C5L2表达与肝癌患者预后分析

田 璐 吴 炯 朱 捷 高姚怡 张爱伦 王蓓丽 张春燕 潘柏申 郭 玮

(复旦大学附属中山医院检验科 上海 200032)

C5L2表达与肝癌患者预后分析

田 璐 吴 炯 朱 捷 高姚怡 张爱伦 王蓓丽 张春燕 潘柏申 郭 玮△

(复旦大学附属中山医院检验科 上海 200032)

目的 探讨C5L2表达在肝细胞肝癌(hepatocellular carcinoma ,HCC)患者预后判断中的价值。方法回顾性分析2012年10月至2013年9月在复旦大学附属中山医院接受根治性切除术的175例原发性HCC患者。研究C5L2表达与临床基本参数之间的关系,运用COX回归模型分析各临床参数对术后复发的影响,通过Kaplan-Meier法评估C5L2和AFP联合应用的预后判断效能。在5种肝癌细胞系中检测C5L2表达,筛选Hep3B和Huh7分别对C5L2进行下调和上调,检测细胞侵袭迁移能力的改变并初步探索其机制。 结果 C5L2表达与性别、肿瘤大小和复发相关,C5L2低表达患者复发率更高,多因素分析显示C5L2低表达是肝癌复发的独立危险因素,C5L2和AFP联合应用可以更加有效地判断肝癌预后。在Hep3B中敲除C5L2可促进细胞侵袭迁移,伴随β-catenin和MMP2表达上调;相反,在Huh7中过表达C5L2可抑制细胞侵袭迁移,伴随β-catenin和MMP2表达下调。结论 C5L2可作为判断肝癌预后的辅助指标,帮助临床制定更有效的HCC治疗和监测方案。

C5L2; 肝细胞肝癌; 预后

肝细胞癌(hepatocellular carcinoma,HCC)是最常见的恶性肿瘤之一,最新统计显示,全球范围内肝癌发生率在恶性肿瘤中位于第4位,病死率居第2位[1]。目前对HCC切除术后的患者仍缺乏有效的治疗手段,联合局部介入及放疗的综合治疗法虽然提高了部分患者的生存率[2],但治疗后5年内高达70%的复发转移率依然是改善HCC预后难以逾越的瓶颈[3],寻找行之有效的预测及干预手段是改善疾病预后的关键。

C5L2(GPR77)基因位于染色体19,q13.33-13.34,是由337个氨基酸组成的7次跨膜G蛋白偶联受体[4],广泛表达于免疫细胞[5-6](中性粒细胞,淋巴细胞,单核细胞,不成熟的树突状细胞,巨噬细胞)和非免疫细胞[5-9](脂肪细胞,皮肤成纤维细胞,血管平滑肌细胞,星形胶质细胞,神经元,心、肝、脾、肺等组织细胞)。目前关于C5L2的研究较少并存在一定争议,有研究认为C5L2是一种诱导性表达受体[8],另有研究认为C5L2能够与一些炎性介质发生复杂的相互作用[10-11],还有研究表明C5L2能够与C5aR和β-arrestin结合对其信号进行负调控[11-12]。C5L2的作用在不同种属、不同疾病、不同细胞类型中不尽相同[8,13-14],而C5L2在肝癌中的作用还未见报道。

本研究旨在探讨C5L2在与肝癌患者临床特征的相关性,评估其在判断肝癌患者预后中的价值,并在体外初步探索C5L2影响肝癌细胞侵袭迁移的潜在机制。

资 料 和 方 法

研究对象 选择2012年10月至2013年9月在复旦大学附属中山医院接受根治性切除术的175例原发性HCC患者。入组标准:(1)年龄≥18岁;(2)术前未行放化疗;(3) 首次接受根治性切除术;(4) 术后病理诊断为原发性HCC;(5)有完整的临床资料及随访数据。排除标准:(1)既往或合并有其他恶性肿瘤;(2)术前感染;(3)术前无影像学资料;(4)合并血液、免疫系统疾病。所有样本获取及使用过程获复旦大学附属中山医院伦理委员会批准。

