The influence of glucose regulatory protein 78 on prognosis and tumor cell proliferation of hepatocellular carcinoma
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摘要:目的
分析葡萄糖调节蛋白78（glucose regulatory protein 78,GRP78）对肝癌预后及肿瘤细胞增殖的影响。
方法采用实验研究方法和回顾性队列研究方法。采用肝癌组织芯片,体外培养Huh7、Hep3B肝癌细胞和LO2正常肝细胞,结合免疫组织化学染色、细胞转染、实时荧光定量聚合酶链式反应（qRT‑PCR）、Western blot检测、细胞增殖实验、细胞克隆形成实验及高通量转录组学检测分析肝癌细胞GRP78表达情况。Hep3B、Huh7细胞转染GRP78基因特异性shRNA慢病毒设为GRP78⁃shRNA组,转染阴性对照shRNA慢病毒设为对照⁃shRNA组。观察指标：（1）肝癌组织和癌旁组织GRP78表达及其与临床病理特征的关系。（2）肝癌病人预后及影响因素分析。（3）抑制GRP78表达对肝癌细胞增殖的影响。（4）抑制GRP78表达对肝癌细胞p53、p21、CDK2、CDK4、CDK6基因和蛋白表达的影响。（5）HA15对肝癌细胞增殖和p53、p21、CDK2、CDK4、CDK6基因和蛋白表达的影响。正态分布的计量资料以x±s表示,组间比较采用t检验或方差分析。重复测量数据采用重复测量方差分析。计数资料以绝对数表示,组间比较采用χ²检验。单因素和多因素分析采用COX比例风险回归模型。采用Kaplan‑Meier法计算生存时间并绘制生存曲线,采用Log‑rank检验进行生存分析。
结果（1）肝癌组织和癌旁组织GRP78表达及其与临床病理特征的关系：肝癌组织芯片免疫组织化学染色结果显示,GRP78在90例肝癌组织中低表达53例,高表达37例,GRP78在90例癌旁组织中低表达84例,高表达6例,肝癌组织与癌旁组织比较,差异有统计学意义（P<0.05）。（2）肝癌病人预后及影响因素分析：90例病人均获得随访,随访时间为5～56个月,中位随访时间为49个月。53例GRP78低表达肝癌病人中位总体生存时间和中位疾病无进展生存时间分别为56个月和53个月,37例GRP78高表达肝癌病人上述指标分别为32个月和19个月,两者比较,差异均有统计学意义（χ²=17.482,12.097,P<0.05）。单因素分析结果显示：丙氨酸氨基转移酶（ALT）、肿瘤病理学分级、GRP78表达是影响肝癌病人3年总体生存率和疾病无进展生存率的相关因素（风险比=2.168,2.161,3.784和2.254,0.893,3.493,95%可信区间为1.061~4.432,1.069~4.368,1.793~7.989和1.096~4.636,0.438~1.818,1.631~7.252,P<0.05）。多因素分析结果显示：ALT>40 U/L、肿瘤病理学分级为Ⅲ~Ⅳ级、GRP78高表达是影响肝癌病人3年总体生存率和疾病无进展生存率的独立危险因素（风险比=2.317,2.039,3.740和2.194,2.177,2.927,95%可信区间为1.150~4.671,1.201~3.462,2.116~6.612和1.408~4.593,1.093~4.336,1.492~5.742,P<0.05）。（3）抑制GRP78表达对肝癌细胞增殖的影响：①qRT‑PCR检测结果显示,GRP78 mRNA在Huh7、Hep3B、LO2细胞中的相对表达量分别为3.06±0.33、4.42±0.60、1.00±0.02,Huh7、Hep3B细胞分别与LO2细胞比较,差异均有统计学意义（t=6.19,5.42,P<0.05）。②Western blot检测结果显示：GRP78蛋白在Huh7、Hep3B、LO2细胞中的相对表达量分别为1.65±0.01、1.77±0.01、0.99±0.02,Huh7、Hep3B细胞分别与LO2细胞比较,差异均有统计学意义（t=75.09,108.10,P<0.05）。③细胞增殖实验检测结果显示：Huh7细胞GRP78‑shRNA组和对照‑shRNA组24、48、72、96 h细胞增殖率分别为111.51%±0.35%、144.85%±0.68%、188.71%±3.62%、282.51%±5.25%和190.08%±0.58%、285.76%±2.69%、459.51%±4.29%、597.88%±12.25%,两组比较,差异有统计学意义（F组间=1 360.000,F时间=668.500,F交互=197.600,P<0.05）。Hep3B细胞GRP78‑shRNA组和对照‑shRNA组上述指标分别为124.47%±0.25%、153.25%±1.25%、195.45%±3.19%、282.51%±10.76%和179.69%±0.33%、322.67%±2.46%、486.27%±5.82%、622.35%±12.58%,两组比较,差异有统计学意义（F组间=1 222.000,F时间=706.200,F交互=179.600,P<0.05）。④细胞克隆形成实验结果显示：Huh7细胞GRP78‑shRNA组和对照‑shRNA组细胞数分别为（125±3）个和（435±17）个,两组比较,差异有统计学意义（t=17.86,P<0.05）；Hep3B细胞GRP78‑shRNA组和对照‑shRNA组上述指标分别为（138±3）个和（388±7）个,两组比较,差异有统计学意义（t=32.29,P<0.05）。（4）抑制GRP78表达对肝癌细胞p53、p21、CDK2、CDK4、CDK6基因和蛋白表达的影响：高通量转录组学检测结果显示,Huh7细胞GRP78‑shRNA组相对于对照‑shRNA组的p53、p21、CDK2、CDK4、CDK6表达率分别为19%、334%、398%、41%、49%。①qRT⁃PCR检测结果显示：Huh7细胞GRP78‑shRNA组和对照‑shRNA组中,GRP78、p53、p21、CDK2、CDK4、CDK6 mRNA的相对表达量分别为0.17±0.03,4.05±0.71,3.73±0.47,0.49±0.09,0.48±0.06,0.36±0.07和1.00±0.05,1.03±0.17,1.00±0.07,1.01±0.09,1.02±0.14,1.00±0.03,两组比较,差异均有统计学意义（t=14.62,4.17,5.72,4.26,3.49,8.82,P<0.05）。Hep3B细胞GRP78‑shRNA组和对照‑shRNA组上述指标分别为0.11±0.01,4.28±0.43,4.19±0.22,0.44±0.01,0.25±0.03,0.68±0.04和1.01±0.09,1.02±0.15,1.00±0.06,1.01±0.09,1.01±0.08,1.15±0.02,两组比较,差异均有统计学意义（t=10.19,7.14,13.79,6.37,9.42,9.61,P<0.05）。②Western Blot检测结果显示：Huh7细胞GRP78‑shRNA组和对照‑shRNA组中,GRP78、p53、p21、CDK2、CDK4、CDK6蛋白的相对表达量分别为0.45±0.01,1.98±0.05,2.31±0.12,0.75±0.03,0.69±0.04,0.82±0.03和1.01±0.05,1.03±0.01,1.00±0.02,1.00±0.01,1.01±0.02,1.00±0.03,两组比较,差异均有统计学意义（t=11.07,14.56,11.30,11.29,10.55,11.37,P<0.05）。Hep3B细胞GRP78⁃shRNA组和对照‑shRNA组上述指标分别为0.61±0.03,1.98±0.16,2.55±0.12,0.85±0.03,0.78±0.01,0.54±0.02和1.00±0.03,1.05±0.02,1.05±0.01,1.05±0.02,1.00±0.02,1.00±0.02,两组比较,差异均有统计学意义（t=10.97,13.40,12.35,11.06,12.45,13.78,P<0.05）。（5）HA15 对肝癌细胞增殖和p53、p21、CDK2、CDK4、CDK6基因和蛋白表达的影响：HA15半抑制浓度（IC50）实验结果显示,Huh7、Hep3B细胞48 h IC50分别为9.98 μmol/L、13.70 μmol/L。①分别以9.98 μmol/L和13.70 μmol/L HA15作用Huh7、Hep3B细胞,细胞增殖实验检测结果显示：HA15⁃Huh7细胞和正常Huh7细胞24、48、72、96 h细胞增殖率分别为112.81%±0.27%、154.71%±1.45%、237.66%±16.77%、294.40%±14.92%和133.67%±0.49%、352.93%±2.31%、557.17%±4.89%、662.60%±13.31%,两者比较,差异有统计学意义（F组间=766.800,F时间=518.200,F交互=133.300,P<0.05）；HA15⁃Hep3B细胞和正常Hep3B细胞上述指标分别为121.27%±2.32%、203.85%±3.18%、240.80%±3.02%、286.50%±7.10%和239.14%±1.02%、362.00%±5.44%、539.37%±10.80%、694.79%±17.13%,两者比较,差异有统计学意义（F组间=594.300,F时间=317.900,F交互=78.600,P<0.05）。②qRT‑PCR检测结果显示：HA15‑Huh7细胞和正常Huh7细胞中,GRP78、p53、p21、CDK2、CDK4、CDK6 mRNA的相对表达量分别为0.27±0.05,3.64±0.28,4.13±0.41,0.51±0.07,0.39±0.03,0.17±0.02和1.02±0.14,1.00±0.03,1.00±0.05,1.01±0.08,1.01±0.09,1.03±0.17,两者比较,差异均有统计学意义（t=5.00,9.25,7.63,4.73,6.82,5.01,P<0.05）；HA15⁃Hep3B细胞和正常Hep3B细胞上述指标分别为0.28±0.03,3.49±0.78,4.31±0.53,0.38±0.05,0.36±0.04,0.24±0.03和1.01±0.11,1.03±0.18,1.01±0.08,1.00±0.06,1.02±0.15,1.00±0.06,两者比较,差异均有统计学意义（t=6.26,3.08,6.21,7.97,4.26,11.08,P<0.05）。③Western blot检测结果显示：HA15‑Huh7细胞和正常Huh7细胞中,GRP78、p53、p21、CDK2、CDK4、CDK6蛋白的相对表达量分别为0.52±0.05,1.94±0.08,1.58±0.02,0.89±0.00,0.86±0.02,0.74±0.01和1.02±0.03,1.00±0.03,1.02±0.02,1.04±0.03,1.00±0.01,1.01±0.02,两者比较,差异均有统计学意义（t=11.54,10.28,11.03,12.81,13.67,10.09,P<0.05）。HA15‑Hep3B细胞和正常Hep3B细胞上述指标分别为0.57±0.02,1.67±0.04,1.41±0.04,0.82±0.03,0.70±0.02,0.74±0.01和1.03±0.01,0.98±0.03,1.00±0.03,1.03±0.03,1.01±0.01,1.04±0.01,两者比较,差异均有统计学意义（t=10.81,11.54,12.26,13.62,14.23,10.17,P<0.05）。
结论GRP78高表达是影响肝癌病人3年总体生存率和疾病无进展生存率的独立危险因素,抑制GRP78表达可抑制肝癌细胞增殖活性及影响p53、p21、CDK2、CDK4、CDK6基因和蛋白表达。
Abstract:ObjectiveTo investigate the influence of glucose regulatory protein 78 (GRP78) on prognosis and tumor cell proliferation of hepatocellular carcinoma.
