【作者】 郑平；

【导师】 李建明；

【作者基本信息】 南方医科大学 ， 病理学， 2010， 硕士

【摘要】 结直肠癌是常见的恶性肿瘤之一,转移是导致患者死亡的最重要的原因,也是结直肠癌术后复发的直接原因。结直肠癌的演进是多因素,多水平,多通路的复杂过程。因此,筛选其发生、转移相关基因并阐明其相关分子机制,将对临床结直肠癌的诊断、预防、治疗具有重要意义。促肝细胞再生磷酸酶-3(phosphatase of regenerating liver-3, PRL-3),属于蛋白质酪氨酸磷酸酶(protein tyrosine phosphatase)家族(PRL-1、PRL-2、PRL-3)成员,其蛋白的相对分子量约为19KD,它通过调节酪氨酸残基的磷酸化状态实现对蛋白质活性的调控。目前证实PRL-3与人结直肠癌的发生、进展、转移及预后密切相关。PRL-3在正常结直肠上皮中几乎不表达,在原发性结直肠癌肿瘤组织中不表达或低表达,而在结直肠癌转移灶(尤其是肝脏)中高表达。PRL-3的上调与肿瘤的转移密切相关。PRL-3是细胞粘附和上皮间叶转化的重要的调节因子,通过增强整合素相关Src信号及PI-3K/AKT信号通路促进EMT (epithelial msenchymal transition)的发生,调节RhoGTP酶家族信号通路、细胞周期等。我们的前期课题实验结果发现PRL-3可直接调节钙粘附蛋白(Cadherin)促进EMT发生。除此之外,PRL-3也是重要的细胞周期调节因子。但其相互作用的底物仍然不太清楚。Integrin a 1是最先报道的与PRL-3相互作用蛋白,我们课题组通过酵母双杂交技术及免疫共沉淀方法筛选并证实PRL-3另一相互作用蛋白CDH22；国外通过蛋白组学方法成功筛选出PRL-3部分作用底物及相互作用蛋白——ezrin、cytokeratin 8、the elongation factor 2 (EF2)。为了进一步了解PRL-3在结直肠癌发生、转移中的功能和作用,我们运用蛋白组学策略检测PRL-3瞬时敲低24小时、48小时及稳定敲低结直肠癌细胞后蛋白水平的改变,运用基质辅助激光解析电离飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer,MALDI-TOF MS)技术鉴定出39个差异表达蛋白；其中我们发现致癌基因Stathmin随着PRL-3的敲低而表达下调。Stathmin是一种小分子磷蛋白,分子量约17KD,为微管不稳定蛋白,与肿瘤的浸润、转移密切相关,在本研究中我们将通过免疫共沉淀、免疫荧光、构建干扰及过表达载体后生物学功能等方面对两者之间的相互作用作进一步研究。生长状态良好的人结直肠癌细胞株SW480、瞬时敲低PRL-3 24小时及48小时的SW480细胞、稳定敲低PRL-3的SW480细胞克隆共四组,分别获取细胞全蛋白后,行双向凝胶电泳技术筛选PRL-3相关的差异蛋白点,通过MALDI-TOF MS分析鉴定相关蛋白。随机获取149例新鲜人结直肠癌组织石蜡包埋后4μm切片,免疫组织化学染色检测Stathmin的表达,Western Blot分析不同人结直肠癌细胞株中Stathmin的表达,Western Blot分析人结直肠癌组织与其配对正常粘膜组织中Stathmin的表达。设计人Stathmin特异性引物,克隆至真核绿色荧光蛋白表达载体pEGFP-C1,应用PCR、酶切及DNA测序进行鉴定,确认后转染结直肠癌细胞SW480及HT29,应用Western Blot检测Stathmin蛋白的表达；设计人Stathmin基因特异性RNAi靶序列,克隆至pGPU6/GFP/Neo siRNA表达载体,应用PCR及DNA测序进行鉴定,确认后转染入结直肠癌细胞SW620及Lovo,应用Western Blot检测不同干扰片段对Stathmin蛋白表达的影响。利用MTT法、平板克隆形成实验检测Stathmin过表达对结直肠癌细胞SW480、HT29体外增殖能力的影响及Stathmin敲低后对结直肠癌细胞SW620及Lovo体外增殖的影响；应用运动小室实验检测Stathmin过表达对结直肠癌细胞SW480、HT29运动和迁移能力的影响及Stathmin敲低后对结直肠癌细胞SW620及Lovo迁移能力的影响；应用细胞粘附实验测定Stathmin过表达对结直肠癌细胞SW480、HT29粘附能力的影响及Stathmin敲低对结直肠癌细胞SW620、Lovo粘附能力的影响。生长状态良好的人结直肠癌细胞株SW620、Lovo、SW480、HT29免疫荧光标记后共聚焦分析PRL-3与Stathmin在结直肠癌细胞内的共定位；收集人结直肠癌细胞及结直肠癌组织蛋白后行免疫共沉淀法进一步鉴定PRL-3与Stathmin在结直肠癌细胞及组织中的相互作用。免疫荧光及Western Blot观察PRL-3与Stathmin过表达对结直肠癌细胞SW480、HT29中α-tubulin及乙酰化α-tubulin的分布和表达的影响,敲低PRL-3与Stathmin对结直肠癌细胞SW620、Lovoα-tubulin及乙酰化α-tubulin的分布和表达的影响。在四个比较组中,通过二维聚丙烯酰胺凝胶电泳(Two—Dimensional Polyacrylamide Gel Electrophoresis,2-DE)结合MALDI-TOF MS技术成功鉴定了39个有差异的蛋白点,其中21个蛋白点在瞬时转染24小时、48小时及稳定敲低的SW480细胞株中均表达下调；9个蛋白点在瞬时转染24小时、48小时及稳定敲低的SW480细胞株中均表达上调；3个蛋白点只在稳定敲低的SW480细胞株中表达下调；6个蛋白点只在稳定敲低的SW480细胞株中表达上调。有意义的是,癌基因Stathmin/OP18随着PRL-3的表达下调而下调。免疫组织化学染色结果显示Stathmin定位于结直肠癌细胞胞浆；正常结直肠组织Stathmin不表达或弱表达,结直肠癌组织中Stathmin的表达明显高于正常结直肠组织；在淋巴结与肝脏转移灶中Stathmin强表达。Stathmin的表达与性别、年龄、肿瘤的大小及范围差异无统计学意义(P>0.05)；但是,Stathmin的表达与结直肠癌患者肿瘤的分化(χ2=6.656,P=0.010)、浸润(χ2=7.762,P=0.05)、淋巴结转移(χ2=33.888,P=0.000)、Dukes分级(χ2=37.480,P=0.000)、TNM分期(χ2=42.155,P=0.000)之间差异有统计学意义。Kaplan-Meier分析方法及Log-rank test发现Stathmin的表达与结直肠癌患者的生存时间存在显著相关性(χ2=53.205,P=0.000)；采用多变量COX回归分析患者生存时间与肿瘤浸润、分化、Duke’s分级、TNM分期、Stathmin表达的关系。结果显示Stathmin表达可作为结直肠癌患者预后的一个潜在的独立预测因子(χ2=48.012,P=0.000)。在随机挑选的4对结直肠癌组织及其配对正常粘膜组织中Western Blot结果显示Stathmin表达在原发结直肠癌组织中明显高于其配对正常粘膜组织；在发生转移的原发结直肠癌组织中Stathmin的表达明显高于未发生转移的原发结直肠癌组织。