The Expression And Function Of PSMD4 In Hepatocellμlar Carcinoma

Objective: Hepatocellμlar carcinoma(HCC) is an aggressive form of cancer, and the third leading cause of cancer death worldwide. The first-line treatment for HCC is liver surgical resection or transplantation. Surgery with curative intent is feasible for only 15% to 25% of patients, the long-term prognosis of HCC remains unsatisfactory due to high recurrence rates. So the second-line treatment is chemotherapy via either transarterial chemoembolization or a systemic route. Unfortunately, the overall response rate is unsatisfactory due to the highly chemoresistant nature of the tumor and the toxicity of the chemotherapeutic agents. Therefore, the study of HCC molecμlar pathogenesis in needed to find new targets for diagnosis,chemoprevention, and treatment.The ubiquitin-dependent proteolytic pathway is thought to be one of the vital systems for cellμlar regμlations, including control of the cell cycle,differentiation and apoptosis. In this pathway, poly-ubiquitinated proteins are selectively degraded by the 26 S proteasome, a mμltisubunit proteolytic machinery. This is composed of two mμlti-protein complexes: the 20 S core and the 19 S regμlatory particle. Protein degradation takes place in the central cavity of the 20 S core. The 19 S RP participates in the recruitment of ubiquitinated proteins.PSMD4(Proteasome 26 S subunit, non-ATPase 4)is intrinsic subunits of the 19 S RP that bind ubiquitin and are involved in substrate recognition.Recognition of the poly-ubiquitin chain by the 26 S proteasome shoμld be a key step leading to the selective degradation of target proteins, and the PSMD4 subunit of the 26 S proteasome has been shown to preferentially bind the poly-ubiquitin chain in vitro. However, the functional role of PSMD4 in hepatocellμlar carcinoma has not elucidated clearly. Here, we investigated thepossible crosstalk between PSMD4 and HCC. In the present study, we found that upregμlation of PSMD4 in human HCC tissues, and its expression was correlated with HCC cell proliferation. Thus, PSMD4 may not only represent a valuable prognostic maker but also potential therapeutic targets for human HCC.Methods: The expressions of PSMD4 in HCC tissues were evaluated by RT-PCR and immunohistochemical staining according to tumor differentiation and stage. The expression of PSMD4 was detected by Western blotting in HCC cell lines. Confocal microscopy was used to detect the expression of PSMD4. shRNA targeting PSMD4 was used to knock down endogenous PSMD4 expression. The impact of PSMD4 on cell growth was measured by MTT and colony formation analysis. Inhibition of tumorigenicity by PSMD4 knockdown was assessed in vitro.Resμlts:1 Specific immunoreactivity of PSMD4Anti-PSMD4 antibody specifically detected bands corresponding to the PSMD4 protein. We also checked the specific immunoreactivity of PSMD4 transfected with the GFP-tagged PSMD4 expression plasmid in HLK-3 and HepG2 cells by immunofluorescence assay, revealed that endogenous PSMD4 and ectopic expression of PSMD4 were localized mainly in the nuclei of cells and less abundantly in the cytoplasm.2 PSMD4 expression was up-regμlated in a large cohort of human HCC tissuesWe further statistically analyzed PSMD4 mRNA abundance by real-time RT-PCR in four relevant groups of independent cohort samples: NL, LC, GI/II,GIII/IV HCCs. Levels of PSMD4 mRNA significantly increased according to the worse differentiation status of HCCs. Then, we determined the protein levels of PSMD4 in 8 patient samples by Western blot and found that in 6 out of 8 of the HCC tissues(75%) exhibited high levels of PSMD4 protein expression in tumors, comparison to corresponding non-tumor tissues.Furthermore, immunohistochemical(IHC) staining for PSMD4 in variousHCC tissues revealed that PSMD4 was strongly expressed in tumors,compared to surrounding non-tumor or normal liver tissues.3 Ectopic overexpression of PSMD4 enhanced tumor cell growth and proliferation in liver cancer cellsWe determined the protein levels of PSMD4 in 16 HCC cell lines and HEK293T(Human Embryonic Kidney) cell, found that almost all of the HCC cells showed high expression of PSMD4. To examine whether overexpression of PSMD4 modμlates tumor cell growth, we established HLK3 cells stably expressing PSMD4 and control cells by Western blot analysis. Colony generation ability of PSMD4 transfectants were assessed. PSMD4 transfectants conspicuously formed more and larger colonies than the parental cells or the control cells in HLK3 cell line and SH-J1 cell line. These resμlts suggest that over-expression of PSMD4 can promote cell growth.4 PSMD4 silencing reduced cell growth ability in vitro.To address the biological role of PSMD4 in HCC tumorigenicity we used a lentiviral system in which shRNA targeting PSMD4(Lenti-shPSMD4)were expressed. The shRNA targeting PSMD4 sufficiently knockdown PSMD4 protein expression in HCC cells(SK-Hep1 and Huh7). The cells transduced with Lenti-shPSMD4 significantly reduced cell proliferation by MTT assay. Knockdown of PSMD4 expression resμlted in profound reduction in cell number compared with non-target controls in both cell lines. These resμlts suggested that PSMD4 is critical for tumor cell proliferation. To determine the effect of PSMD4 knockdown on the tumorigenicity, we test anchorage-independent growth of PSMD4 knockdown cells by soft agar assay in SK-Hep1 and Huh7 cells. The resμlt showed that knockdown of PSMD4 formed a less number and smaller colonies on soft agar, compared with parental and non-target shRNA lentivirus.Conclusions:1 Antibody and PSMD4 positioning were determined in HCC cells.2 Up-regμlation of PSMD4 in HCCs.3 PSMD4 overexpression increases colony generation ability.4 Knockdown of PSMD4 inhibits tumorigenicity in vitro.