Molecular Mechanisms Of Effect Of PTEN And Profilin 1 On Proliferation And Migration In Humam Hepatocelluar Carcinoma Cells

Hepatocelluar Carcinoma (HCC) is a common malignant tumor in China. Metastasis and recurrence are the major causes for its bad prognosis. Because of the limitations in conventional therapy methods, it is necessary to make research into the molecular mechanisms of HCC development and migration. In many types of cancers, the activation of signaling pathways associated with PPARγ (peroxisome proliferator activated receptor γ) and atRA (all-trans retinoic acid) has been shown to have widely antitumor effects, such as inhibiting proliferation and invasion, inducing differentiation and apoptosis, et al. Although such effects have been reported in many studies, the potential mechanisms still remain to be further investigated. Therefore further study on molecular mechanisms of these pathways will supply new insights and methods to HCC therapy. The purpose of our study is to elucidate the effect of these pathways on proliferation and migration of hepatocarcinoma cells.Part 1Up-regulation of PTEN was involved in inhibition of migration by activation of PPARγ signaling in human hepatocelluar carcinoma cellsBEL-7404 cell was shown to have lower ability of migration after it was treated with rosiglitazone (a selective ligand of PPAR) as well as rosiglitazone treatment did not interfere in its proliferation. The phosphorylation of Akt and FAK were significantly attenuated in cells treated with rosiglitazone. However, the phosphorylation of ERK did not significantly change. Because both Akt and FAK are on the downstream of tumor suppressor PTEN, we detected the expression of PTEN in cells treated with rosiglitazone and found rosiglitazone could increase PTEN protein expression in a time- and dose- dependent manner, which depended on the activation of PPARγ. Rosiglitazone treatment also increased PTEN mRNA level. Furthermore, PTEN overexpression resulted in the inhibition of cell migration and PTEN knock-down blocked the effect of rosiglitazone on cell migration. It suggested that PTEN was required for rosiglitazone-induced migration inhibition in BEL-7404 cells. In conclusion, our results demonstrated that PTEN played a critical role in rosiglitazone-induced migration inhibition in BEL-7404 cell.Part 2 Mechanism of PTEN negative feedback regulation in human hepatocarcinomacell line SMMC-7721To further investigate mechanism of PTEN regulation, GFP-PTEN expression plasmids was introduced into SMMC-7721 cell (human hepatocarcinoma cell line). We observed that higher amount of PTEN led to lower endogenic PTEN both in protein level and mRNA level. These results implied there might be a feedback regulation of PTEN in SMMC-7721 cell. Furthermore, activated Akt blocked the inhibition effect of ectopic PTEN on endogenic PTEN and PI3K inhibitor LY294002 treatment resulted in decreasing PTEN both in protein and mRNA levels. These results suggested the feedback regulation of PTEN depended on inhibition of PDK/Akt pathway. LY294002-induced inhibition of PTEN was also found in ES-2 and Changs cells, suggesting the feedback regulation of PTEN might exist in other types of cell. Using fragment deletion plasmids of PTEN, we found that the inhibition effect of ectopic PTEN on endogenic PTEN depended on its Carboxyl-terminal region.Part 3Up-regulation of Profilin 1 was involved in atRA-induced inhibition of proliferation and migration in human hepatocelluar carcinoma cellsOur previous research showed that atRA could increase profilin 1 protein expression. Using western blot and immunohistochemical stain, we also found the protein level of profilin 1 was lower in hepatocarcinoma tissues than corresponding matched paracancerous tissues. Futhermore, we constructed SiRNA plasmid targeting PFN1 to perform further research. Using contracted SiRNA plasmid, we found RNAi-based PFN1 knockdown could rescue the inhibitory effect of ATRA on cell proliferation and migration. These results suggest that profilin 1 plays a role in the inhibitory effect of atRA on cell migration suppression in SMMC-7721.