高表达CAMSAP2通过上调TGF-β信号促进胃癌细胞的侵袭和转移.pdf

高表高表达达CAMSAPCAMSAP2 2通过上调通过上调TGF-TGF-信号促进胃癌细胞的侵袭和信号促进胃癌细胞的侵袭和转转移移左芦根1，6，王 炼1，2，杨 子1，2，李骏杰2，5，王文锋2，5，李 静3，王月月3，宋 雪4，张小凤4，耿志军4蚌埠医学院第一附属医院1胃肠外科，3检验科，4中心实验室，安徽 蚌埠 233004；2蚌埠医学院临床医学院，安徽 蚌埠 233030；5组织移植安徽省重点实验室，安徽 蚌埠 233030；6炎症相关性疾病基础与转化研究安徽省重点实验室，安徽 蚌埠 233030High expression of CAMSAP2 promotes invasion and metastasis of gastric cancer cellsby upregulating TGF-signalingZUO Lugen1,WANG Lian1,2,YANG Zi1,2,LI Junjie2,5,WANG Wenfeng2,5,LI Jing3,WANG Yueyue3,SONG Xue4,ZHNAG Xiaofeng4,GENG Zhijun41Department of Gastrointestinal Surgery,3Clinical Laboratory,4Central Laboratory,First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;2College of Clinical Medicine,Bengbu Medical College,Bengbu 233030,China;5Anhui Provincial Key Laboratory ofTissue Transplantation,Bengbu Medical College,Bengbu 233030,China;6Anhui Province Key Laboratory of Basic and TranslationalResearch of Inflammation-related Diseases,Bengbu 233030,China摘要：目的 阐明钙调蛋白调控的光谱相关蛋白2（CAMSAP2）在胃癌中的表达及其对疾病进展和预后的影响，并分析可能的机制。方法 利用公共癌症数据初步分析CAMSAP2的表达和胃癌进展及预后的关系，另纳入2013年10月2017年10月在我院完成胃癌根治术的106例患者进行验证。利用生物信息学预测CAMSAP2的生物学功能。采用CAMSAP2过表达质粒、特异性干扰分别处理胃癌细胞系（MGC803）分为CAMSAP2上调组、CAMSAP2下调组，将未经处理的MGC803设为空白对照组，体外观察CAMSAP2对胃癌细胞上皮-间质转化（EMT）和侵袭的影响，并建立裸鼠胃原位种植瘤模型进行在体验证，同时分析CAMSAP2调控胃癌的分子机制。结果 CAMSAP2在胃癌组织中的表达显著高于癌旁组织（P0.05），并和外周血CEA、CA19-9水平正相关（P0.001）。Cox回归分析显示CAMSAP2高表达是影响胃癌患者术后5年生存率的独立危险因素（HR：2.969；95%CI：1.031-8.548）。富集分析提示CAMSAP2的作用可能与EMT和TGF-信号有关。免疫印迹、划痕和Transwell实验发现：体外上调CAMSAP2可促进胃癌细胞表达Vimentin和N-cadherin，同时抑制E-cadherin的表达，并促进胃癌细胞迁移和侵袭（P0.05）；下调CAMSAP2则结果相反（P0.05）。在体上调CAMSAP2可促进裸鼠胃原位种植瘤的转移（P0.05），促进胃癌组织中Vimentin和N-cadherin的表达，同时抑制E-cadherin的表达（P0.05）；下调CAMSAP2则结果相反（P0.05）。体内外实验中，上调CAMSAP2可升高胃癌组织或胃癌细胞中TGF-和p-Smad2/3的水平（P0.05），而下调CAMSAP2则结果相反（P0.05）。结论 CAMSAP2在胃癌中高表达并和肿瘤恶性进展以及预后不良相关，其可能通过上调TGF-信号参与了胃癌的EMT过程。关键词：胃癌；CAMSAP2；TGF-；预后；上皮-间质转化Abstract:Objective To investigate the expression of calmodulin-regulated spectrin-associated protein 2(CAMSAP2)in gastriccancer and its effect on gastric cancer cell invasion and metastasis.Methods The association of CAMSAP2 expression levelswith progression and prognosis of gastric cancer was analyzed using public cancer data and in 106 patients receiving radicalgastrectomy in our hospital from October,2013 to October,2017.The biological functions of