Inhibitory effect of tumor suppressor gene PTEN on hepatocellular carcinoma cell line HHCC proliferation and its mechanisms of action / 中华肿瘤杂志

ABSTRACT

<p><b>OBJECTIVE</b>To study the effect of tumor suppressor gene PTEN on proliferation and cell cycle of hepatocellular carcinoma cell line HHCC.</p><p><b>METHODS</b>Firstly, eukaryotic expression vectors of wild type and mutated type of PTEN gene were constructed, named as pEGFP-WT-PTEN and pEGFP-PTEN; G129R, respectively. Lipofectamine 2000 was used to transfect the constructed expression vectors into hepatocellular carcinoma cell line HHCC which was PTEN protein negative. G418 was used to select the cell clones constantly expressing PTEN protein. Flow cytometry was used to assay the cell cycle of HHCC transfected by above mentioned eukaryotic expression vectors and non-transfected cell line HHCC. Intrinsic 473-phosphorylated AKT representing the level of active AKT was assayed by Western blot. The non-transfected HHCC served as control.</p><p><b>RESULTS</b>The proliferation of HHCC constantly expressing PTEN protein was obviously inhibited compared with HHCC cells transfected with mutated PTEN gene and empty vectors, and non-transfected HHCC cells. The number of HHCC cells transfected with wild type PTEN gene at G(1) phase, G(2) phase and S phase was 70.8%, 6.8% and 22.4%, respectively. Compared with control group transfected with empty vector, the number of G(1) phase HHCC cells constantly expressing wild type-PTEN protein was significantly higher than that of control. The number of cells in G(2) and S phase was significantly lower than that of control. However, the number of cells in G(1) phase, G(2) phase and S phase of HHCC transfected with mutant PTEN was 63.2%, 10.1% and 26.7%, respectively. There was no significant difference compared with control group. Western blot result showed that the intrinsic level of 473-phosphorylated AKT of HHCC constantly expressing wild type PTEN protein was down-regulated, and that of HHCC transfected with mutated PTEN gene was equal to that of control.</p><p><b>CONCLUSION</b>Wild type PTEN gene can inhibit the proliferation of hepatocellular carcinoma cells at G(1) phase. The mechanism is possibly related with intrinsic activity of AKT, which is down-regulated by wild type PTEN.</p>