C5L2表達與肝癌患者預后分析

田 璐 吳 炯 朱 捷 高姚怡 張愛倫 王蓓麗 張春燕 潘柏申 郭 瑋

(復旦大學附屬中山醫院檢驗科 上海 200032)

C5L2表達與肝癌患者預后分析

田 璐 吳 炯 朱 捷 高姚怡 張愛倫 王蓓麗 張春燕 潘柏申 郭 瑋△

(復旦大學附屬中山醫院檢驗科 上海 200032)

目的 探討C5L2表達在肝細胞肝癌(hepatocellular carcinoma ,HCC)患者預后判斷中的價值。方法回顧性分析2012年10月至2013年9月在復旦大學附屬中山醫院接受根治性切除術的175例原發性HCC患者。研究C5L2表達與臨床基本參數之間的關系,運用COX回歸模型分析各臨床參數對術后復發的影響,通過Kaplan-Meier法評估C5L2和AFP聯合應用的預后判斷效能。在5種肝癌細胞系中檢測C5L2表達,篩選Hep3B和Huh7分別對C5L2進行下調和上調,檢測細胞侵襲遷移能力的改變并初步探索其機制。 結果 C5L2表達與性別、腫瘤大小和復發相關,C5L2低表達患者復發率更高,多因素分析顯示C5L2低表達是肝癌復發的獨立危險因素,C5L2和AFP聯合應用可以更加有效地判斷肝癌預后。在Hep3B中敲除C5L2可促進細胞侵襲遷移,伴隨β-catenin和MMP2表達上調;相反,在Huh7中過表達C5L2可抑制細胞侵襲遷移,伴隨β-catenin和MMP2表達下調。結論 C5L2可作為判斷肝癌預后的輔助指標,幫助臨床制定更有效的HCC治療和監測方案。

C5L2; 肝細胞肝癌; 預后

肝細胞癌(hepatocellular carcinoma,HCC)是最常見的惡性腫瘤之一,最新統計顯示,全球范圍內肝癌發生率在惡性腫瘤中位于第4位,病死率居第2位[1]。目前對HCC切除術后的患者仍缺乏有效的治療手段,聯合局部介入及放療的綜合治療法雖然提高了部分患者的生存率[2],但治療后5年內高達70%的復發轉移率依然是改善HCC預后難以逾越的瓶頸[3],尋找行之有效的預測及干預手段是改善疾病預后的關鍵。

C5L2(GPR77)基因位于染色體19,q13.33-13.34,是由337個氨基酸組成的7次跨膜G蛋白偶聯受體[4],廣泛表達于免疫細胞[5-6](中性粒細胞,淋巴細胞,單核細胞,不成熟的樹突狀細胞,巨噬細胞)和非免疫細胞[5-9](脂肪細胞,皮膚成纖維細胞,血管平滑肌細胞,星形膠質細胞,神經元,心、肝、脾、肺等組織細胞)。目前關于C5L2的研究較少并存在一定爭議,有研究認為C5L2是一種誘導性表達受體[8],另有研究認為C5L2能夠與一些炎性介質發生復雜的相互作用[10-11],還有研究表明C5L2能夠與C5aR和β-arrestin結合對其信號進行負調控[11-12]。C5L2的作用在不同種屬、不同疾病、不同細胞類型中不盡相同[8,13-14],而C5L2在肝癌中的作用還未見報道。

本研究旨在探討C5L2在與肝癌患者臨床特征的相關性,評估其在判斷肝癌患者預后中的價值,并在體外初步探索C5L2影響肝癌細胞侵襲遷移的潛在機制。

資 料 和 方 法

研究對象 選擇2012年10月至2013年9月在復旦大學附屬中山醫院接受根治性切除術的175例原發性HCC患者。入組標準:(1)年齡≥18歲;(2)術前未行放化療;(3) 首次接受根治性切除術;(4) 術后病理診斷為原發性HCC;(5)有完整的臨床資料及隨訪數據。排除標準:(1)既往或合并有其他惡性腫瘤;(2)術前感染;(3)術前無影像學資料;(4)合并血液、免疫系統疾病。所有樣本獲取及使用過程獲復旦大學附屬中山醫院倫理委員會批準。