在不同的结直肠癌细胞株中,在转移能力较强的细胞株SW620、Lovo、HCT116中Stathmin的表达较强；而在低转移潜能的细胞株SW480、HT29、LS174T中Stathmin的表达较弱。MTT法检测结果表明结直肠癌细胞SW480(F=22.236,P=0.000)、HT29(F=93.138,P=0.000)Stathmin过表达后细胞体外生长增殖能力较对照组相比,其生长能力明显增强,差异具有统计学意义。Stathmin瞬时敲低对Lovo细胞(F=19.599,P=0.000)、SW620细胞(F=16.791,P=0.000)体外增殖能力影响与对照组相比,前者细胞的增殖能力明显大于后者,且差异具有统计学意义。平板克隆形成实验显示Stathmin过表达后SW480细胞(F=113.958,P=0.000)、HT29细胞(F=33.477,P=0.000)细胞克隆形成能力明显增强,Stathmin瞬时敲低后Lovo细胞(F=31.172,P=0.001)、SW620细胞(F=30.258,P=0.001)细胞克隆形成能力显著下降,差异具有显著的统计学意义。粘附实验结果表明,Stathmin过表达后SW480细胞(F=24.567,P=0.000)、HT29细胞(F=90.259,P=0.000)粘附能力明显增强,Stathmin瞬时敲低后Lovo细胞(F=11.088,P=0.000)、SW620细胞(F=12.387,P=0.000)粘附能力显著下降,差异具有显著的统计学意义。Transwell小室表明,Stathmin过表达后SW480细胞(F=36.154,P=0.000)、HT29细胞(F=6.568,P=0.033)迁移能力明显增强,Stathmin瞬时敲低后Lovo细胞(F=38.866,P=0.000)、SW620细胞(F=52.068,P=0.000)迁移能力显著下降,差异具有显著的统计学意义。免疫共沉淀分析结果表明,在不同结直肠细胞株中和不同结直肠癌组织中PRL-3与Stathmin存在直接相互作用；在不同结直肠癌细胞株SW480、SW620、LOVO、HT29细胞中PRL-3与Stathmin共定位分析也支持二者存在相互作用。微管是细胞骨架的主要构成成分之一,由α-tubulin及β-tubulin组成微管蛋白二聚体。体内细胞可通过改变微管蛋白的表达,转录后修饰等方式来调节微管的动力学平衡。我们知道Stathmin的主要功能是调节微管的稳定性,因此我们通过观察tubulin及乙酰化tubulin来观察微管的改变。结果表明在不同人结直肠癌细胞株的对照组中,α-tubulin弥散分布于整个细胞,然而,在人结直肠癌细胞株SW480、HT29中,上调PRL-3及Stathmin的表达,α-tubulin的分布呈现聚集倾向；在人结直肠癌细胞株SW620、LOVO中,下调PRL-3及Stathmin的表达,α-tubulin的分布无显著差异性改变。在细胞株SW480、HT29中Stathmin过表达后,acetylated-α-tubulin的表达下调；在细胞株SW620、Lovo中Stathmin表达敲低后,acetylated-α-tubulin的表达上调。上调或下调PRL-3的表达结果与Stathmin一致。Western Blot检测不同人结直肠癌细胞株转染PRL-3与Stathmin过表达或干扰载体后α-tubulin和乙酰化α-tubulin的蛋白表达,发现结直肠癌细胞中PRL-3与Stathmin过表达或干扰后其α-tubulin表达无明显变化,而PRL-3与Stathmin过表达的结直肠癌细胞SW480、HT29乙酰化α-tubulin的表达较对照组相比明显降低,PRL-3与Stathmin敲低的结直肠癌细胞SW620、Lovo乙酰化α-tubulin的表达较对照组相比明显增高,与免疫荧光观察结果一致。1.应用蛋白组学策略成功筛选出39个PRL-3调节相关蛋白,其中Stathmin/OP18蛋白随着PRL-3表达的下调而下调；2. Stathmin/OP18在结直肠癌组织及淋巴结和转移灶组织中的表达明显高于正常结直肠粘膜组织；发生转移的结直肠癌组织中stathmin的表达明显高于未发生转移的结直肠癌组织,可作为结直肠癌患者预后的一个潜在的独立预测因子；3. Stathmin/OP18促进结直肠癌细胞的增殖、迁移、粘附；4.在结直肠癌细胞中Stathmin是PRL-3相互作用蛋白,二者共同作用促进微管解聚促进结直肠癌进展和转移。更多还原

【Abstract】 Colorectal cancer (CRC) is a common malignant tumor in the world. The incidence and death rate of CRC in China is increasing year by year during the past decade. Metastasis is the main cause affecting the therapeutic efficacy and leading to the death of patients with CRC. However,the mechanims of metastasis of CRC is still not well-known. To find genes associated with metastasis and elucidate their functions in CRC will be helpful for clinical diagnosis, prognosis analysis and possible target treatment of CRC.PRL-3 (Phosphatase of Regenerating Liver-3), a key gene associated with progression and metastasis of CRC, belongs to a protein tyrosine phosphatase family with only there members like PRL-1, PRL-2 and PRL-3. PRL-3 is a small molecule about 19KD. Many evidences have been reported in support of the role of PRL-3 in progression, metastasis and prognosis of CRC. PRL-3 is overexpressed in many kinds of cancer, such as ovarian cancers, gastric cancers, human gliomas, non-small cell lung cancer, and nasopharyngeal carcinoma. However, the role of PRL-3 in tumor progression and metastasis is still not very clear.PRL-3 was suggested