CN116125070B - Trim21作为进展期结肠癌的诊断标志物及其应用 - Google Patents
Trim21作为进展期结肠癌的诊断标志物及其应用 Download PDFInfo
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- CN116125070B CN116125070B CN202211007181.1A CN202211007181A CN116125070B CN 116125070 B CN116125070 B CN 116125070B CN 202211007181 A CN202211007181 A CN 202211007181A CN 116125070 B CN116125070 B CN 116125070B
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Abstract
本发明涉及TRIM21作为进展期结肠癌的诊断标志物及其应用。本发明在小鼠转移模型中验证抗抑郁药维拉佐酮(Vilazodone)对结直肠癌肿瘤远处转移的影响。通过尾静脉注射肠癌细胞和TRIM21敲除细胞系，发现维拉佐酮(Vilazodone)给药可显著抑制结直肠癌细胞肺转移，其抑制效应以TRIM21正常表达的细胞更为显著。研究结果证明维拉佐酮(Vilazodone)为TRIM21蛋白的配体，通过和TRIM21有效结合，促进TRIM21功能和Hippo通路的上调，有效抑制肠癌细胞的肺内转移；从而为进展期和转移性结直肠癌的治疗提供新的靶点和理论依据。
Description
技术领域
本发明属于生物医药技术领域，尤其是指一种TRIM21作为进展期结肠癌的诊断标志物及其应用。
背景技术
结直肠癌以高发病率为特点，在全球范围内癌症相关死亡原因中排名第二。研究表明，非转移性结直肠癌患者的五年生存率为90％，而远处转移的患者五年生存率仅为14％；转移是导致结直肠患者预后差的最重要的原因。随着高通量测序技术发展，基因转录组技术揭示结直肠癌患者具有不同的基因表达分型，其中，以上皮-间质转化(epithelial-to-mesenchymal transition，EMT)为特征的一组结直肠癌患者，临床出现肿瘤间质浸润、患者易复发等特点，与其他亚型相比患者生存和预后更差。相较于基因组学的多样化，蛋白质组学在不同结直肠癌患者及不同的病变阶段均显示出更为明显的异质性。一项基于146名原发性结直肠癌患者多组学测序的研究显示，结直肠癌患者原发灶和转移灶间存在较大差异的蛋白质表达和激酶活性，并且这种差异性与抗肿瘤药物的疗效密切相关。因此，发现结直肠癌进展过程中重要的调控蛋白和机制，能够为肿瘤分子标记物的开发、进展期结直肠癌个体化治疗提供理论依据。
泛素化修饰是蛋白质稳定性调控的重要方式，介导蛋白酶体依赖性的蛋白质降解。其中，Tripartite motif(TRIM)-containing protein 21(TRIM21)是细胞中广泛存在的E3泛素连接酶，通过促进靶蛋白的泛素化和稳态维持，参与调控细胞重要的生理过程；包括，细胞分裂、氧化还原反应等。不同类型肿瘤中TRIM21的调控作用具备多样性，受细胞类型和疾病阶段所影响，发挥着促癌或抑癌的作用。除了上述的对于蛋白稳定性的调节，TRIM21还可通过K63介导的泛素化对靶蛋白的结合作用和亚细胞定位进行调节。在一项结直肠癌小鼠体内成瘤模型中，TRIM21的下调与肿瘤发生密切相关，提示其在结直肠癌中的潜在抑癌作用。然而，目前TRIM21的研究还局限于肿瘤发生的初始阶段，在结直肠癌进展和转移中的作用极为局限。
