Lowered HGK expression inhibits cell invasion and adhesion in hepatocellular carcinoma cell line HepG2

Abstract

Aim: To investigate the effects of RNA interference targeting hepatocyte progenitor kinase-like kinase (HGK) in the invasion and adhesion of hepatocellular carcinoma (HCC) cell line HepG2.

Methods: Three paired insert DNA fragments specific to HGK gene and one negative control DNA fragment were synthesized and inserted into RNAi-Ready pSIREN-RetroQ-ZsGreen vector. Western blotting assay and real-time reverse transcriptase polymerase chain reaction (RT-PCR) were used to screen the vector with a highest inhibitory rate. The vector was used to generate recombinant retrovirus specific to HGK. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide (MTT) assay was used to examine cell growth; wound closure assay and cell adhesion assay were employed to investigate cell migration and adhesion respectively; and transwell assay and three-dimensional culture invasion assay were used to detect cell invasion. The expressions of matrix metalloproteinase (MMP)-2, MMP-9 and nuclear factor (NF)-κB were detected by Western blotting assay.

Results: The real time RT-PCR and Western blotting assay showed that cells transfected with retrovirus mediating RNAi targeting of HGK (RV-shHGK)-1 vector had the strongest inhibition of HGK protein, with an inhibition rate of 76%, and this vector was used to generate recombinant retrovirus RV-shHGK-1. Cell adhesion assay and MTT assay found that cell adhesion and growth of the cells infected with RV-shHGK-1 were significantly lower than those of the control cells (P < 0.05). Wound closure assay, transwell assay and three-dimensional culture invasion assay showed that the cell invasiveness was significantly less in HGK knockdown cells than in the control cells (P < 0.05). The expressions of MMP-2, MMP-9 and NF-κB were inhibited in HepG2 cells infected with RV-shHGK-1.

Conclusion: Down-regulation of HGK can obviously inhibit the migration and invasion of HepG2 cells in vitro. HGK may be a new therapeutic target for treatment of HCC.

Figure 1

Selection of the most effective hepatocyte progenitor kinase-like kinase specific shRNA expression vector in 293T cells. A: Phase contrast and GFP expression under a fluorescent microscope after 48 h in 293T cells; B: Protein mRNA and hepatocyte progenitor kinase-like kinase (HGK) levels after HepG2 cells were treated with different vectors detected by real-time reverse transcriptase polymerase chain reaction and Western boltting assay. The vector retrovirus mediating RNAi targeting of HGK (RV-shHGK)-1 significantly inhibited HGK expression in HepG2 cells, ^aP < 0.05 vs HepG2 cells and HepG2 cells transfected with RV-shHGK-C vector. 1: 293T cells; 2: Transfection of RV-shHGK-1 vector in 293T cells; 3: Transfection of RV-shHGK-2 vector; 4: Transfection of RV-shHGK-3 vector; 5: Transfection of RV-shHGK-C vector (original magnification × 200).

Figure 2

Hepatocyte progenitor kinase-like kinase expression suppressed by RV-shHGK-1 retrovirus in HepG2 cells. A: HepG2 cells infected with retrovirus mediating RNAi targeting of hepatocyte progenitor kinase-like kinase (RV-shHGK)-1 or RV-shHGK-C (multiplicity of infection = 4), GFP expression and the phase contrast images after 48 h (original magnification × 200); B: Protein and mRNA levels of hepatocyte progenitor kinase-like kinase (HGK) after HepG2 cells were treated with different retrovirus detected by real-time reverse transcriptase polymerase chain reaction and Western boltting assay. The retrovirus RV-shHGK-1 significantly inhibited HGK expression in HepG2 cells, ^aP < 0.05 vs HepG2 cells and HepG2 cells infected with RV-shHGK-C retrovirus.

Figure 3

Down-regulation of hepatocyte progenitor kinase-like kinase inhibits HepG2 cell growth and adhesion (methyl thiazolyl tetrazolium assay). A: HepG2 cell growth was significantly suppressed by retrovirus mediating RNAi targeting of hepatocyte progenitor kinase-like kinase (RV-shHGK)-1 vs RV-shHGK-C group; B: RV-shHGK-1 inhibited HepG2 adhesion to fibronectin (FN), laminin (LN) and collagen IV. ^aP < 0.05 vs control.

Figure 4

Down-regulation of hepatocyte progenitor kinase-like kinase inhibits HepG2 cell invasion. A: HepG2 cell invasiveness was significantly suppressed by retrovirus mediating RNAi targeting of hepatocyte progenitor kinase-like kinase (RV-shHGK)-1 vs RV-shHGK-C group (original magnification × 200, ^aP < 0.05); B: The blue-stained cells are those invading the ECMatrix and migrating through the polycarbonate membrane to the lower surface of the membrane (original magnification × 200, ^aP < 0.05 vs HepG2 cells infected with RV-shHGK-C); C: HepG2 cells infected with RV-shHGK-1 do not form the structure patterned network of interconnected loops (original magnification × 200).

Figure 5

Hepatocyte progenitor kinase-like kinase, matrix metalloproteinase-2, matrix metalloproteinase-9 and nuclear factor-κB expressions in HepG2 cells (Western blotting). A: Retrovirus mediating RNAi targeting of hepatocyte progenitor kinase-like kinase (RV-shHGK)-1 significantly inhibited matrix metalloproteinase (MMP)-2, MMP-9 and nuclear factor (NF)-κB in HepG2 cells. MMP-2 protein expression in each group (Western blotting); B: Densitometric analysis hepatocyte progenitor kinase-like kinase (HGK), MMP-2, MMP-9 and NF-κB protein expression in HepG2 cells infected with RV-shHGK-1 or RV-shHGK-C, respectively. ^aP < 0.05 vs HepG2 cells infected with RV-shHGK-C.