcripts. Data are shown from 3 independent experiments each performed in triplicate as mean ± SD. *p < 0.05 vs. respective controls.

Figure 6

Effects of miR-199a-5p and DDR1 expression on hepatoma cell invasion and proliferation. (A) HepG2 and SNU-182 cells were transfected with 10 nM miR-199a-5p precursor or 100 nM DDR1-siRNA, and cell invasion across a membrane with 8 μm pores coated with Matrigel was assessed. Cells transfected with control miRNA precursor or control siRNA were used as controls. Data are expressed as invasion index and normalized to cell number using WST-1 assay. Data are shown from 3 separate experiments performed each in triplicate as mean ± SD. *p < 0.01 vs. respective controls. (B) HepG2 and SNU-182 cells were transfected with 10 nM miR-199a-5p precursor or 100 nM DDR1-siRNA, and cell and the proliferation was assessed after 72 hours using WST-1 assay. Cells transfected with control miRNA precursor or control siRNA were used as controls. Data are expressed as proliferation index. Data are shown from 3 separate experiments performed each in triplicate as mean ± SD.