MethodsThe experimental study and retrospective cohort study were conducted. Based on hepatocellular carcinoma tissue chip, in vitro culture of Huh7 and Hep3B hepatoma cells and LO2 normal hepatic cell, and combined with immunohistochemical staining, cell transfection, quantitative real‑time polymerase chain reaction (qRT‑PCR), Western blot detection, cell proliferation experiments, cell clone formation experiments and high‑throughput transcription histological analysis, the GRP78 expression in hepatoma cells was analyzed. Huh7 and Hep3B hepatoma cells being transfected with the GRP78 gene-specific shRNA lentiviruses or the negative control shRNA lentivirus were set as the GRP78 gene-specific shRNA lentivirus group and the negative control shRNA lentivirus group respectively. Observation indicators: (1) GRP78 expression in hepatocellular carcinoma tissue and adjacent tissue and its correlation with the clinicopathological characteristics of hepatocellular carcinoma patients; (2) analysis of factors affecting the prognosis of hepatocellular carcinoma patients; (3) effects of inhibiting of GRP78 expression on the proliferation of hepatoma cells; (4) effects of inhibiting of GRP78 expression on the gene and protein expression of p53, p21, CDK2, CDK4, and CDK6 in hepatoma cells; (5) effects of HA15 on the proliferation and the gene and protein expression of p53, p21, CDK2, CDK4, and CDK6 in hepatoma cells. Measurement data of the normal distribution were expressed as Mean±SD, and comparison of groups was conducted using the t test or ANOVA. Repeated measurement data were analyzed using repeated ANOVA. Count data were expressed as absolute numbers, and comparisons between groups was conducted using the chi‑square test. COX proportional hazards regression model was used for univariate and multivariate analysis. The Kaplan‑Meier method was used to calculate the survival time and draw survival curve, and the Log‑rank test was used for generative analysis.
Results(1) GRP78 expression in hepatocellular carcinoma tissue and adjacent tissue and its correlation with the clinicopathological characteristics of hepatocellular carcinoma patients: results of immunohistochemical staining of hepatocellular carcinoma tissue chip showed that GRP78 was low‑expressed in 53 cases and high-expressed in 37 cases of the 90 hepatocellular carcinoma tissues. GRP78 was low‑expressed in 84 cases and high‑expressed in 6 cases of the 90 paracancerous tissues. There was a significant difference in GRP78 expression between hepatocellular carcinoma tissues and paracancerous tissues (P<0.05). (2) Analysis of factors affecting the prognosis of hepatocellular carcinoma patients: all 90 patients were followed up for 5 to 56 months, with a median follow-up time of 49 months. The median overall survival time and median disease progression‑free survival time were 56 months and 53 months in the 53 hepatocellular carcinoma patients with GRP78 as low‑expressed, versus 32 months and 19 months in the 37 hepatocellular carcinoma patients with GRP78 as high‑expressed, respec-tively, showing significant differences (χ²=17.482, 12.097, P<0.05). Results of univariate analysis showed that alanine aminotransferase (ALT), tumor pathological grading and GRP78 expression were related factors affecting the 3-year overall survival rate and disease progression-free survival rate of hepatocellular carcinoma patients (hazard ratio=2.317, 2.039, 3.740 and 2.194, 2.177, 2.927, 95% confidence interval as 1.150‒4.671, 1.201‒3.462, 2.116‒6.612 and 1.048‒4.593, 1.093‒4.336, 1.492‒5.742, P<0.05). Results of multivariate analysis showed that ALT >40 U/L, tumor pathological grading as Ⅲ-Ⅳ grade and GRP78 as high‑expressed were independent risk factors affecting the 3-year overall survival rate and disease progression-free survival rate of hepatocellular carcinoma patients (hazard ratio=2.438, 2.245, 3.223 and 3.046, 2.473, 3.307, 95% confidence interval as 1.114‒5.334, 1.047‒4.814, 1.396‒7.440 and 1.337‒6.940, 1.141‒5.360, 1.399‒7.819, P<0.05). (3) Effects of inhibiting of GRP78 expression on the proliferation of hepatoma cells: ①results of qRT‑PCR showed that the relative expression of GRP78 messenger RNA (mRNA) in Huh7, Hep3B, and LO2 cells were 3.06±0.33, 4.42±0.60 and 1.00±0.02. There were significant differences in GRP78 mRNA expression between Huh7 and LO2 cells or Hep3B and LO2 cells (t=6.19, 5.42, P<0.05). ②Results of Western Blot detection showed that the relative expression of GRP78 protein in Huh7, Hep3B, and LO2 cells were 1.65±0.01, 1.77±0.01 and 0.99±0.02. There were significant differences in GRP78 protein expression between Huh7 and LO2 cells or Hep3B and LO2 cells (t=75.09, 108.10, P<0.05). ③Results of cell proliferation experiments showed that the growth rates in Hu7 GRP78 gene‑specific shRNA lentiviruses group cells and Hu7 negative control shRNA lentivirus group cells at 24, 48, 72 and 96 hours were 111.51%±0.35%, 144.85%±0.68%, 188.71%±3.62%, 282.51%±5.25% and 190.08%±0.58%, 285.76%±2.69%, 459.51%±4.29%, 597.88%±12.25%, showing signifi-cant differences (Fgroups=1 360.000, Ftime=668.500, Finteraction=197.600, P<0.05). The growth rates in Hep3B GRP78 gene‑specific shRNA lentiviruses group cells and Hep3B negative control shRNA lentivirus group cells at 24, 48, 72 and 96 hours were 124.47%±0.25%, 153.25%±1.25%, 195.45%±3.19%, 282.51%±10.76% and 179.69%±0.33%, 322.67%±2.46%, 486.27%±5.82%, 622.35%±12.58%, showing significant differences (Fgroups=1 222.000, Ftime=706.200, Finteraction=179.600, P<0.05). ④Results