| Objective:Ubiquitination is one of the most important post-translational modifications of intracellular protein,which mediates the degradation of specific protein substrate via proteasome or modulates the activity of substrate in a non-protein degradation manner,whereby participating in a variety of cellular processes,including cell cycle,signaling transduction and DNA damage repair.Upon activated by ubiquitin-activiting enzyme(E1),the ubiquitin molecule is transferred via ubiquitin-conjugation enzyme(E2)to the substrate,which is specifically recognized by ubiquitin ligase(E3).E3 plays a critical role during ubiquitination process in that it determines the specificity of the ubiquitination-modified substrate.The CUL4 A ligase belongs to the RNIG-finger E3 ligase family,and it has recently been reported to be taking part in the process of accurately modulating cell cycle,DNA repair and apoptosis at the post-translational level,therefore aberrant expression of CUL4 A is involoved in a number of malignancies.The aim of this study is to determine the expression level of CUL4 A in gastric cancer as well as its correlation with clinic-pathologicalcal data,and to investigate the roles of CUL4 A in response to chemotharepy in gastric cancer.Methods:1.We collected the total protein of 34 gastric tumor samples and the paired adjacent normal gastric mucosa in Tianjin Medical University Cancer Institute and Hospital to determine CUL4 A exression at both mRNA and protein levels though qRT-PCR and western blotting.Moreover,we collected 167 gastric tumor samples and the paired adjacent normal gastric mucosa from the patients who underwent surgery treatment during January 2003 to June 2009 in the same hospital to carry on the following studies:(1)Prepare the tissue microarray to determine the expression of CUL4 A in both tumor tissues and normal controls by immunohistochemical staining(IHC);(2)Collect the clinical and pathological characteristics of all patients for detailed statistical analysis;(3)Analyze the relationship among the clinicopathological characteristics,prognosis of the patients and the expression of CUL4 A.(4)Perform a subgroup analysis of the patients who have received chemotherapy.2.Construct the CUL4 A tetracycline-induced knockdown plasmid(Tet-On shCul4a)using pLKO-Tet-On lentiviral vector,and the newly constructed plasmid was confirmed by restriction enzyme digestion and sequencing.The lentiviral particles carrying Tet-On shCul4 a were synthesized by transfection with LipofectamineTM3000 into 293 T cells.The gastric cancer cell lines were subsequently infected to generate stable gastric cancer cell lines carrying Tet-On shCul4 a.The expression of CUL4 A protein in these cell lines were determined by western blotting.3.WST-8 was used to test the sensitivity of CUL4 A silenced gastric cancer cell line to 5-FU and cisplatin.Cell cycle was analyzed using PI staining.The levels of cell cycle related proteins,including the known substrate proteins of CUL4 A ligase were determined by western blotting.The comet assay was used to evaluate the DNA repair ability of CUL4 A knockdown gastric cells.Moreover,the levels of some known and potential substrate proteins of CUL4 A ligase,including EME1,were determined.Furthermore,Co-IP and competitive ubiquitination assay were carried out to confirm EME1 as a substrate protein of CUL4 A ligase.Apoptotic analysis and invasion ability of gastric cancer cells were also performed upon CUL4 A knockdown.Result:1.The expression of CUL4 A in gastric cancer tissues:(1)CUL4A positive stainings were located in the nucleus and cytoplasm,and CUL4 A expression was significantly higher in the tumor tissue than that in normal gastric tissue(P<0.001).(2)The relative low expression of CUL4 A in tumor was associated with the degree of tumor differentiation and Laurn classification,which were independent risk factors in prognosis.(3)The results of subgroup analysis showed that the expression of CUL4 A was negatively correlated with the chemotherapy response and was an independent risk factor for poor prognosis in gastric cancer patients.2.The roles of CUL4 A in modulating the chemotharepy response in gastric cancer:(1)Knockdown of CUL4 A in gastric cancer cells enhanced the tolerance of 5-FU,at least partially,due to the G1 phase arrest cause by p21,which is a a substrate protein of CUL4 A ligase,since further knockdown of p21 in those cells reversed the 5-FU resistance phenotype.(2)Knockdown of CUL4 A in gastric cancer cells resulted in cisplatin resistance due to the enhanced DNA repair capacity obtained from increased EME1 level in these cells.(3)The increase of EME1 level upon tetracycline-induced CUL4 A knockdown was induction-time dependent.Co-IP result showed that there was a direct interaction between EME1 and CUL4A-DDB1 complex.Furthermore,competitive ubiquination assay showed decreased EME1 ubiquitination level following the increased dosages of dominant negative CUL4A(? CUL4A).Together,these results suggested that EME1 was a novel substrate of CUL4 A ligase in gastric cancer cells.Conclusions:1.The relative low expression of CUL4 A in tumor tissue is an independent risk factor for poor prognosis of gastric cancer patients,which is associated with increased chemotherapy resistance.2.Knockdown of CUL4 A in gastric cancer cells results in enhanced tolerance of 5-FU and cisplatin,which at least partially caused by CUL4 A regulated cell cycle arrest and enhanced DNA damage repair ability via modulating the levels of its substate protein P21 and EME1. |
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