PRL-3和CDH22双基因表达抑制的结直肠癌细胞模型的建立及初步研究
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摘要
研究背景和目的
结直肠癌是常见的恶性肿瘤之一,近十年来,结直肠癌在我国的发病率有逐年上升的趋势。转移是影响患者治疗效果和导致患者死亡的主要原因,也是结直肠癌术后复发的直接原因。
肿瘤的发生、发展是多种基因突变累积以及相互作用形成的基因网络调控的结果,而这些突变基因的差异表达是决定肿瘤恶性表型多样性的分子基础对这些基因进行功能研究是了解肿瘤发生发展过程的重要策略,这不仅可以探究造成基因表型差异的遗传原因或外界原因,而且对肿瘤的诊断及治疗具有非常重要的意义。因此,筛查结直肠癌转移特异性基因,探讨与结直肠癌转移相关的因素,系统阐述转移分子机制,将为结直肠癌的转移治疗提供理想的靶点。
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家族成员,现被认为是结直肠癌转移相关的少数特异性表达分子之一。随着对PRL-3研究的不断深入,它与多种肿瘤发生、转移之间的关系将逐步得到证实。但迄今为止,PRL-3的底物、其促进结直肠癌发生发展的机制以及它所参与的信号途径仍未明确。前期我们课题组应用酵母双杂交技术发现PRL-3新型相互作用蛋白质CDH22,并应用GST-pull down、免疫共沉淀及共定位技术进行了证实。CDH22(又称为PB-cadherin)与E-钙粘附蛋白同属于钙粘附蛋白家族经典型亚类成员。在结构上具有5个胞外结构域结构,除跨膜区外还具有胞内区,在钙粘附蛋白相关的信号通路中起着重要作用。目前研究发现,钙粘附蛋白家族中有许多成员都与肿瘤的发生发展关系密切。我们课题组前期实验结果显示PRL-3通过诱导E-cadherin及CDH22表达下调、促进snail表达增加参与结直肠癌细胞上皮间叶转化的形成;结果表明,PRL-3促进细胞核内β-Catenin的聚集是激活Wnt通路的的重要途径。然而具体的分子生物学机制仍需进一步探讨。既然PRL-3蛋白与CDH22存在直接的相互作用,那么二者可能共同参与某一信号转导通路或某一疾病发病过程。
近年来研究发现β-catenin及其通路相关基因的改变在结直肠肿瘤发生、发展过程中具有重要作用。β-catenin蛋白分子量约为92kD～95kD,其作用涉及两个相对独立的过程,既是细胞间黏附连接的主要结构成分,参与构成E-cadherin/β-catenin复合体,维持正常上皮的极性和完整性;又是Wnt信号通路的中枢成分。在正常成熟细胞胞浆内的β-catenin大部分与细胞膜上E-cadherin胞内肽段结合,参与细胞粘连、生长、增殖等过程;少部分与胞浆内APC、GSK-3β及Axin蛋白结合成多蛋白复合体,通过磷酸化与去磷酸化过程来降解β-catenin。因而胞浆内游离β-catenin水平极低,不能进入细胞核调控相应基因表达。Wnt信号通路是一条十分保守的信号传导通路,在胚胎的发育过程及多种肿瘤的发生发展中起作用。Wnt通路发生异常的关键是胞浆内游离的β-catenin积累,与Tcf-4结合形成复合物进入核内,激活下游靶基因(如c-myc、cyclinD1、gastrin等)转录,从而启动肿瘤生长程序。β-catenin介导的β-catenin/E-cadherin复合体功能异常以及Wnt信号传导通路异常激活以及均与结直肠癌的发生、发展关系密切。
在β-catenin通路相关基因中,处于上游的E-cadherin及处于核心地位的β-catenin正常表达定位于上皮细胞膜,结直肠腺瘤、结直肠腺瘤恶变和结直肠癌组织E-cadherin、β-catenin胞膜表达不同程度缺失,β-catenin呈胞浆(胞核)异位表达。下游通路中的靶基因cyclinD1是调节细胞进入细胞周期中增殖期的主要因子,其过度表达和异常调控均可导致细胞周期发生异常,从而导致肿瘤的发生。正常结直肠黏膜cyclinD1表达均为阴性,结直肠癌组织中cyclinD1主要呈细胞核表达,少数肿瘤细胞呈细胞质表达。探讨β-catenin通路相关基因在结直肠癌形成过程中的变化规律,将有助于研究肿瘤恶性表型多样性的分子机制。同时也应认识到,人体是一个由多条信号转导途径交织成网络、共同作用的有机整体,在研究β-catenin/Wnt途径的同时,研究其他途径的作用以及β-catenin/Wnt通路与其他因素之间的相互影响,将为肿瘤侵袭转移的分子机制提供一个新的解释。