Hippo信号通路是沟通细胞外环境和内在信号的关键信号，在器官发育，组织再生和损伤修复的方面发挥重要的调控作用。Hippo信号通路由关键的激酶：MST1/2和LATs1/2级联调控，促使重要的癌基因YAP和旁系同源物TAZ发生磷酸化，导致其胞质滞留或发生降解，从而使得与之结合的转录因子TEAD家族的活性下降。在肿瘤发生过程中，Hippo通路的失活、YAP入核、与TEAD结合，将开启下游靶基因的转录，促进肿瘤的发生和转移。在APC突变介导的结直肠癌发生小鼠模型中，肿瘤组织较正常组织相比，YAP蛋白的表达显著升高。值得注意的是，Hippo通路中的激酶MST1/2进一步的表达缺失，以促进肿瘤细胞去分化的机制加速小鼠肠道肿瘤进展。此外，YAP和TAZ还可通过上调促转移因子整合素ITGAV而促进肿瘤细胞的侵袭和转移。由此，抑制YAP和TAZ的活性和下游通路是进展期结直肠癌患者靶向药物开发的关键。
目前，TRIM21对于肿瘤调控机制的研究集中于肿瘤的发生阶段，对于结直肠癌进展期(浸润和转移阶段)的调节机制尚未揭示。此外，TRIM21在各种细胞中维持着蛋白质稳态和介导重要的生理过程；TRIM21的单克隆抗体对其调控作用进行广泛的抑制，导致相应细胞正常生理功能的发挥受抑制，因此其单克隆抗体的应用有限。
另一方面，在针对YAP蛋白靶向药物的研究和开发中，维替泊芬(Verteporfin)虽然可以有效抑制其与共转录因子TEAD的结合，有效抑制肿瘤细胞的生长。然而，维替泊芬与其他靶向转录因子的药物类似，由于其广泛的转录阻断作用和对靶基因的影响，在实际应用时对肿瘤病人造成了明显的毒性和副作用。因此，针对YAP和TAZ上游的Hippo通路进行靶向干预，对肿瘤细胞的过度生长和转移进行有效性的抑制，将为进展期结直肠癌的治疗带来新方向。
发明内容
为解决上述技术问题，本发明提供了一种TRIM21作为进展期结肠癌的诊断标志物及其在治疗中的应用。
本发明的第一个目的在于提供一种结直肠癌的诊断标志物，所述诊断标志物为TRIM21；所述结直肠癌为进展期。
在本发明的一个实施例中，所述TRIM21的氨基酸序列如SEQ ID NO:1所示。
SEQ ID NO:1：
本发明的第二个目的在于提供一种试剂盒，包括所述的诊断标志物。
本发明的第三个目的在于提供所述的诊断标志物、所述的试剂盒在制备用于进展期直肠癌的诊断、辅助诊断或预后分析的试剂中的应用。
本发明的第四个目的在于提供所述的诊断标志物、所述的试剂盒在制备预防和/或治疗进展期结直肠癌制剂中的应用；其中，维拉佐酮为所述生物标志物的配体，可与其特异性结合，实现以下至少一种目的：
(1)增强TRIM21和Hippo通路关键激酶MST2的相互作用；
(2)增强TRIM21介导的MST2蛋白的K63泛素化；
(3)激活并上调Hippo信号通路；
(4)抑制癌基因YAP蛋白的活性；
(5)抑制结直肠癌细胞远处转移。
在本发明的一个实施例中，所述维拉佐酮的剂量为10-40mg/kg。其中，每隔至少两天腹腔注射一次，共计6周。
在本发明的一个实施例中，所述试剂还包括药学上或药理上可接受的载体、盐、酯、水合物、溶剂化物、结晶形式、对映异构体、立体异构体、醚、代谢物和前药。
在本发明的一个实施例中，所述载体选自崩解剂、稀释剂、润滑剂、粘合剂、湿润剂、矫味剂、填充剂、助悬剂、表面活性剂和防腐剂中的一种或多种。
在本发明的一个实施例中，所述盐包括无机酸盐、有机酸盐、烷基磺酸盐和芳基磺酸盐中的至少一种。
在本发明的一个实施例中，所述试剂的剂型为片剂、胶囊剂、软胶囊剂、颗粒剂、丸剂、口服液、乳剂、干混悬剂、干浸膏剂或注射剂。
本发明的上述技术方案相比现有技术具有以下优点：