to be involved in several kinds of signal pathways. PRL-3 regulates integrin/Src signaling pathway, Rho family GTPases, Ang-II signaling, Cell cycle et al. Enhanced Src signaling and PI-3K/Akt signaling contribute to epithelial msenchymal transition (EMT). Recently, we find that PRL-3 promotes EMT by regulating cadherin directly. PRL-3 is also an important cell cycle regulator and a direct p53 target gene. PRL-3 regulates invasion and metastasis by directly affecting the cytoskeleton and also through the transcriptional regulation of target genes. But, the proteins regulated by PRL-3 are still largely unknown.Several strategies can be used to find out the novel targets of PRL-3. The first one is to screen for PRL-3 interacting proteins. Integrin a 1 was first reported as a new interacting protein of PRL-3. Using this strategy, we identified CDH22 as a new PRL-3 interacting protein. Yeast two hybrid system and co-immunoprecipitation assay are two basic methods to find PRL-3 interacting proteins. The second strategy is based on proteomic methods to find out a great number of proteins regulated by PRL-3. Using this strategy, ezrin, cytokeratin 8 and the elongation factor 2 (EF2) are also found as substrates or interacting proteins of PRL-3.In order to further understand PRL-3 functions and its role in progression or metastasis of CRC, we performed proteomic analysis to detect changes in the protein levels in two kinds of cell models with PRL-3 transient or stable knockdown. We identified 39 differential spots regulated by PRL-3 using MALDI-TOF MS technique. We found that Stathmin, a key oncoprotein, was regulated by PRL-3Stathmin is a small molecule phosphoprotein about 17KD. It belongs to an unstable microtubule protein, and is associated with tumor invasion and metastasis. In this paper, we study the role of Stathmin in CRC, and we found a new link between PRL-3 and Stathmin, which contributed to progression and metastasis of CRC.Four groups were included in the following experiments:human colorectal cancer cell line SW480 as a control, SW480 cell line with PRL-3 transient knockdown for 24 hours or 48hours, SW480 cell line with PRL-3 stable knockdown. The cell pellets were lysed in sample buffer (7M urea,2M thiourea,0.2%(w/v) Bio-Lyte, pH 3-10,65mM DTT,4%(w/v) CHAPS) by sonication on ice. Lysates were centrifuged at 14 OOOxg for 1h. The supernatants used for 2-DE were stored at-80℃. Protein concentration was determined by Bradford method. Differentially expressed proteins were identified using 2D gel electrophoresis and mass spectrometry.2D gel electrophoresis was performed using IPG strip (17cm, pH 3-10, NL). Proteins were separated according to charge, and molecular weight. The gels were then underwent silver staining to visualize proteins. Images were obtained by scanning gels with scanner and analyzed using the PDQuest software v7.1.1. Differentially expressed spots were excised for in-gel digestion. Peptide mass mapping was performed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF MS).Fresh or paraffin-embedded tissue