RNA干扰(RNA interference,RNAi)技术的出现为基因功能研究提供了一种新的、高效和特异的功能基因组研究策略,已经应用于功能基因组学、药物靶点筛选和细胞信号传导通路分析等方面。
本研究采用RNAi技术建立PRL-3和CDH-22双基因表达下调的结直肠癌细胞模型,然后对其生物学特性的改变进行了初步的研究,通过比较其与同一亲本来源的对照组细胞的形态、生物学特性、基因表达、蛋白差异等,探讨PRL-3、CDH22与β-catenin/Wnt信号通路相关基因的相互作用,本研究有助于更深入了解肿瘤侵袭转移的分子机制。
方法
1.靶向人CDH22基因RNAi慢病毒的包装、滴度测定
2.慢病毒对SW480/PRL-3-细胞的二次感染
将5×10~4个SW480/PRL-3-细胞接种于24孔板,37℃,5%CO2培养,16h后其融合达60%—70%,用靶向人CDH22基因的RNAi慢病毒以MOI为40感染细胞,添加polybrene,终浓度为8mg/ml。28h后去除病毒液,更换为含10%胎牛血清的RPMI1640培养液继续培养。
3.有限稀释法获得单克隆细胞株,对所得的各单克隆细胞株进行扩大培养
4.应用荧光定量PCR及Western blot检测各克隆PRL-3及CDH22基因mRNA和蛋白的表达水平,综合二者结果,鉴定出在mRNA及蛋白表达水平PRL-3及CDH22基因均显著降低的克隆株,从而建立PRL-3和CDH22双基因表达沉默的结直肠癌细胞模型,命名为SW480/PRL-3-/CDH22-。
5.细胞蜡块、流式细胞术、MTT法、比较SW480/PRL-3-/CDH22-与对照组SW480、SW480/Mock、SW480/PRL-3-在细胞形态、细胞生长周期、细胞增殖能力方面的差异。
6.免疫细胞化学检测SW480/PRL-3-/CDH22-及其同一亲本来源的对照组细胞株β-catenin/Wnt信号转导通路相关蛋白表达情况。
结果
1.慢病毒滴度测定收集病毒上清液,测定慢病毒的病毒滴度为8×10~5U/ml。
2.细胞二次感染后进行克隆化培养:经有限稀释法共获得17个单克隆细胞株,对所得的各单克隆细胞株进行扩大培养。
3.PRL-3和CDH22双基因表达沉默的结直肠癌细胞模型的建立及鉴定
荧光定量PCR的结果显示,挑取的各个单克隆都有不同程度CDH22基因的表达,通过公式计算,发现克隆2的干扰效率最高,表达量仅为对照的25%,即干扰效率为75%,其次是克隆1,表达量仅为对照的27%,即干扰效率为73%。荧光定量PCR结果显示,挑取的各个单克隆均有不同程度PRL-3基因的表达,通过公式计算,发现克隆4的干扰效率最高,表达量仅为对照的27%,即干扰效率为73%,其次是克隆1,表达量仅为对照的30%,即干扰效率为70%。通过荧光定量PCR初步得到4个可能符合实验预期目的克隆株。
Western Blot进一步检测这4个克隆株PRL-3及CDH22蛋白表达水平。综合考虑荧光定量PCR、Western Blot的结果,证实PRL-3及CDH22稳定表达敲低的细胞克隆为克隆1,命名为SW480/PRL-3-/CDH22-。
4.PRL-3和CDH22双基因表达抑制的结直肠癌细胞的体外生物学特性的改变
细胞蜡块HE染色显示:SW480/PRL-3-较SW480/PRL-3-/CDH22-、SW480、SW480/Mock细胞胞浆丰富,细胞连接紧密,SW480/PRL-3-/CDH22-、SW480、SW480/Mock3组细胞之间差异不显著。应用MTT法,我们检测SW480、SW480/Mock、SW480/PRL-3-、SW480/PRL-3-/CDH22-细胞体外增殖能力,经析因方差分析,四组差异具有显著性(F=952.067,P=0.000);经LSD法多重比较,结果表明,SW480细胞增殖速度最快,SW480/Mock、SW480/PRL-3-/CDH22-细胞次之,而SW480/PRL-3-细胞的增殖速度最慢。应用流式细胞术分析细胞周期变化,SW480、SW480/Mock、SW480/PRL-3-/CDH22-及SW480/PRL-3-四种细胞的S期细胞平均比例分别为46.6%、38.3%、30.9%和20.8%,统计分析结果显示四种细胞的S期差异有统计学意义(F=42.994,P=0.000)。SW480/PRL-3-细胞大部分阻滞在G1期,S期所占比例明显减少。
