【作者】 卢瑗瑗；

【导师】 樊代明； 王新；

【作者基本信息】 第四军医大学 ， 内科学， 2011， 博士

【摘要】 【背景】结直肠癌是世界范围内发病率和死亡率均高的恶性肿瘤，在我国位于消化系统肿瘤发病率的第三位。传统的肿瘤标志物CEA、CA199等敏感性较差，在早期患者中阳性率低，不利于结直肠癌的早期筛查。近年来随着基因组和蛋白质组研究技术的发展，一些有价值的分子标志物被陆续鉴定，但经大样本量临床验证并成功用于临床实践者仍然较少。因此，寻找和鉴定新的分子标志物并经临床研究得到验证和转化，将有助于结直肠癌的早期诊断、预后判断和治疗监测，对结直肠癌的诊疗意义重大。本实验室樊代明院士等在上世纪80年代采用结肠癌转移淋巴结分离的癌细胞匀浆免疫BALB/c小鼠，利用淋巴细胞杂交瘤技术制备了一组抗结直肠癌单克隆抗体，命名为MC(colorectal monoclonal antibody)系列。其中，MC3Ab识别的抗原MC3-Ag在结直肠癌组织中的表达阳性率达90%以上，且其表达与TNM分期、有否转移密切相关，是结直肠癌灵敏度高、特异性强的候选生物标志物。本实验室前期对MC3-Ag在结直肠癌中的表达、临床诊断中的价值和作为靶标分子在治疗中的应用等方面进行了有意义的研究和探索，发表了多篇有影响的国际国内论文。但是MC3-Ag作为结直肠癌相关新抗原，一直未能得到鉴定，其编码基因尚不清楚。本研究应用蛋白质组学研究方法和技术成功分离和鉴定MC3-Ag，随后研究了其在结直肠癌中的表达和作为预后判断标志物的价值。功能学实验研究了MC3-Ag对于结直肠癌多种恶性生物学行为如无限增殖、凋亡抵抗和侵袭转移的影响，并对MC3-Ag的相互作用蛋白、下游分子事件和信号通路进行深入研究。【目的】1、分离和鉴定结直肠癌单克隆抗体MC3Ab所识别的抗原MC3-Ag2、明确MC3-Ag/Txl-2对于结直肠癌多种恶性生物学行为的调控3、研究MC3-Ag/Txl-2不同选择性剪接体在结直肠癌中的功能差异4、探讨MC3-Ag/Txl-2调控结直肠癌恶性生物学行为的分子机制【方法】1.采用蛋白质组学技术包括免疫沉淀、双向凝胶电泳(two-dimensional gelelectrophoresis,2-DE)和基质辅助激光解吸电离飞行时间质谱(matrix-assistedlaser desorption/ionization time of flight mass spectrometry, MALDI-TOF-MS)分离和鉴定MC3-Ag，配对免疫组织化学、免疫细胞化学、免疫荧光/激光共聚焦和Western blot验证鉴定结果；组织芯片检测了MC3-Ag在正常组织和多种肿瘤组织中的表达谱。2.免疫组织化学研究MC3-Ag/Txl-2在243例结肠癌组织中的表达及其与临床病理参数间的关系，并获取随访资料进行单因素和多因素生存分析。构建Txl-2siRNA载体，稳定转染入高侵袭性结肠癌细胞SW620中并筛选获得Txl-2表达下调的细胞株。MTT实验、平板集落形成实验和流式细胞术检测细胞周期观察Txl-2对结肠癌细胞增殖能力的影响；Annexin V/PI凋亡染色研究Txl-2对细胞凋亡的影响；损伤刮擦实验、黏附实验、Transwell侵袭实验和软琼脂集落形成实验研究Txl-2对于结肠癌细胞迁移、黏附、侵袭和转移能力的影响。体内裸鼠成瘤和尾静脉转移实验验证体外实验结果。3.提取结肠癌组织、癌旁组织及正常组织mRNA,半定量RT-PCR检测Txl-2各选择性剪接体mRNA和总mRNA的表达情况。构建Txl-2各选择性剪接体Txl-2a、b和c的正义表达载体，稳定转染入无内源性Txl-2表达的结肠癌细胞系LoVo中，体外和体内功能实验分别观察Txl-2各亚型蛋白对无限增殖、凋亡抵抗和侵袭转移等恶性生物学行为的影响，研究它们的功能差异；对Txl-2b结构域关键催化位点进行位点特异性突变，研究介导恶性表型的关键结构域和特异性位点。4.利用酵母双杂交系统，通过AD载体和BD载体的共转染明确Txl-2各选择性剪接体与小G蛋白家族成员Ran的相互作用情况;免疫共沉淀实验进一步验证；间接免疫荧光/激光共聚焦观察Txl-2与Ran在结肠癌细胞和组织中的共定位情况。应用siRNAOligos、抗体和相关信号通路抑制剂等干预，Western blot检测相关分子和信号通路的改变。【结果】1．蛋白质组学研究鉴定并验证MC3Ab识别的抗原MC3-Ag是Txl-2蛋白应用结肠癌细胞裂解液和组织匀浆进行免疫沉淀，富集MC3-Ag，发现MC3Ab抗体识别30kDa左右的2个条带。免疫沉淀产物经2-DE后分别进行银染和Western blot鉴定，发现MC3抗体恒定识别分子量32kDa和28kDa、等电点pI~5的两个蛋白质点。将两个蛋白质点进行胶内酶解和MALDI-TOF-MS分析，得到相应的肽质量指纹图谱(peptide massfingerprinting, PMF)，经ProFound搜索引擎生物信息学分析，确认2个点是Thioredoxin like-2(Txl-2)蛋白的2个选择性剪接体(Swiss-Prot: Q86XW9-2和Q86XW9-3)。应用MC3Ab和anti-Txl-2Ab进行比对验证，配对免疫组织化学、免疫细胞化学发现两者染色模式一致；免疫荧光/激光共聚焦证实两个抗体染色的共定位；Western blot发现两个抗体能够在免疫沉淀产物和细胞裂解液中识别同样的条带,从而进一步证实MC3Ab识别的抗原MC3-Ag为Txl-2蛋白。组织芯片明确了MC3-Ag/Txl-2在多种肿瘤组织和正常组织中的表达谱。2.下调Txl-2的表达能够显著抑制结肠癌细胞的无限增殖、凋亡抵抗和侵袭转移等恶性表型MC3-Ag/Txl-2在243例结直肠癌组织中的免疫组化研究发现，MC3-Ag/Txl-2的表达与肿瘤分化程度呈显著负相关，随着TNM分期增高而表达水平逐渐增高，在转移性结直肠癌组织中高表达。单因素和多因素生存分析表明MC3-Ag/Txl-2是结直肠癌患者预后判断的独立指标，其表达量越高则预后越差。成功构建Txl-2siRNA载体，稳定转染入SW620细胞，获得表达抑制50%和70%以上的细胞株。功能学研究发现，下调Txl-2的表达能够显著抑制结肠癌细胞生长，平板集落形成能力减弱，细胞周期G0/G1期阻滞；抑制Txl-2的表达能够显著增加去血清或药物诱导的细胞凋亡比例；在高侵袭性细胞SW620中下调Txl-2的表达能够显著抑制结肠癌细胞的黏附、迁移和侵袭能力。体内裸鼠成瘤实验和尾静脉转移实验进一步验证了体外实验结果。3. Txl-2的3个选择性剪接体差异调控结肠癌细胞恶性生物学行为在18例结肠癌组织、癌旁组织和正常组织中检测了Txl-2总mRNA和各选择性剪接体mRNA的表达情况，发现Txl-2总mRNA的表达水平在肿瘤组织中表达增高；在3个选择性剪接体中，Txl-2b和Txl-2c的mRNA在肿瘤中表达增高，而Txl-2a表达率较低（2/18例）。成功构建Txl-2各选择性剪接体的特异性正义表达载体，稳定转染入LoVo细胞中，获得稳定表达的细胞株。体内和体外功能实验表明，Txl-2的3种选择性剪接体差异调控结肠癌细胞恶性生物学行为：Txl-2b是发挥主要促癌作用的亚型，Txl-2a更多还原

