Study Of The Inhibition Function Of PTEN Gene On Hepatocellular Carcinoma And Its Mechanisms

PTEN gene is a new rumor suppressor gene found in 1997. Many tumors have been found to have mutations or deletions of PTEN gene, which is closely related with the genesis of tumor. PTEN is a dual specificity phosphatase: It has both lipid phosphatase activity and protein phosphatase activity, and it is also the first found tumor suppressor gene to have lipid phosphatase activity up to now. PTEN may function to regulate cell migration/adhesion through its protein phosphatase activity and to regulate cellular growth and induce apoptosis through its lipid phosphatase activity.Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with short course, quick progress, high fatality rate and great harm to the health of humans beings. Since the genesis of HCC is a very complicated multi-steped course and a lot of changed tumor suppressor genes involved in it, deep study of the tumor suppressor genes is of great significance to clarify the cause of HCC and help to the prevention and cure of this disease.We first studied the expression of PTEN mRNA and its protein in HCC by in situ hybridization and immunohistochemistry. The results are as follows: (1) Of the 56 cases of hepatocellular carcinoma, positive rate of PTEN mRNA andits protein was 62.5%(35/56) and 71.4%(40/56) respectively. (2) Of the 17 cases of liver cirrhosis, positive rate of PTEN mRNA and its protein was 88.2% (15/17) and 94.1%(16/17) respectively. (3) Of the 21 cases of pericarcinomatous tissues, positive rate of PTEN mRNA and its protein was 90.5%( 19/21) and 100%(21/21) respectively. Significant difference (PO.05) was observed between HCC and pericarcinomatous tissues and liver cirrhosis. The lowest differential HCC showed the lowest positive rate: 42.9%(9/21) and 57.1%( 12/21) respectively. A significant difference (PO.05) was observed between I , II and Ell grades of HCC in the expression of PTEN mRNA and its protein. Low expression rate of PTEN mRNA and its protein was observed in HCC, and the lower differential HCC showed the lower positive rate. These suggested that the low expression of PTEN mRNA and its protein might play a certain role in the progression of HCC and their deletions were mainly in the late stage.The ukaryotic expression vector pBabe-Puro-PTEN of PTEN was introduced into human hepatocellular carcinoma cell line HHCC by gene trasfection mediated by lipofectamine. In situ hybridization , immunohistochemistry and western blot were used to detect the PTEN mRNA and its protein in the fourth passage of HHCC cells which transfected with pBabe-Puro-PTEN. Then we observed the effect of PTEN gene on HHCC cell growth and proliferation ability by measuring the growth curve and plate-clone formation. We found the growth slowed down and the ability to form colony decreased hi HHCC cells transfected with pBabe-Puro-PTEN. We concluded that exogenous PTEN gene might have the ability to suppress the growth and decease the malignant phenotype of human hepatocellular carcinoma cell line HHCC.The mechanisms of the function of PTEN gene were further analysed through flow cytometry, phase contrast microscope, HE staining, electronic microscope, DNA electrophoresis , TRAP-PCR-ELISA and western blot. Theresult of flow cytometry showed that the distribution of HHCC/pBabe-Puro cells in GI phase was about 46.7% while HHCC/pBabe-Puro-PTEN cells in GI phase arrived to 56.1%. This proved the existing of GI phase arrest; In addition, there appeared an apoptotic peak in HHCC/pBabe-Puro-PTEN cells and the apoptotic rate is 11.7%. Apoptosis were also demonstrated by phase contrast microscope, HE staining , electronic microscope and DNA electrophoresis. TRAP-PCR-ELISA showed that the telomerase activity decreased in HHCC/pBabe-Puro-PTEN cells and the absorbance value of "A45o-A69o" was 1.484 ?.08. While the HHCC cells and HHCC/pBabe-Puro cells expressed high level of telomerase activity with the absorbance value of "A4so-A69o" being 2.786?.06 and 2.843 + 0.09 respectively. The level of p27Kipl protein was increased in the PTEN...