5.PRL-3和CDH22双基因表达抑制的结直肠癌细胞及对照组细胞株β-catenin/Wnt信号转导通路相关蛋白表达情况
细胞免疫化学实验显示:
1) SW480及SW480/Mock细胞E-cadherin在胞膜表达呈不同程度缺失;SW480/PRL-3-/CDH22-细胞胞膜呈间断性或异位表达,但并未出现明显的膜表达缺失;SW480/PRL-3-细胞几乎所有细胞E-cadherin成明显的胞膜着色。
2) SW480及SW480/Mock细胞β-catenin膜表达缺失明显;SW480/PRL-3-/CDH22-细胞β-catenin从细胞浆转移到细胞核膜及核周区域;SW480/PRL-3-细胞β- catenin重新定位于细胞膜。
3) CyclinD1阳性着色定位于细胞核。SW480及SW480/Mock细胞约85%的细胞CyclinD1呈核阳性表达,SW480/PRL-3-/CDH22-细胞约50%细胞呈核阳性表达,SW480/PRL-3-细胞约30%的细胞呈核阳性表达。
4) 4组细胞株CK均呈细胞膜阳性表达,无显著差异。
5) 4组细胞株Vimentin均未见阳性表达。
6) Gsk-3β在4组细胞中表现为胞浆表达。其表达强度依次为:SW480/PRL-3-/CDH22-、SW480/PRL-3-、SW480/mock、SW480。
结论
1.转染一种基因慢病毒干扰载体的细胞株可再次接受靶向另一基因干扰序列的慢病毒颗粒的转染,为研究慢病毒二次感染细胞并稳定干扰各靶基因的表达提供有实用价值的实验方法。
2.成功建立PRL-3和CDH22双基因表达抑制的结直肠癌细胞模型。
3.PRL-3、CDH22表达呈现不同水平时,Wnt/β-catenin信号转导通路相关蛋白表达强度及部位发生改变。单独下调PRL-3基因,CDH22表达上调,细胞株细胞膜E-cadherin恢复连续线性表达,细胞质或细胞核中的β-catenin重新定位于细胞膜,细胞核cyclinD1低表达。细胞之间连接变紧密,细胞增殖相对减慢,细胞阻滞在G1期,S期所占比例明显减少。
4.PRL-3基因可通过下调CDH22表达而诱导β-Catenin向细胞核聚集,从而激活Wnt信号靶基因cyclinD1的表达,导致结直肠癌细胞增殖加快。
BACKGROUND & OBJECTIVE
Colorectal cancer(CRC) is a common malignant tumor which severely threatens human health.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 cancer patients.
The tumor is caused by accumulated gene mutations of cells and is regulated by genetic networks resulted from gene mutations,which are the molecular basis leading to various phenotypic diversity of tumor malignancy.Functional study on these genes is becoming the essential strategy to know the mechanisms of tumor.Therefore, screening of specific genes for metastasis of CRC and elucidating the molecular mechanism of metastasis will provide us the possible new targets for treatment of CRC.
