巨大肝细胞癌根治术后近期复发转移的临床与相关蛋白组学初步研究
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摘要
目的
应用同位素标记相对和绝对定量蛋白组学技术（iTRAQ）鉴定和筛选与巨大肝细胞癌根治术后近期复发转移相关的差异蛋白，试图找到术后近期复发转移预测新指标和治疗新靶点。
方法
1.回顾性分析166例巨大肝细胞癌患者的临床病理资料，根据术后近期复发转移时间，分为术后6个月内复发转移组和6～12个月内复发转移组，应用Logistic回归模型分不同时间段对临床病理因素进行单因素和多因素分析，并建立术后近期复发转移概率预测数学模型。
2.蛋白组学实验：应用巨大肝细胞癌手术切除组织标本作为实验对象，根据临床随访复发转移与否分为3组（随访时间24个月）：术后6个月内复发转移组（n=20），术后6～12个月内复发转移组（n=20）及未复发转移组；对这些组织标本进行蛋白提取、定量和酶解，应用iTRAQ试剂标记（117标记6个月内复发转移组，119标记6～12个月内复发转移组，121标记未复发转移组）二维液相色谱-串联质谱（2DLC-MS/MS）联用技术进行分析。利用ProteinPilot软件（版本4.2）对MS/MS肽段进行分析，在Intemational Swissprot(20111015，human)数据库中鉴定蛋白质并定量。鉴定蛋白的比值117:121或119:121大于2倍或小于0.5倍（P<0.05）认为二者之间存在表达差异。应用MetaCore（GeneGo公司）软件对差异表达蛋白进行GO分析，包括亚细胞定位、分子功能和生物学过程。应用IPA (Ingenuity Pathway Analysis)软件分析差异表达蛋白参与的信号通路和蛋白间相互作用的网络。
3.应用蛋白组学实验部分的组织标本和分组标准，采用免疫印迹（WesternBlot）、免疫组化和实时荧光定量PCR(Q-PCR)技术从蛋白和mRNA水平检测差异蛋白Galectin-4、S100A12和Alpha-methylacyl-CoAracemase（AMACR）表达情况，验证质谱的准确性。
4.采用构建组织芯片结合免疫组化方法检测感兴趣的目标蛋白Galectin-4在73例巨大肝癌术后1年内复发转移与73例未复发转移的癌组织及配对的癌旁组织和10例正常肝组织中的表达情况，并探讨Galectin-4蛋白表达与各临床病理参数及与复发转移的关系。
结果
1.术后6个月内复发转移率为36.7%（61/166），术后6~12个月内复发转移率为27.1%（45/166）；Logistic单因素分析提示术前血清AFP值≥1210ng/ml（X_4）、肿瘤包膜(X_7)、镜下脉管癌栓（X_(10)）、术后癌细胞病理分级（X_(11)）与术后6个月内复发转移有关，术前血清AFP值≥1210ng/ml(X_4)和镜下脉管癌栓（X_(10)）与术后6～12个月复发转移有关；多因素分析提示，术后6个月内复发转移的独立危险因素为术前血清AFP值≥1210ng/ml（X_4）、肿瘤有包膜(X_7)和镜下脉管癌栓（X_(10)）；术后6～12个月内复发转移的独立危险因素为术前血清AFP值≥1210ng/ml（X_4）和镜下脉管癌栓（X_(10)）；术后6个月内复发转移概率的预测方程为P=Y/(1+Y)，Y=EXP(-0.852+0.753X_4-1.273X_7+2.524X_(10))；术后6～12个月复发转移概率的预测方程为P=Y/(1+Y)，Y=EXP(-0.804-0.897X_4+0.923X_(10))。
2.利用ProteinPilot软件（版本4.2）对MS/MS肽段进行分析，并在Intemational Swissprot(20111015，human)数据库中搜索，共鉴定出1407种蛋白；基于筛选差异表达蛋白的条件，共筛选出了87个差异表达蛋白；与未复发转移组比较，6个月内复发转移组差异表达蛋白有46个，其中15个蛋白表达上调，31个蛋白表达下调，6～12月内复发转移组差异表达蛋白有49个，其中33个蛋白表达上调，16个蛋白表达下调；6个月内复发转移组、6～12月内复发转移组共同的差异表达蛋白有8个，分别是Galectin-4、Lactotransferrin、Eukaryotictranslation initiation factor3subunit B、Biliverdin reductase A、60kDa SS-A/Roribonucleoprotein、Apolipoprotein A-II、Protein S100A12和Alpha-methylacyl-CoAracemase（AMACR）。