免疫组化芯片染色 HCC 组织切片在二甲苯脱蜡、酒精水化后,置于抗原修复液中微波修复9 min,在3%的过氧化氢溶液中浸泡15 min,PBS 漂洗3 次,5% BSA 室温封闭30 min,一抗孵育4 ℃ 过夜。次日PBS 漂洗3 次,HRP标记山羊抗小鼠IgG室温孵育60 min,DAB 显色液显色8 min后苏木精复染20 s,酒精脱水,二甲苯透明,中性树胶封片。

结果判定 所有组织标本均由2位病理科医师独立进行评分,如不一致,请第3位病理医师评分,以两个相同结果为准。染色强度评分标准:不着色0 分,浅黄色1分,棕黄色2分,黄褐色3 分。阳性细胞所占比例评分标准:阳性细胞数<10%者0分,10%～40%者1分,40%～70% 者2分,≥70%者3分。通过乘积法计算,其中0～3分为低表达,4～9分为高表达。

实时荧光定量PCR 采用Trizol法提取细胞总RNA,逆转录成cDNA,实时荧光定量PCR方法进行基因相对定量分析。PCR引物序列如下:ACTB:(forward)5’-AGCGAGCATCCCCCAA-AGTT-3’,(reverse) 5’-GGGCACGAAGGCTC-ATCATT-3’;C5L2:(forward) 5’-GACTGCCAT-CCGGTTTCTTTTTG-3’,(reverse)5’-TGGCC-AGGAGTGCGGAGTTC-3’; β-catenin :(forward)5’-TGCAACTAAACAGGAAGGGATGGA-3’,(reverse)5’-GATGGCAGGCTCAGTGATGTCT-TC-3’; MMP2:(forward)5’-CCAGATGTGGCC-AACTACAA-3’,(reverse)5’- GGCATCATCCA-CTGTCTCTG-3’。

Western blot 采用RIPA裂解液提取细胞总蛋白,并用BCA法测定蛋白浓度。进行SDS不连续凝胶电泳,电泳结束后转膜,再加入BSA室温封闭60 min。将不同相对分子质量条带对应加入稀释好的一抗孵育过夜。次日用TBST将膜条洗涤3次后孵育二抗1 h,最后再用TBST洗涤3次后放入曝光仪中成像。

细胞转染 将处于对数生长期的细胞以每孔50万的密度铺入6孔培养板中,待细胞融合度达到70%～80%时进行转染。对照组将5 μL NC溶于245 μL Opti- mem无血清培养基中,实验组将5 μL si-C5L2或pcDNA3.1-C5L2溶于245 μL Opti-mem无血清培养基中,与配置好的转染试剂1∶1混合静置20 min,加入无血清细胞培养液中,置于37 ℃培养箱4～6 h后换入完全培养液。

侵袭与迁移 用无血清培养基重悬细胞至1×105/mL,接种到Transwell小室内(侵袭实验需要加一步:在小室中铺50 μL Matrigel胶,37 ℃培养箱中放置4～6 h使其凝固),每个小室加0.2 mL细胞悬液,下层加入0.5 mL含10% FBS的完全培养液,置于37 ℃培养箱中培养48～72 h,用棉签小心擦去Transwell上室细胞,多聚甲醛固定并用结晶紫染色,PBS洗净后晾干,封片后于显微镜下观察并计数。

统计学分析 采用SPSS 19.0软件进行统计学分析。等级变量比较采用χ2检验或Fisher精确检验,COX比例风险模型行单因素和多因素分析。Graphpad prism 5绘制Kaplan-Meier曲线。P<0.05为差异有统计学意义。