本发明发现了TRIM21在结直肠癌侵袭和转移中的作用及机制。新颖的发现TRIM21可以与Hippo通路中的关键激酶MST2相结合，促进MST2形成同源二聚体，从而帮助MST2的磷酸化及Hippo级联信号的激活。具有很大转化意义的是，通过基于受体生物大分子结构的虚拟筛选技术，发现了抗重度抑郁药物维拉佐酮(Vilazodone)与TRIM21蛋白的有效结合，此作用促进TRIM21对MST2的K63泛素化而增强其二聚体的形成和Hippo通路的激活。在肠癌细胞中，维拉佐酮(Vilazodone)可有效抑制YAP蛋白入核及其下游转录活性，并很大程度抑制YAP蛋白依赖的肿瘤发生和转移机制的激活。因此，在TRIM21的表达可能作为结直肠癌患者进展和转移的分子标记物，用于术后病理的分型及预后的评估；维拉佐酮(Vilazodone)通过靶向Hippo通路有效抑制YAP蛋白的功能，对进展期结直肠癌患者的治疗具有潜在价值。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中
图1是本发明实施例TRIM21在结直肠癌中的差异性表达。(A)TRIM21在0-2期和3-4期结直肠癌患者(GSE39582)中的mRNA水平。(B)免疫组化显示TRIM21在伴和不伴有淋巴结转移患者的结肠癌原发灶表达。TRIM21染色评分差异显示于右图。
图2是本发明实施例TRIM21表达对结直肠癌患者预后的影响；其中，图中，总生存(Overall Survival)，无复发生存(Recurrence-free Survival)和疾病相关存活(Disease-specific Survival)均采用Kaplan–Meier进行统计学检验，TRIM21高表达和低表达的区分进行了最佳分组检测。
图3是本发明实施例TRIM21敲除对于小鼠结直肠癌转移的影响；其中，左图显示结直肠癌肺部转移HE染色切片；右图为DMetrix图像软件统计分析的肺部转移灶累计面积。
图4是本发明实施例TRIM21敲除对结直肠癌类器官侵袭能力的影响；左图所示典型的TRIM21感染阴性对照和TRIM21特异性shRNA后类器官形态；右图为侵袭性表型类器官所占比例的统计结果。
图5是本发明实施例TRIM21过表达对肠癌细胞肺转移的影响；其中，左图所示典型的肺转移HE染色切片；右图为累计肺部转移面积的统计结果。
图6是本发明实施例TRIM21表达对YAP亚细胞定位的影响；左图所示在RKO细胞中敲除TRIM21后YAP的免疫荧光定位；右图所示在HCT116细胞中过表达TRIM21后YAP的免疫荧光染色。
图7是本发明实施例TRIM21转基因小鼠肠癌的YAP表达；其中，Western免疫印迹检测野生型小鼠(Trim21+/+)和TRIM21敲除小(Trim21-/-)的蛋白表达。
图8是本发明实施例TRIM21敲除小鼠肠隐窝类器官的YAP定位；其中，免疫荧光检测野生型小鼠(Trim21+/+)和TRIM21敲除小(Trim21-/-)的YAP定位。通过Image J对YAP蛋白荧光强度和定位检测，F-actin显示肠上皮细胞骨架。
图9是本发明实施例TRIM21敲除小鼠的YAP靶基因表达；其中，荧光实时定量PCR显示TRIM21，YAP及下游靶基因在不同小鼠类器官中的mRNA相对表达水平。
图10是本发明实施例维拉佐酮(Vilazodone)与TRIM21蛋白的亲和力分析；其中，(A)SPR分析技术显示不同浓度维拉佐酮(Vilazodone)对TRIM21结合作用的影响。(B)Biacore T-200软件分析维拉佐酮(Vilazodone)作用的解离常数(KD)。(C)荧光滴定法检测维拉佐酮(Vilazodone)与TRIM21的结合。(D)Origin软件计算维拉佐酮(Vilazodone)作用的解离常数(KD)。