CAMSAP2 were predicted usingbioinformatics analysis.Gastric cancer MGC803 cells with CAMSAP2 overexpression and knockdown were observed forepithelial-mesenchymal transition(EMT),migration and invasion.A nude mouse model bearing orthotopic gastric cancer cellxenografts was established for verifying the results and exploring the underlying molecular mechanism.Results Gastric cancertissues expressed high levels of CAMSAP2,which were positively correlated with CEA and CA19-9(P0.001).Cox regressionanalysis showed that CAMSAP2 expression level was an independent risk factor affecting the 5-year survival rate of gastriccancer patients(HR=2.969,95%CI:1.031-8.548).Enrichment analysis suggested that CAMSAP2 was involved in epithelial-mesenchymal transition(EMT)and TGF-signaling.In gastric cancer cells,CAMSAP2 overexpression significantly increasedthe expressions of vimentin and N-cadherin,inhibited the expression of E-cadherin,and enhanced cell migration and invasion(P0.05);CAMSAP2 knockdown produced the opposite effects in the cells(P0.05).In the tumor-bearing mice,xenograftsoverexpressing CAMSAP2 showed enhanced metastasis(P0.05),increased vimentin and N-cadherin expressions and loweredE-cadherin expression(P0.05),and the xenografts with CAMSAP2 knockdown showed the opposite changes(P0.05).Boththe in vivo and in vitro experiments showed that CAMSAP2 overexpression increased and CAMSAP2 knockdown lowered thelevels of TGF-and p-Smad2/3 in the gastric cancer cells(P1个；膈肌、肠系膜、腹膜后10个）、转移性结节50mm3、腹水形成（体积5 mL）、黄疸、肠梗阻和恶病质；（3）腹水形成体积5 mL，记2分。1.6.3 免疫印迹检测胃癌EMT进程及其可能的分子机制 将胃原位肿瘤组织剪碎，使用RIPA裂解液裂解并提蛋白。其余步骤和抗体同1.5.2。1.7 统计学方法数据分析采用SPSS 26.0软件。计数资料的组间比较用2检验。两组间计量资料的比较用t检验。三组间比较用单因素方差分析。相关性分析使用Spearman检验。生存曲线（K-M）用于分析术后生存率，Cox回归模型用于预测影响胃癌患者术后5年生存率的危险因素。P0.05为差异具有统计学意义。2 结果2.1 CAMSAP2在胃癌组织中表达升高分析Timer、GEPIA和HPA数据库发现CAMSAP2在胃癌等多种癌症中的表达升高（P0.05，图1AC）。免疫组化技术检测本机构的胃癌手术标本发现：CAMSAP2在胃癌组织中的表达水平显著高于癌旁组织（P0.05，图1D、E）。543210STAD(num(T)=408;num(N)=211)CAMSAP2 expression level(log2 TPM)6420AdjacentCancerAdjacentCancer200 m200 m40 m40 m150 m150 m20 m20 mRelative CAMSAP2 IOD76543210CancerAdjacent*ABCDE图1 CAMSAP2在胃癌组织中表达升高Fig.1 Expression of CAMSAP2 is elevated in gastric cancer tissue.A:Pan-cancer expression analysis.B,C:Expression ofCAMSAP2 in gastric cancer and adjacent tissues.D,E:Immunohistochemical staining for CAMSAP2 in gastric cancer andadjacent tissues.