【Abstract】 BackgroundColorectal cancer (CRC) is a leading cause of cancer related morbidity andmortality worldwide. Traditional CRC biomarkers such as carcinoembryonicantigen (CEA) and CA199are not suitable for screening and early diagnosis dueto low sensitivities. Genomic and proteomic researches have been advancingrapidly in recent years, facilitating the discovery of a range of promising CRCbiomarkers. However, few has been further validated and applied to the clinicalpractice after translational study. Hence, identification of novel biomarkers forCRC and translation the study data into patient care may open the door to a moreaccurate and target specific personalized medicine with improved patientsurvival.Using a homogenate preparation of colon cancer tissues from metastaticlymph nodes, Fan et al. developed a series of CRC-specific monoclonalantibodies through hybridoma technique in1980s. Among those antibodies, MC3is a specific and sensitive antibody that reacts with an unknown antigenpresent in up to90%cancer tissues. MC3-Ag is an ideal candidate biomarker forCRC for its expression is correlated to TNM (tumor-node-metastasis) stagingand its overexpression is observed in metastatic CRC. Tremendous works hasbeen done in our laboratory concerning MC3-Ag on its expression profile inCRC, its diagnostic and predictive value as a novel biomarker and also thetargeted therapy, with a lot of papers published. However, MC3has not yetgained recognition and acceptance, primarily because the target antigen of theMC3antibody had never been identified.In the present study, proteomics approaches were applied to successfullyisolate and identify the MC3Ab immunoreactive protein MC3-Ag. Thefollowing study went on to validate its diagnostic and prognostic value.Functional studies revealed its specific regulation of multiple malignantbehaviors of CRC including proliferation, anti-apoptosis and metastasis. Finally,the interaction protein with MC3-Ag was identified and the downstreammolecular events and pathways were further explored.Objectives1. To isolate and identify MC3-Ag specific to CRC2. To study the impact of MC3-Ag/Txl-2on multiple malignant behaviors ofCRC3. To examine the differential functions of MC3-Ag/Txl-2alternative splicedvariants in CRC4. To explore the underlying mechanisms mediated the function ofMC3-Ag/Txl-2in CRC Methods1Proteomics approaches including immunoprecipitation, two-dimensional gelelectrophoresis (2-DE) and matrix-assisted laser desorption/ionization time offlight mass spectrometry (MALDI-TOF-MS) were applied to isolate and identifyMC3-Ag. Paired immunohistochemical study, immunofluorescence andconfocal microscopy and Western blot analysis were done for further validation.Tissue arrays were applied to examine the MC3-Ag expression profiles innormal tissues and multiple tumor tissues.2Immunohistochemical study was done in243cases of clinical samples tostudy the association of MC3-Ag/Txl-2expression with clinicopathologicalparameters and further validate its prognostic