免疫組化芯片染色 HCC 組織切片在二甲苯脫蠟、酒精水化后,置于抗原修復液中微波修復9 min,在3%的過氧化氫溶液中浸泡15 min,PBS 漂洗3 次,5% BSA 室溫封閉30 min,一抗孵育4 ℃ 過夜。次日PBS 漂洗3 次,HRP標記山羊抗小鼠IgG室溫孵育60 min,DAB 顯色液顯色8 min后蘇木精復染20 s,酒精脫水,二甲苯透明,中性樹膠封片。

結果判定 所有組織標本均由2位病理科醫師獨立進行評分,如不一致,請第3位病理醫師評分,以兩個相同結果為準。染色強度評分標準:不著色0 分,淺黃色1分,棕黃色2分,黃褐色3 分。陽性細胞所占比例評分標準:陽性細胞數<10%者0分,10%～40%者1分,40%～70% 者2分,≥70%者3分。通過乘積法計算,其中0～3分為低表達,4～9分為高表達。

實時熒光定量PCR 采用Trizol法提取細胞總RNA,逆轉錄成cDNA,實時熒光定量PCR方法進行基因相對定量分析。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裂解液提取細胞總蛋白,并用BCA法測定蛋白濃度。進行SDS不連續凝膠電泳,電泳結束后轉膜,再加入BSA室溫封閉60 min。將不同相對分子質量條帶對應加入稀釋好的一抗孵育過夜。次日用TBST將膜條洗滌3次后孵育二抗1 h,最后再用TBST洗滌3次后放入曝光儀中成像。

細胞轉染 將處于對數生長期的細胞以每孔50萬的密度鋪入6孔培養板中,待細胞融合度達到70%～80%時進行轉染。對照組將5 μL NC溶于245 μL Opti- mem無血清培養基中,實驗組將5 μL si-C5L2或pcDNA3.1-C5L2溶于245 μL Opti-mem無血清培養基中,與配置好的轉染試劑1∶1混合靜置20 min,加入無血清細胞培養液中,置于37 ℃培養箱4～6 h后換入完全培養液。

侵襲與遷移 用無血清培養基重懸細胞至1×105/mL,接種到Transwell小室內(侵襲實驗需要加一步:在小室中鋪50 μL Matrigel膠,37 ℃培養箱中放置4～6 h使其凝固),每個小室加0.2 mL細胞懸液,下層加入0.5 mL含10% FBS的完全培養液,置于37 ℃培養箱中培養48～72 h,用棉簽小心擦去Transwell上室細胞,多聚甲醛固定并用結晶紫染色,PBS洗凈后晾干,封片后于顯微鏡下觀察并計數。

統計學分析 采用SPSS 19.0軟件進行統計學分析。等級變量比較采用χ2檢驗或Fisher精確檢驗,COX比例風險模型行單因素和多因素分析。Graphpad prism 5繪制Kaplan-Meier曲線。P<0.05為差異有統計學意義。

結 果

C5L2表達與肝癌患者臨床基本參數的關系 本研究共入選175例原發性肝癌患者,年齡32～87歲,其中男性134 例,女性41例。對比兩組患者在性別、年齡、肝硬化、腫瘤數量、腫瘤大小、腫瘤包膜、衛星灶、血管侵犯、手術分級、Child-Pugh評分、BCLC分期、術前甲胎蛋白(alpha-fetoprotein,AFP)、谷丙轉氨酶(alanine aminotransferase,ALT)、谷氨酰轉肽酶( glutamete transpeptidase,GGT)、天門冬氨酸氨基轉移酶(aspartate aminotransferase,AST)及復發之間的關系。結果顯示,C5L2低表達患者男性偏多(P=0.008)、腫瘤體積更大(P=0.029)、復發率更高(P=0.010,表1)。

表1 C5L2表達和臨床基本特征的關系

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

C5L2在肝癌預后判斷中的價值 定期對患者的情況進行跟蹤隨訪,通過Kaplan-Meier法繪制生存曲線,采用log-rank檢驗評估C5L2的預后價值。結果顯示,C5L2低表達組的復發率(45.0%)明顯高于高表達組(25.2%),并且差異具有統計學意義(P=0.039 4,圖 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.