图11是本发明实施例YAP蛋白在维拉佐酮(Vilazodone)作用下的亚细胞定位；免疫荧光显示在SW620细胞中加入10μM维拉佐酮(Vilazodone)3小时后的YAP细胞定位；右图为Image J对YAP强度和定位的定量分析。
图12是本发明实施例维拉佐酮(Vilazodone)作用下的YAP转录活性分析；其中，(A)HCT116细胞在10μM维拉佐酮(Vilazodone)处理20小时后TEAD4荧光素酶报告基因检测；(B)热图显示SW480细胞在不同浓度维拉佐酮(Vilazodone)处理后相关基因的mRNA水平。
图13是本发明实施例维拉佐酮(Vilazodone)对TRIM21与MST2相互作用影响及泛素化分析；其中，(A)维拉佐酮(Vilazodone)处理RKO细胞3小时后TRIM21的免疫共沉淀结果；(B)维拉佐酮(Vilazodone)处理HCT116细胞3小时后MST2的泛素化检测；(C)维拉佐酮(Vilazodone)处理HCT116细胞MST2的二聚体结合分析；(D)维拉佐酮(Vilazodone)处理HCT116细胞Hippo信号磷酸化检测。
图14是本发明实施例维拉佐酮(Vilazodone)对肠癌细胞侵袭及迁移的影响。其中，(A)HCT116细胞在10μM维拉佐酮(Vilazodone)给药26小时后的侵袭和迁移染色图片。(B)显微镜观测计数所得侵袭和迁移的细胞数量。
图15是本发明实施例维拉佐酮(Vilazodone)给药对结直肠癌细胞肺转移影响。其中，(A)HE染色的典型小鼠肺部转移切片；(B)Dmetrix图像软件定量分析小鼠肺部转移灶的累计面积。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例本发明所涉及方法均采用本领域的常规方法，具体实验方法的步骤：
(1)原代鼠肠隐窝类器官培养
选取6-8周小鼠进行解剖，取20cm小肠沿着长轴剪开，精细分离成2mm的小块。无菌PBS清洗组织20次以清除肠道毒素。加入温和解离试剂室温孵育15分钟后，加入10mL的0.1％BSA的冷PBS将组织吹散，重力沉降后将含有肠隐窝悬液通过70μL细胞滤网。使用210xg离心转速得到肠隐窝组织并弃去上清，用DMEM/F-12培养基重悬隐窝，将重悬液滴加在细胞计数板上观察。将5000个肠隐窝用鼠类器官培养基进行重悬，并配置成50％的基质胶细胞悬液，垂直加于24孔板形成半球面，待基质胶凝固后加入培养基。培养两天后观察肠隐窝出芽，一周后加入解离试剂传代，将第二代肠隐窝类器官用于后续实验。
(2)原代人肠癌组织类器官培养
结肠癌根治术后病人组织进行新鲜取材，将直径约1.5cm的肿瘤组织进行分离，手术剪切断为直径5mm的小块。加入10mL温和细胞解离试剂，水平摇床室温孵育60分钟以解离肿瘤细胞。加入1mL冰冷的DMEM+1％BSA将组织上下吹打20次，将细胞悬液通过70μm细胞筛网并转移至15mL离心管中，继续加入DMEM+1％BSA润洗细胞筛网。滴加10μL细胞悬液于细胞计数板，显微镜下进行观察。将含有5000个肿瘤细胞团块的悬液离心，沉淀所得细胞用DMEM/F-12培养基与基质胶1：1混合。将50μL基质胶-细胞混悬液滴加于24孔板，凝固后加入人类器官培养基。人肠癌组织类器官3天后出芽，增殖后进行后续实验。
(3)免疫共沉淀(Co-IP)
