Preliminary Studies On The Correlation Of P27^kip1 And P27^kip1-related Molecule JAB1 With Human Hepatocellular Carcinoma

ObjectiveInvestigate the expression of JAB1 and p27 protein in human hepatocellular carcinoma (HCC) and its clinicopathologic significance. The expression of JAB1 and p27 were analyzed and compared with that of proliferative cell nuclear antigen (ki67) in these specimens. Clone human p27kip1 gene, construct its eukaryotic expressive vectors, Evaluate the effect of arsenic trioxide(As2O3) (a chemotherapeutic drug) on activity, cell cycle and apoptosis of SMMC-7721 cells, and preliminarily investigate the role of p27kip1 linked protein network during the process.Methods1. Immunohistochemical study for JAB1, p27 was performed on 76 cases of hepatocellular carcinoma tissue and adjacent nontumorous tissues, 10 cases of native liver tissue adjacent to hepatic hemangioma and 10cases of fresh HCC tissue and the adjacent liver tissue .Fresh specimens from 6 cases of HCC and the adjacent liver tissue were also collected for Western blot analysis.2. Human p27kip1 complete cDNA was amplified by RT-PCR method, and was reconstructed into the eukaryotic expressive vector pcDNA3.1 and pEGFP-N2 to form the p27kip1 sense or antisense expressive plasmid and p27-EGFP fusing protein expressive plasmid. The reconstructed DNA was identified by enzyme digestion and DNA sequencing.3. The influence of As2O3 on the activity, cell cycle or apoptosis of hepatomacellular carcinoma SMMC-7721 cell, cells was detected by MTT colorimetry, Flow cytometry and Hochest staining. The expression of p27kip1 and p27kip1-related molecule was investigated by Western blot. The subcellular localization of p27kip1 and p27kip1-related molecule were detected by immunoflurescence.Results1. Western blot analysis showed that in most HCC, JAB1 expression was higher than that in adjacent liver tissue. Statistically significant difference in expressions of JAB1 and p27kip1 were noted in the three groups studied (P<0.001).The expression of JAB1 in HCC was significantly higher than that in other two groups.The expression of JAB1 was correlated with histological differentiation, serum alpha-fetal protein levels and metastasis (P<0.05).The levels of p27kip1 is lower in cancer tissues than in nontumorous tissues. There was a inverse correlation between JAB1 and p27 in HCC. Positive correlation was found between JAB1 and ki67 in HCC (P<0.001).2. The cloned DNA was confirmed to be human p27kip1 cDNA.3. As2O3 significantly inhibited the growth of SMMC-7721 cell, compared with untreated control cell. A striking increase in p27kip1 expression and a reciprocal decrease in p27kip1-related protein (JAB1, CRM1) expression were found in As2O3-treated SMMC-7721 cell. Meanwhile, As2O3 decreased the protein levels of Cdk2 and cyclinE .The location of p27kip1 and JAB1 were transferred from cytoplasm to nucleus which detected by immunoflurescence.CRM1 was mainly located in cytoplasm and no significant changing of subcellular localization has been detected during this time.Conculusions1.Decreased expression of p27kip1 and increased expression of JAB1 were closely correlated with the aggressiveness of HCC. There was a inverse correlation between JAB1 and p27kip1 in HCC, and positive correlation was found between JAB1 and ki67. overexpression of JAB1 may reduce the protein level of p27kip1 by accelerating p27kip1 degradation via nuclear export mechanism and causes disruption of cell cycle control and enhancement of the proliferative activity, indicating JAB1 playes an important role in oncogenesis and development of HCC.2. We successfully cloned human p27kip1 gene and constructed its eukaryotic expressive plasmids, which was an ideal model for further study.3. Arsenic trioxide attenuated JAB1, CRM1 expression, thereby disturbing the location and expression of p27kip1, and then may participate in regulating the growth of human hepatoma cell through interfering with the function of p27kip1.