*P0.05 vs adjacent tissue.J South Med Univ,2023,43(9):1460-1468http:/www.j-14622.2 CAMSAP2的表达量和胃癌恶性进展参数正相关UALCAN数据库显示CAMSAP2的表达量与胃癌的癌症分期和肿瘤分级显著正相关（P0.05，图2A、B）。以免疫组化所测得CAMSAP2相对表达量的中位数（4.93）为界，将本机构106例患者分为CAMSAP2高表达组（n=53）和低表达组（n=53）。如表 1 所示，CAMSAP2高表达组患者中CEA5 g/L、CA19-937kU/L、T3-T4 stage及N2-N3 stage的患者比例显著高于CAMSAP2低表达组（P0.05）。此外，Spearman相关分析表明 CAMSAP2 的表达与患者外周血 CEA 和CA19-9的水平正相关（P0.001，图2C、D）。NormalStage1Stage2Stage3Stage4(n=34)(n=18)(n=123)(n=169)(n=41)Expression of CAMSAP2 in STAD based onindividual cancer stages302520151050Transcript per million2520151050Transcript per millionExpression of CAMSAP1L1 in STAD basedon tumor grade图2 CAMSAP2的表达量和胃癌恶性进展参数的相关性Fig.2 Correlation between CAMSAP2 expression level and progression of gastric cancer.A,B:CAMSAP2 expression is correlated with stage and grade of gastric cancer.C,D:CAMSAP2expression level is correlated with peripheral blood CEAand CA19-9 levels.*P0.05 vs Normal.*NormalGrade 1Grade 2Grade 3(n=34)(n=12)(n=148)(n=246)CAMSAP2 IOD value706050403020100048CA19-9(kU/L)P0.001r=0.663P0.001r=0.733CAMSAP2 IOD value14121086420048CEA(g/L)ABCDFactorsGenderAge(year)CEA(g/L)CA19-9(kU/L)Tumor size(cm)Cancer cell typeT stageN stageMaleFemale606055373755AdenocarcinomaOther1-23-40-12-3n45613670495757494165426450564957CAMSAP2 expression(n,%)Low(n=53)25(55.6%)28(45.9%)19(52.8%)34(48.6%)41(83.7%)12(21.1%)39(68.4%)14(28.6%)21(51.2%)32(49.2%)25(59.5%)28(43.8%)43(86%)10(17.9%)34(69.4%)19(33.3%)High(n=53)20(44.4%)33(54.1%)17(47.2%)36(51.4%)8(16.3%)45(78.9%)18(31.6%)35(71.4%)20(48.8%)33(50.8%)17(40.5%)36(56.3%)7(14%)46(82.1%)15(30.6%)38(66.7%)20.9650.16841.33016.7370.0402.52449.06313.701P0.3260.6820.0010.0010.8420.1120.0010.001表1 CAMSAP2的表达量和胃癌恶性进展参数正相关Tab.1 CAMSAP2 expression level is positively correlated with progression of gastric cancerhttp:/www.j-J South Med Univ,2023,43(9):1460-146814632.3 CAMSAP2高表达影响术后5年生存率KM-plotter数据库表明：胃癌组织CAMSAP2高表达的患者生存期显著缩短（P0.05，图3A）。通过K-M曲线对本研究纳入患者的生存数据进行分析发现，CAMSAP2高表达的胃癌患者术后5年生存率显著低于CAMSAP2低表达组的患者（P0.001，图3B）。