3.87个差异表达蛋白生物信息学分析，发现大部分定位细胞质，分子功能主要是蛋白结合，生物学过程主要体现在代谢和调控。差异蛋白富集度最高的生物通路是EIF2信号通路，发现差异蛋白富集度最高的蛋白网络是以ERK1/2为核心的调控网络，共有14个差异表达蛋白参与。
4.部分差异表达蛋白验证结果显示，与未复发转移癌组织比较，Galectin-4蛋白和相应的mRNA表达在6个月内复发转移癌组织、6～12月内复发转移癌组织中均下调，与蛋白组学结果一致；S100A12和AMACR蛋白与相应的mRNA表达在6个月内复发转移组表达下调，6～12月内复发转移组表达上调，与蛋白组学结果一致。
5. Galectin-4mRNA的表达在巨大肝癌组织中表达上调，在癌旁和正常肝组织中表达均下调，各组间差异具有统计学意义（P<0.05）；扩大临床组织样本量采用组织芯片结合免疫组化的方法检测Galectin-4在巨大肝癌中表达情况，结果显示Galectin-4在巨大肝癌组织中阳性表达率为39.0%（57/146），明显低于癌旁组织71.9%（105/146）和正常肝组织80%（8/10），差异具有统计学意义（P均<0.05）；Galectin-4在12个月内复发转移组阳性表达率为19.1%（14/73），明显低于未复发转移组58.9%（43/73），两组间比较差异有统计学意义（P=0.000）；Galectin-4蛋白阴性表达与性别、年龄、有无合并肝硬化、是否合并乙肝病毒感染、肿瘤包膜、癌细胞病理分化无关，而与术前血清AFP值和镜下脉管癌栓有关（P均<0.05）；Kaplan-Meier生存曲线分析显示Galectin-4阴性表达组3年生存率24.0%低于阳性表达组34.8%（χ~2=6.589，P=0.010）；Galectin-4阴性表达组1年无瘤生存率53.1%明显低于阳性表达组83.6%（χ~2=5.836，P=0.016）。
结论
1.巨大肝细胞癌术后12个月复发转移率高；术前血清AFP值、肿瘤包膜、微血管侵犯是巨大肝细胞癌根治术后近期复发转移的危险因素。
2.巨大肝细胞癌术后6个月内复发转移和6～12月内复发转移的影响因素不同，复发转移概率预测的数学模型不同。
3.经验证确认的差异表达蛋白Galectin-4、S100A12、AMACR有望成为巨大肝细胞癌术后近期复发转移潜在的预警蛋白，其分子机制需进一步研究。
4. Galectin-4蛋白在巨大肝癌组织中表达下调；Galectin-4表达与术后近期复发转移有关，Galectin-4低表达提示3年生存率和1年无瘤生存率低，可为巨大肝癌预后判断提供有效依据。
Objective
By using iTRAQ-label-based quantitative proteomic technique, the present studyaims to screen and identify the differential protein expressions associated with earlyrecurrence/metastasis of huge hepatocellular carcinoma(HHCC) following radicalresection,in order to screen new predicting biomarkers and potential therapeutictargets.
Methods
1. A total of166patients with primary HCC larger than10cm were recruitedfrom January2006to December2010. These patients underwent radical resection andthe clinicopathology was retrospectively analyzed.Univariable and multivariableLogistic regression analyses were performed and a mathematic model predicting therecurrence/metastasis of HHCC at different time points was constructed.
