IL-17RA表達(dá)對(duì)肝細(xì)胞癌(HCC)患者的預(yù)后意義及其對(duì)HCC細(xì)胞株生物學(xué)特性的影響王妍;謝曉鶯;陳榮新;任正剛;張嵐【期刊名稱(chēng)】《《復(fù)旦學(xué)報(bào)（醫(yī)學(xué)版）》》【年(卷),期】2019(046)005【總頁(yè)數(shù)】7頁(yè)(P625-630,636)【關(guān)鍵詞】肝細(xì)胞癌(HCC);IL-17RA;預(yù)后【作者】王妍;謝曉鶯;陳榮新;任正剛;張嵐【作者單位】復(fù)旦大學(xué)附屬中山醫(yī)院肝腫瘤內(nèi)科上海200032【正文語(yǔ)種】中文【中圖分類(lèi)】R735.7肝癌在全球常見(jiàn)腫瘤中排名第六位,而在腫瘤相關(guān)死因排名中位居第四,每年有84100078200075%～85%為肝細(xì)胞癌(hepatocellularcarcinoma,HCC)HCC患者獲益,但其價(jià)格昂貴、客觀(guān)反應(yīng)率低[2FOLFOX4HCC(8.15%,HCC系統(tǒng)化療失敗的主要原因,晚期HCC的治療仍是一大難題。炎癥與腫瘤密不可分,在眾多炎癥因子中,Th17細(xì)胞及其分泌的IL-17A在多種動(dòng)物模型和人類(lèi)腫瘤研究中均被證實(shí)影響腫瘤的發(fā)生、發(fā)展及患者預(yù)后IL-17RAIL-17AHCCHCCIL-17RA在HCCHCC細(xì)胞株生物學(xué)特征的影響。資料和方法2008112HCC的患者1633訪(fǎng)患者生存情況,統(tǒng)計(jì)患者的總生存期(overallsurvival,OS末),次隨訪(fǎng)時(shí)間為2016年4月。24μm。針對(duì)IL-17RA使用二步法進(jìn)行免疫組化染色(IL-17A公司,1∶150),著色面積(0:0分;1%～15%:1分;16%～50%:2分;>50%:3:01232>4A:Negative;B:Weak;C:Positive;D:Strong.Scalebar:51肝癌組織芯片17RAFig1ImmunohistochemicalstainingofIL-17RAinHCCspecimenstissuemicroarray細(xì)胞瞬時(shí)轉(zhuǎn)染處理及轉(zhuǎn)染效率驗(yàn)證高侵襲性HCC細(xì)胞系MHCC-97H 和Huh7復(fù)旦大學(xué)附屬中山醫(yī)院肝癌研究所提供。siRNA由吉馬基因公司設(shè)計(jì)序列為GCGUCAGGUUUGAGUU-UCUTT- 3’。轉(zhuǎn)染前一天在6孔板中鋪板,24h后用Lipo2000進(jìn)行轉(zhuǎn)染培養(yǎng)箱中轉(zhuǎn)染4～6h后更換新鮮培養(yǎng)基。用qRT-PCR檢干擾組和對(duì)照組IL-17RAmRNA 水平上游引物TCACGGGCATCTCC- 3’;下游引物3’),WesternblotIL-17RA:IL-17AAbcam公司,1∶1000)。80%200無(wú)菌槍頭在培養(yǎng)板中央輕輕劃痕,4833侵襲實(shí)驗(yàn)使用Transwell侵襲小室將基質(zhì)膠于4℃化為液預(yù)冷實(shí)驗(yàn)所需槍頭培養(yǎng)基將工作濃度基質(zhì)膠100加于小室上室后放入培養(yǎng)箱凝固2h。細(xì)胞消化后計(jì)數(shù)并調(diào)整細(xì)胞濃度上室接種5×105個(gè)細(xì)胞懸液100μL(無(wú)血DMEM 培養(yǎng))下室加入含20%FBS的DMEM 高糖培養(yǎng)基600μL,培24～48h后取出小室用棉簽擦去上室面細(xì)胞漂洗、固定、結(jié)晶紫染色顯微鏡下拍照觀(guān)察并計(jì)數(shù)腫瘤細(xì)胞侵襲情況。實(shí)驗(yàn)重復(fù)3次取其平均數(shù)作為實(shí)驗(yàn)結(jié)果。965×10310204080μmol/L348h后加入CCK8試劑并測(cè)OD3SPSS19.0Kaplan-MeierCox回歸模型。實(shí)驗(yàn)數(shù)據(jù)采用GraphPad6.0軟件t檢驗(yàn),P<0.05結(jié)果IL-17RAHCC患者術(shù)后生存期1631979平均(52.32±12.29)1372613580%、60%51%,(64.87±3.39)IL-17RA高表達(dá)組和IL-17RA術(shù)前甲胎蛋白、乙肝表面抗原、腫瘤直徑、個(gè)數(shù)、有無(wú)肉眼或鏡下癌栓等因素差異進(jìn)一步分析上述因素及IL-17RA的表達(dá)對(duì)患者術(shù)后OS的影響。