value. Txl-2siRNA vector wasconstructed and stably transfected into the highly invasive colon cancer cell lineSW620. MTT assay, colony formation assay, cell cycle analysis by flowcytometry (FCM) were done to examine the impact of Txl-2on cell proliferation;Annexin V/PI double staining and FCM were done to examine the serumdeprivation-induced and chemodrug-induced apoptosis. Wound healing assay,adhesion assay, Transwell invasion assay and soft agar assay were done toexamine the function of Txl-2on tumor cell invasive and metastatic potential.Further in vivo assays including nude mice tumorigenesis assay and tail veinassay of metastasis were carried out to verify the results from in vitro studies.3RT-PCR was done to examine the mRNA of three Txl-2alternative splicedvariants and total mRNA level from18cases of colon cancer tissues, adjacenttissues and normal tissues. Isoform-specific cDNA expression vectors wereconstructed and stably transfected into colon cancer cells. The differentialfunctions of each individual Txl-2isoform in CRC were investigated.Site-directed mutation of catalytic active sites was manipulated to determine the functional domain and critical sequences.4Yeast two-hybrid system and coimmunoprecipitation were utilized toidentify the Txl-2interaction protein. Immunofluorescence and confocalmicroscopy were done to observe the colocalization of Txl-2and Ran.Interventions by siRNA oligos, antibody and pathway inhibitors were done toinvestigate the therapeutic effect and determine the downstream events andspecific signal pathway.Results1. MC3-Ag was identified as Thioredoxin like-2(Txl-2) by proteomic studyImmunoprecipitation of MC3-Ag using SW480cell lysates or fresh CRCtissue homogenates showed that MC3Ab consistently identified two proteinbands around30kDa. Then the immunoprecipitates were subjected to2-DEfollowed by silver staining or Western blot respectively. The results showed thatMC3antibody consistently detected two immunoreactive spots at the basic pHrange near pH5with Mr of32and28kDa, respectively. Then the two spotswere subjected to in-gel trypsin digestion and MALDI-TOF-MS analysis. ThePMFs were subjected to database searches by ProFound and the two MC3immunoreactive spots were identified as two isoforms of Txl-2(Swiss-Prot:Q86XW9-2and Q86XW9-3). The following paired immunohistochemical study,immunofluorescence and confocal microscopy and Western blot analysis usingboth MC3Ab and anti Txl-2Ab further validated the that Txl-2is the antigenfor MC3Ab. Additionally, expression profiles of MC3-Ag in normal tissues andmultiple tumor tissues were examined by tissue arrays.2. Knockdown of Txl-2significantly suppressed colon cell proliferation,invasion and metastasis, and sensitized tumor cells to apoptosis. Immunohistochemical study in243cases of CRC tissues revealed thatMC3-Ag/Txl-2expression was correlated with TNM staging and its expressionwas significantly upregulated in metastatic CRC. The following survivalanalysis indicated that MC3-Ag/Txl-2could serve as an independent prognosisbiomarker