2. Proteomics study:the resection tissue samples of HHCC were applied to thisexperiment. The specimens were divided into3groups according to clinical follow-uprecurrence/metastasis or not(follow-up time are24months) after receiving HHCCresection:a recurrence/metastasis time≤6months group (n=20),a recurrence/metastasistime between6and12months group (n=20) and a non-recurrence/metastasis>12months group (n=20).After quantification and enzymolysis of the protein extract fromthe specimens of the3groups, Protein expression profile was assessed by using thetechnique of iTRAQ (reagent117,119and121were used to label the recurrence time≤6months group, the recurrence time between6and12months group and thenon-recurrence>12months group, respectively)-2DLC-MS/MS.The MS/MS peptideswere analyzed by using ProteinPilot software (version4.2), proteins were identifiedand quantitatived by using the Intemational Swissprot database (20111015, human).For117:121or119:121differentially expressed proteins, the fold change cut-offratio>2or <0.5was considered as the existence of significant differential expression(P<0.05). The bio-informative analysis software Ingenuity Pathways Analysis (IPA)was used to conduct bio-informative analysis, including GO function assessment, signaling pathway analysis and transcription regulation networks analysis of thedifferentially expressed protein.
3. Application of proteomics experimental part of the tissue samples andgrouping criteria,3(Galectin-4,S100A12and Alpha-methylacyl-CoA racemase) ofthe differentially expressed proteins identified in Part II were selected to verify theirexpressions.Western Blot and immunohistochemical assessment were used to detectthe expression of the protein. Real time-quantitative PCR (Q-PCR) technique wasused to detect the mRNA expression of the differentially-expressed protein.
4. The Q-PCR technique was applied to examine the mRNA expression ofGalectin-4in40HHCC compared with paired adjacent tissues and6healthy livertissues.The techniques of tissue microarray and immunohistochemical staining wereemployed to examine the expression profiles of Galectin-4in specimens (bothcarcinoma tissues and peri-carcinoma tissues) collected from the73patients withrecurrence/metastasis (≤12months) HHCC and73patients with non-recurrent/metastasis HHCC,as well as the expression profiles in10healthy liver tissues.Inaddition,the implications of Galectin-4and its correlations with other clinical patholo-gical parameters and recurrence/metastasis were also studied.
Results
1. The recurrence/metastasis rate was36.7%(61/166) within6months，at6~12months after surgery, the recurrence/metastasis rate was27.1%(45/166). Univariableanalysis showed pre-operative AFP of≥1210ng/ml (X_4),tumor capsule (X_7),post-operative pathological grade (X_(11)) and microscopic tumor thrombus (X_(10)) wererelated to the recurrence/metastasis within6months after surgery. Pre-operativeserum AFP of≥1210ng/ml (X_4) and microscopic tumor thrombus (X_(10)) wereassociated with the recurrence/metastasis within6~12months after surgery.Multivariable analysis revealed the risk factors of recurrence within6months aftersurgery included pre-operative AFP of≥1210ng/ml (X_4),without tumor capsule (X_7)and microscopic tumor thrombus (X_(10)),while those within6~12months after surgerywere pre-operative serum AFP of≥1210ng/ml (X_4) and microscopic tumor thrombus(X_(10)). The equation for the prediction of recurrence/metastasis within6months was P=Y/(1+Y),Y=EXP(-0.852+0.753X_4-1.273X_7+2.524X_(10)) and that within6~12months was P=Y/(1+Y), Y=EXP(-0.804-0.897X_4+0.923X_(10)).