圖1 原發性肝癌患者生存曲線分析

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

COX回歸模型分析復發相關危險因素 采用COX比例風險模型對各種風險因素進行分析。單因素分析顯示肝硬化(HR:2.064,95%CI:1.011～4.215,P=0.047)、腫瘤大小(HR:1.852,95%CI:1.095～3.135,P=0.022)、腫瘤包膜(HR:2.448,95%CI:1.044～5.737,P=0.039)、衛星病灶血管侵犯(HR:4.057,95%CI:2.022～8.138,P=0.000)、術前AST(HR:1.940,95%CI:1.137～3.308,P=0.015)和C5L2表達(HR:0.477,95%CI:0.282～0.808,P=0.006)是影響HCC復發的危險因素。多因素分析發現僅血管侵犯(HR:3.043,95%CI:1.434～6.457,P=0.004)和C5L2低表達(HR:0.589,95%CI:0.338～0.976,P=0.043)是影響HCC復發的獨立危險因素(表2)。

表2 復發相關因素的COX比例風險模型單因素和多因素分析

HR is hazard ratio abbreviation.NA:Not applicable.

C5L2和AFP聯合應用在肝癌預后判斷中的價值 C5L2與AFP聯合應用對肝癌復發的陽性預測率為56.4%,陰性預測率為75.0%,總體有效率為70.9%,均高于單獨應用AFP。AFP和C5L2聯合應用對肝癌預后判斷的靈敏度降低而特異性提高(表3)。Kaplan-Mier生存曲線顯示,高AFP低C5L2組的術后復發率(56.4%)遠遠高于低AFP高C5L2組(27.3%),差異具有統計學意義(P=0.001 6,圖1B)。因此,聯合應用兩指標可以更好地協助醫師判斷患者預后。

表3 C5L2和AFP對預后判斷的效能評估

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

C5L2敲除和過表達效率驗證 檢測5種常見肝癌細胞系Huh7、MHHC97H、LM3、7721、Hep3B和1種正常肝細胞系L02中C5L2的表達量。qRT-PCR結果顯示Hep3B細胞系C5L2表達量最高,Huh7細胞系最低(圖2A)。

選擇Hep3B細胞系對C5L2表達進行下調,設計3條特異siRNA干擾序列,si-C5L2-1、si-C5L2-2和si-C5L2-3,并以si-NC為陰性對照,qRT-PCR結果顯示3條序列均能使C5L2表達降低,其中以si-C5L2-2敲除效率最高(圖2B)。同時,在Huh7細胞系通過pcDNA3.1-C5L2對該基因進行上調,以空質粒pcDNA3.1為陰性對照,qRT-PCR結果顯示pcDNA3.1-C5L2能夠顯著上調目標基因(圖2C)。

上調或下調C5L2影響肝癌細胞侵襲遷移能力采用Transwell檢測上述兩種細胞侵襲遷移能力,結果顯示敲低C5L2后Hep3B的侵襲遷移能力增強(圖3A),過表達C5L2后Huh7的侵襲遷移能力減弱(圖3B)。選取上、中、下、左、右5個視野進行計數并分析,差異均具有統計學意義(si-C5L2vs.si-NC,P<0.01;pcDNA3.1-C5L2vs.pcDNA3.1-NC,P<0.01),證實C5L2能夠體外抑制細胞侵襲遷移能力。

C5L2通過MMP2影響細胞侵襲遷移能力 Wnt 信號通路是促進細胞侵襲遷移的重要通路之一,β鏈接蛋白(β-catenin)的激活能夠促進基質金屬蛋白酶2 (matrix metalloproteinase 2,MMP2的分泌,從而通過降解細胞外基質和基底膜增強腫瘤細胞的侵襲力。通過qRT-PCR和WB檢測,在下調C5L2的Hep3B細胞系中β-catenin (P=0.003)和MMP2 (P=0.007)的水平升高(圖4A、C),而上調C5L2的Huh7細胞系中β-catenin (P=0.009) 和MMP2 (P=0.037)水平下降(圖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.