结 果

C5L2表达与肝癌患者临床基本参数的关系 本研究共入选175例原发性肝癌患者,年龄32～87岁,其中男性134 例,女性41例。对比两组患者在性别、年龄、肝硬化、肿瘤数量、肿瘤大小、肿瘤包膜、卫星灶、血管侵犯、手术分级、Child-Pugh评分、BCLC分期、术前甲胎蛋白(alpha-fetoprotein,AFP)、谷丙转氨酶(alanine aminotransferase,ALT)、谷氨酰转肽酶( glutamete transpeptidase,GGT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)及复发之间的关系。结果显示,C5L2低表达患者男性偏多(P=0.008)、肿瘤体积更大(P=0.029)、复发率更高(P=0.010,表1)。

表1 C5L2表达和临床基本特征的关系

(1)Fisher exact test.AFP:Alpha-fetoprotein;ALT:Alanine aminotransferase;GGT:Glutamete transpeptidase;AST:Aspartate aminotransferase.

C5L2在肝癌预后判断中的价值 定期对患者的情况进行跟踪随访,通过Kaplan-Meier法绘制生存曲线,采用log-rank检验评估C5L2的预后价值。结果显示,C5L2低表达组的复发率(45.0%)明显高于高表达组(25.2%),并且差异具有统计学意义(P=0.039 4,图 1)。

A:Kaplan-Meier analysis for time to recurrence in the whole cohort;B:Kaplan-Meier analysis for time to recurrence with combined AFP and C5L2 groups.

图1 原发性肝癌患者生存曲线分析

Fig 1 Kaplan-Meier analysis for time to recurrence in HCC patients

COX回归模型分析复发相关危险因素 采用COX比例风险模型对各种风险因素进行分析。单因素分析显示肝硬化(HR:2.064,95%CI:1.011～4.215,P=0.047)、肿瘤大小(HR:1.852,95%CI:1.095～3.135,P=0.022)、肿瘤包膜(HR:2.448,95%CI:1.044～5.737,P=0.039)、卫星病灶血管侵犯(HR:4.057,95%CI:2.022～8.138,P=0.000)、术前AST(HR:1.940,95%CI:1.137～3.308,P=0.015)和C5L2表达(HR:0.477,95%CI:0.282～0.808,P=0.006)是影响HCC复发的危险因素。多因素分析发现仅血管侵犯(HR:3.043,95%CI:1.434～6.457,P=0.004)和C5L2低表达(HR:0.589,95%CI:0.338～0.976,P=0.043)是影响HCC复发的独立危险因素(表2)。

表2 复发相关因素的COX比例风险模型单因素和多因素分析

HR is hazard ratio abbreviation.NA:Not applicable.

C5L2和AFP联合应用在肝癌预后判断中的价值 C5L2与AFP联合应用对肝癌复发的阳性预测率为56.4%,阴性预测率为75.0%,总体有效率为70.9%,均高于单独应用AFP。AFP和C5L2联合应用对肝癌预后判断的灵敏度降低而特异性提高(表3)。Kaplan-Mier生存曲线显示,高AFP低C5L2组的术后复发率(56.4%)远远高于低AFP高C5L2组(27.3%),差异具有统计学意义(P=0.001 6,图1B)。因此,联合应用两指标可以更好地协助医师判断患者预后。

表3 C5L2和AFP对预后判断的效能评估

PPR:Positive predictive rate;NPR:Negative predictive rate.

C5L2敲除和过表达效率验证 检测5种常见肝癌细胞系Huh7、MHHC97H、LM3、7721、Hep3B和1种正常肝细胞系L02中C5L2的表达量。qRT-PCR结果显示Hep3B细胞系C5L2表达量最高,Huh7细胞系最低(图2A)。

选择Hep3B细胞系对C5L2表达进行下调,设计3条特异siRNA干扰序列,si-C5L2-1、si-C5L2-2和si-C5L2-3,并以si-NC为阴性对照,qRT-PCR结果显示3条序列均能使C5L2表达降低,其中以si-C5L2-2敲除效率最高(图2B)。同时,在Huh7细胞系通过pcDNA3.1-C5L2对该基因进行上调,以空质粒pcDNA3.1为阴性对照,qRT-PCR结果显示pcDNA3.1-C5L2能够显著上调目标基因(图2C)。