2. A total of1407proteins were identified through the searching ofInternational Swissprot (20111015,human) database. Based on the conditions ofdifferential expression protein identification, a total of87proteins were found. Incontrast to the non-recurrence/metastasis group, there were46proteins expresseddifferentially proteeins (15up-regulated expressionand31down-regulated expression)in the recurrence/metastasis time≤6months group.Compare with the non-recurrence/metastasisgroup, there were49proteins expressed differentially (33up-regulatedexpression and16down-regulated expression) in the recurrence/metastasis timebetween6and12months group.In relative to the non-recurrence/metastasis group,theother two groups (recurrence/metastasis time≤6months+the recurrence/metastasistime between6and12months) had8differentially-expressed proteins in common,namely, Galectin-4, Lactotransferrin, Eukaryotic translation initiation factor3subunitB,Biliverdin reductase A,60kDa SS-A/Ro ribonucleoprotein,Apolipoprotein A-II,Protein S100A12and Alpha-methylacyl-CoA racemase（AMACR）.
3. The87differentially-expressed proteins were analysised by GO database,suggesting that most of the differentially expressed proteins were localized in theextracellular domain; they played roles in the functions of protein binding,they weremainly involved in metabonomics and regulation. Accoding to IPA software analysis,the most important biological pathway is EIF2signaling pathway,the highest proteinconcentration degree of protein network is regulated and controlled by ERK1/2,a toalof14proteins were involved in.
4. Part of differentially expressed proteins validation results show that, Whencompared with the non-recurrence/metastasis group, the expressions of bothGalectin-4mRNA and protein in the other two groups (recurrence/metastasis time≤6months+the recurrence/metastasis time between6and12months) were alldown-regulated,the expressions of both S100A12and AMACR mRNA and protein inthe recurrence/metastasis time≤6months group were down-regulated while those inthe recurrence/metastasis time between6and12months group were up-regulated.The results were accordance with the results of proteomic study.
5. Galectin-4mRNA expression levels in HHCC were higer than para-carcinoma tissues and normal tissues rsepectively with statistical significance(P<0.05). Expanding clinical samples using tissue chips combined with immunohisto-chemical method to detect Galectin-4expressed in HHCC.The positive rate ofGalectin-4in the carcinoma tissues was39.0%(57/146), which was significantlylower than those in the peri-carcinoma tissues (71.9%,105/146) and the healthy livertissues (80%,8/10); and differences were statistically significant (P were <0.05).Galectin-4protein was expressed in19.1%(14/73) of the carcinoma tissues from therecurrence (≤12months) group, while this ratio in the non-recurrence group (58.9%,43/73) was much higher. The difference between these two groups was statisticallysignificant (P=0.000).The negative expression of Galectin-4was independent ofpatient’s age,gender,complication of cirrhosis,HBV infection,pathological grading.andhepatic capsule (P were>0.05), but associated with pre-operative serum AFP value,and microscopic tumor thrombus (P were <0.05).Kaplan-Meier survival curveanalysis showed that the3-year survival ratio in the negative expression of Galectin-424.0%was lower than the positive group34.8%(χ~2=6.589,P=0.010),the1-yeardisease free survival rate in the negative expression of Galectin-4(53.1%) was muchlower than the positive group83.6%,(χ~2=5.8369, P=0.016).
Conclusions
1. The recurrence/metastasis rate is high within12month after resection ofHHCC. Pre-operative AFP levels,tumor capsule and microvascular invasion are riskfactors of early recurrence/metastasis of huge HCC after surgery.
2. The effect factors at different time points of early recurrence/metastasis ofhuge HCC after surgery are different,the equation for prediction of HHCCrecurrence/metastasis at different time points varies.
3. The differentially expressed proteins include Galectin-4,S100A12,AMACRwhich have been confirmded were expected to be potential warning protein predictingearly recurrence/metastasis of HHCC after resection.
4. Galectin-4mRNA expression was up-regulated in HHCC comparing with para-carcinoma tissues and normal tissues.While the protein expression wasdown-regulated in the huge HCC tissue.Galectin-4is associated with earlyrecurrence/metastasis of HHCC,Galectin-4lower expression suggests lower3yearsurvival rates and1year disease-free survival rate,Galectin–4can provide effectivebasis for HHCC prognosis judgment.