of the cell clone formation experiments showed that the number of cells in Hu7 GRP78 gene‑specific shRNA lentiviruses group cells and Hu7 negative control shRNA lentivirus group cells were 125±3 and 435±17, showing a significant difference (t=17.86, P<0.05). The number of cells in Hep3B GRP78 gene‑specific shRNA lentiviruses group cells and Hep3B negative control shRNA lentivirus group cells were 138±3 and 388±7, showing a significant difference (t=32.29, P<0.05). (4) Effects of inhibiting of GRP78 expression on the gene and protein expression of p53, p21, CDK2, CDK4, and CDK6 in hepatoma cells: results of high‑throughput transcription histological analysis showed that the relative expression rates of p53, p21, CDK2, CDK4, and CDK6 were 19%, 334%, 398%, 41% and 49% in the Hu7 GRP78 gene‑specific shRNA lentiviruses group cells comparing to the Hu7 negative control shRNA lentivirus group cells. ①Results of qRT-PCR showed that the relative expression of GRP78, p53, p21, CDK2, CDK4, and CDK6 mRNA were 0.17±0.03, 4.05±0.71, 3.73±0.47, 0.49±0.09, 0.48±0.06, 0.36±0.07 in the Hu7 GRP78 gene‑specific shRNA lentiviruses group cells, versus 1.00±0.05, 1.03±0.17, 1.00±0.07, 1.01±0.09, 1.02±0.14, 1.00±0.03 in the Hu7 negative control shRNA lentivirus group cells, showing significant differences (t=14.62, 4.17, 5.72, 4.26, 3.49, 8.82, P<0.05). The relative expression of GRP78, p53, p21, CDK2, CDK4, and CDK6 mRNA were 0.11±0.01, 4.28±0.43, 4.19±0.22, 0.44±0.01, 0.25±0.03, 0.68±0.04 in Hep3B GRP78 gene‑specific shRNA lentiviruses group cells, versus 1.01±0.09, 1.02±0.15, 1.00±0.06, 1.01±0.09, 1.01±0.08, 1.15±0.02 in Hep3B negative control shRNA lentivirus group cells, showing significant differences (t=10.19, 7.14, 13.79, 6.37, 9.42, 9.61, P<0.05). ②Results of Western Blot detection showed that the relative expression of GRP78, p53, p21, CDK2, CDK4, and CDK6 protein were 0.45±0.01, 1.98±0.05, 2.31±0.12, 0.75±0.03, 0.69±0.04, 0.82±0.03 in the Hu7 GRP78 gene‑specific shRNA lentiviruses group cells, versus 1.01±0.05, 1.03±0.01, 1.00±0.02, 1.00±0.01, 1.01±0.02, 1.00±0.03 in the Hu7 negative control shRNA lentivirus group cells, showing significant differences (t=11.07, 14.56, 11.30, 11.29, 10.55, 11.37, P<0.05). The relative expression of GRP78, p53, p21, CDK2, CDK4, and CDK6 protein were 0.61±0.03, 1.98±0.16, 2.55±0.12, 0.85±0.03, 0.78±0.01, 0.54±0.02 in Hep3B GRP78 gene‑specific shRNA lentiviruses group cells, versus 1.00±0.03, 1.05±0.02, 1.05±0.01, 1.05±0.02, 1.00±0.02, 1.00±0.02 in Hep3B negative control shRNA lentivirus group cells, showing significant differences (t=10.97, 13.40, 12.35, 11.06, 12.45, 13.78, P<0.05). (5) Effects of HA15 on the proliferation and the gene and protein expression of p53, p21, CDK2, CDK4, and CDK6 in hepatoma cells: results of 50% inhibiting concentration (IC50) test of HA15 showed that the IC50 of HA15 for Huh7 and Hep3B cells at 48 hours were 9.98 μmol/L and 13.70 μmol/L. ①Huh7 and Hep3B cells were treated with 9.98 μmol/L and 13.70 μmol/L of HA15. Results of cell proliferation experiments showed that the growth rates at 24, 48, 72, and 96 hours were 112.81%±0.27%, 154.71%±1.45%, 237.66%±16.77%, 294.40%±14.92% in the HA15‑Huh7 cells, versus 133.67%±0.49%, 352.93%±2.31%, 557.17%±4.89%, 662.60%±13.31% in the normal Huh7 cells, showing a significant difference (Fgroups=766.800, Ftime=518.200, Finteraction=133.300, P<0.05). The growth rates at 24, 48, 72, and 96 hours were 121.27%±2.32%, 203.85%±3.18%, 240.80%±3.02%, 286.50%±7.10% in the HA15-Hep3B cells, versus 239.14%±1.02%, 362.00%±5.44%, 539.37%±10.80%, 694.79%±17.13% in the normal Hep3B cells, showing a signifi-cant difference (Fgroups=594.300, Ftime=317.900, Finteraction=78.600, P<0.05). ②Results of qRT-PCR showed that the relative expression of GRP78, p53, p21, CDK2, CDK4, and CDK6 mRNA were 0.27±0.05, 3.64±0.28, 4.13±0.41, 0.51±0.07, 0.39±0.03, 0.17±0.02 in the HA15‑Huh7 cells, versus 1.02±0.14, 1.00±0.03, 1.00±0.05, 1.01±0.08, 1.01±0.09, 1.03±0.17 in the normal Huh7 cells, showing significant differences (t=5.00, 9.25, 7.63, 4.73, 6.82, 5.01, P<0.05). The relative expression of GRP78, p53, p21, CDK2, CDK4, and CDK6 mRNA were 0.28±0.03, 3.49±0.78, 4.31±0.53, 0.38±0.05, 0.36±0.04, 0.24±0.03 in the HA15-Hep3B cells, versus 1.01±0.11, 1.03±0.18, 1.01±0.08, 1.00±0.06, 1.02±0.15, 1.00±0.06 in the normal Hep3B cells, showing significant differences (t=6.26, 3.08, 6.21, 7.97, 4.26, 11.08, P<0.05). ③Results of Western Blot detection showed that the relative expression of GRP78, p53, p21, CDK2, CDK4, and CDK6 protein were 0.52±0.05, 1.94±0.08, 1.58±0.02, 0.89±0.00, 0.86±0.02, 0.74±0.01 in the HA15-Huh7 cells, versus 1.02±0.03, 1.00±0.03, 1.02±0.02, 1.04±0.03, 1.00±0.01, 1.01±0.02 in the normal Huh7 cells, showing significant differences (t=11.54, 10.28, 11.03, 12.81, 13.67, 10.09, P<0.05). The relative expression of GRP78, p53, p21, CDK2, CDK4, and CDK6 protein were 0.57±0.02, 1.67±0.04, 1.41±0.04, 0.82±0.03, 0.70±0.02, 0.74±0.01 in the HA15‑Hep3B cells, versus 1.03±0.01, 0.98±0.03, 1.00±0.03, 1.03±0.03, 1.01±0.01, 1.04±0.01 in the normal Huh7 cells, showing significant differences (t=10.81, 11.54, 12.26, 13.62, 14.23, 10.17, P<0.05).
ConclusionsHigh expression of GRP78 is an independent risk factor affecting the overall survival and disease progression-free survival of hepatocellular carcinoma patients. Inhibiting of GRP78 expression can reduce cell proliferation and the expression of p53, p21, CDK2, CDK4, and CDK6 mRNA and proteins in hepatoma cells.
