Department of Gastroenterology, The First People's Hospital of Anqing, Anqing, Anhui 246000, China.
Department of Laboratory Medicine, Yijishan Hospital of Wannan Medical College, Wuhu, Anhui 241001, China.
Chin Med J (Engl). 2021 Nov 3;134(22):2721-2729. doi: 10.1097/CM9.0000000000001851.
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.
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.
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 ± 0.12 vs. 0.23 ± 0.05, 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 ± 0.07 vs. 1.01 ± 0.11, t = 8.09, P = 0.001; protein level: 0.61 ± 0.03 vs. 0.93 ± 0.07, t = 7.19, P = 0.002) and Hep3b cells (mRNA level: 0.55 ± 0.11 vs. 1.04 ± 0.15, t = 4.51, P = 0.011; protein level: 0.64 ± 0.10 vs. 0.95 ± 0.08, 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 ± 0.14 vs. 0.97 ± 0.16, t = 3.13, P = 0.036; OD value of 4 days: 1.03 ± 0.07 vs. 1.50 ± 0.12, t = 5.97, P = 0.004) and Hep3b (OD value of 3 days: 0.69 ± 0.14 vs. 1.10 ± 0.11, t = 3.91, P = 0.017; OD value of 4 days: 1.12 ± 0.12 vs. 1.48 ± 0.13, 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 ± 3.41]% vs. [58.72 ± 3.78]%, t = 9.34, P = 0.001) and Hep3b cells (EdU positive rate: [44.13 ± 7.02]% vs. [61.79 ± 3.96]%, t = 3.79, P = 0.019). FCM assay suggested that CCT4 knockdown induced apoptosis in HCC cells (apoptosis rate of Huh7: [9.10 ± 0.80]% vs. [3.66 ± 0.64]%, t = -9.18, P = 0.001; apoptosis rate of Hep3b: [6.69 ± 0.72]% vs. [4.20 ± 0.86]%, 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 ± 0.06 vs. 0.44 ± 0.05, t = -4.69, P = 0.009) and B-cell lymphoma-2 (Bcl-2) interacting mediator of cell death (Bim; 0.96 ± 0.06 vs. 0.61 ± 0.09, t = -5.65, P = 0.005) accumulation. The upregulation of securin inhibited cell growth by downregulating cyclin D1 (0.65 ± 0.05 vs. 1.04 ± 0.07, t = 8.12, P = 0.001), and the accumulation of Bim inhibited Bcl-2 (0.77 ± 0.04 vs. 0.87 ± 0.04, t = 3.00, P = 0.040) and activated caspase 9 (caspase 9: 0.77 ± 0.04 vs. 0.84 ± 0.05, t = 1.81, P = 0.145; cleaved caspase 9: 0.64 ± 0.06 vs. 0.16 ± 0.07, t = 1.81, P = 0.001), which led to elevated apoptosis.
Overall, these results showed that CCT4 played an important role in HCC pathogenesis through, at least partly, interacting with Cdc20.
伴侣蛋白包含 T 复合物（CCT）蛋白在酵母和哺乳动物的细胞周期相关蛋白降解中发挥重要作用。CCT 蛋白的一种亚型 CCT4 在肝细胞癌（HCC）进展中的作用尚未完全阐明。本研究旨在探讨 CCT4 在 HCC 中的作用机制。
本研究利用 UALCAN 平台分析 CCT4 与 HCC 的关系，并分析 CCT4 与 HCC 患者总生存期（OS）的相关性。Western blot（WB）检测 HCC 肿瘤组织和正常组织中 CCT4 的表达。采用慢病毒载体敲低 CCT4 的表达，并用定量聚合酶链反应（qPCR）和 WB 检测 HCC 细胞系中 CCT4 的水平。细胞计数试剂盒-8（CCK-8）和 5-乙炔基-2'-脱氧尿苷（EdU）检测细胞增殖，流式细胞术（FCM）检测 HCC 细胞的凋亡。通过共免疫沉淀（co-IP）实验和 WB 实验探索 CCT4 调节 HCC 细胞生长的机制。至少重复三次实验并计算数据，结果表示为均数±标准差。采用学生 t 检验、配对 t 检验和 Kaplan-Meier 分析比较不同组之间的差异。
我们发现 CCT4 在 HCC 组织中表达上调，与不良预后相关（P＜0.001）。与正常组织相比，CCT4 在 HCC 肿瘤组织中表达显著增加（0.98±0.12 比 0.23±0.05，t=7.73，P＜0.001）。用 CCT4 短发夹 RNA（shRNA）转染后，Huh7（mRNA 水平：0.41±0.07 比 1.01±0.11，t=8.09，P=0.001；蛋白水平：0.61±0.03 比 0.93±0.07，t=7.19，P=0.002）和 Hep3b 细胞（mRNA 水平：0.55±0.11 比 1.04±0.15，t=4.51，P=0.011；蛋白水平：0.64±0.10 比 0.95±0.08，t=4.32，P=0.012）中 CCT4 的 mRNA 和蛋白水平均降低。CCK8 检测表明，CCT4 敲低抑制了 Huh7（细胞活力：3 天的 OD 值：0.60±0.14 比 0.97±0.16，t=3.13，P=0.036；4 天的 OD 值：1.03±0.07 比 1.50±0.12，t=5.97，P=0.004）和 Hep3b（细胞活力：3 天的 OD 值：0.69±0.14 比 1.10±0.11，t=3.91，P=0.017；4 天的 OD 值：1.12±0.12 比 1.48±0.13，t=3.55，P=0.024）细胞的增殖。EdU 检测表明，CCT4 敲低抑制了 Huh7（EdU 阳性率：[31.25±3.41]%比[58.72±3.78]%，t=9.34，P=0.001）和 Hep3b（EdU 阳性率：[44.13±7.02]%比[61.79±3.96]%，t=3.79，P=0.019）细胞的增殖。FCM 检测提示 CCT4 敲低诱导 HCC 细胞凋亡（Huh7 的凋亡率：[9.10±0.80]%比[3.66±0.64]%，t=-9.18，P=0.001；Hep3b 的凋亡率：[6.69±0.72]%比[4.20±0.86]%，t=-3.84，P=0.018）。我们还发现，CCT4 通过与 Cdc20 相互作用来调节后期促进复合物（APC）Cdc20 活性。此外，CCT4 敲低诱导 securin（0.65±0.06 比 0.44±0.05，t=-4.69，P=0.009）和 B 细胞淋巴瘤-2（Bcl-2）相互作用的细胞死亡介体（Bim；0.96±0.06 比 0.61±0.09，t=-5.65，P=0.005）积累。securin 的上调通过下调细胞周期蛋白 D1（0.65±0.05 比 1.04±0.07，t=8.12，P=0.001）抑制细胞生长，而 Bim 的积累抑制 Bcl-2（0.77±0.04 比 0.87±0.04，t=3.00，P=0.040）并激活半胱天冬酶 9（caspase 9：0.77±0.04 比 0.84±0.05，t=1.81，P=0.045；cleaved caspase 9：0.64±0.06 比 0.16±0.07，t=1.81，P=0.001），从而导致细胞凋亡增加。
总之，这些结果表明，CCT4 通过与 Cdc20 相互作用在 HCC 发病机制中发挥重要作用。