将转染的结直肠癌细胞用非变性RIPA裂解液(50mM Tris-HCl PH7.4；1％NP-40；0.25％Na-deoxycholate；150mM NaCl；蛋白酶抑制剂)裂解细胞，高速离心后收集上清。从中取5％上清液加入等体积的2×SDS缓冲液做为总蛋白样品对照(Input)。剩余的95％的上清液加入5-10μL特异性一抗，于4℃摇床孵育2小时。随后加入30μL protein A/G琼脂糖微珠，4℃摇床孵育1-2小时；4℃2500rpm离心5分钟后弃去上清；用1mL冰凉的细胞裂解液洗涤沉淀至少3次，每次5分钟；于4℃2000rpm离心5分钟取沉淀。向离心管中加入适量的2×SDS缓冲液于100℃金属浴变性。随后进行SDS-PAGE凝胶电泳分离蛋白，经一抗和HRP偶联二抗孵育后，ECL系统进行显影。
(4)表面等离子共振技术(Surface plasmon resonance，SPR)
采用Biacore T200分子相互作用仪和CM5传感芯片(GE Healthcare)在室温25℃条件下，对TRIM21蛋白和维拉佐酮(Vilazodone)的动态相互作用进行检测。将纯化的TRIM21蛋白固定于CM5传感芯片，维拉佐酮(Vilazodone)以一定浓度梯度(0.78μM，1.56μM，3.125μM，6.25μM和12.5μM)于流动条件下通过芯片表面，测定其与TRIM21蛋白相互作用的信号强度。结果采用Biacore T200 evaluation software v2.0软件(GE Healthcare)进行曲线拟合并计算TRIM21蛋白和维拉佐酮(Vilazodone)结合的解离常数(KD)。
(5)AOM/DSS化学诱导小鼠发生肠肿瘤模型
苏州大学动物资源中心SPF房繁育的TRIM21野生型小鼠(TRIM21+/+)以及敲除TRIM21基因的小鼠(TRIM21-/-)，每组10-15只，取6-8周雄性，置于SPF环境下饲养。腹腔注射10mg/kg的氧化偶氮甲烷(AOM)生理盐水溶液；一周后，饮水中继续给予1.5％葡聚糖硫酸钠(DSS)，持续一周。随后间隔两周，小鼠正常条件饲养不予任何处理。同样方式循环3次。于不同的诱导阶段处死小鼠，分离出小鼠的结直肠组织，肝脏和肺组织进行观察。并且继续进行免疫组织化学和Western Blot分析，对目的蛋白的定位和表达进行检测。
(6)裸鼠体内结肠癌细胞转移模型
在SPF房和无菌条件下，收集处于对数生长期的待接种的对照组细胞(包括shRNA敲除和TRIM21过表达的稳定细胞株)，用无血清培养液洗涤，倒置显微镜下计数细胞活性>95％，调整细胞浓度为1×107/mL，将细胞重悬于PBS缓冲液中。将每只实验组和对照组BALB/c小鼠尾静脉注射0.2mL细胞悬液，观察小鼠一般状况。待注射15-60天后，颈椎脱臼处死并解剖小鼠，取小鼠双侧肺组织观察转移情况；并且进行福尔马林固定石蜡包埋，或制备冰冻切片，采用免疫荧光和免疫组织化学对表达蛋白和肠癌分子标记物检测，评估转移情况。
实验结果
(1)Trim21蛋白在进展期结直肠癌的表达检测
本发明为明确Trim21蛋白在结直肠癌进展中的表达情况，通过高通量基因表达GEO数据库对566名不同分期的结直肠癌患者Trim21的转录水平进行检测，结果发现：Trim21在进展期(3-4期)患者的表达明显低于非进展期(0-2期)患者(图1A)。进一步在收集的182例原发性结直肠癌肿瘤组织，通过免疫组织化学染色发现Trim21在伴有淋巴结转移的患者的表达明显低于不伴有淋巴结转移的患者(图1B)。这些临床数据显示，Trim21在结直肠癌进展期肿瘤组织中明显下调，与肿瘤浸润和转移具有相关性。
进一步对747例结直肠癌患者预后生存的分析中，TRIM21的低表达的结直肠癌患者的总生存，无复发生存及疾病相关存活率均较TRIM21高表达组明显下降；从而说明TRIM21蛋白表达降低与患者的预后不良相关(图2)。
(2)TRIM21在小鼠及类器官模型中的功能分析
