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


CONCLUSIONS
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.
Han
Su-Xia
SX
Oncology Center of the First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, China. hsummer22099@yahoo.cn
Zhu
Qing
Q
Ma
Jin-Lu
JL
Zhao
Jing
J
Huang
Chen
C
Jia
Xi
X
Zhang
Dan
D
D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009361:Neoplasm Invasiveness; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism
D016326:Extracellular Matrix Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; C117090:MAP4K4 protein, human; D017346:Protein Serine-Threonine Kinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9