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原发性肝癌是我国常见恶性肿瘤,其中肝细胞癌（简称肝癌）发病率在全世界恶性肿瘤中排名第6,病死率排名第4[1‑2]。肝癌病人术后5年总体生存率<40.0%,复发率为40.0%~70.0%,即使复发后再次切除或行肝移植,病人3年总体生存率仅为52.6%[3‑4]。化疗、靶向治疗等辅助治疗仍无法满足临床需求,因此,寻找更有效的肝癌预后生物分子和治疗靶点具有重要临床意义[5‑6]。葡萄糖调节蛋白78（glucose regulatory protein 78,GRP78）是热休克蛋白70家族重要成员,发挥维持细胞稳态作用[7‑10]。GRP78在促进肿瘤生长、迁移和耐药过程中发挥重要作用,但具体分子机制尚未阐明[11‑14]。HA15是GRP78靶向抑制剂,在肿瘤细胞中具有诱导自噬和凋亡作用[15⁃16]。本研究采用肝癌组织芯片及体外培养肝癌细胞和正常肝细胞实验,分析GRP78对肝癌预后及肿瘤细胞增殖的影响。
材料与方法
一、实验材料与方法
采用实验研究方法和回顾性队列研究方法。
（一）实验材料
1.肝癌组织芯片购自上海芯超生物科技有限公司（HLivH180Su15）,包含90例肝癌及90例对应癌旁组织标本,其中男80例,女10例；中位年龄为52岁,年龄范围为31~78岁。
2.肝癌细胞Huh7和Hep3B（1101 HUM⁃PUMC 000376和1101 HUM‑PUMC000679）购自中国科学院细胞库；人正常肝细胞LO2（BNCC100012）购自北京北纳创联生物技术研究院。
3.3种GRP78基因特异性shRNA慢病毒（1341668、1341665、13416B6）、阴性对照shRNA慢病毒（133F00A）及病毒转染试剂聚凝胺（1341658）购自上海吉凯基因化学技术有限公司。
4.GRP78靶向抑制剂HA15（21621）购自Med Chem Express公司上海分部。
5.细胞增殖毒性检测试剂盒（cell counting kit‑8,CCK‑8）,货号为CK04,购自东仁化学科技（上海）有限公司。
6.GRP78、GAPDH单克隆兔抗人抗体（66574⁃1‑ Ig、60004‑1‑Ig）及辣根过氧化物酶标记山羊抗兔二抗（PR30009）购自武汉三鹰生物技术有限公司。
7.p53、p21、CDK2、CDK4、CDK6单克隆兔抗人抗体（2524T、2947T、2546T、12790T、3136T）购自赛信通上海生物试剂有限公司。
8.胎牛血清（LTBEUFBS500）购自上海诺娃医药科技有限公司。
9.DMEM培养基（8121090）、细胞总RNA提取液TRIzol（229012）购自赛默飞世尔科技（中国）有限公司。
10.细胞总蛋白提取液RIPA（P0013B）、二喹啉甲酸（bicinchoninic acid,BCA）试剂盒（OS28）、增强型化学发光（efficient chemiluminescence,ECL）试剂盒（1121200302）、二氨基联苯胺（diamnobenzidine,DAB）染色试剂盒（P0202‑1）购自上海碧云天生物技术有限公司。
11.聚偏二氟乙烯（ployvinylidene fluoride,PVDF）膜（IBFP0785C）购自默克化工（上海）有限公司。
12.TBST缓冲液、结晶紫染色液（20210126、G1062）购自北京索莱宝科技有限公司。
13.Prime script RT试剂盒、SYBR Premix Ex Taq RT‑PCR试剂盒（047A、820A）购自宝日医生物技术（北京）有限公司。
14.引物由上海生工生物工程股份有限公司合成,序列如下：GRP78基因引物序列为上游：5′‑AA⁃UACAGCAAUUAGUAAAGTT‑3′,下游：5′‑CUUUA⁃CUAAUUGCUGUAUUTT‑3′。GAPDH基因引物序列为上游：5′‑CCATCACCATCTTCCAGG‑3′,下游：5′⁃ATGAGTCCTTCCACGATAC‑3′。p21基因引物序列为上游：5′‑GATGGAACTTCGACTTTGTCAC‑3′,下游：5′‑GTCCACATGGTCTTCCTCTG‑3′。CDK2基因引物序列为上游：5′‑CCTGGGCTGCAAATATTATT⁃CC‑3′,下游：5′‑TGGCTTGTAATCAGGCATAGAA‑3′。CDK4基因引物序列为上游：5′‑TTTTGAGCATCC⁃CAATGTTGTC‑3′,下游：5′‑TCGACGAAACATCTC⁃TTGATCT‑3′。CDK6基因引物序列为上游：5′‑CG⁃AACAGACAGAGAAACCAAAC‑3′,下游：5′‑CTCG⁃GTGTGAATGAAGAAAGTC‑3′。
（二）实验方法
1.免疫组织化学染色：采用DAB染色方法,操作步骤参照试剂盒说明书,GRP78抗体以1∶2 500稀释,空白对照一抗为PBS液。由2位独立的病理科医师进行双盲阅片,评估染色结果。
2.细胞培养及转染：Hep3B、Huh7及LO2细胞采用含10%胎牛血清的DMEM培养基,添加100 U/mL青霉素和100 μg/L链霉素,置于37 ℃、5% CO2培养箱中培养。取对数生长期细胞,均匀接种于6孔培养板中,当细胞生长至覆盖生长面约60%面积时进行细胞转染。筛选干扰效率最高的慢病毒进行后续实验。细胞分组：Hep3B、Huh7细胞株转染GRP78基因特异性shRNA慢病毒设为GRP78‑shRNA组,转染阴性对照shRNA慢病毒设为对照‑shRNA组。转染时用含有6 μg/mL聚凝胺的2 mL新鲜细胞培养基替换旧培养基,同时加入10 μL GRP78基因特异性shRNA慢病毒或5 μL阴性对照shRNA慢病毒；转染48 h后,提取细胞总RNA,分析基因表达；转染72 h后,提取细胞总蛋白,分析蛋白表达。
3.RNA提取和实时荧光定量聚合酶链式反应（quantitative real‑time polymerase chain reaction,qRT⁃PCR）检测：qRT‑PCR反应条件为95 ℃预变性5 min,95 ℃变性10 s,60 ℃退火20 s,72 ℃延伸20 s,40个循环；95 ℃延伸10 min。
4.Western blot检测：采用ECL试剂盒显影法,操作步骤参照说明书,GRP78、p53、p21、CDK2、CDK4、CDK6抗体以1∶2 000稀释,GAPDH抗体以1∶3 000稀释,含辣根过氧化物酶标记山羊抗兔二抗按1∶3 000稀释,以GAPDH作为内参照,采用ImageJ软件（V1.8.0.112）分析GRP78、p53、p21、CDK2、CDK4、CDK6蛋白表达量。
5.细胞增殖实验：取对数生长期细胞,均匀接种于96孔培养板中,当细胞生长至覆盖生长面约95%面积时；于0、24、48、72、96 h采用CCK⁃8检测细胞增殖率。
6.HA15半抑制浓度（50% inhibiting concentra⁃tion,IC50）实验：取对数生长期细胞,均匀接种于96孔培养板中；24 h后分别向细胞中加入含有0、1、2、4、8、10、16、20、21、22、23、24、25、26、27、28、29、30、40 μmol/L HA15新鲜细胞培养基；继续培养48 h后采用CCK‑8检测细胞增殖率。
7.细胞克隆形成实验：取对数生长期细胞,均匀接种于6孔板中,每孔接种500个细胞,于37 ℃、5%CO2培养箱中培养2周；当可通过肉眼观察到细胞群落时,采用4%多聚甲醛固定细胞,经结晶紫染色液核染色后,进行细胞计数。
8.高通量转录组学检测：分别收集GRP78⁃shRNA组和对照‑shRNA组Huh7细胞,并采用TRIzol从细胞中提取总RNA；经信使RNA（messenger RNA,mRNA）纯化、片段化后逆转录合成cDNA第一链,以第一链cDNA为模板合成第二链cDNA；采用Agilent 2100生物分析仪（美国安捷伦科技有限公司）进行测序检测。
二、观察指标和评价标准
观察指标：（1）肝癌组织和癌旁组织GRP78表达及其与临床病理特征的关系,包括性别、年龄、HBV、ALT、GGT、AFP、TBil、美国癌症联合会（AJCC）分期、肿瘤长径、肿瘤数目、肝硬化、肿瘤病理学分级。（2）肝癌病人预后及影响因素分析：性别、年龄、HBV、ALT、GGT、AFP、TBil、AJCC分期、肿瘤长径、肿瘤数目、肝硬化、肿瘤病理学分级、GRP78表达。（3）抑制GRP78表达对肝癌细胞增殖的影响。（4）抑制GRP78表达对肝癌细胞p53、p21、CDK2、CDK4、CDK6基因和蛋白表达的影响。（5）HA15 对肝癌细胞增殖和p53、p21、CDK2、CDK4、CDK6基因和蛋白表达的影响。
评价标准：免疫组织化学染色结果以染色强度及阳性细胞百分比进行评分,0分为阴性表达,1~4分为弱表达,5~8分为中表达,8~12分为高表达,将阴性表达、弱表达和中表达统一为低表达。
三、统计学分析
应用SPSS 25.0统计软件进行分析。正态分布的计量资料以x±s表示,组间比较采用t检验或方差分析。重复测量数据采用重复测量方差分析。计数资料以绝对数表示,组间比较采用χ²检验。单因素和多因素分析采用COX比例风险回归模型。采用Kaplan⁃Meier法计算生存时间并绘制生存曲线,采用Log⁃rank检验进行生存分析。P<0.05为差异有统计学意义。
结果
一、肝癌组织和癌旁组织GRP78表达及其与临床病理特征的关系
肝癌组织芯片免疫组织化学染色结果显示：GRP78在90例肝癌组织中低表达53例（阴性表达、弱表达、中表达分别为0、16、37例）,高表达37例；GRP78在90例癌旁组织中低表达84例（阴性表达、弱表达、中表达分别为36、46、2例）,高表达6例,肝癌组织与癌旁组织比较,差异有统计学意义（P<0.05）。GRP78免疫组织化学染色主要位于细胞质,少量见于细胞核。见图1。
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图 1 葡萄糖调节蛋白78在肝癌癌旁组织和癌组织中的表达 1A：葡萄糖调节蛋白78在癌旁组织中表达 免疫组织化学染色 低倍放大；1B：葡萄糖调节蛋白78在癌旁组织中表达 免疫组织化学染色 中倍放大；1C：葡萄糖调节蛋白78在癌组织中表达 免疫组织化学染色 低倍放大；1D：葡萄糖调节蛋白78在癌组织中表达 免疫组织化学染色 中倍放大Figure 1. Expression of glucose regulatory protein (GRP) 78 in adjacent tissues and tumor tissues of hepatocellular carcinoma 1A: Expression of GRP78 in adjacent tissues of hepatocellular carcinoma Immunohistochemical staining Low magnification; 1B: Expression of GRP78 in adjacent tissues of hepatocellular carcinoma Immunohistochemical staining Medium magnification; 1C: Expression of GRP78 in tumor tissues of hepatocellular carcinoma Immunohistochemical staining Low magnification; 1D: Expression of GRP78 in tumor tissues of hepatocellular carcinoma Immunohistochemical staining Medium magnification53例GRP78低表达与37例GRP78高表达肝癌病人HBV、肿瘤病理学分级比较,差异均有统计学意义（P<0.05）,性别、年龄、ALT、GGT、AFP、TBil、AJCC分期、肿瘤长径、肿瘤数目、肝硬化比较,差异均无统计学意义（P>0.05）。见表1。