for CRC patients. Txl-2expression was significantly suppressed bystable transfection of siRNA vectors in highly invasive SW620cells.Knockdown of Txl-2expression significantly suppressed cell proliferation withreduced ability of colony formation and cell cycle G0/G1arrest. Inhibition ofTxl-2expression significantly increased the cell apoptotic ratio induced byserum-deprivation or chemodrugs. Knockdown of Txl-2also significantlyinhibited cell migration, adhesion, invasion and anchorage-independent growth.In vivo assay including nude mice tumor formation assay and tail veinmetastasis assay further validated the in vitro results.3. Three isoforms of Txl-2exerted divergent impacts on multiple malignantbehaviors of colon cancer cells.mRNA level of each Txl-2isoform and total Txl-2mRNA level wereexamined in18cases of clinical samples. Total Txl-2mRNA was elevated incolon cancer tissues compared to adjacent tissues and normal mucosa. Among3isoforms, mRNA of Txl-2b and Txl-2c were significantly upregulated in coloncancer tissues while Txl-2a was rarely expressed in all samples (2/18cases).EGFP recombinant expression vectors of each Txl-2isoform were constructedand stably transfected into LoVo cells. Both in vitro and in vivo studiesindicated that3isoforms of Txl-2exerted divergent impacts on multiplemalignant behaviors of colon cancer cells. Txl-2b contributes to thephysiopathology of colon cancer progression and tumorigenesis with its role onfavoring different aspects of colon cancer progression; Txl-2a overexpression produced a less significant modulation of function whereas Txl-2c exhibited arelatively inhibitive effect. Site-directed mutation in catalytic active sitesrevealed that Trx domain is critical for Txl-2mediated invasion and metastasis.4. Txl-2b interacted with the small GTPase Ran and promoted tumor cellmetastasis through PI3K pathwayYeast two-hybrid system verification and co-immunoprecipitation wereutilized to identify the small GTPase family member Ran as a Txl-2b interactingprotein, but not for Txl-2a and Txl-2c. Immunofluorescence and confocalmicroscopy revealed that Txl-2was colocalized with Ran. Transfection ofsiRNA-Ran oligos could partially reverse the Txl-2b mediated tumor metastasis.Further study revealed that PI3K activation participated in Txl-2-Ran-MMPmediated tumor cell invasion and metastasis. Besides, Txl-2was also found tointeract with microtubules and influence colon cancer cell metastasis throughcell shape remodeling.【Conclusion】In the present study, we successfully identified Txl-2as the antigen of themonoclonal antibody MC3associated with CRC. Txl-2exerts multiple impactson tumor cell malignant phenotypes with isoform-specific modulation. Txl-2bdirectly interacts with Ran and PI3K pathway participates in Txl-2b-Ran-MMPmediated tumor invasion and metastasis. The current study provides a novelbiomarker and target molecule for diagnosis and treatment of CRC and opensthe door to a novel understanding of cancer specific alternative splicing and itsfunctioning mechanisms.更多还原