單因素分析結(jié)果顯示術(shù)前AFP≤20ng/mL組和AFP>20ng/mL 組的平均生存時(shí)間分別為68.83個(gè)月和59.60個(gè)月腫瘤直徑≤10cm和>10cm組的平均生存時(shí)間分為71.45個(gè)月和35.02個(gè)月未合并癌栓和合并癌栓組的平均生存時(shí)間76.6344.64(P<0.001),IL-17RA68.3753.36>10cm(HR=1.820,P=0.028)、合并癌栓(HR=2.087,P=0.003)IL-17RA高表達(dá)(HR=1.579,P=0.042)是影響肝癌患者術(shù)后總生存期的獨(dú)立危險(xiǎn)因素(圖2,表2)。1IL-17RATab1ClinicalcomparisonbetweenIL-17RAlowgroupandIL-17RAhighgroup(n)FactorsIL-17RAlow(n=99)IL-17RAhigh(n=64)PAge(y)>651810≤6581540.673GenderMale8255Female1790.597Totalbilirubin(μmol/L)≤17.18054>17.119100.651Albumin(g/L)≥359160<35840.662Prothrombintime(s)≤149155>14890.222AFP(ng/mL)≤204520>2054440.071HbsAgNegative158Positive84560.635Tumordiameter(cm)≤108349>1016150.333TumornumbersSingle8156Multiple1880.387ThrombusNo6434Yes35300.642圖2肝癌患者術(shù)后生存曲線(xiàn)Fig2SurvivalcurveofHCCunderwentresection表2肝癌患者術(shù)后總體生存期的預(yù)后因素分析Tab2PrognosticfactorsofoverallsurvivalforHCCunderwentresectionFactorsnUnivariateMultivariateOS(mo)PHR(95%CI)PAge(y)>652866.38≤6513554.770.321GenderMale13763.31Female2655.730.121Totalbilirubin(μmol/L)≤17.113466.10>17.12956.130.455Albumin(g/L)≥3515166.04<351246.130.285Prothrombintime(s)≤1414665.86>141752.890.504AFP(ng/mL)≤206568.83>209859.600.0431.061(0.647-1.741)0.814HbsAgNegative2362.39Positive14064.520.657Tumordiameter(cm)≤1013271.45>103135.020.0001.820(1.065-3.109)0.028TumornumbersSingle13767.51Multiple2648.560.063ThrombusNo9876.63Yes6544.640.0002.087(1.275-3.414)0.003IL-17RALow9968.37High6453.360.0091.579(1.016-2.452)0.042下調(diào)IL-17RA表達(dá)后HCC細(xì)胞體外遷移和侵襲能力下降用siRNA瞬時(shí)轉(zhuǎn)染人HCC細(xì)胞株MHCC-97H 和Huh7,下調(diào)IL-17RA表達(dá)后分別采用qRT-PCR和Westernblot方法檢測(cè)干擾效率。結(jié)果顯示與對(duì)照組相比干擾組細(xì)胞在的mRNA 和蛋白質(zhì)水平均明顯下降圖。瞬時(shí)轉(zhuǎn)染siRNA后用劃痕實(shí)驗(yàn)觀(guān)察下調(diào)IL-17RA對(duì)MHCC-97H 和Huh7細(xì)胞遷移能力的影響結(jié)果顯示干擾組細(xì)胞遷移能力較對(duì)照組明顯下降用Tranwell侵襲實(shí)驗(yàn)觀(guān)察下調(diào)IL-17RA對(duì)MHCC-97H 和Huh7細(xì)胞侵襲能力的影響結(jié)果顯示干擾組細(xì)胞侵襲能力較對(duì)照組明顯下降圖?？