图3 CAMSAP2高表达影响术后5年生存率Fig.3 A high CAMSAP2 expression is associated with a reduced 5-year survival rate of gastriccancer patients after radical gastrectomy.A:CAMSAP2 expression level affects overall survivalof gastric cancer patients.B:Kaplan-Meier survival analysis for 5-year survival rate of thepatients after radical gastrectomy.Cumulative survival rate(%)100500OS0204060Survival(month)Logrank 2=72.190P0.001Low CAMSAP2High CAMSAP2Probability1.00.80.60.40.20.0HR=1.58(1.33-1.87)Logrank P=1.2e-07LowHighExpression050100150Survival(month)AB2.4 CAMSAP2高表达是影响胃癌患者术后5年生存率的独立危险因素如表2所示，经Cox单因素和多因素生存分析发现以下因素是影响胃癌根治术后5年生存率的独立危险因素，包括：CAMSAP2 高表达、CEA5 g/L、CA19-937kU/L、T3-T4stage及N2-N3 stage。FactorsGender(male vs female)Age(60 vs 60)CAMSAP2 expression(high vs low)CEA(5 g/L vs 5 g/L)CA19-9(37 kU/L vs 37 kU/L)Tumor size(5 cm vs 5 cm)Cancer cell type(adenocarcinoma vs other)T stage(T1-T2 vs T3-T4)N stage(N0-N1 vs N2-N3)Univariate analysisHR1.1350.76514.0434.8773.8560.8791.50610.4574.09995%CI0.662-1.9460.443-1.3236.503-30.3252.555-9.3122.177-6.8290.511-1.5130.855-2.6534.879-22.4142.184-7.692P0.6460.3380.0010.0010.0010.6430.1560.0010.001Multivariate analysisHR2.9692.3212.0464.2564.21995%CI1.031-8.5481.038-5.1861.075-3.8961.697-10.6752.050-8.684P0.0440.0400.0290.0020.001表2 影响胃癌根治术后5年生存率的危险因素分析Tab.2 Analysis of risk factors affecting 5-year survival rate of gastric cancer patients after radical gastrectomy2.5 基因富集分析预测 CAMSAP2 和 EMT 过程及TGF-信号有关KEGG富集分析结果显示CAMSAP2的生物学功能可能和EMT及TGF-信号有关（图4）。2.6 CAMSAP2体外促进胃癌细胞的EMT、迁移和侵袭利 用 慢 病 毒 转 染 技 术 调 控 胃 癌 细 胞 系 中CAMSAP2的表达，分为上调、下调和对照组（P0.05，图5A、B）。免疫印迹结果显示，上调CAMSAP2促进Vimentin和N-cadherin的表达并抑制E-cadherin的表达，下调则相反（P0.05，图5C、D）。划痕和Transwell实验表明，CAMSAP2的上调促进MGC803细胞的迁移和侵袭，CAMSAP2的下调则反之（P0.05，图5EG）。2.7 CAMSAP2在体内促进胃癌的进展各组小鼠胃原位肿瘤和肿瘤腹腔脏器转移情况如图6A，CAMSAP2上调组原位肿瘤体积明显高于对照组，而下调组则明显低于对照组（P0.05，图6B）。同时，CAMSAP2上调组裸鼠肿瘤的宏观播散评分明显高于对照组，而下调组则相反（P0.05，图6C）。此外，免J South Med Univ,2023,43(9):1460-1468http:/www.j-1464疫印迹结果显示：下调CAMSAP2可降低Vimentin和N-cadherin的表达并增加E-cadherin的表达，上调则结果相反（P0.05，图6D、E）。2.8 CAMSAP2可能通过上调TGF-信号促进胃癌细胞的EMT进程体外实验数据显示，CAMSAP2上调组胃癌细胞中 TGF-和 p-Smad2/3 的水平显著高于对照组，而CAMSAP2下调组则显著低于对照组（P0.05，图7A、B）。同时，体内研究数据与体外研究数据一致（P0.05，图7C、D）。3 讨论本研究中，我们通过生物信息学和本机构临床病例的联合分析发现：CAMSAP2在胃癌组织中高表达，且与疾病恶性进展和患者预后不良相关；在此基础上，开展细胞和动物实验进一步验证，CAMSAP2在体内外环境下均可上调TGF-信号，从而促进胃癌细胞的EMT进程以及侵袭迁移等恶性行为。既往报道显示，CAMSAP2在肝癌和结直肠癌中的表达均显著增高，但在胃癌中未见相关报道 15,16。我们通过对公共肿瘤数据库和本机构的临床病理标本进行分析，发现CAMSAP2在胃癌组织中的表达水平显著高于癌旁组织；通过分析CAMSAP2表达与胃癌临床病理参数间的关系，发现CAMSAP2高表达和外周血CEA和CA19-9水平，以及T stage和N stage呈正相关。进一步采用Cox回归模型联合TCGA数据库分析发现CAMSAP2高表达是影响胃癌患者术后5年生存率的独立危险因素。