考虑到TRIM21蛋白在进展期结直肠癌患者表达降低，为了明确其在肿瘤进展中的作用方式，本发明建立了裸鼠尾静脉注射转移模型。通过利用shRNA(序列5’-AGAAGUUGGAAGUGGAAAU-3’)将结直肠癌细胞中的TRIM21进行稳定敲除，与对照细胞同时进行尾静脉注射，TRIM21敲除的肿瘤细胞通过血液循环在肺组织形成的转移灶的累计面积明显高于对照组(图3)。
接着，将结直肠癌肿瘤切除术后取得的新鲜肿瘤组织进行分离，消化和获取原代细胞，在基质胶中培养形成肿瘤细胞类器官。通过在肿瘤细胞中进行shRNA慢性毒感染敲除TRIM21蛋白，结果显示：TRIM21的表达受抑可显著促使类器官形成浸润型表型，诱导肿瘤细胞突破基质胶向周围进行侵袭(图4)。
反之，通过在结直肠癌细胞系中建立TRIM21高表达的稳定细胞系后，在裸鼠中进行尾静脉注射建立转移模型。结果同样显示：TRIM21的上调可明显抑制肿瘤细胞在肺内形成转移灶(图5)。结合动物和肿瘤类器官模型，TRIM21敲除可很大程度促进肿瘤细胞的局部浸润和远处转移。
(3)TRIM21蛋白对癌蛋白YAP活性的影响
为了探寻TRIM21调控结直肠癌侵袭和转移的分子机制，通过蛋白质互作质谱分析筛选出可能被TRIM21调控的重要信号通路Hippo/YAP。由于癌蛋白YAP功能的发挥依赖于其细胞核-细胞质的定位。因此，通过在两种不同类型肠癌细胞系(RKO和HCT116)中改变TRIM21的表达，对YAP亚细胞定位进行免疫荧光染色分析，从而评估YAP蛋白的活性的影响。结果显示：通过shRNA抑制TRIM21的表达可促进YAP蛋白的核内募集；反之，通过质粒高表达TRIM21则可促使YAP蛋白部分出核(图6)。由此得出TRIM21下调可显著促进YAP蛋白的核内募集。
接着，本发明在小鼠模型中观测TRIM21对YAP总蛋白的影响。结果显示：TRIM21敲除的小鼠肿瘤组织中YAP蛋白的水平明显高于对照小鼠的肿瘤组织(图7)；从而在体内模型中证实TRIM21敲除可导致肠癌组织YAP蛋白的上调。
继续在小鼠肠类器官中验证TRIM21对YAP蛋白的调控作用。通过将TRIM21基因敲除小鼠和对照小鼠进行肠隐窝分离和提取，在基质胶中培养形成典型的肠类器官组织。针对类器官YAP蛋白进行免疫荧光染色及定位分析，结果显示：TRIM21稳定敲除小鼠的YAP主要定位于细胞核，而野生型小鼠的YAP定位于细胞质(图8)；从而说明TRIM21的敲除可显著促使小鼠肠类器官上皮细胞YAP蛋白入核。
接着，对YAP蛋白共转录所调控的靶基因CTGF，Amphiregulin，cIAP1和ANKRD1在类器官中的表达进行检测，结果显示：TRIM21敲除小鼠的肠隐窝类器官中YAP靶基因的表达显著高于对照组小鼠类器官，而YAP蛋白自身的转录并无明显差异。此结果提示：TRIM21的表达抑制可通过上调YAP活性，增加YAP下游靶基因的转录水平(图9)。
综上，体内外研究证明TRIM21可通过抑制YAP蛋白的细胞核内分布，抑制其共转录因子活性及下游靶基因的转录。
(4)维拉佐酮(Vilazodone)对TRIM21及Hippo/YAP信号的靶向作用分析
TRIM21对YAP活性的重要调控作用使得药物研究聚焦于靶向TRIM21蛋白上。通过对TRIM21蛋白的结构域研究发现在其“螺旋卷曲结构域”与“pry-spry结构域”之间的区域为药物最佳的靶点。通过对FDA批准的化合物进行结合能力检测和验证分析后，发现用于成人重症抑郁症治疗的药物维拉佐酮(Vilazodone)与TRIM21有直接的结合作用。通过表面等离子共振(SPR)生物分子相互作用的检测技术发现，维拉佐酮(Vilazodone)与TRIM21的结合呈现出剂量的依赖性，其解离常数(KD)为12.198μM(图10A,B)。接着，本发明采用荧光滴定法继续验证维拉佐酮(Vilazodone)与TRIM21蛋白的结合。结果显示：维拉佐酮(Vilazodone)以浓度依赖性的方式降低TRIM21的蛋白荧光(图10C)；拟合所得解离常数(KD)约为4μM(图10D)。综上，维拉佐酮(Vilazodone)作为TRIM21的配体，以浓度依赖性的方式有效地与TRIM21结合。