应用同位素标记相对和绝对定量蛋白组学技术（iTRAQ）鉴定和筛选与巨大肝细胞癌根治术后近期复发转移相关的差异蛋白，试图找到术后近期复发转移预测新指标和治疗新靶点。
方法
1.回顾性分析166例巨大肝细胞癌患者的临床病理资料，根据术后近期复发转移时间，分为术后6个月内复发转移组和6～12个月内复发转移组，应用Logistic回归模型分不同时间段对临床病理因素进行单因素和多因素分析，并建立术后近期复发转移概率预测数学模型。
2.蛋白组学实验：应用巨大肝细胞癌手术切除组织标本作为实验对象，根据临床随访复发转移与否分为3组（随访时间24个月）：术后6个月内复发转移组（n=20），术后6～12个月内复发转移组（n=20）及未复发转移组；对这些组织标本进行蛋白提取、定量和酶解，应用iTRAQ试剂标记（117标记6个月内复发转移组，119标记6～12个月内复发转移组，121标记未复发转移组）二维液相色谱-串联质谱（2DLC-MS/MS）联用技术进行分析。利用ProteinPilot软件（版本4.2）对MS/MS肽段进行分析，在Intemational Swissprot(20111015，human)数据库中鉴定蛋白质并定量。鉴定蛋白的比值117:121或119:121大于2倍或小于0.5倍（P<0.05）认为二者之间存在表达差异。应用MetaCore（GeneGo公司）软件对差异表达蛋白进行GO分析，包括亚细胞定位、分子功能和生物学过程。应用IPA (Ingenuity Pathway Analysis)软件分析差异表达蛋白参与的信号通路和蛋白间相互作用的网络。
3.应用蛋白组学实验部分的组织标本和分组标准，采用免疫印迹（WesternBlot）、免疫组化和实时荧光定量PCR(Q-PCR)技术从蛋白和mRNA水平检测差异蛋白Galectin-4、S100A12和Alpha-methylacyl-CoAracemase（AMACR）表达情况，验证质谱的准确性。
4.采用构建组织芯片结合免疫组化方法检测感兴趣的目标蛋白Galectin-4在73例巨大肝癌术后1年内复发转移与73例未复发转移的癌组织及配对的癌旁组织和10例正常肝组织中的表达情况，并探讨Galectin-4蛋白表达与各临床病理参数及与复发转移的关系。
结果
1.术后6个月内复发转移率为36.7%（61/166），术后6~12个月内复发转移率为27.1%（45/166）；Logistic单因素分析提示术前血清AFP值≥1210ng/ml（X_4）、肿瘤包膜(X_7)、镜下脉管癌栓（X_(10)）、术后癌细胞病理分级（X_(11)）与术后6个月内复发转移有关，术前血清AFP值≥1210ng/ml(X_4)和镜下脉管癌栓（X_(10)）与术后6～12个月复发转移有关；多因素分析提示，术后6个月内复发转移的独立危险因素为术前血清AFP值≥1210ng/ml（X_4）、肿瘤有包膜(X_7)和镜下脉管癌栓（X_(10)）；术后6～12个月内复发转移的独立危险因素为术前血清AFP值≥1210ng/ml（X_4）和镜下脉管癌栓（X_(10)）；术后6个月内复发转移概率的预测方程为P=Y/(1+Y)，Y=EXP(-0.852+0.753X_4-1.273X_7+2.524X_(10))；术后6～12个月复发转移概率的预测方程为P=Y/(1+Y)，Y=EXP(-0.804-0.897X_4+0.923X_(10))。
2.利用ProteinPilot软件（版本4.2）对MS/MS肽段进行分析，并在Intemational Swissprot(20111015，human)数据库中搜索，共鉴定出1407种蛋白；基于筛选差异表达蛋白的条件，共筛选出了87个差异表达蛋白；与未复发转移组比较，6个月内复发转移组差异表达蛋白有46个，其中15个蛋白表达上调，31个蛋白表达下调，6～12月内复发转移组差异表达蛋白有49个，其中33个蛋白表达上调，16个蛋白表达下调；6个月内复发转移组、6～12月内复发转移组共同的差异表达蛋白有8个，分别是Galectin-4、Lactotransferrin、Eukaryotictranslation initiation factor3subunit B、Biliverdin reductase A、60kDa SS-A/Roribonucleoprotein、Apolipoprotein A-II、Protein S100A12和Alpha-methylacyl-CoAracemase（AMACR）。