表 1 53例GRP78低表达肝癌病人与37例GRP78高表达肝癌病人临床病理特征比较（例)Table 1. Comparison of clinicopathological features between 53 hepatocellular carcinoma patients with GRP78 low expression and 37 hepatocellular carcinoma patients with GRP78 high expressionGRP78表达 例数 性别 年龄 乙型肝炎病毒 丙氨酸氨基转移酶 γ‑谷氨酰转移酶 甲胎蛋白 男 女 ≤60岁 >60岁 阴性 阳性 ≤40 U/L >40 U/L ≤49 U/L >49 U/L ≤400 μg/L >400 μg/L 低表达 53 48 5 32 21 17 36 31 22 27 26 25 28 高表达 37 32 5 18 19 2 35 19 18 17 20 22 15 χ²值 0.070 1.214 9.306 0.450 0.218 1.319 P值 >0.05 >0.05 <0.05 >0.05 >0.05 >0.05 注： GRP78为葡萄糖调节蛋白78二、肝癌病人预后及影响因素分析
90例病人均获得随访,随访时间为5～56个月,中位随访时间为49个月。53例GRP78低表达肝癌病人中位总体生存时间和中位疾病无进展生存时间分别为56个月和53个月,37例GRP78高表达肝癌病人上述指标分别为32个月和19个月,两者比较,差异均有统计学意义（χ²=17.482,12.097,P<0.05）。见图2,3。
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图 2 53例葡萄糖调节蛋白78低表达和37例葡萄糖调节蛋白78高表达肝癌病人总体生存曲线Figure 2. Overall survival curves of 53 hepatocellular carcinoma patients with low expression of glucose regulatory protein 78 and 37 hepatocellular carcinoma patients with high expression of glucose regulatory protein 78![]()
图 3 53例葡萄糖调节蛋白78低表达和37例葡萄糖调节蛋白78高表达肝癌病人疾病无进展生存曲线Figure 3. Disease progression free survival curves of 53 hepatocellular carcinoma patients with low expression of glucose regulatory protein 78 and 37 hepatocellular carcinoma patients with high expression of glucose regulatory protein 78单因素分析结果显示：ALT、肿瘤病理学分级、GRP78表达是影响肝癌病人3年总体生存率和疾病无进展生存率的相关因素（P<0.05）,性别、年龄、HBV、GGT、AFP、TBil、AJCC分期、肿瘤长径、肿瘤数目、肝硬化不是影响肝癌病人3年总体生存率和疾病无进展生存率的相关因素（P>0.05）。见表2。
表 2 影响90例肝癌病人3年总体生存率和疾病无进展生存率的单因素分析Table 2. Univariate analysis of 3⁃year overall survival and 3⁃year disease progression free survival of 90 hepatocellular carcinoma patients临床病理因素 赋值 例数 3年总体生存率（%） 风险比（95%可信区间） P值 3年疾病无进展生存率（%） 风险比（95%可信区间） P值 性别 男 1 80 65.0 1.276（0.388~4.200） >0.05 45.0 1.186（0.360~3.980） >0.05 女 0 10 80.0 50.0 年龄（岁） ≤60 0 50 70.0 1.149（0.571~2.311） >0.05 56.0 0.960（0.472~1.953） >0.05 >60 1 40 62.5 32.5 乙型肝炎病毒 阴性 0 19 68.4 2.170（0.825~5.708） >0.05 47.4 2.158（0.822~5.663） >0.05 阳性 1 71 66.2 45.1 丙氨酸氨基转移酶（U/L） ≤40 0 50 68.0 2.168（1.061~4.432） <0.05 50.0 2.254（1.096~4.636） <0.05 >40 1 40 65.0 40.0 γ‑谷氨酰转移酶（U/L） ≤49 0 44 72.7 1.537（0.761~3.104） >0.05 54.6 1.537（0.761~3.104） >0.05 >49 1 46 60.9 37.0 甲胎蛋白（μg/L） ≤400 0 47 70.2 0.537（0.258~1.115） >0.05 48.9 0.582（0.274~1.237） >0.05 >400 1 43 62.8 41.9 总胆红素（μmol/L） 3.5~20.0 0 74 66.2 1.159（0.669~2.009） >0.05 44.6 1.139（0.644~2.014） >0.05 >20.0 1 16 68.8 50.0 美国癌症联合会分期 1期 0 63 73.0 1.480（0.723~3.033） >0.05 50.8 1.252（0.598~2.618） >0.05 2~3期 1 27 51.9 33.3 肿瘤长径（cm） <5 0 55 72.7 0.767（0.369~1.593） >0.05 52.7 0.908（0.434~1.899） >0.05 ≥5 1 35 57.1 34.3 肿瘤数目 单发 0 79 69.6 0.805（0.428~1.514） >0.05 49.4 1.081（0.550~2.123） >0.05 多发 1 11 45.5 18.2 肝硬化 无 0 9 77.8 0.717（0.251~2.052） >0.05 66.7 1.062（0.530~2.130） >0.05 有 1 81 65.4 43.2 肿瘤病理学分级 Ⅰ~Ⅱ级 0 54 85.2 2.161（1.069~4. 368） <0.05 59.3 0.893（0.438~1.818） <0.05 Ⅲ~Ⅳ级 1 36 38.9 25.0 葡萄糖调节蛋白78表达 低表达 0 53 83.0 3.784（1.793~7.989） <0.05 58.5 3.493（1.631~7.252） <0.05 高表达 1 37 43.2 27.0 多因素分析结果显示：ALT>40 U/L、肿瘤病理学分级为Ⅲ~Ⅳ级、GRP78高表达是影响肝癌病人3年总体生存率和疾病无进展生存率的独立危险因素（P<0.05）。见表3,4。
表 3 影响90例肝癌病人3年总体生存率的多因素分析Table 3. Multivariate analysis of 3⁃year overall survival of 90 hepatocellular carcinoma patients临床病理因素 b值 标准误 Wald值 风险比 95%可信区间 P值 丙氨酸氨基转移酶>40 U/L 0.840 0.358 5.524 2.317 1.150~4.671 <0.05 肿瘤病理学分级Ⅲ~Ⅳ级 0.712 0.270 6.954 2.039 1.201~3.462 <0.05 葡萄糖调节蛋白78高表达 1.319 0.291 20.601 3.740 2.116~6.612 <0.05 表 4 影响90例肝癌病人3年疾病无进展生存率的多因素分析Table 4. Multivariate analysis of 3⁃year disease progression free survival of 90 hepatocellular carcinoma patients临床病理因素 b值 标准误 Wald值 风险比 95%可信区间 P值 丙氨酸氨基转移酶>40 U/L 0.786 0.377 4.345 2.194 1.048~4.593 <0.05 肿瘤病理学分级Ⅲ~Ⅳ级 0.778 0.352 4.894 2.177 1.093~4.336 <0.05 葡萄糖调节蛋白78高表达 1.074 0.344 9.759 2.927 1.492~5.742 <0.05 三、抑制GRP78表达对肝癌细胞增殖的影响
qRT‑PCR检测结果显示：GRP78 mRNA在Huh7、Hep3B、LO2细胞中的相对表达量分别为3.06±0.33、4.42±0.60、1.00±0.02,Huh7、Hep3B细胞分别与LO2细胞比较,差异均有统计学意义（t=6.19,5.42,P< 0.05）。
Western blot检测结果显示：GRP78蛋白在Huh7、Hep3B、LO2细胞中的相对表达量分别为1.65±0.01、1.77±0.01、0.99±0.02,Huh7、Hep3B细胞分别与LO2细胞比较,差异均有统计学意义（t=75.09,108.10,P<0.05）。
qRT‑PCR检测结果显示：转染3种GRP78基因特异性shRNA慢病毒（GRP78⁃shRNA组）和转染阴性对照shRNA慢病毒（对照⁃shRNA组）Hu7细胞中,GRP78 mRNA的相对表达量分别为0.46±0.11,0.17±0.01,0.38±0.04和1.02±0.15,GRP78‑shRNA组分别与对照‑shRNA组比较,差异均有统计学意义（t=3.13,5.82,4.23,P<0.05）。
细胞增殖实验检测结果显示：Huh7细胞GRP78⁃shRNA组和对照⁃shRNA组24、48、72、96 h细胞增殖率分别为111.51%±0.35%、144.85%±0.68%、188.71%±3.62%、282.51%±5.25%和190.08%±0.58%、285.76%±2.69%、459.51%±4.29%、597.88%±12.25%,两组比较,差异有统计学意义（F组间=1 360.000,F时间=668.500,F交互=197.600,P<0.05）。Hep3B细胞GRP78⁃shRNA组和对照⁃shRNA组上述指标分别为124.47%±0.25%、153.25%±1.25%、195.45%±3.19%、282.51%±10.76%和179.69%±0.33%、322.67%±2.46%、486.27%±5.82%、622.35%±12.58%,两组比较,差异有统计学意义（F组间=1 222.000,F时间=706.200,F交互=179.600,P<0.05）。