梢?jiàn)下調(diào)IL-17RA表達(dá)可以抑制MHCC-97H 和Huh7細(xì)胞的遷移和侵襲能力。下調(diào)IL-17RA表達(dá)后奧沙利鉑對(duì)HCC細(xì)胞增殖的抑制率提高用siRNA瞬時(shí)轉(zhuǎn)染HCC細(xì)胞株MHCC-97H 和Huh7下調(diào)IL-17RA表達(dá)后分別用不同濃度的奧沙利鉑作用于干擾組和對(duì)照組細(xì)胞用CCK8方法檢測(cè)奧沙利鉑對(duì)腫瘤細(xì)胞增殖的抑制率。結(jié)果顯示當(dāng)奧沙利鉑作用濃度為40μmol/L及80μmol/L時(shí)對(duì)干擾組細(xì)胞抑制率明顯高于對(duì)照組圖?？梢?jiàn)下調(diào)IL-17RA的表達(dá)可以提高奧沙利鉑對(duì)肝癌細(xì)胞增殖的抑制率。A:IL-17RAproteinlevelwasdecreasedinMHCC-97HorHuh7withsiRNAcomparedwithcontrolcellsdetectedbyWesternblot;B:IL-17RAmRNAlevelwasdecreasedinMHCC-97HorHuh7withsiRNAcomparedwithcontrolcellsdetectedbyqRT-PCR圖3siRNA瞬時(shí)轉(zhuǎn)染MHCC-97H 和細(xì)胞株后IL-17RA表達(dá)Fig3IL-17RAexpressioninMHCC-97HandHuh7afterdown-regulatedIL-17RAbysiRNAA:ThemigrationrateofMHCC-97HandHuh7wasdecreasedwithsiRNAcomparedwithcontrolcells;B:ThenumberofinvasioncellsofMHCC-97HandHuh7wasdecreasedwithsiRNAcomparedwithcontrolcells.(1)vs.control5.4siRNA瞬時(shí)轉(zhuǎn)染下調(diào)HCC細(xì)胞IL-17RA表達(dá)后MHCC-97H 和Huh7細(xì)胞的遷移和侵襲能力下降Fig4TheabilitiesmigrationandinvasiveofMHCC-97HandHuh7weredecreasedafterdown-regulatedtheexpressionofIL-17RA圖5siRNA瞬時(shí)轉(zhuǎn)染下調(diào)HCC細(xì)胞IL-17RA表達(dá)后奧沙利鉑對(duì)肝癌細(xì)胞的抑制Fig5TheinhibitionrateofoxaliplatinontumorwasincreasedwithsiRNAcomparedwithcontrolcells討論IL-17IL-17A、IL-17B、IL-17C、IL-17DIL-17E(IL-25IL-17F,其中Th17細(xì)胞分泌的IL-17A和IL-17F形成二聚體,通過(guò)激活效應(yīng)細(xì)胞上的含有IL-17RA和IL-17RC受體復(fù)合物發(fā)揮效應(yīng)[7]。IL-17A/IL-17RA軸在結(jié)腸癌、宮頸癌、肺癌等腫瘤的發(fā)生、轉(zhuǎn)移以及化療耐藥等方面都發(fā)揮了重要作用[8]。IL-17A既能直接活化腫瘤細(xì)胞STAT3信號(hào)通路促進(jìn)腫瘤的生長(zhǎng)轉(zhuǎn)移[9]也,通過(guò)微環(huán)CD8+T細(xì)胞、MDSCIL-17NF-ERKG-CSFVEGF抑制劑的敏感性IL-17G-CSFTANs表面NOS2、BV8S100a8S100a9的表達(dá)而使其促進(jìn)乳腺癌轉(zhuǎn)移IL-17RAIL-17A[12][13HCCIL-17A/IL-17RA軸在HCC中的作用機(jī)制提供了基礎(chǔ)和依據(jù)。