3.87个差异表达蛋白生物信息学分析，发现大部分定位细胞质，分子功能主要是蛋白结合，生物学过程主要体现在代谢和调控。差异蛋白富集度最高的生物通路是EIF2信号通路，发现差异蛋白富集度最高的蛋白网络是以ERK1/2为核心的调控网络，共有14个差异表达蛋白参与。
4.部分差异表达蛋白验证结果显示，与未复发转移癌组织比较，Galectin-4蛋白和相应的mRNA表达在6个月内复发转移癌组织、6～12月内复发转移癌组织中均下调，与蛋白组学结果一致；S100A12和AMACR蛋白与相应的mRNA表达在6个月内复发转移组表达下调，6～12月内复发转移组表达上调，与蛋白组学结果一致。
5. Galectin-4mRNA的表达在巨大肝癌组织中表达上调，在癌旁和正常肝组织中表达均下调，各组间差异具有统计学意义（P<0.05）；扩大临床组织样本量采用组织芯片结合免疫组化的方法检测Galectin-4在巨大肝癌中表达情况，结果显示Galectin-4在巨大肝癌组织中阳性表达率为39.0%（57/146），明显低于癌旁组织71.9%（105/146）和正常肝组织80%（8/10），差异具有统计学意义（P均<0.05）；Galectin-4在12个月内复发转移组阳性表达率为19.1%（14/73），明显低于未复发转移组58.9%（43/73），两组间比较差异有统计学意义（P=0.000）；Galectin-4蛋白阴性表达与性别、年龄、有无合并肝硬化、是否合并乙肝病毒感染、肿瘤包膜、癌细胞病理分化无关，而与术前血清AFP值和镜下脉管癌栓有关（P均<0.05）；Kaplan-Meier生存曲线分析显示Galectin-4阴性表达组3年生存率24.0%低于阳性表达组34.8%（χ~2=6.589，P=0.010）；Galectin-4阴性表达组1年无瘤生存率53.1%明显低于阳性表达组83.6%（χ~2=5.836，P=0.016）。
结论
1.巨大肝细胞癌术后12个月复发转移率高；术前血清AFP值、肿瘤包膜、微血管侵犯是巨大肝细胞癌根治术后近期复发转移的危险因素。
2.巨大肝细胞癌术后6个月内复发转移和6～12月内复发转移的影响因素不同，复发转移概率预测的数学模型不同。
3.经验证确认的差异表达蛋白Galectin-4、S100A12、AMACR有望成为巨大肝细胞癌术后近期复发转移潜在的预警蛋白，其分子机制需进一步研究。
4. Galectin-4蛋白在巨大肝癌组织中表达下调；Galectin-4表达与术后近期复发转移有关，Galectin-4低表达提示3年生存率和1年无瘤生存率低，可为巨大肝癌预后判断提供有效依据。
Objective
By using iTRAQ-label-based quantitative proteomic technique, the present studyaims to screen and identify the differential protein expressions associated with earlyrecurrence/metastasis of huge hepatocellular carcinoma(HHCC) following radicalresection,in order to screen new predicting biomarkers and potential therapeutictargets.
Methods
1. A total of166patients with primary HCC larger than10cm were recruitedfrom January2006to December2010. These patients underwent radical resection andthe clinicopathology was retrospectively analyzed.Univariable and multivariableLogistic regression analyses were performed and a mathematic model predicting therecurrence/metastasis of HHCC at different time points was constructed.