上调或下调C5L2影响肝癌细胞侵袭迁移能力采用Transwell检测上述两种细胞侵袭迁移能力,结果显示敲低C5L2后Hep3B的侵袭迁移能力增强(图3A),过表达C5L2后Huh7的侵袭迁移能力减弱(图3B)。选取上、中、下、左、右5个视野进行计数并分析,差异均具有统计学意义(si-C5L2vs.si-NC,P<0.01;pcDNA3.1-C5L2vs.pcDNA3.1-NC,P<0.01),证实C5L2能够体外抑制细胞侵袭迁移能力。

C5L2通过MMP2影响细胞侵袭迁移能力 Wnt 信号通路是促进细胞侵袭迁移的重要通路之一,β链接蛋白(β-catenin)的激活能够促进基质金属蛋白酶2 (matrix metalloproteinase 2,MMP2的分泌,从而通过降解细胞外基质和基底膜增强肿瘤细胞的侵袭力。通过qRT-PCR和WB检测,在下调C5L2的Hep3B细胞系中β-catenin (P=0.003)和MMP2 (P=0.007)的水平升高(图4A、C),而上调C5L2的Huh7细胞系中β-catenin (P=0.009) 和MMP2 (P=0.037)水平下降(图4B、D)。

A:The mRNA fold change of 5 HCC cell lines;B:The knockdown efficiency of si-C5L2s in Hep3B cell line;C:The overexpressionefficiency of C5L2 in Huh7 cell line.Pvalues were calculated using independentt-test.si-C5L2vs.si-NC ,(1)P<0.05;(2)P<0.01.pcDNA3.1-C5L2vs.pcDNA3.1-NC,(3)P<0.01.

图2 各种肝癌细胞系中C5L2 mRNA水平及敲除和过表达C5L2转染效率验证

Fig 2 The mRNA level of C5L2 in several HCC cell lines and efficiency of knockdown and overexpression of C5L2

A:The effects of down-regulation of C5L2 on invasiveness and motility;B:The effects of up-regulation of C5L2 on invasiveness and motility.Pvalues were calculated using independentt-test.(1)si-C5L2vs.si-NC ,P<0.01;pcDNA3.1-C5L2vs.(2)pcDNA3.1-NC,P<0.01.

图3 敲除或过表达C5L2对细胞侵袭和迁移能力的影响

Fig 3 The effects of knockdown and overexpression of C5L2 on invasiveness and motility

A and B:Levels of β-catenin and MMP2 proteins were determined by Western blot;C and D:Levels of β-catenin and MMP2 mRNAs were determined by qRT-PCR.Pvalues were calculated using independentt-test.(1)si-C5L2vs.si-NC ,P<0.01;(2)pcDNA3.1-C5L2vs.pcDNA3.1-NC,P<0.05.

图4 C5L2抑制β-catenin通路并降低MMP2水平

Fig 4 C5L2 inhibits β-catenin pathway and decreases MMP2 level

讨 论

肝癌作为一种高病死率、高复发率的恶性肿瘤,其发生、发展和转移机制相当复杂。术后复发转移是影响肝癌治疗效果的重要因素,研究复发转移相关因素具有十分重要的临床意义。随着科技的发展,HCC的诊疗手段不断改进,患者的预后状况和生活质量都有所提高。但目前应用于预后判断的指标灵敏度和特异性还有待提高。因此,寻找能够充分反映HCC生物学特性的新型辅助指标,是HCC治疗的基础和关键。

补体系统参与机体抵抗外来病原体的固有免疫和适应性免疫过程[15]。在瀑布样补体级联活化过程中会产生C3a、C4a、C5a等分子片段[6]。C5a及其两个受体C5aR和C5L2与炎症的发生发展有密切关系[16],也有研究表明C5L2能促进动脉粥样硬化和狭窄[17-19]。近些年,炎症对于肿瘤的影响也日益受到关注,而关于C5L2在肿瘤中发挥何种作用研究较少。C5L2作为C5a的第二受体,其功能及信号通路的研究尚存在一定争议。有研究认为C5L2能抑制C5a-C5aR复合物而发挥抗炎症作用[20-21],也有研究认为C5L2能够促进炎性因子的表达[18],还有研究认为C5L2/C5aR比值与ALP、ALT、AST等指标相关[11]。