细胞克隆形成实验结果显示：Huh7细胞GRP78⁃shRNA组和对照‑shRNA组细胞数分别为（125±3）个和（435±17）个,两组比较,差异有统计学意义（t=17.86,P<0.05）；Hep3B细胞GRP78⁃shRNA组和对照‑shRNA组上述指标分别为（138±3）个和（388±7）个,两组比较,差异有统计学意义（t=32.29,P<0.05）。
四、抑制GRP78表达对肝癌细胞p53、p21、CDK2、CDK4、CDK6基因和蛋白表达的影响
高通量转录组学检测结果显示：Huh7细胞GRP78⁃shRNA组相对于对照⁃shRNA组的p53、p21、CDK2、CDK4、CDK6表达率分别为19%、334%、398%、41%、49%。
qRT‑PCR检测结果显示：Huh7细胞GRP78⁃shRNA组和对照‑shRNA组中,GRP78、p53、p21、CDK2、CDK4、CDK6 mRNA的相对表达量分别为0.17±0.03,4.05±0.71,3.73±0.47,0.49±0.09,0.48±0.06,0.36±0.07和1.00±0.05,1.03±0.17,1.00±0.07,1.01±0.09,1.02±0.14,1.00±0.03,两组比较,差异均有统计学意义（t=14.62,4.17,5.72,4.26,3.49,8.82,P<0.05）。Hep3B细胞GRP78‑shRNA组和对照‑shRNA组上述指标分别为0.11±0.01,4.28±0.43,4.19±0.22,0.44±0.01,0.25±0.03,0.68±0.04和1.01±0.09,1.02±0.15,1.00±0.06,1.01±0.09,1.01±0.08,1.15±0.02,两组比较,差异均有统计学意义（t=10.19,7.14,13.79,6.37,9.42,9.61,P<0.05）。
Western blot检测结果显示：Huh7细胞GRP78⁃shRNA组和对照‑shRNA组中,GRP78、p53、p21、CDK2、CDK4、CDK6蛋白的相对表达量分别为0.45±0.01,1.98±0.05,2.31±0.12,0.75±0.03,0.69±0.04,0.82±0.03和1.01±0.05,1.03±0.01,1.00±0.02,1.00±0.01,1.01±0.02,1.00±0.03,两组比较,差异均有统计学意义（t=11.07,14.56,11.30,11.29,10.55,11.37,P<0.05）。Hep3B细胞GRP78‑shRNA组和对照‑shRNA组上述指标分别为0.61±0.03,1.98±0.16,2.55±0.12,0.85±0.03,0.78±0.01,0.54±0.02和1.00±0.03,1.05±0.02,1.05±0.01,1.05±0.02,1.00±0.02,1.00±0.02,两组比较,差异均有统计学意义（t=10.97,13.40,12.35,11.06,12.45,13.78,P<0.05）。
五、HA15对肝癌细胞增殖和p53、p21、CDK2、CDK4、CDK6基因和蛋白表达的影响
HA15 IC50实验结果显示：Huh7、Hep3B细胞48 h IC50分别为9.98 μmol/L、13.70 μmol/L。
分别以9.98 μmol/L和13.70 μmol/L HA15作用Huh7、Hep3B细胞,细胞增殖实验检测结果显示：HA15⁃Huh7细胞和正常Huh7细胞24、48、72、96 h细胞增殖率分别为112.81%±0.27%、154.71%±1.45%、237.66%±16.77%、294.40%±14.92%和133.67%±0.49%、352.93%±2.31%、557.17%±4.89%、662.60%±13.31%,两者比较,差异有统计学意义（F组间=766.800,F时间=518.200,F交互=133.300,P<0.05）；HA15⁃Hep3B细胞和正常Hep3B细胞上述指标分别为121.27%±2.32%、203.85%±3.18%、240.80%±3.02%、286.50%±7.10%和239.14%±1.02%、362.00%±5.44%、539.37%±10.80%、694.79%±17.13%,两者比较,差异有统计学意义（F组间=594.300,F时间=317.900,F交互=78.600,P<0.05）。
qRT‑PCR检测结果显示：HA15‑Huh7细胞和正常Huh7细胞中,GRP78、p53、p21、CDK2、CDK4、CDK6 mRNA的相对表达量分别为0.27±0.05,3.64±0.28,4.13±0.41,0.51±0.07,0.39±0.03,0.17±0.02和1.02±0.14,1.00±0.03,1.00±0.05,1.01±0.08,1.01±0.09,1.03±0.17,两者比较,差异均有统计学意义（t=5.00,9.25,7.63,4.73,6.82,5.01,P<0.05）。HA15⁃Hep3B细胞和正常Hep3B细胞上述指标分别为0.28±0.03,3.49±0.78,4.31±0.53,0.38±0.05,0.36±0.04,0.24±0.03和1.01±0.11,1.03±0.18,1.01±0.08,1.00±0.06,1.02±0.15,1.00±0.06,两者比较,差异均有统计学意义（t=6.26,3.08,6.21,7.97,4.26,11.08,P<0.05）。
Western blot检测结果显示：HA15‑Huh7细胞和正常Huh7细胞中,GRP78、p53、p21、CDK2、CDK4、CDK6蛋白的相对表达量分别为0.52±0.05,1.94±0.08,1.58±0.02,0.89±0.00,0.86±0.02,0.74±0.01和1.02±0.03,1.00±0.03,1.02±0.02,1.04±0.03,1.00±0.01,1.01±0.02,两者比较,差异均有统计学意义（t=11.54,10.28,11.03,12.81,13.67,10.09,P<0.05）。HA15⁃Hep3B细胞和正常Hep3B细胞上述指标分别为0.57±0.02,1.67±0.04,1.41±0.04,0.82±0.03,0.70±0.02,0.74±0.01和1.03±0.01,0.98±0.03,1.00±0.03,1.03±0.03,1.01±0.01,1.04±0.01,两者比较,差异均有统计学意义（t=10.81,11.54,12.26,13.62,14.23,10.17,P<0.05）。
讨论
一、GRP78表达与恶性肿瘤细胞增殖及病人预后的关系
肝癌是我国常见恶性肿瘤,发病率高,预后差,虽然近年来分子靶向、免疫治疗等多种手段应用于肝癌临床实践,但总体疗效仍不满意[17‑25]。GRP78可影响肿瘤细胞增殖、迁移和凋亡,在多种肿瘤中呈高表达[26‑35]。本研究结果显示：GRP78表达在肝癌与癌旁组织中比较,差异有统计学意义；GRP78高表达是影响肝癌病人3年总体生存率和疾病无进展生存率的独立危险因素；抑制肝癌细胞GRP78表达可抑制细胞增殖活性。这提示GRP78与肝癌进展和预后密切相关。
二、GRP78调控肿瘤细胞增殖相关分子机制
目前关于GRP78促进肿瘤增殖的分子机制研究结果显示：PI3K/AKT通路发挥重要作用[36]。Clarke等[37]的研究结果显示：GRP78可通过PI3K/AKT通路促进胰腺癌细胞增殖和侵袭。Sánchez等[38]的研究结果显示：抑制肝癌细胞中GRP78表达,可抑制肿瘤细胞增殖活性,并同时抑制肿瘤细胞中p53蛋白的表达。Li等[39]的研究结果显示：抑制小鼠神经母细胞瘤GRP78表达,可促进p53蛋白的表达。Huang等[40]的研究结果显示：抑制GRP78表达可通过p53信号通路促进正常肝细胞LO2凋亡。Arnaudeau等[41]的研究结果显示：GRP78可调节p53蛋白的活性。本研究结果显示：抑制肝癌细胞GRP78表达可抑制p53、p21、CDK2、CDK4、CDK6基因和蛋白表达。
已有的研究结果显示：HA15可通过抑制细胞ATP酶活性诱导自噬和凋亡[16,42]。Bian等[43]的研究结果显示：GRP78高表达与白血病的化疗耐药性相关,抑制GRP78表达可提高肿瘤细胞的药物敏感性。本研究结果显示：HA15能够抑制肝癌细胞GRP78、p53、p21、CDK2、CDK4、CDK6基因和蛋白表达及细胞增殖活性。笔者认为：靶向抑制肝癌细胞GRP78表达可能通过p53及其相关分子p21、CDK2、CDK4、CDK6发挥抑制肿瘤细胞增殖的作用,详细的分子间信号关系尚需进一步分析。
综上,GRP78高表达是影响肝癌病人3年总体生存率和疾病无进展生存率的独立危险因素,抑制GRP78表达可抑制肝癌细胞增殖活性及影响p53、p21、CDK2、CDK4、CDK6基因和蛋白表达。
所有作者均声明不存在利益冲突马海东, 曹洁, 高龙, 等. 葡萄糖调节蛋白78对肝癌预后及肿瘤细胞增殖的影响[J]. 中华消化外科杂志, 2021, 20(12): 1294-1305. DOI: 10.3760/cma.j.cn115610-20211008-00492.http://journal.yiigle.com/LinkIn.do?linkin_type=cma&DOI=10.3760/cma.j.cn115610-20211008-00492
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图 1 葡萄糖调节蛋白78在肝癌癌旁组织和癌组织中的表达 1A：葡萄糖调节蛋白78在癌旁组织中表达 免疫组织化学染色 低倍放大；1B：葡萄糖调节蛋白78在癌旁组织中表达 免疫组织化学染色 中倍放大；1C：葡萄糖调节蛋白78在癌组织中表达 免疫组织化学染色 低倍放大；1D：葡萄糖调节蛋白78在癌组织中表达 免疫组织化学染色 中倍放大
Figure 1. Expression of glucose regulatory protein (GRP) 78 in adjacent tissues and tumor tissues of hepatocellular carcinoma 1A: Expression of GRP78 in adjacent tissues of hepatocellular carcinoma Immunohistochemical staining Low magnification; 1B: Expression of GRP78 in adjacent tissues of hepatocellular carcinoma Immunohistochemical staining Medium magnification; 1C: Expression of GRP78 in tumor tissues of hepatocellular carcinoma Immunohistochemical staining Low magnification; 1D: Expression of GRP78 in tumor tissues of hepatocellular carcinoma Immunohistochemical staining Medium magnification