PRL-3(Phosphatase of Regenerating Liver-3 ) belongs to a subclass of protein tyrosine phosphatases(PTPs) family,which is now considered as a key gene associated with metastatic colorectal carcinomas(CRCs).However,the substrates of PRL-3,the role of PRL-3 and its signal pathways in CRC have not been well-identified.
In our previous studies,CDH22 was found as a candidate of PRL-3-interacting proteins by yeast two-hybrid screening systems.Interaction between PRL-3 and CDH22 was proved by GST-pull down assay,co-immunoprecipitation assay and co-localization analysis.Both CDH22(PB-cadherin) and E-cadherin belong to the classical subgroups of cadherin family.Both of them have five extracellular domains, one transmembrane domain and an intracellular region in structure.The intracellular domain of cadherin family has an important role in the regulation of cadherin related signaling pathways of colorectal cancer.It has been found that many members of the cadherin family are correlated with the occurrence and development of tumor.
We found previously that PRL-3 induces epithelial mesenchymal transition by down-regulation of E-cadherin,CDH22 and up-regulation of snail expression.And we also observed that PRL-3 can activate Wnt pathway by leading to nuclear accumulation ofβ-Catenin.A further study how PRL-3 affects the accumulation ofβ-Catenin is needed.As we found,there is a direct interaction between PRL-3 and CDH22,the possible roles of PRL-3 and CDH22 in related signaling pathway are observed in our study.
Recent studies found thatβ-catenin and related genes play the important roles in colorectal tumor.In normal mature cell,β-catenin is composed as the main structural components of intercellular adhesion and forms the E-cadherin-β-catenin complex to maintain the polarity and integrity of normal epithelium.Most parts ofβ-catenin are combined with intracellular domain of E-cadherin in the cytoplasm.Meanwhile,few parts ofβ-catenin are combined with APC,GSK-3βand Axin proteins to form multi-protein complex,which degradesβ-catenin through phosphorylation and dephosphorylation.Therefore,there remains only very few freeβ-catenin in the cytoplasm,it is very hard for the freeβ-catenin to accumulate in the nucleus and result in activation of Wnt signaling pathway.Wnt signal pathway is an evolutionary conservative signal pathway that acts in the development of embryos and many kinds of tumors.Freeβ-catenin accumulates in cytoplasm,combines with Tcf-4 to form a complex,enters into the cell nucleus,activates transcription of the target genes (C-myc,cyclinD1,gastri,et al.) and initiates the program of tumors.Dysfunction ofβ-catenin as well as abnormal activation of Wnt signaling pathway are closely related to the development and progression of CRC.
In this study,we established a subclone of colorectal cancer cells with knockdown of both PRL-3 and CDH22 expression using lentiviral RNAi technique. Morphological changes and biological characteristics of cells were compared within the subclone and its parent subclone.We focused on changes ofβ-catenin/Wnt signal pathway regulated by PRL-3,CDH22 and their interaction.Our study helps to further understand the molecular mechanisms of PRL-3 in tumor invasion and metastasis of CRC.
METHODS
1.A recombinant lentivirus targeting against CDH22 was produced and titered.
2.Second infection of SW480/PRL-3-cells with lentivirus targeting against CDH22. Fivex10~4 SW480/ PRL-3 cells per well were plated in a 24-well plate and incubated at 37℃.When the cells were grown to 60-70%confluence,recombinant lentivirus targeting against CDH22 with MOI at 40 was added.Then,the medium containing virus was removed 28 hours later.
3.Single cell derived clone was established by limiting dilution method.
4.Real-time quantitative PCR and Western blot were used to measure mRNA and protein expression levels of PRL-3 and CDH22 in each clone.Two clones with downregulation of both PRL-3 and CDH22 in mRNA and protein expression level were identified and established.
5.Morphological changes and biological characteristics of SW480,SW480/Mock, SW480/PRL-3-/CDH22-and SW480/PRL-3-cells were analyzed using HE staining, flow cytometry and MIT assay.