接着，本发明运用免疫荧光染色分析维拉佐酮对(Vilazodone)TRIM21调控作用的影响。肠癌细胞系中加入维拉佐酮(Vilazodone)后，YAP蛋白由核内分布转变为胞质分布(图11)。为了进一步明确维拉佐酮(Vilazodone)对于YAP转录活性的影响，通过荧光素酶报告基因分析，加入维拉佐酮(Vilazodone)可显著抑制YAP蛋白对TEAD4的转录调控(图12A)。并且，通过实时定量荧光PCR反应，维拉佐酮(Vilazodone)显著降低了YAP靶基因(CTGF，Survivin，Amphiregulin)及上皮-间质转化标记物N-cadherin的表达(图12B)。此研究说明维拉佐酮(Vilazodone)通过靶向TRIM21蛋白介导YAP蛋白的出核和转录活性的抑制。
为了进一步揭示维拉佐酮(Vilazodone)对Hippo/YAP信号的调控机制，通过免疫共沉淀技术在肠癌细胞内检测相关蛋白的相互作用。结果显示，维拉佐酮(Vilazodone)可显著增强TRIM21和Hippo通路关键激酶MST2的相互作用(图13A)。通过对MST2蛋白进行免疫沉淀，维拉佐酮(Vilazodone)可很大程度增强TRIM21介导的MST2蛋白的K63泛素化(图13B)。MST激酶可以通过同源二聚体的形成、发生自我磷酸化而促进Hippo通路的激活；通过加入维拉佐酮(Vilazodone)后，MST2蛋白发生同源聚合的能力明显增加(图13C)。在对Hippo信号转导进行检测时，维拉佐酮(Vilazodone)可显著上调TRIM21所介导的Hippo信号的激活，包括MST1和LATS1的磷酸化(图13D)。综上，细胞内分子机制研究揭示维拉佐酮(Vilazodone)通过与TRIM21结合增强其与MST2的相互作用和K63泛素化，进一步促进MST2激酶形成二聚体，从而激活Hippo级联激酶的磷酸化。
为了分析维拉佐酮(Vilazodone)对肿瘤生物学功能的影响，本发明通过细胞迁移和侵袭试验检测其效应。结果显示：维拉佐酮(Vilazodone)可有效抑制肠癌细胞的迁移及侵袭能力；而TRIM21的敲除可有效削弱维拉佐酮(Vilazodone)对肠癌细胞迁移及侵袭力的影响(图14)。
进一步在小鼠转移模型中验证维拉佐酮(Vilazodone)对肿瘤进展的影响。通过尾静脉注射肠癌细胞和TRIM21敲除细胞系，维拉佐酮(Vilazodone)给药可显著抑制结直肠癌细胞的肺转移，其抑制效应以TRIM21正常表达的细胞更为显著(图15)。研究结果证明维拉佐酮(Vilazodone)通过靶向TRIM21和上调Hippo通路，有效抑制肠癌细胞的肺内转移；从而为进展期结直肠癌的治疗提供新的靶点和理论依据。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (1)
1.检测结直肠癌肿瘤组织中TRIM21蛋白表达水平的免疫组织化学检测试剂在制备用于辅助诊断结直肠癌淋巴结转移的试剂中的应用，所述TRIM21的氨基酸序列如SEQ ID NO:1所示；所述TRIM21蛋白在伴有淋巴结转移的患者中的表达水平明显低于不伴有淋巴结转移的患者。
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