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图 2 53例葡萄糖调节蛋白78低表达和37例葡萄糖调节蛋白78高表达肝癌病人总体生存曲线
Figure 2. Overall survival curves of 53 hepatocellular carcinoma patients with low expression of glucose regulatory protein 78 and 37 hepatocellular carcinoma patients with high expression of glucose regulatory protein 78
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图 3 53例葡萄糖调节蛋白78低表达和37例葡萄糖调节蛋白78高表达肝癌病人疾病无进展生存曲线
Figure 3. Disease progression free survival curves of 53 hepatocellular carcinoma patients with low expression of glucose regulatory protein 78 and 37 hepatocellular carcinoma patients with high expression of glucose regulatory protein 78
表 1 53例GRP78低表达肝癌病人与37例GRP78高表达肝癌病人临床病理特征比较（例)
Table 1 Comparison of clinicopathological features between 53 hepatocellular carcinoma patients with GRP78 low expression and 37 hepatocellular carcinoma patients with GRP78 high expression
GRP78表达 例数 性别 年龄 乙型肝炎病毒 丙氨酸氨基转移酶 γ‑谷氨酰转移酶 甲胎蛋白 男 女 ≤60岁 >60岁 阴性 阳性 ≤40 U/L >40 U/L ≤49 U/L >49 U/L ≤400 μg/L >400 μg/L 低表达 53 48 5 32 21 17 36 31 22 27 26 25 28 高表达 37 32 5 18 19 2 35 19 18 17 20 22 15 χ²值 0.070 1.214 9.306 0.450 0.218 1.319 P值 >0.05 >0.05 <0.05 >0.05 >0.05 >0.05 注： GRP78为葡萄糖调节蛋白78
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表 2 影响90例肝癌病人3年总体生存率和疾病无进展生存率的单因素分析
Table 2 Univariate analysis of 3⁃year overall survival and 3⁃year disease progression free survival of 90 hepatocellular carcinoma patients
临床病理因素 赋值 例数 3年总体生存率（%） 风险比（95%可信区间） P值 3年疾病无进展生存率（%） 风险比（95%可信区间） P值 性别 男 1 80 65.0 1.276（0.388~4.200） >0.05 45.0 1.186（0.360~3.980） >0.05 女 0 10 80.0 50.0 年龄（岁） ≤60 0 50 70.0 1.149（0.571~2.311） >0.05 56.0 0.960（0.472~1.953） >0.05 >60 1 40 62.5 32.5 乙型肝炎病毒 阴性 0 19 68.4 2.170（0.825~5.708） >0.05 47.4 2.158（0.822~5.663） >0.05 阳性 1 71 66.2 45.1 丙氨酸氨基转移酶（U/L） ≤40 0 50 68.0 2.168（1.061~4.432） <0.05 50.0 2.254（1.096~4.636） <0.05 >40 1 40 65.0 40.0 γ‑谷氨酰转移酶（U/L） ≤49 0 44 72.7 1.537（0.761~3.104） >0.05 54.6 1.537（0.761~3.104） >0.05 >49 1 46 60.9 37.0 甲胎蛋白（μg/L） ≤400 0 47 70.2 0.537（0.258~1.115） >0.05 48.9 0.582（0.274~1.237） >0.05 >400 1 43 62.8 41.9 总胆红素（μmol/L） 3.5~20.0 0 74 66.2 1.159（0.669~2.009） >0.05 44.6 1.139（0.644~2.014） >0.05 >20.0 1 16 68.8 50.0 美国癌症联合会分期 1期 0 63 73.0 1.480（0.723~3.033） >0.05 50.8 1.252（0.598~2.618） >0.05 2~3期 1 27 51.9 33.3 肿瘤长径（cm） <5 0 55 72.7 0.767（0.369~1.593） >0.05 52.7 0.908（0.434~1.899） >0.05 ≥5 1 35 57.1 34.3 肿瘤数目 单发 0 79 69.6 0.805（0.428~1.514） >0.05 49.4 1.081（0.550~2.123） >0.05 多发 1 11 45.5 18.2 肝硬化 无 0 9 77.8 0.717（0.251~2.052） >0.05 66.7 1.062（0.530~2.130） >0.05 有 1 81 65.4 43.2 肿瘤病理学分级 Ⅰ~Ⅱ级 0 54 85.2 2.161（1.069~4. 368） <0.05 59.3 0.893（0.438~1.818） <0.05 Ⅲ~Ⅳ级 1 36 38.9 25.0 葡萄糖调节蛋白78表达 低表达 0 53 83.0 3.784（1.793~7.989） <0.05 58.5 3.493（1.631~7.252） <0.05 高表达 1 37 43.2 27.0
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表 3 影响90例肝癌病人3年总体生存率的多因素分析
Table 3 Multivariate analysis of 3⁃year overall survival of 90 hepatocellular carcinoma patients
临床病理因素 b值 标准误 Wald值 风险比 95%可信区间 P值 丙氨酸氨基转移酶>40 U/L 0.840 0.358 5.524 2.317 1.150~4.671 <0.05 肿瘤病理学分级Ⅲ~Ⅳ级 0.712 0.270 6.954 2.039 1.201~3.462 <0.05 葡萄糖调节蛋白78高表达 1.319 0.291 20.601 3.740 2.116~6.612 <0.05
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表 4 影响90例肝癌病人3年疾病无进展生存率的多因素分析
Table 4 Multivariate analysis of 3⁃year disease progression free survival of 90 hepatocellular carcinoma patients
临床病理因素 b值 标准误 Wald值 风险比 95%可信区间 P值 丙氨酸氨基转移酶>40 U/L 0.786 0.377 4.345 2.194 1.048~4.593 <0.05 肿瘤病理学分级Ⅲ~Ⅳ级 0.778 0.352 4.894 2.177 1.093~4.336 <0.05 葡萄糖调节蛋白78高表达 1.074 0.344 9.759 2.927 1.492~5.742 <0.05
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[1] 中华人民共和国国家卫生健康委员会医政医管局.原发性肝癌诊疗规范(2019年版)[J].中华消化外科杂志,2020,19(1):1‑20. DOI: 10.3760/cma.j.issn.1673-9752.2020.01.001. [2] TorreLA, BrayF, SiegelRL, et al. Global cancer statistics,2012[J]. CA Cancer J Clin,2015,65(2):87‑108. DOI:10.33 22/caac.21262.
[3] 应倩,汪媛.肝癌流行现况和趋势分析[J].中国肿瘤,2020,29(3):185‑191. DOI: 10.11735/j.issn.1004-0242.2020.03.A005. [4] WangC, VegnaS, JinH, et al. Inducing and exploiting vulnerabilities for the treatment of liver cancer[J]. Nature,2019,574(7777):268‑272. DOI:10.1038/s41586-019-16 07-3.
[5] 万赤丹,王国斌.原发性肝癌治疗进展[J].腹部外科.2019, (1):1‑6. DOI: 10.3969/j.issn.1003-5591.2019.01.001. [6] AnwanwanD, SinghSK, SinghS, et al. Challenges in liver cancer and possible treatment approaches[J]. Biochim Biophys Acta Rev Cancer,2020,1873(1):188314. DOI:10. 1016/j.bbcan.2019.188314.
[7] IbrahimIM, AbdelmalekDH, ElfikyAA. GRP78: a cell′s response to stress[J]. Life Sci,2019,226:156‑163. DOI:10. 1016/j.lfs.2019.04.022.
[8] LiaoY, LiuY, XiaX, et al. Targeting GRP78‑dependent AR-V7 protein degradation overcomes castration-resistance in prostate cancer therapy[J]. Theranostics,2020,10(8):3366‑3381. DOI: 10.7150/thno.41849.
[9] YuanXP, DongM, LiX, et al. GRP78 promotes the invasion of pancreatic cancer cells by FAK and JNK[J]. Mol Cell Biochem,2015,398(1/2):55‑62. DOI: 10.1007/s11010-014-2204-2.