6.Protein expressions of genes involved inβ-catenin/Wnt signal pathway of SW480,SW480/Mock,SW480/PRL-3-/CDH22-and SW480/PRL-3-cells were detected using immuocytochemistry assay.
RESULTS
The main results are as follows:
1.The titer of the recombinant lentivirus targeting against CDH22 was about 8×10~5 U/ml.
2.Seventeen single cell derived cell clones were selected and established.
3.A subclone of colorectal cancer cells with knockdown of PRL-3 and CDH22 expression was established.
Real-time reverse transcription polymerase chain reaction(PCR) assays showed that two clones were selected as CDH22 stable knockdown subclones,Expression level of CDH22 mRNA in clone2 decreased to about 25%of the level in Mock group. In clone 1,expression level of CDH22 mRNA is equivalent to 27%of the level in Mock group.Western Blot experiment was performed to detect expression levels of PRL-3 and CDH22 protein in these cell clones.Real time PCR and Western Blot results showed that both PRL-3 and CDH22 in clone 1 were knocked down.Clone 1 designated as SW480/PRL-3-/CDH22-.
4.Morphological changes and biological characteristics of SW480,SW480/Mock, SW480/PRL-3-/CDH22-and SW480/PRL-3-cells
We observed that most of SW480 and SW480/Mock cells were larger and exhibited an epithelial-type appearance.Some of SW480 and SW480/Mock cells were smaller and displayed a rounder,fibroblast or spindle-shaped morphology.After PRL-3 was knocked-down,the cells grew like paving stones.The morphology of SW480/PRL-3-/CDH22-was closer to that of SW480 cells.
In vitro proliferation of SW480/PRL-3-cells decreased significantly compared with that of SW480 and SW480/MOCK cells as determined by in vitro MTT assay (F=952.067,P=0.000).However,in vitro proliferation of SW480/PRL-3-/CDH22-increased to some extent compared with that of SW480/PRL-3-cells.Flow cytometry analysis indicated that most of SW480/PRL-3-cells or SW480/PRL-3-/CDH22-were blocked in G1 phase of the cell cycle.Average ratios of SW480,SW480/mock, SW480/PRL-3-/CDH22-and SW480/PRL-3-cells in S phase were 46.6%,38.3%, 30.9%and 20.8%respectively(F=42.994,P=0.000).
5.Protein expressions of genes involved inβ-catenin/Wnt signal pathway
1) E-cadherin expression
Loss of membrane expression of E-cadherin was observed in SW480 and SW480/Mock cells.Membrane expression of E-cadherin recovered in SW480/PRL-3-cells.In SW480/PRL-3-/CDH22-cells,membrane expression of E-cadherin was partially missing.Intracellular accumulation of E-cadherin was observed in parts of SW480/PRL-3-/CDH22-cells.
2)β-catenin expression
Loss of membrane expression ofβ-catenin was observed in SW480 and SW480/Mock cells.Membrane expression ofβ-catenin recovered in SW480/PRL-3-cells.In SW480/PRL-3-/CDH22-cells,intracellular or perinuclear accumulation ofβ-catenin was observed in parts of SW480/PRL-3-/CDH22-cells.
3) cyclinD1 expression
CyclinD1 expression is located in the cellular nucleus.In SW480 and SW480/Mock cells,85%of cells showed nuclear positive staining of cyclinD1.In SW480/PRL-3-cells,only about 30%of cells showed nuclear positive staining of cyclinD1.The positive cell percentage increased to about 50%in SW480/PRL-3-/CDH22-.
4) CK expression
Membrane expressions of CK protein were observed in SW480,SW480/Mock, SW480/PRL-3-/CDH22-and SW480/PRL-3-cells.
5) Vimentin expression
No positive expressions of Vimentin were detected in SW480,SW480/Mock, SW480/PRL-3-/CDH22-and SW480/PRL-3-cells.
6) GSK-3βexpression
GSK-3βexpression is located in the cytoplasm.Moderate staining of GSK-3βwas found in SW480 and SW480/Mock cells.The positive cell percentage of GSK-3βexpression increased significantly compared with that in SW480/ Mock and SW480.