MMP是一类能降解基质细胞多种蛋白成分的水解酶,在肿瘤侵袭迁移过程中发挥着重要作用。近年来诸多研究表明,MMP2与乳腺癌[22]、小细胞肺癌[23]、前列腺癌[24]、胰腺癌[25]、胆囊癌[26]等癌细胞侵袭力相关;在肝癌研究当中,有报道表明缓激肽通过TRPM7和MMP2促进HCC发生侵袭迁移[27],证实了MMP2的表达与肿瘤细胞的侵袭迁移能力呈正相关。亦有研究表明,MMP2的表达受wnt通路β-catenin的调控,β-catenin-TCF/LEF复合物能够介导MMP的产生,并促进细胞侵袭[28],TRAF4能够诱导β-catenin及其下游靶分子cyclinD1、c-myc、Bcl-2、MMP-9和MMP-2的表达[29]。经典Wnt 通路通过核内β-catenin的累积,激活Wnt相关靶基因,在肝癌的发生过程中起到非常重要的作用[30]。 有研究报道,40%～70%的肝癌细胞核内出现β-catenin的聚集,表明Wnt信号在肝癌发生过程中发挥了重要作用[31-32]。也有研究报道,在肝癌发生的众多机制中β-catenin基因的突变占8%～30%[33-34]。

本研究首次报道了C5L2与肝癌复发的相关性,临床基本特征分析结果显示C5L2表达与性别、肿瘤大小和复发相关,C5L2低表达组的患者复发率更高,并且COX比例风险模型多因素分析得出C5L2低表达是复发的独立危险因素。在Hep3B和Huh7细胞系中,通过下调和上调C5L2的表达,证实了C5L2能够抑制肝癌细胞侵袭迁移,并且通过抑制β-catenin减少MMP2分泌而发挥作用。因此,C5L2低表达患者需要更加密切的临床观察和随访,以便及早发现肝癌的复发转移,其作为一个辅助指标可为临床医师提供更多的信息,从而改善患者生活质量、提高患者预后状况。

[1] SIEGEL RL,MILLER KD,JEMAL A.Cancer statistics,2016[J].CACancerJClin,2016,66(1):7-30.

[2] LIN J,WU L,BAI X,etal. Combination treatment including targeted therapy for advanced hepatocellular carcinoma[J].Oncotarget,2016,7(43):71036-71051.

[3] FORNER A,LLOVET JM,BRUIX J.Hepatocellular carcinoma[J].Lancet,2012,379(9822):1245-1255.

[4] CAIN SA,MONK PN.The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74)[J].JBiolChem,2002,277(9):7165-7169.

[5] OHNO M,HIRATA T,ENOMOTO M,etal.A putative chemoattractant receptor,C5L2,is expressed in granulocyte and immature dendritic cells,but not in mature dendritic cells[J].MolImmunol,2000,37(8):407-412.

[6] CHEN NJ,MIRTSOS C,SUH D,etal. C5L2 is critical for the biological activities of the anaphylatoxins C5a and C3a[J].Nature,2007,446(7132):203-207.

[7] HU M,LIU B,JAWAD S,etal.C5a contributes to intraocular inflammation by affecting retinal pigment epithelial cells and immune cells[J].BrJOphthalmol,2011,95(12):1738-1744.

[8] CROKER DE,HALAI R,FAIRLIE DP,etal. C5a,but not C5a-des Arg,induces upregulation of heteromer formation between complement C5a receptors C5aR and C5L2[J].ImmunolCellBiol,2013,91(10):625-633.

[9] KALANT D,MACLAREN R,CUI W,etal.C5L2 is a functional receptor for acylation-stimulating protein[J].JBiolChem, 2005,280(25):23936-23944.