2. Proteomics study:the resection tissue samples of HHCC were applied to thisexperiment. The specimens were divided into3groups according to clinical follow-uprecurrence/metastasis or not(follow-up time are24months) after receiving HHCCresection:a recurrence/metastasis time≤6months group (n=20),a recurrence/metastasistime between6and12months group (n=20) and a non-recurrence/metastasis>12months group (n=20).After quantification and enzymolysis of the protein extract fromthe specimens of the3groups, Protein expression profile was assessed by using thetechnique of iTRAQ (reagent117,119and121were used to label the recurrence time≤6months group, the recurrence time between6and12months group and thenon-recurrence>12months group, respectively)-2DLC-MS/MS.The MS/MS peptideswere analyzed by using ProteinPilot software (version4.2), proteins were identifiedand quantitatived by using the Intemational Swissprot database (20111015, human).For117:121or119:121differentially expressed proteins, the fold change cut-offratio>2or <0.5was considered as the existence of significant differential expression(P<0.05). The bio-informative analysis software Ingenuity Pathways Analysis (IPA)was used to conduct bio-informative analysis, including GO function assessment, signaling pathway analysis and transcription regulation networks analysis of thedifferentially expressed protein.
3. Application of proteomics experimental part of the tissue samples andgrouping criteria,3(Galectin-4,S100A12and Alpha-methylacyl-CoA racemase) ofthe differentially expressed proteins identified in Part II were selected to verify theirexpressions.Western Blot and immunohistochemical assessment were used to detectthe expression of the protein. Real time-quantitative PCR (Q-PCR) technique wasused to detect the mRNA expression of the differentially-expressed protein.
4. The Q-PCR technique was applied to examine the mRNA expression ofGalectin-4in40HHCC compared with paired adjacent tissues and6healthy livertissues.The techniques of tissue microarray and immunohistochemical staining wereemployed to examine the expression profiles of Galectin-4in specimens (bothcarcinoma tissues and peri-carcinoma tissues) collected from the73patients withrecurrence/metastasis (≤12months) HHCC and73patients with non-recurrent/metastasis HHCC,as well as the expression profiles in10healthy liver tissues.Inaddition,the implications of Galectin-4and its correlations with other clinical patholo-gical parameters and recurrence/metastasis were also studied.
Results
1. The recurrence/metastasis rate was36.7%(61/166) within6months，at6~12months after surgery, the recurrence/metastasis rate was27.1%(45/166). Univariableanalysis showed pre-operative AFP of≥1210ng/ml (X_4),tumor capsule (X_7),post-operative pathological grade (X_(11)) and microscopic tumor thrombus (X_(10)) wererelated to the recurrence/metastasis within6months after surgery. Pre-operativeserum AFP of≥1210ng/ml (X_4) and microscopic tumor thrombus (X_(10)) wereassociated with the recurrence/metastasis within6~12months after surgery.Multivariable analysis revealed the risk factors of recurrence within6months aftersurgery included pre-operative AFP of≥1210ng/ml (X_4),without tumor capsule (X_7)and microscopic tumor thrombus (X_(10)),while those within6~12months after surgerywere pre-operative serum AFP of≥1210ng/ml (X_4) and microscopic tumor thrombus(X_(10)). The equation for the prediction of recurrence/metastasis within6months was P=Y/(1+Y),Y=EXP(-0.852+0.753X_4-1.273X_7+2.524X_(10)) and that within6~12months was P=Y/(1+Y), Y=EXP(-0.804-0.897X_4+0.923X_(10)).