samples from CRC patients at Nanfang Hospital in our university were used in the study. Clinicopathological classification and staging of these samples were performed according to the General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum, and Anus along with the International Union Against Cancer classification. Immunohistochemical staining of protein expression of Stathmin in 149 human tissue samples of CRC was done using a Dako EnVision System. The sections were incubated overnight with the rabbit monoclonal anti-stathmin at a dilution of 1:500. The immunohistochemically stained tissue sections were analyzed separately by two pathologists without knowing the patients’ clinical characteristics. Staining for Stathmin was assessed using a relatively simple, reproducible scoring method. The staining intensity was scored on a scale of 0 to 3 as negative (0), weak (1), medium (2), or strong (3). The extent of the staining, defined as the percentage of positive staining areas of tumor cells in relation to the whole tumor area, was scored on a scale of 0 to 4:0(0%),1(1-25%),2(26-50%), 3(51-75%) and 4 (76-100%). An overall protein expression score (overall score range, 0-12) was calculated by multiplying the intensity and positivity scores.Different human colorectal cancer cell lines or colorectal tumor tissues were homogenized in RIPA lysis buffer (50mM Tris containing 150mM NaCl,0.1% SDS, 1% Triton X-100,1% sodium deoxycholate, pH 7.2) with 0.2% protease and phosphatase inhibitor cocktail on ice for 15 min and centrifuged at 14,500×g for 30 min. The protein concentration of the supernatants was determined by BCA Assay, and aliquots of the protein samples were stored at-80℃. Equal amounts of proteins were separated electrophoretically on 13% SDS/polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (PVDF). The membrane was incubated at 4℃overnight with an anti-PRL-3 rabbit polyclonal antibody (1:100) or an anti-stathmin rabbit monoclonal antibody (1:20,0000).Expression of PRL-3 or Stathmin was determined with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (1:5000) and enhanced chemiluminescence. An anti-P-Actin goat monoclonal antibody (1:1000) was used to confirm equal loading.The human Stathmin-specific primers was cloned into the eukaryotic green fluorescent protein expression vector pEGFP-C1, the recombinant plasmid of pEGFP-C1-Stathmin was identified by PCR, restriction enzyme digestion analysis and DNA sequencing.The pEGFP-C1-Stathmin plasmid was transfected into SW480 cells and HT29 cells. Stathmin expression in transfectant was determined by Western Blot. Four different stathmin-specific short hairpin RNAs were cloned into pGPU6/GFP/Neo siRNA expression vector. After identification by PCR analysis and DNA sequencing, the recombinant vectors were transfected into colorectal cancer cells SW620 and LOVO,then Stathmin expression were identified by Western Blot.Effects of Stathmin overexpression and knock-down on cell proliferation was assessed by MTT assay, plate colony formation assay in vitro. Effects of Stathminoverexpression