葡萄糖调节蛋白78对肝癌预后及肿瘤细胞增殖的影响

The influence of glucose regulatory protein 78 on prognosis and tumor cell proliferation of hepatocellular carcinoma

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。

Objective To investigate the influence of glucose regulatory protein 78(GRP78)on prognosis and tumor cell proliferation of hepatocellular carcinoma.Methods The experimental study and retrospective cohort study were conducted.Based on hepatocellular carcinoma tissue chip,in vitro culture of Huh7 and Hep3B hepatoma cells and L02 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 MeaA?±5D,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,respectively,showing significant differences(X^(2)=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 asⅢ-Ⅵgrade 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=2A38,2.245,3.223 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 L02 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 L02 cells or Hep3B and L02 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 L02 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 L02 cells or Hep3B and L02 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%45.25%and 190.08%±0.58%,285.76%±2.69%,459.51%±4.29%,59788%±12.25%,showing signifi-cant diferences(=1360.000,F=668.500,Fnram=197600,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 diferences(F.=1222.000,Fa=706.200,FEnerero=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 signifcant 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 9.98μmol/L and 13.70 umol/L.①Huh7 and Hep3B cells were treated with 9.98 pmol/L and 13.70μumol/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(F=766.800,Fu,=518.200,F 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 Hep3 B cells,showing a signifi-cant difference(F=594.300,Fm=317.900,Feset,=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 rela tive 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).Conclusions High 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.