[10] GAO H,Neff TA,GUO RF,etal.Evidence for a functional role of the second C5a receptor C5L2[J].FASEBJ,2005,19(8):1003-1005.

[11] CROKER DE,HALAI R,KAESLIN G,etal.C5a2 can modulate ERK1/2 signaling in macrophages via heteromer formation with C5a1 and beta-arrestin recruitment[J].ImmunolCellBiol, 2014,92(7):631-639.

[12] LI R,COULTHARD LG,WU MC,etal. C5L2:a controversial receptor of complement anaphylatoxin,C5a[J].FASEBJ, 2013,27(3):855-864.

[13] HUBER-LANG M,SARMA JV,RITTIRSCH D,etal.Changes in the novel orphan,C5a receptor (C5L2),during experimental sepsis and sepsis in humans[J].JImmunol, 2005,174(2):1104-1110.

[14] PIAO C,CAI L,QIU S,etal.Complement 5a enhances hepatic metastases of colon cancer via monocyte chemoattractant protein-1-mediated inflammatory cell infiltration[J].JBiolChem, 2015,290(17):10667-10676.

[15] GARCIA BL,ZWARTHOFF SA,ROOIJAKKERS SH,etal. Novel evasion mechanisms of the classical complement pathway[J].JImmunol,2016,197(6):2051-2060.

[16] CROKER DE,MONK PN,HALAI R,etal.Discovery of functionally selective C5aR2 ligands:novel modulators of C5a signalling[J].ImmunolCellBiol, 2016,94(8):787-795.

[17] SELLE J,ASARE Y,KOHNCKE J,etal.Atheroprotective role of C5ar2 deficiency in apolipoprotein E-deficient mice[J].ThrombHaemost,2015,114(4):848-858.

[18] VIJAYAN S,ASARE Y,GROMMES J,etal. High expression of C5L2 correlates with high proinflammatory cytokine expression in advanced human atherosclerotic plaques[J].AmJPathol, 2014,184(7):2123-2133.

[19] AN G,REN G,AN F,etal. Role of C5a-C5aR axis in the development of atherosclerosis[J].SciChinaLifeSci,2014,57(8):790-794.

[20] WANG R,LU B,GERARD C,etal.C5L2,the second C5a Anaphylatoxin receptor suppresses LPS-induced acute lung injury[J].AmJRespirCellMolBiol,2016,55(5):657-666.

[21] BOSMANN M,HAGGADONE MD,ZETOUNE FS,etal.The interaction between C5a and both C5aR and C5L2 receptors is required for production of G-CSF during acute inflammation[J].EurJImmunol,2013,43(7):1907-1913.

[22] JACOB A,JING J,LEE J,etal. Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells [J].JCellSci, 2013,126(Pt 20):4647-4658.

[23] YANG CJ,LIU YP,DAI HY ,etal. Nuclear HDAC6 inhibits invasion by suppressing NF-kB/MMP2 and is inversely correlated with metastasis of non-small cell lung cancer[J].Oncotarget,2015,6(30):30263-30276.

[24] CHEN Q,ZHAO X,ZHANG H,etal. MiR-130b suppresses prostate cancer metastasis through down-regulation of MMP2[J].MolCarcinog,2015,54(11):1292-1300.

[25] SHEN M,WU MY,CHEN LP,etal.Cantharidin represses invasion of pancreatic cancer cells through accelerated degradation of MMP2 mRNA[J].SciRep, 2015,5:11836.

[26] PERES R,FURUYA H,PAGANO I,etal. Angiogenin contributes to bladder cancer tumorigenesis by DNMT3b-mediated MMP2 activation[J].Oncotarget,2016,7(28):43109-43123.

[27] CHEN Y,YU Y,SUN S,etal.Bradykinin promotes migration and invasion of hepatocellular carcinoma cells through TRPM7 and MMP2[J].ExpCellRes, 2016,349(1):68-76.

[28] BROWN-CLAY JD,SHENOY DN,TIMOFEEVA O ,etal.PBK/TOPK enhances aggressive phenotype in prostate cancer via β-catenin-TCF/LEF-mediated matrix metalloproteinases production and invasion[J].Oncotarget, 2015,6(17):15594-15609.