and knock-down on cell motility and migration were assessed in vitro by Transwell chamber and adhesion assay. SW480, Lovo, HT29 and SW620 cells were seeded on glass coverslips, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline. Cells were stained using mouse polyclonal anti-PRL-3 or rabbit monoclonal anti-Stathmin, followed with the appropriate FITC-labeled donkey anti-rabbit IgG or TRITC-labeled donkey anti-mouse IgG/DyLight594 secondary antibodies.Approximately lml of whole cell lysate or tissue extract (about lmg) were pre-cleared using sepharose-coupled rabbit or mouse IgG at 4℃for 1h, then centrifuged at 12,000×g for 3min. Protein G Plus/Protein A Agarose and 5μg of primary antibody anti-PRL-3 or an anti-Stathmin rabbit monoclonal antibody were added to the supernatant, mixed and incubated at 4℃overnight. The immune complexes were pulled down with protein G Plus/Protein A Agarose. The sepharose beads were washed five times with PBS and then proteins were eluted by incubation in 2×SDS-loading buffer at 100℃for 5 min and aliquots were subjected to SDS-PAGE and western blot analysis. A rabbit monoclonal antibody against Stathmin or a rabbit polyclonal antibody against PRL-3 was used for western blot analysis. 4.3 Immunofluorescence detection of Expression of MTs in CRCsSW480, Lovo, HT29 and SW620 cells with PRL-3/Stathmin overexpressed or knocked-down were seeded on glass coverslips, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline. Cells were incubated with mouse anti-a-tubulin antibody or anti-acetylated-α-tubulin at 4℃overnight after blocking with 1% BSA for 30min. Subsequently, the cells were incubated with TRITC-labeled donkey anti-mouse IgG/DyLight594 for 1h at 37℃. Processed coverslips were mounted in 75% glycerol/PBS mounting medium. Fluorescence was analyzed on confocal laser scanning microscopewith FV-10 ASW 1.7 Viewer image analysis software. We successfully identified 39 differential expression spots by two-dimensional gel electrophoresis and MALDI-TOF MS methods. Among these spots,21 proteins were down-regulated in both PRL-3 transient and stable knockdown clones,9 proteins were up-regulated in both PRL-3 transient and stable knockdown clones; we also observed 3 proteins were only down-regulated in PRL-3 stable knockdown clone and 6 proteins were only up-regulated in PRL-3 stable knockdown clone.Using functional distribution and category enrichment analysis of Gofact, we found that the proteins involved in process of cell cycle, cell death, biosynthesis, protein biosynthesis and translation were significantly enriched in these identified PRL-3 associated proteins. According to the components of these proteins, intracellular location including cilium, cytoplasm, cytosol, microtubule organizing center, and cytoskeleton were significantly enriched.we analyzed stathmin protein levels in an independent set of 149 paraffin-embedded, archival primary CRC tissues by immunohistochemical staining. Stathmin protein was localized in the cytoplasm of cancer cells. Higher expression of stathmin was observed