2. A total of1407proteins were identified through the searching ofInternational Swissprot (20111015,human) database. Based on the conditions ofdifferential expression protein identification, a total of87proteins were found. Incontrast to the non-recurrence/metastasis group, there were46proteins expresseddifferentially proteeins (15up-regulated expressionand31down-regulated expression)in the recurrence/metastasis time≤6months group.Compare with the non-recurrence/metastasisgroup, there were49proteins expressed differentially (33up-regulatedexpression and16down-regulated expression) in the recurrence/metastasis timebetween6and12months group.In relative to the non-recurrence/metastasis group,theother two groups (recurrence/metastasis time≤6months+the recurrence/metastasistime between6and12months) had8differentially-expressed proteins in common,namely, Galectin-4, Lactotransferrin, Eukaryotic translation initiation factor3subunitB,Biliverdin reductase A,60kDa SS-A/Ro ribonucleoprotein,Apolipoprotein A-II,Protein S100A12and Alpha-methylacyl-CoA racemase（AMACR）.
3. The87differentially-expressed proteins were analysised by GO database,suggesting that most of the differentially expressed proteins were localized in theextracellular domain; they played roles in the functions of protein binding,they weremainly involved in metabonomics and regulation. Accoding to IPA software analysis,the most important biological pathway is EIF2signaling pathway,the highest proteinconcentration degree of protein network is regulated and controlled by ERK1/2,a toalof14proteins were involved in.
4. Part of differentially expressed proteins validation results show that, Whencompared with the non-recurrence/metastasis group, the expressions of bothGalectin-4mRNA and protein in the other two groups (recurrence/metastasis time≤6months+the recurrence/metastasis time between6and12months) were alldown-regulated,the expressions of both S100A12and AMACR mRNA and protein inthe recurrence/metastasis time≤6months group were down-regulated while those inthe recurrence/metastasis time between6and12months group were up-regulated.The results were accordance with the results of proteomic study.
5. Galectin-4mRNA expression levels in HHCC were higer than para-carcinoma tissues and normal tissues rsepectively with statistical significance(P<0.05). Expanding clinical samples using tissue chips combined with immunohisto-chemical method to detect Galectin-4expressed in HHCC.The positive rate ofGalectin-4in the carcinoma tissues was39.0%(57/146), which was significantlylower than those in the peri-carcinoma tissues (71.9%,105/146) and the healthy livertissues (80%,8/10); and differences were statistically significant (P were <0.05).Galectin-4protein was expressed in19.1%(14/73) of the carcinoma tissues from therecurrence (≤12months) group, while this ratio in the non-recurrence group (58.9%,43/73) was much higher. The difference between these two groups was statisticallysignificant (P=0.000).The negative expression of Galectin-4was independent ofpatient’s age,gender,complication of cirrhosis,HBV infection,pathological grading.andhepatic capsule (P were>0.05), but associated with pre-operative serum AFP value,and microscopic tumor thrombus (P were <0.05).Kaplan-Meier survival curveanalysis showed that the3-year survival ratio in the negative expression of Galectin-424.0%was lower than the positive group34.8%(χ~2=6.589,P=0.010),the1-yeardisease free survival rate in the negative expression of Galectin-4(53.1%) was muchlower than the positive group83.6%,(χ~2=5.8369, P=0.016).
Conclusions
1. The recurrence/metastasis rate is high within12month after resection ofHHCC. Pre-operative AFP levels,tumor capsule and microvascular invasion are riskfactors of early recurrence/metastasis of huge HCC after surgery.
2. The effect factors at different time points of early recurrence/metastasis ofhuge HCC after surgery are different,the equation for prediction of HHCCrecurrence/metastasis at different time points varies.
3. The differentially expressed proteins include Galectin-4,S100A12,AMACRwhich have been confirmded were expected to be potential warning protein predictingearly recurrence/metastasis of HHCC after resection.
4. Galectin-4mRNA expression was up-regulated in HHCC comparing with para-carcinoma tissues and normal tissues.While the protein expression wasdown-regulated in the huge HCC tissue.Galectin-4is associated with earlyrecurrence/metastasis of HHCC,Galectin-4lower expression suggests lower3yearsurvival rates and1year disease-free survival rate,Galectin–4can provide effectivebasis for HHCC prognosis judgment.
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