[10] SatoM, YaoVJ, ArapW, et al. GRP78 signaling hub a recep-tor for targeted tumor therapy[J]. Adv Genet,2010,69:97-114. DOI: 10.1016/S0065-2660(10)69006-2.
[11] GopalU, PizzoSV. Cell surface GRP78 signaling: an emer-ging role as a transcriptional modulator in cancer[J]. J Cell Physiol,2021,236(4):2352‑2363. DOI: 10.1002/jcp.30030.
[12] LiuK, TsungK, AttenelloFJ. Characterizing cell stress and GRP78 in glioma to enhance tumor treatment[J]. Front Oncol,2020,10:608911. DOI: 10.3389/fonc.2020.608911.
[13] RaiterA, LipovetzkiJ, LubinI, et al. GRP78 expression in peripheral blood mononuclear cells is a new predictive marker for the benefit of taxanes in breast cancer neo-adjuvant treatment[J]. BMC Cancer,2020,20(1):333. DOI: 10.1186/s12885-020-06835-z.
[14] ElfikyAA, BaghdadyAM, AliSA, et al. GRP78 targeting: hitting two birds with a stone[J]. Life Sci,2020,260:118317. DOI: 10.1016/j.lfs.2020.118317.
[15] RuggieroC, Doghman‑BouguerraM, RoncoC, et al. The GRP78/BiP inhibitor HA15 synergizes with mitotane action against adrenocortical carcinoma cells through conver-gent activation of ER stress pathways[J]. Mol Cell Endocrinol,2018,474:57‑64. DOI: 10.1016/j.mce.2018.02.010.
[16] CerezoM, LehraikiA, MilletA, et al. Compounds trigger-ing ER stress exert anti‑melanoma effects and overcome BRAF inhibitor resistance[J]. Cancer Cell,2016,29(6):805-819. DOI: 10.1016/j.ccell.2016.04.013.
[17] 樊嘉,高强.肝癌分子分型指导精准诊断与治疗的研究前沿[J].中华消化外科杂志,2020,19(1):28-31. DOI: 10.3760/cma.j.issn.1673-9752.2020.01.003. [18] 雷海科,李小升,赵玉兰,等.原发性肝癌患者生存预后及影响因素分析(附3106例报告)[J].中华消化外科杂志,2020,19(2):179-184. DOI:10.3760/cma.j.issn.1673-9752.2020. 02.010. [19] 徐新明,陈健,陆荣柱,等.姜黄素通过内质网应激信号通路诱导肝癌SMMC‑7721细胞凋亡的作用研究[J].东南大学学报:医学版,2018,37(1):12‑17. DOI: 10.3969/j.issn.1671-6264.2018.01.003. [20] MassarwehNN, El‑SeragHB. Epidemiology of hepato-cellular carcinoma and intrahepatic cholangiocarcinoma[J]. Cancer Control,2017,24(3):1073274817729245. DOI: 10.1177/1073274817729245.
[21] 刘连新,蔡伟.肝细胞癌免疫治疗的现状和展望[J].中华消化外科杂志,2020,19(2):119-122. DOI:10.3760/cma.j.issn. 1673-9752.2020.02.002. [22] 于世伟,孙厚斌,尹力楠,等.自体肿瘤免疫细胞辅助介入治疗中晚期原发性肝癌[J].局解手术学杂志,2016,25(10):762-765. DOI: 10.11659/jjssx.01E016048. [23] 孙俪洋,高恒军,牛哲禹,等.免疫检查点抑制剂治疗原发性肝癌的研究进展[J].中国现代普通外科进展,2019,22(3):203-206. DOI: 10.3969/j.issn.1009-9905.2019.03.009. [24] 张宁宁,陆伟.肝细胞癌的靶向治疗[J].临床肝胆病杂志,2021,37(8):1753-1757. DOI:10.3969/j.issn.1001-5256. 2021.08.003. [25] 王涛,陈东风.肝细胞癌的临床诊治:从指南到临床实践[J].临床肝胆病杂志,2021,37(8):1745-1747. DOI: 10.3969/j.issn.1001-5256.2021.08.001. [26] BanHS, HanTS, HurK, et al. Epigenetic alterations of heat shock proteins (HSPs) in cancer[J]. Int J Mol Sci,2019,20(19):4758. DOI: 10.3390/ijms20194758.
[27] LangBJ, Guerrero‑GiménezME, PrinceTL, et al. Heat shock proteins are essential components in transformation and tumor progression: cancer cell intrinsic pathways and beyond[J]. Int J Mol Sci,2019,20(18). DOI:10.3390/ijms20 184507.
[28] UlianichL, InsabatoL. Endoplasmic reticulum stress in endometrial cancer[J]. Front Med(Lausanne),2014,1:55. DOI: 10.3389/fmed.2014.00055.
[29] WangK, ZhangZ, WangM, et al. Role of GRP78 inhibiting artesunate‑induced ferroptosis in KRAS mutant pancrea-tic cancer cells[J]. Drug Des Devel Ther,2019,13:2135-2144. DOI: 10.2147/DDDT.S199459.
[30] MisraUK, Gonzalez‑GronowM, GawdiG, et al. A novel re-ceptor function for the heat shock protein Grp78: silen-cing of Grp78 gene expression attenuates alpha2M*-induced signalling[J]. Cell Signal,2004,16(8):929‑938. DOI: 10.1016/j.cellsig.2004.01.003.
[31] GopalU, MoweryY, YoungK, et al. Targeting cell surface GRP78 enhances pancreatic cancer radiosensitivity through YAP/TAZ protein signaling[J]. J Biol Chem,2019,294(38):13939‑13952. DOI: 10.1074/jbc.RA119.009091.
[32] HardyB, RaiterA, YakimovM, et al. Colon cancer cells expressing cell surface GRP78 as a marker for reduced tumorigenicity[J]. Cell Oncol(Dordr),2012,35(5):345‑354. DOI: 10.1007/s13402-012-0094-4.
[33] PootrakulL, DatarRH, ShiSR, et al. Expression of stress response protein Grp78 is associated with the develop-ment of castration‑resistant prostate cancer[J]. Clin Cancer Res,2006,12(20Pt 1):5987‑5993. DOI:10.1158/1078-04 32.CCR-06-0133.
[34] ZhengY, LiuP, WangN, et al. Betulinic acid suppresses breast cancer metastasis by targeting GRP78‑mediated glycolysis and ER stress apoptotic pathway[J]. Oxid Med Cell Longev,2019,2019:8781690. DOI:10.1155/2019/878 1690.
[35] DuT, LiH, FanY, et al. The deubiquitylase OTUD3 stabi-lizes GRP78 and promotes lung tumorigenesis[J]. Nat Commun,2019,10(1):2914. DOI:10.1038/s41467-019-10 824-7.
[36] CasasC. GRP78 at the centre of the stage in cancer and neuroprotection[J]. Front Neurosci,2017,11:177. DOI:10. 3389/fnins.2017.00177.
[37] ClarkeWR, AmundadottirL, JamesMA. CLPTM1L/CRR9 ectodomain interaction with GRP78 at the cell surface signals for survival and chemoresistance upon ER stress in pancreatic adenocarcinoma cells[J]. Int J Cancer,2019,144(6):1367‑1378. DOI: 10.1002/ijc.32012.
[38] SánchezDI, González‑FernándezB, CrespoI, et al. Melato-nin modulates dysregulated circadian clocks in mice with diethylnitrosamine‑induced hepatocellular carcinoma[J]. J Pineal Res,2018, 65(3):e12506. DOI: 10.1111/jpi.12506.
[39] LiW, WangW, LiY, et al. Proteomics analysis of normal and senescent NG108‑15 cells: GRP78 plays a negative role in cisplatin‑induced senescence in the NG108‑15 cell line[J]. PLoS One,2014,9(3):e90114. DOI: 10.1371/journal.pone.0090114.
[40] HuangQ, ZhangJ, MartinFL, et al. Perfluorooctanoic acid induces apoptosis through the p53‑dependent mitochon-drial pathway in human hepatic cells: a proteomic study[J]. Toxicol Lett,2013,223(2):211‑220. DOI:10.1016/j.tox let.2013.09.002.
[41] ArnaudeauS, ArboitP, BischofP, et al. Glucose‑regulated protein 78: a new partner of p53 in trophoblast[J]. Proteomics,2009,9(23):5316‑5327. DOI:10.1002/pmic. 200800865.
[42] SinghP, ZengX, ChenX, et al. Quantitation of polymeric-microneedle‑delivered HA15 in tissues using liquid chro-matography‑tandem mass spectrometry[J]. J Pharm Biomed Anal,2020,185:113230. DOI:10.1016/j.jpba.2020. 113230.
[43] BianT, TagmountA, VulpeC, et al. CXL146, a novel 4H-chromene derivative, targets GRP78 to selectively elimi-nate multidrug‑resistant cancer cells[J]. Mol Pharmacol,2020,97(6):402‑408. DOI: 10.1124/mol.119.118745.
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