in CRC tissue compared with that in normal counterparts. We found stronger expression of stathmin was found in both metastasis in lymph nodes and in livers. No significant association were found between stathmin expression and age, gender, tumor size, tumor site (P>0.05). Interestingly, we observed that stathmin expression was positively correlated with tumor differentiation(x2=6.656,P=0.010), tumor invasion(χ2=7.762,P=0.000),lymph node status(χ2=33.888,P=0.000), Dukes classification(χ2=37.480,P=0.000)and TNM staging (χ2=42.155,P=0.000) of CRC patients. Using Kaplan-Meier analysis method, we found that the protein expression of stathmin in CRC was significantly correlated with overall survival (χ2=53.205,P=0.000) of CRC patients. To determine whether the expression of stathmin was an independent prognostic factor of outcomes, multivariate survival analysis including invasion, differentiation, Dukes classification, TNM staging and stathmin expression, was done. Results showed that the expression of stathmin protein was a potential independent prognostic factor of outcomes of CRC patients(χ2=48.012,P=0.000).Western Blot showed that expression of stathmin was impaired when PRL-3 was transiently and stably knocked down; meanwhile,we performed western blot analysis to measure the protein expression of stathmin in fresh CRC tissue samples and their paired normal mucosal counterparts. We found that protein expression of stathmin was higher in most of the primary CRC tissue samples than that in their normal counterparts. Interestingly, protein expression of stathmin in primary tumor samples with metastasis (mCRC) is stronger that that in primary CRC tissues without metastasis (nmCRC). In CRC cell lines, strong expressions of stathmin were found in SW620, Lovo, HCT115, relatively low expressions of stathmin were observed in SW480, HT29 and LS174T. In our following functional study of stathmin, we study the effects of stathmin in CRC by downregulating expression of stathmin in SW620 or Lovo and by upregulating expression of stathmin in SW480 or HT29. 3 Stathmin Promotes Proliferation, Sdhesion And Migration of Human CRC CellsAccording to expression levels of stathmin in CRC cell lines, we study the effects of stathmin in CRC by down-regulating expression of stathmin in SW620 or Lovo and by up-regulating expression of stathmin in SW480 or HT29. A significantly increased proliferation detected by in vitro MTT assay was found in SW480 (F=22.236,P=0.000)and HT29(F=93.138,P=0.000) cells after stathmin expression in these cells was up-regulated. An impaired proliferation was found in SW620(F=16.791,P=0.000) and Lovo(F=19.599,P=0.000) cells after stathmin expression in these cells was down-regulated. Stathmin overexpression in SW480(F=113.958,P=0.000) and HT29 (F=33.477,P=0.000)cells had a significant enhanced ability to form colonies in plates; meanwhile, stathmin knockdown in SW620(F=30.258,P=0.000) and Lovo(F=31.172,P=0.000) cells had a decreased ability to form colonies in plates.Both gain-of-function and loss-of-function analyses revealed