CCT4 suppression inhibits tumor growth in hepatocellular carcinoma by interacting with Cdc20.

BACKGROUND: The chaperonin containing t-complex (CCT) proteins play an important role in cell cycle-related protein degradation in yeast and mammals. The role of the chaperonin containing t-complex 4 (CCT4), one subtype of CCT proteins, in the progress of hepatocellular carcinoma (HCC) was not fully elucidated. Here, we aimed to explore the mechanisms of CCT4 in HCC. METHODS: In this study, we used the UALCAN platform to analyze the relationship between CCT4 and HCC, and the association of CCT4 with the overall survival (OS) of HCC patients was also analyzed. CCT4 expression in HCC tumor tissues and normal tissues was also determined by western blot (WB) assay. Lentivirus vector was used to knock down the CCT4 expression, and quantitative polymerase chain reaction and WB were used to determine the level of CCT4 in HCC cell lines. Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to detect the cell proliferation, and flow cytometry (FCM) was performed to evaluate the effect of CCT4 on the apoptosis of HCC cells. Co-immunoprecipitation (co-IP) assay and WB were used to explore the mechanisms of CCT4 regulating the growth of HCC. Data were calculated from at least three replicate experiments and expressed as mean ± standard deviation. Student's t test, paired t test, and Kaplan-Meier analysis were used to compare across different groups. RESULTS: We found CCT4 was upregulated in HCC tissues compared with normal tissues, and its high expression was associated with poor prognosis (P < 0.001). CCT4 was significantly increased in HCC tumor tissues compared with normal tissues (0.98 ±â€Š0.12 vs. 0.23 ±â€Š0.05, t = 7.73, P < 0.001). After being transfected with CCT4 short-hairpin RNA (shRNA), CCT4 was decreased in mRNA level and protein level in both Huh7 (mRNA level: 0.41 ±â€Š0.07 vs. 1.01 ±â€Š0.11, t = 8.09, P = 0.001; protein level: 0.61 ±â€Š0.03 vs. 0.93 ±â€Š0.07, t = 7.19, P = 0.002) and Hep3b cells (mRNA level: 0.55 ±â€Š0.11 vs. 1.04 ±â€Š0.15, t = 4.51, P = 0.011; protein level: 0.64 ±â€Š0.10 vs. 0.95 ±â€Š0.08, t = 4.32, P = 0.012). CCK8 assay indicated that CCT4 knockdown inhibited cell proliferation in both Huh7 (OD value of 3 days: 0.60 ±â€Š0.14 vs. 0.97 ±â€Š0.16, t = 3.13, P = 0.036; OD value of 4 days: 1.03 ±â€Š0.07 vs. 1.50 ±â€Š0.12, t = 5.97, P = 0.004) and Hep3b (OD value of 3 days: 0.69 ±â€Š0.14 vs. 1.10 ±â€Š0.11, t = 3.91, P = 0.017; OD value of 4 days: 1.12 ±â€Š0.12 vs. 1.48 ±â€Š0.13, t = 3.55, P = 0.024) cells. EdU assay showed that CCT4 knockdown inhibited the cell proliferation in both Huh7 (EdU positive rate: [31.25 ±â€Š3.41]% vs. [58.72 ±â€Š3.78]%, t = 9.34, P = 0.001) and Hep3b cells (EdU positive rate: [44.13 ±â€Š7.02]% vs. [61.79 ±â€Š3.96]%, t = 3.79, P = 0.019). FCM assay suggested that CCT4 knockdown induced apoptosis in HCC cells (apoptosis rate of Huh7: [9.10 ±â€Š0.80]% vs. [3.66 ±â€Š0.64]%, t = -9.18, P = 0.001; apoptosis rate of Hep3b: [6.69 ±â€Š0.72]% vs. [4.20 ±â€Š0.86]%, t = -3.84, P = 0.018). We also found that CCT4 could regulate anaphase-promoting complex (APC)Cdc20 activity via interacting with Cdc20. Furthermore, CCT4 knockdown induced securin (0.65 ±â€Š0.06 vs. 0.44 ±â€Š0.05, t = -4.69, P = 0.009) and B-cell lymphoma-2 (Bcl-2) interacting mediator of cell death (Bim; 0.96 ±â€Š0.06 vs. 0.61 ±â€Š0.09, t = -5.65, P = 0.005) accumulation. The upregulation of securin inhibited cell growth by downregulating cyclin D1 (0.65 ±â€Š0.05 vs. 1.04 ±â€Š0.07, t = 8.12, P = 0.001), and the accumulation of Bim inhibited Bcl-2 (0.77 ±â€Š0.04 vs. 0.87 ±â€Š0.04, t = 3.00, P = 0.040) and activated caspase 9 (caspase 9: 0.77 ±â€Š0.04 vs. 0.84 ±â€Š0.05, t = 1.81, P = 0.145; cleaved caspase 9: 0.64 ±â€Š0.06 vs. 0.16 ±â€Š0.07, t = 1.81, P = 0.001), which led to elevated apoptosis. CONCLUSIONS: Overall, these results showed that CCT4 played an important role in HCC pathogenesis through, at least partly, interacting with Cdc20.

Tumor markers in serum and ascites in the diagnosis of benign and malignant ascites.

OBJECTIVE: To evaluate the values of 4 tumor markers in serum and ascites and their ascites/serum ratios in the identification and diagnosis of benign and malignant ascites. MATERIALS AND METHODS: A total of 76 patients were selected as subjects and divided into malignant ascites group (45 cases) and benign ascites group (31 cases). Samples of ascites and serum of all hospitalized patients were collected before treatment. The levels of carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), cancer antigen 125 (CA125) and carbohydrate antigen 19-9 (CA19-9) were detected by chemiluminescence (CLIA) . RESULTS: CEA, AFP and CA19-9 in both serum and ascites as well as CA125 in ascites were evidently higher in the malignant ascites group than in the benign ascites group (P<0.01). Malignant ascites was associated with elevated ascites/serum ratios for AFP and CA125 (P<0.01). The areas under receiver operating characteristic (AUROCs) of CEA and CA125 in ascites and the ratios of ascites/serum of AFP, CEA, CA125 and CA19-9 were all >0.7, suggesting certain values, while those of ascites CA19-9 and serum CEA were 0.697 and 0.629 respectively, indicating low accuracy in the identification and diagnosis of benign and malignant ascites. However, the AUROCs of the remaining indexes were <0.5, with no value for identification and diagnosis. Compared with single index, the sensitivity of combined detection increased significantly (P<0.05), in which the combined detection of CEA, CA19-9 and CA125 in ascites as well as the ratio of ascites/serum of CEA, CA19-9, CA125 and AFP had the highest sensitivity (98.4%) but with relevantly low specificity. Both sensitivity and specificity of combined detection should be comprehensively considered so as to choose the most appropriate index. CONCLUSIONS: Compared with single index, combined detection of tumor markers in serum and ascites can significantly improve the diagnostic sensitivity and specificity.