CONCLUSION
1.RNAi lentivirus targeting against another gene can be used to establish double-gene knockdown cell line after the primary gene of the cell line was knocked down.
2.A subclone of colorectal cancer cell line with knockdown of PRL-3 and CDH22 is established using lentiviral RNAi technique.
3.In vitro proliferation of SW480 decreases significantly after PRL-3 is knocked down.Most of SW480 cells are blocked in G1 phase of the cell cycle after PRL-3 is knocked down.
4.Interaction between PRL-3 and CDH22 contributes to activation of Wnt/β-catenin signal pathway by affecting nuclear accumulation ofβ-catenin,which results in transcription of cyclinD1..
结直肠癌是常见的恶性肿瘤之一,近十年来,结直肠癌在我国的发病率有逐年上升的趋势。转移是影响患者治疗效果和导致患者死亡的主要原因,也是结直肠癌术后复发的直接原因。
肿瘤的发生、发展是多种基因突变累积以及相互作用形成的基因网络调控的结果,而这些突变基因的差异表达是决定肿瘤恶性表型多样性的分子基础对这些基因进行功能研究是了解肿瘤发生发展过程的重要策略,这不仅可以探究造成基因表型差异的遗传原因或外界原因,而且对肿瘤的诊断及治疗具有非常重要的意义。因此,筛查结直肠癌转移特异性基因,探讨与结直肠癌转移相关的因素,系统阐述转移分子机制,将为结直肠癌的转移治疗提供理想的靶点。
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家族成员,现被认为是结直肠癌转移相关的少数特异性表达分子之一。随着对PRL-3研究的不断深入,它与多种肿瘤发生、转移之间的关系将逐步得到证实。但迄今为止,PRL-3的底物、其促进结直肠癌发生发展的机制以及它所参与的信号途径仍未明确。前期我们课题组应用酵母双杂交技术发现PRL-3新型相互作用蛋白质CDH22,并应用GST-pull down、免疫共沉淀及共定位技术进行了证实。CDH22(又称为PB-cadherin)与E-钙粘附蛋白同属于钙粘附蛋白家族经典型亚类成员。在结构上具有5个胞外结构域结构,除跨膜区外还具有胞内区,在钙粘附蛋白相关的信号通路中起着重要作用。目前研究发现,钙粘附蛋白家族中有许多成员都与肿瘤的发生发展关系密切。我们课题组前期实验结果显示PRL-3通过诱导E-cadherin及CDH22表达下调、促进snail表达增加参与结直肠癌细胞上皮间叶转化的形成;结果表明,PRL-3促进细胞核内β-Catenin的聚集是激活Wnt通路的的重要途径。然而具体的分子生物学机制仍需进一步探讨。既然PRL-3蛋白与CDH22存在直接的相互作用,那么二者可能共同参与某一信号转导通路或某一疾病发病过程。
近年来研究发现β-catenin及其通路相关基因的改变在结直肠肿瘤发生、发展过程中具有重要作用。β-catenin蛋白分子量约为92kD～95kD,其作用涉及两个相对独立的过程,既是细胞间黏附连接的主要结构成分,参与构成E-cadherin/β-catenin复合体,维持正常上皮的极性和完整性;又是Wnt信号通路的中枢成分。在正常成熟细胞胞浆内的β-catenin大部分与细胞膜上E-cadherin胞内肽段结合,参与细胞粘连、生长、增殖等过程;少部分与胞浆内APC、GSK-3β及Axin蛋白结合成多蛋白复合体,通过磷酸化与去磷酸化过程来降解β-catenin。