本研究進(jìn)一步通過(guò)siRNAHCC細(xì)胞株IL-17RAHCCIL-17RAHCC細(xì)胞株IL-17RAHCC既往多項(xiàng)研究證實(shí),IL-17A/IL-17RAIL-17A通過(guò)上調(diào)磷酸化ERK1/2促進(jìn)乳腺癌的增殖及對(duì)多西他賽治療的耐藥IL-17AIL-17A5-FUIL-6的表達(dá)有許多關(guān)于奧沙利鉑化療療效的臨床研究正在進(jìn)行HCC細(xì)胞株IL-17RA的表達(dá)可以提高奧沙利HCCIL-17A/IL-17RA參考文獻(xiàn)【相關(guān)文獻(xiàn)】BRAYF,FERLAYJ,SOERJOMATARAMI,etal.Globalcancerstatistics2018:GLOBOCANestimatesofincidenceandmortalityworldwidefor36cancersin185countries[J].CACancerJClin,2018,68(6):394-424.EL-KHOUEIRYAB,SANGROB,YAUT,etal.Nivolumabinpatientswithadvancedhepatocellularcarcinoma(CheckMate040):anopen-label,non-comparative,phasedoseescalationandexpansiontrial[J].Lancet,2017,389(10088):2492-2502.QINS,BAIY,LIMHY,etal.Randomized,multicenter,open-labelstudyofoxaliplatinfluorouracil/leucovorinversusdoxorubicinaspalliativechemotherapyinpatientswithadvancedhepatocellularcarcinomafromAsia[J].JClinOncol,2013,31(28):3501-3508.HURTADOCG,WANF,HOUSSEAUF,etal.Rolesforinterleukin17andadaptiveimmunityinpathogenesisofcolorectalcancer[J].Gastroenterology,2018,155(6):1706-1715.HUANGQ,DUJ,FANJ,etal.TheeffectofproinflammatorycytokinesonIL-17RAexpressioninNSCLC[J].MedOncol,2014,31(9):144.ASUKAIK,KAWAMOTOK,EGUCHIH,etal.PrognosticimpactofperitumoralIL-17-positivecellsandIL-17axisinpatientswithintrahepaticcholangiocarcinoma[J].AnnOncol,2015,22(Suppl3):S1524-S1531.GUC,WUL,LIX.IL-17family:cytokines,receptorssignaling[J].Cytokine,2013,64(2):477-485.FABREJ,GIUSTINIANIJ,GARBARC,etal.Targetingthetumormicroenvironment:theprotumoreffectsofIL-17relatedtocancertype[J].IntJMolSci,2016,17(9):E1433.DESIMONEV,FRANZEE,RONCHETTIG,etal.Th17-typecytokines,IL-6andTNF-alphasynergisticallyactivateSTAT3andNF-kBtopromotecolorectalcancercellgrowth[J].Oncogene,2015,34(27):3493-3503.CHUNGAS,WUX,ZHUANGG,etal.Aninterleukin-17-mediatedparacrinenetworkpromotestumorresistancetoanti-angiogenictherapy[J].NatMed,2013,19(9):1114-1123.COFFELTSB,KERSTENK,DOORNEBALCW,etal.IL-17-producinggammadeltaTandneutrophilsconspiretopromotebreastcancermetastasis[J].Nature,2015,522(7556):345-348.IWAKURAY,ISHIGAMEH,SAIJOS,etal.Functionalspecializationofinterleukin-17familymembers[J].Immunity,2011,34(2):149-162.JIANGYX,LIPA,YANGSW,etal.IncreasedchemokinereceptorIL-17RAexpressionassociatedwithpoorsurvivalingastriccancerpatients[J].IntJ