以上数据显示CAMSAP2在胃癌中高表达且影响肿瘤恶性进展和预后，但其作用途径和机制并不明确。新近的研究发现CAMSAP2是一种微管负极靶向蛋白，其可通过促进肿瘤细胞的高效运动，参与肿瘤细胞迁移和侵袭等恶性行为的调控 18-20。我们采用KEGG富集分析发现CAMSAP2在胃癌中的作用可能和调控EMT进程有关。EMT是上皮细胞在形态学上趋向间质细胞表型 21,22，细胞间粘附性减弱并获得迁移侵袭能力的过程 23,24，可参与肿瘤细胞的转移 25,26。然而，CAMSAP2是否参与了胃癌细胞EMT进程尚不明确。我们首先采用慢病毒感染调控CAMSAP2在胃癌细胞系中的表达，免疫印迹检测发现上调CAMSAP2可促进胃癌细胞的EMT进程，同时划痕和Transwell实验结果证实上调CAMSAP2可促进胃癌细胞迁移和侵袭。此外，我们进一步构建裸鼠原位癌移植模型进行在体验证，结果发现上调CAMSAP2对胃癌细胞EMT进程和肿瘤转移均有促进作用。以上结果表明CAMSAP2可促进胃癌细胞EMT进程以及迁移、侵袭等恶性行为，但具体的机制尚不清楚。既往研究表明，TGF-是调控恶性肿瘤EMT的关键信号 27,28，其中TGF-/Smad信号的激活在启动EMT进程中发挥重要作用 29-31，而我们的生信富集分析发现CAMSAP2的作用可能和TGF-信号有关。为进一步验证CAMSAP2调控胃癌细胞的分子机制，我们采用免疫印迹技术于体内和体外研究中发现，上调CAMSAP2可促进TGF-/Smad2/3信号传导，而下调CAMSAP2可抑制 TGF-/Smad2/3 信号传导。以上结果提示CAMSAP2对胃癌细胞EMT进程的促进作用可能和TGF-/Smad信号的激活有关。我们研究具有如下潜在临床意义：首先，我们通过对HPA和KM-plotter等数据库以及本机构收治的胃癌患者数据进行了深入分析，证实CAMSAP2在胃癌组织中高表达且和疾病进展及患者预后相关，这一结果有望为胃癌的临床进展评估以及预后判断提供新的参考指标；此外，我们通过体内和体外研究发现CAMSAP2通过上调TGF-/Smad信号，从而参与了胃癌细胞的EMT过程，这些分子信号领域的研究有望对开发胃癌的治疗方案革新思路。我们研究仍存在以下不足：本研究的纳入患者数量有限，所得临床研究结果有待更大样本量的研究加以验正；此外，我们发现CAMSAP2可能通过激活TGF-信号促进胃癌EMT进程，但也可能忽略了CAMSAP2其他调控机制和途径。综上，本研究发现CAMSAP2在胃癌中高表达且和肿瘤进展、预后不良相关，其可能通过上调TGF-信号参与了胃癌EMT和侵袭迁移的调控。图4 KEGG富集分析结果Fig.4 Results of KEGG enrichment analysis.-1og10(p value)5432Count50100http:/www.j-J South Med Univ,2023,43(9):1460-14681465CAMSAP2-actinsi-CAMSAP2LV-CAMSAP2Control图5 CAMSAP2促进胃癌细胞的EMT、迁移和侵袭Fig.5 CAMSAP2 overexpression promotes EMT,migration and invasion of gastric cancer cells.A,B:Lentiviraltransfection effect.C,D:Expression of EMT markers in MGC803 cells.E:Wound-healing assay.F,G:Cell migrationand invasion of MGC803 cells.Control:normal control;si:siRNA;LV:overexpression.*P0.05 vs Control.N-cadherinVimentinE-cadherinControlsi-CAMSAP2LV-CAMSAP2Protein expression*6420Controlsi-CAMSAP2LV-CAMSAP2Controlsi-CAMSAP2LV-CAMSAP2Controlsi-CAMSAP2LV-CAMSAP2InvasionMigration24 h0 hsi-CAMSAP2LV-CAMSAP2ControlProtein expression43210*si-CAMSAP2LV-CAMSAP2Controlsi-CAMSAP2LV-CAMSAP2ControlMigration rate(%)6420*VimentinN-cadherinE-cadherin-actinsi-CAMSAP2LV-CAMSAP2ControlNumber of invaded cells50403020100*si-CAMSAP2ControlLV-CAMSAP2Number of migrated cells75604530150*BCEFADG50 m50 m50 m50 m50 m50 mJ South Med Univ,2023,43(9):1460-1468http:/www.j-1466Controlsi-CAMSAP2LV-CAMSAP2si-CAMSAP2LV-CAMSAP2ControlVolume(mm3)si-CAMSAP2LV-CAMSAP2Controlsi-CAMSAP2LV-CAMSAP2ControlN-cadherinE-cadherinVimentinProtein