圖2 各種肝癌細胞系中C5L2 mRNA水平及敲除和過表達C5L2轉染效率驗證

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.

圖3 敲除或過表達C5L2對細胞侵襲和遷移能力的影響

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.

圖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

討 論

肝癌作為一種高病死率、高復發率的惡性腫瘤,其發生、發展和轉移機制相當復雜。術后復發轉移是影響肝癌治療效果的重要因素,研究復發轉移相關因素具有十分重要的臨床意義。隨著科技的發展,HCC的診療手段不斷改進,患者的預后狀況和生活質量都有所提高。但目前應用于預后判斷的指標靈敏度和特異性還有待提高。因此,尋找能夠充分反映HCC生物學特性的新型輔助指標,是HCC治療的基礎和關鍵。

補體系統參與機體抵抗外來病原體的固有免疫和適應性免疫過程[15]。在瀑布樣補體級聯活化過程中會產生C3a、C4a、C5a等分子片段[6]。C5a及其兩個受體C5aR和C5L2與炎癥的發生發展有密切關系[16],也有研究表明C5L2能促進動脈粥樣硬化和狹窄[17-19]。近些年,炎癥對于腫瘤的影響也日益受到關注,而關于C5L2在腫瘤中發揮何種作用研究較少。C5L2作為C5a的第二受體,其功能及信號通路的研究尚存在一定爭議。有研究認為C5L2能抑制C5a-C5aR復合物而發揮抗炎癥作用[20-21],也有研究認為C5L2能夠促進炎性因子的表達[18],還有研究認為C5L2/C5aR比值與ALP、ALT、AST等指標相關[11]。

MMP是一類能降解基質細胞多種蛋白成分的水解酶,在腫瘤侵襲遷移過程中發揮著重要作用。近年來諸多研究表明,MMP2與乳腺癌[22]、小細胞肺癌[23]、前列腺癌[24]、胰腺癌[25]、膽囊癌[26]等癌細胞侵襲力相關;在肝癌研究當中,有報道表明緩激肽通過TRPM7和MMP2促進HCC發生侵襲遷移[27],證實了MMP2的表達與腫瘤細胞的侵襲遷移能力呈正相關。亦有研究表明,MMP2的表達受wnt通路β-catenin的調控,β-catenin-TCF/LEF復合物能夠介導MMP的產生,并促進細胞侵襲[28],TRAF4能夠誘導β-catenin及其下游靶分子cyclinD1、c-myc、Bcl-2、MMP-9和MMP-2的表達[29]。經典Wnt 通路通過核內β-catenin的累積,激活Wnt相關靶基因,在肝癌的發生過程中起到非常重要的作用[30]。 有研究報道,40%～70%的肝癌細胞核內出現β-catenin的聚集,表明Wnt信號在肝癌發生過程中發揮了重要作用[31-32]。也有研究報道,在肝癌發生的眾多機制中β-catenin基因的突變占8%～30%[33-34]。

本研究首次報道了C5L2與肝癌復發的相關性,臨床基本特征分析結果顯示C5L2表達與性別、腫瘤大小和復發相關,C5L2低表達組的患者復發率更高,并且COX比例風險模型多因素分析得出C5L2低表達是復發的獨立危險因素。在Hep3B和Huh7細胞系中,通過下調和上調C5L2的表達,證實了C5L2能夠抑制肝癌細胞侵襲遷移,并且通過抑制β-catenin減少MMP2分泌而發揮作用。因此,C5L2低表達患者需要更加密切的臨床觀察和隨訪,以便及早發現肝癌的復發轉移,其作為一個輔助指標可為臨床醫師提供更多的信息,從而改善患者生活質量、提高患者預后狀況。

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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

國家自然科學基金面上項目(81572064);上海市科學技術委員會基金項目(1641195922100);上海市衛生和計劃生育委員會面上項目(201540052);上海市衛生計生系統重要薄弱學科建設項目(2015ZB0201)

R735.7

A

10.3969/j.issn.1672-8467.2017.03.005

2016-09-23;編輯: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).