027, Fisher’s exact test) (Table 1). Presence of tumor microsatellites is an established feature of intrahepatic metastasis of HCC. This finding showed that underexpression
of PTEN was frequent in human HCCs and was related to tumor growth and tumor metastasis. The overall survival rates of the 40 patients were 55.6%, 27.8%, and 16.7% months at 1, 3, and 5 years, respectively. Patients whose tumors had PTEN underexpression had significantly shorter overall survival rates compared with those whose tumors had no PTEN underexpression (median, 15.2 buy Tyrosine Kinase Inhibitor Library and 63.2 months, respectively; P = 0.035, log-rank test) (Fig. 1C). From our clinicopathologic correlation findings, PTEN underexpression was associated with feature of intrahepatic metastasis. Interestingly, we observed a marked reduction of PTEN protein level in a higher metastatic potential HCC cell (H2M) compared with the cell line (H2P) derived from the primary HCC of the same patient and has a lower metastatic potential HCC cell (Fig. 1D).10 To assess the effect of PTEN on HCC cell migration, we knocked down the expression of PTEN Alectinib solubility dmso by shRNA in the HCC cells lines BEL-7402 and SMMC-7721,
which have relatively high levels of PTEN (Fig. 1D). Reduced PTEN expression was confirmed by western blotting; there was 35%-51% reduction of endogenous PTEN protein level in either BEL-7402 or SMMC-7721 cells (Fig. 2A). Using Transwell migration and Matrigel invasion assays (Fig. 2B,C and Supporting Information Fig. 1), we observed that knockdown of PTEN in HCC cells significantly enhanced cell migration and invasion, respectively, in both BEL-7402 and SMMC-7721 cells
(P = 0.011 and 0.020, respectively, for cell migration assay and P = 0.004 and 0.012, respectively, for cell invasion assay, Student t test) (Fig. 2B,C and Supporting Fig. 1). To further delineate the effects of complete PTEN loss on cell migration and invasion, PTEN−/− MEFs were established and the absence of PTEN protein expression was confirmed with western blotting (Fig. 3A). Similar to the PTEN-knockdown HCC cells, the PTEN−/− MEFs showed 上海皓元 a significantly enhanced ability to migrate and invade, as assessed by the Transwell migration and Matrigel cell invasion assays, respectively (P < 0.001 for both, Student t test) (Fig. 3B). We then investigated whether the induced cell invasion by loss of PTEN, as shown in the cell invasion assay, involved degradation of the Matrigel. We evaluated the role of MMP2, a gelatinase responsible for degradation of collagen IV, in the enhanced cell invasion mediated by loss of PTEN. The mRNA levels of MMP2 in the PTEN-knockdown stable clones of SMMC-7721 and BEL-7402 were analyzed by quantitative PCR. MMP2 transcription was up-regulated by 3.5-fold and two-fold in the PTEN-knockdown BEL-7402 and SMMC-7721 cells, respectively (P = 0.002 and 0.006, respectively, Student t test) (Fig. 4A). Consistently, there was 1.