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AIM:To investigate the effects of RNA interference tar-geting hepatocyte progenitor kinase-like kinase(HGK) in the invasion and adhesion of hepatocellular carcinoma(HCC) cell line HepG2.METHODS:Three paired insert DNA fragments specif ic 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 recom-binant retrovirus specif ic 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 me-diating RNAi targeting of HGK(RV-shHGK)-1 vector had the strongest inhibition of HGK protein,with an inhibi-tion 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.
AIM: To investigate the effects of RNA interference tar-gelling hepatocyte progenitor kinase-like kinase (HGK) in the invasion and adhesion of hepatocellular carcinoma (HCC) cell line HepG2.METHODS: Three paired insert DNA fragments specif ic 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 recombination retrovirus specif ic 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 me-diating RNAi targeting of HGK (RV-shHGK) -1 vector had the strongest inhibition of HGK protein, with an inhibi -tion 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 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.