expression3210Score43210201612840*VimentinN-cadherinE-cadherin-actin图6 CAMSAP2在体内促进胃癌的进展Fig.6 CAMSAP2 overexpression promotes gastric cancer progression in nude mice.A:Representative pictures oforthotopic transplantation models.B:Comparison of tumor volume among the groups.C:Dissemination score.D,E:Expression of EMT markers in the tumor tissue.Control:normal control;si:siRNA;LV:overexpression.*P0.05 vs Control.Controlsi-CAMSAP2LV-CAMSAP2ABCDE参考文献：1 Smyth EC,Nilsson M,Grabsch HI,et al.Gastric cancer J .Lancet,2020,396(10251):635-48.2 Sung H,Ferlay J,Siegel RL,et al.Global cancer statistics 2020:GLOBOCAN estimates of incidence and mortality worldwide for 36cancersin185countries J .CAACancerJClin,2021,71(3):209-49.3 Patel TH,Cecchini M.Targeted therapies in advanced gastric cancer J .CurrTreatOptionsinOncol,2020,21(9):70.4 Yoshida K,Yamaguchi K,Okumura N,et al.Is conversion therapypossible in stage IV gastric cancer:the proposal of new biologicalcategoriesofclassification J .GastricCancer,2016,19(2):329-38.5 张 越,季 刚,陶凯雄,等.加速康复外科模式下腹腔镜与开腹胃癌根治术的临床效果:基于中国多中心数据分析 J .南方医科大学学报,2021,41(12):1828-34.6 Yu J,Huang CM,SunYH,et al.Effect of laparoscopic vs open distalgastrectomy on 3-year disease-free survival in patients with locallyadvanced gastric cancer:the CLASS-01 randomized clinical trial J .JAMA,2019,321(20):1983-92.图7 CAMSAP2可能通过TGF-信号促进胃癌EMT进程Fig.7 CAMSAP2 overexpression promotes gastric cancer EMT possibly via TGF-signaling.A,B:Expression of TGF-1,p-Smad2/3 and Smad2/3 in MGC803 cells.C,D:Expression of TGF-1,p-Smad2/3 and Smad2/3 in the tumor tissue.Control:normal control;si:siRNA;LV:overexpression.*P0.05 vs Control.ABCDTGF-1p-Smad2/3Smad2/3-actinsi-CAMSAP2LV-CAMSAP2Controlsi-CAMSAP2LV-CAMSAP2ControlTGF-1p-Smad2/3TGF-1p-Smad2/3Controlsi-CAMSAP2LV-CAMSAP2TGF-1p-Smad2/3Smad2/3-actinProtein expression3210Protein expression43210*Controlsi-CAMSAP2LV-CAMSAP2http:/www.j-J South Med Univ,2023,43(9):1460-14681467 7 Huang CM,Liu H,Hu YF,et al.Laparoscopic vs open distalgastrectomy for locally advanced gastric cancer:five-year outcomesfrom the CLASS-01 randomized clinical trial J .JAMASurg,2022,157(1):9-17.8 Xia X,Zhang ZZ,Zhu CC,et al.Neutrophil extracellular 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