[29] YANG J,WEI D,WANG W ,etal.TRAF4 enhances oral squamous cell carcinoma cell growth,invasion and migration by Wnt- β -catenin signaling pathway[J].IntJClinExpPathol,2015,8(9):11837-11846.

[30] LACHENMAYER A,ALSINET C,SAVIC R,etal.Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib[J].ClinCancerRes,2012,18(18):4997-5007.

[31] WONG CM,FAN ST,NG IO.beta-Catenin mutation and overexpression in hepatocellular carcinoma:clinicopathologic and prognostic significance[J].Cancer,2001,92(1):136-145.

[32] BENAYOUN BA,CABURET S,VEITIA RA.Forkhead transcription factors:key players in health and disease[J].TrendsGenet, 2011,27(6):224-232.

[33] GUICHARD C,AMADDEO G,IMBEAUD S,etal. Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma[J].NatGenet,2012,44(6):694-698.

[34] TANIGUCHI K,ROBERTS LR,ADERCA IN,etal.Mutational spectrum of beta-catenin,AXIN1,and AXIN2 in hepatocellular carcinomas and hepatoblastomas[J].Oncogene,2002,21(31):4863-4871.

Prognostic analysis of C5L2 in patients with hepatocellular carcinoma

TIAN Lu, WU Jiong, ZHU Jie, GAO Yao-yi, ZHANG Ai-lun, WANG Bei-li, ZHANG Chun-yan, PAN Bai-shen, GUO Wei△

(DepartmentofClinicalLaboratoryMedicine,ZhongshanHospital,FudanUniversity,Shanghai200032,China)

Objective To investigate the prognostic value of C5L2 in patients with hepatocellular carcinoma (HCC). Methods The data of 175 patients with HCC who underwent curative resection at Zhongshan Hospital,Fudan University from Oct.,2012 to Sep.,2013 were analyzed retrospectively.The correlation between C5L2 and clinicopathologic characteristics were explored.COX regression model was used to determine the influence of clinical parameters on predicting recurrence,and the prognostic value of combined application of C5L2 and AFP were evaluated by Kaplan-Meier method.Invitro,the expression of C5L2 were tested in 5 HCC cell lines,and Hep3B and Huh7 were chosen for down-regulation and up-regulation of C5L2,respectively,the abilities of invasion and migration were examined by transwell and the potential mechanism was explored. Results The C5L2 expression was correlated to gender,tumor size and recurrence,and the recurrence rate of low C5L2 expression group was higher.Also,the multivariate analysis showed that C5L2 low expression was an independent risk factor for recurrence.Moreover,the combined application of C5L2 and AFP could estimate prognosis more effectively.Knockdown of C5L2 in Hep3B promoted the invasiveness and motility,and

increased the level of β-catenin and MMP2;conversely,overexpression of C5L2 in Huh7 inhibited the invasiveness and motility,and decreased the level of β-catenin and MMP2. Conclusions C5L2 could be regarded as an auxiliary indicator for prognosis of HCC,thereby the evaluation of C5L2 could help with making effective and comprehensive management for HCC patients.

C5L2; hepatocellular carcinoma; prognosis

国家自然科学基金面上项目(81572064);上海市科学技术委员会基金项目(1641195922100);上海市卫生和计划生育委员会面上项目(201540052);上海市卫生计生系统重要薄弱学科建设项目(2015ZB0201)

R735.7

A

10.3969/j.issn.1672-8467.2017.03.005

2016-09-23;编辑:王蔚)

△Corresponding author E-mail:guo.wei@zs-hospital.sh.cn

*This work was supported by the General Program of National Natural Science Foundation of China (81572064),the Fund from Science and Technology Commission of Shanghai Municipality (1641195922100), the General Program of Shanghai Municipal Commission of Health and Family Planning (201540052),and the Important Weak Discipline Construction Project of Shanghai Health and Family Planning System (2015ZB0201).