that stathmin promoted adhesion and migration of CRC cells(P=0.000).Stathmin MT-depolymerizing activity is negatively regulated by stathmin phosphorylation. As a phosphatase, PRL-3 may be involved in stathmin phosphorylation. Our co-immunoprecipitation assays in different CRC cell lines and CRC tissues revealed that PRL-3 could directly interact with stathmin. Co-localization of stathmin and PRL-3 in SW480, SW620, HT29 and Lovo cells was also found. Taken together, our results show that PRL-3 can interact directly with stathmin in the CRC cell systems.As we know, stathmin is a MT destabilizing protein. We observed the effects of PRL-3 or/and stathmin overexpression on distribution and expression ofα-tubulin. In control CRC cells, the distribution ofα-tubulin is dispersed into whole cells. However,α-tubulin aggregates locally after stathminWT or PRL-3 was up-regulated in two human CRC cell line SW480 and HT29. No significant change of distribution ofα-tubulin was observed after stathminWT or PRL-3 was down-regulated in two human CRC cell line Lovo and SW620.Stable MTs can be distinguished by a variety of posttranslational modifications, such as acetylation, poly-glutamylation, and detyrosination. Using specific acetylation antibody forα-tubulin, we explored the amount of stable MTs after stathminWT was up-regulated or down-regulated in human CRC cell lines. Expression of stathmin/EGFP significantly decreased the amount of cellular acetylated/stable MTs detected by acetylationα-tubulin. Consistently, increased the amount of cellular acetylated/stable MTs was observed after stathmin was knocked down in CRC cell lines Lovo and SW620. PRL-3 overexpression or knockdown led to similar effects as stathmin overexpression or knockdown induced. Westernblot analysis showed the similar result as Immunofluorescence detection. Based on the confirmed interaction between PRL-3 and stathmin, we concluded that interaction between PRL-3 and stathmin led to MT destabilization of CRC cells, which contributed to progression and metastasis of CRC.1. Thirty-nine PRL-3 associated proteins were screened and identified successfully. Proteins involved in process of cell cycle, cell death, biosynthesis, protein biosynthesis and translation were significantly enriched in these identified PRL-3 associated proteins.2. The expression of Stathmin/OP18 in CRC tissues is much higher than that in normal counterparts. Stathmin expression in metastatic CRC tissues is significantly higher than that in non-metastatic CRC tissues. It can be used as a potential independent predictor of the prognosis of CRC patients. Overexpression of Stathmin is related to tumor invasion, differentiation, Dukes classification, TNM staging of CRC patients. The expression of stathmin protein is a potential independent prognostic factor for outcomes of CRC patients.3. Stathmin/OP18 promotes proliferation, migration and adhesion of colorectal cancer cells.4. Interaction between PRL-3 and stathmin leads to MT destabilization of CRC cells, which contributes to progression and metastasis of CRC.更多还原