PIM-2 Activates API-5 To Inhibit The Apoptosis Of Hepatoma Carcinoma Cells Through NF-ΚB Pathway

Objective: To explore the relationship among oncogene Pim-2 and its possible downstream factors NF-κB and API-5, so as to illuminate the detailed anti-apoptosis mechanism of Pim-2 in hepatocarcinoma cells, and finally confirm that Pim-2 could activate apoptotic protein API-5 to inhibit the apoptosis of hepatocarcinama cells through NF-κB pathway.Methods: (1) 27 hepatocellular carcinoma tissues (HCC) and 27 corresponding paired noncancerous liver tissues (PNL) were obtained from the surgery specimen in the second affiliated hospital of Chongqing medical university (HCC were diagnosed by pathology. PNL were defined as liver tissues 2cm away from the edge of carcinoma). 11 normal liver tissues (NL) were obtained from non liver disease volunteer patients who receive upper abdominal surgery in the same period in the hospital. Then three experimental groups were set up in the clinical specimen experiment: HCC, PNL and NL. (2) The mRNA levels of Pim-2, NF-κB and API-5 in the tissues were detected by real-time PCR; the protein levels of Pim-2, total API-5 (tAPI-5) and phosphorylated API-5 (pAPI-5) were detected by immunohistochemistry and western blotting; and the NF-κB activity were detected by EMSA. (3) Eukaryotic expression plasmid carrying human Pim-2 gene was transfected into the nontumorous human liver cell line L02. L02 cells transfected with empty vector and the ones without transfection were set up as controls. Pim-2 specific SiRNA were designed and transfected into human liver cancer cell line HepG2. Also, HepG2 cells transfected with empty vector and the ones without transfection were set up as controls (completed in our prior work). (4) Eight experimental groups were set up in the cell experiment: nontumorous human liver cell line (L02), L02 cells transfected with Pim-2 gene (L02/Pim-2), L02 cells transfected with empty vector (L02/Vector), human liver cancer cell line (HepG2), HepG2 cells transfected with Pim-2 SiRNA (HepG2/Pim-2 SiRNA), HepG2 cells transfected with empty vector (HepG2/Vector), L02/Pim-2 cells cultured with NF-κB specific inhibitor parthenolide (L02/Pim-2+parthenolide), and HepG2 cells cultured with parthenolide (HepG2+parthenolide). (5) The mRNA levels of Pim-2, NF-κB and API-5 in the cells were detected by real-time PCR; the protein levels of Pim-2, tAPI-5 and pAPI-5 were detected by western blotting; the NF-κB activity were detected by EMSA; and the cell apoptosis rate were detected by AnnexinV-FITC/PI double staining method.Results: (1) In the clinical specimen experiment, the mRNA levels of Pim-2, NF-κB and API-5 in HCC group were significantly higher than those in PNL and NL groups (p<0.05); the protein expression of Pim-2, tAPI-5 and pAPI-5 were positive in HCC group but negative in PNL and NL groups; the protein levels of Pim-2, tAPI-5 and pAPI-5 in HCC group were significantly higher than those in PNL and NL groups (p<0.05); the activity of NF-κB in HCC group was significantly higher than those in PNL and NL groups (p<0.05). (2) In the cell experiment, after the transfection of Pim-2 gene, the mRNA and protein levels of Pim-2 were significantly higher in L02/Pim-2 cells than those in L02 cells and L02/vector cells (p<0.05). After the transfection of Pim-2 SiRNA, the mRNA and protein levels of Pim-2 were significantly lower in HepG2/Pim-2 SiRNA cells than those in HepG2 cells and HepG2/vector cells (p<0.05). In these cell groups, the mRNA levels of NF-κB and API-5 were parallel to the mRNA level of Pim-2. The protein levels of tAPI-5 and pAPI-5, as well as the activity of NF-κB, were parallel to the protein level of Pim-2. When parthenolide were added in L02/Pim-2 cells and HepG2 cells which highly expressed Pim-2, the mRNA and protein levels of Pim-2 had no significant changes, while the mRNA levels of NF-κB and API-5, the protein levels of tAPI-5 and pAPI-5, as well as the activity of NF-κB, were all significantly decreased (p<0.05). It was not the Pim-2 but pAPI-5 levels that affected the cell apoptosis rates directly. The cell apoptosis rates were antiparallel to the level of pAPI-5 in all the cell groups.Conclusions: (1) From the clinical specimen experiments, we could infer that Pim-2, NF-κB and API-5 may be related factors in some signal transduction pathway which is associated with the genesis and deveiopment of liver cancer. (2) The cell experiments convince that Pim-2 could activate API-5 through phosphorylation mechanism so as to inhibit the apoptosis of hepatocarcinoma cells, and NF-κB may be the key regulating factor on this signal transduction pathway.