因而胞浆内游离β-catenin水平极低,不能进入细胞核调控相应基因表达。Wnt信号通路是一条十分保守的信号传导通路,在胚胎的发育过程及多种肿瘤的发生发展中起作用。Wnt通路发生异常的关键是胞浆内游离的β-catenin积累,与Tcf-4结合形成复合物进入核内,激活下游靶基因(如c-myc、cyclinD1、gastrin等)转录,从而启动肿瘤生长程序。β-catenin介导的β-catenin/E-cadherin复合体功能异常以及Wnt信号传导通路异常激活以及均与结直肠癌的发生、发展关系密切。
在β-catenin通路相关基因中,处于上游的E-cadherin及处于核心地位的β-catenin正常表达定位于上皮细胞膜,结直肠腺瘤、结直肠腺瘤恶变和结直肠癌组织E-cadherin、β-catenin胞膜表达不同程度缺失,β-catenin呈胞浆(胞核)异位表达。下游通路中的靶基因cyclinD1是调节细胞进入细胞周期中增殖期的主要因子,其过度表达和异常调控均可导致细胞周期发生异常,从而导致肿瘤的发生。正常结直肠黏膜cyclinD1表达均为阴性,结直肠癌组织中cyclinD1主要呈细胞核表达,少数肿瘤细胞呈细胞质表达。探讨β-catenin通路相关基因在结直肠癌形成过程中的变化规律,将有助于研究肿瘤恶性表型多样性的分子机制。同时也应认识到,人体是一个由多条信号转导途径交织成网络、共同作用的有机整体,在研究β-catenin/Wnt途径的同时,研究其他途径的作用以及β-catenin/Wnt通路与其他因素之间的相互影响,将为肿瘤侵袭转移的分子机制提供一个新的解释。
RNA干扰(RNA interference,RNAi)技术的出现为基因功能研究提供了一种新的、高效和特异的功能基因组研究策略,已经应用于功能基因组学、药物靶点筛选和细胞信号传导通路分析等方面。
本研究采用RNAi技术建立PRL-3和CDH-22双基因表达下调的结直肠癌细胞模型,然后对其生物学特性的改变进行了初步的研究,通过比较其与同一亲本来源的对照组细胞的形态、生物学特性、基因表达、蛋白差异等,探讨PRL-3、CDH22与β-catenin/Wnt信号通路相关基因的相互作用,本研究有助于更深入了解肿瘤侵袭转移的分子机制。
方法
1.靶向人CDH22基因RNAi慢病毒的包装、滴度测定
2.慢病毒对SW480/PRL-3-细胞的二次感染
将5×10~4个SW480/PRL-3-细胞接种于24孔板,37℃,5%CO2培养,16h后其融合达60%—70%,用靶向人CDH22基因的RNAi慢病毒以MOI为40感染细胞,添加polybrene,终浓度为8mg/ml。28h后去除病毒液,更换为含10%胎牛血清的RPMI1640培养液继续培养。
3.有限稀释法获得单克隆细胞株,对所得的各单克隆细胞株进行扩大培养
4.应用荧光定量PCR及Western blot检测各克隆PRL-3及CDH22基因mRNA和蛋白的表达水平,综合二者结果,鉴定出在mRNA及蛋白表达水平PRL-3及CDH22基因均显著降低的克隆株,从而建立PRL-3和CDH22双基因表达沉默的结直肠癌细胞模型,命名为SW480/PRL-3-/CDH22-。
5.细胞蜡块、流式细胞术、MTT法、比较SW480/PRL-3-/CDH22-与对照组SW480、SW480/Mock、SW480/PRL-3-在细胞形态、细胞生长周期、细胞增殖能力方面的差异。
6.免疫细胞化学检测SW480/PRL-3-/CDH22-及其同一亲本来源的对照组细胞株β-catenin/Wnt信号转导通路相关蛋白表达情况。
结果
1.慢病毒滴度测定收集病毒上清液,测定慢病毒的病毒滴度为8×10~5U/ml。
2.细胞二次感染后进行克隆化培养:经有限稀释法共获得17个单克隆细胞株,对所得的各单克隆细胞株进行扩大培养。
3.PRL-3和CDH22双基因表达沉默的结直肠癌细胞模型的建立及鉴定
荧光定量PCR的结果显示,挑取的各个单克隆都有不同程度CDH22基因的表达,通过公式计算,发现克隆2的干扰效率最高,表达量仅为对照的25%,即干扰效率为75%,其次是克隆1,表达量仅为对照的27%,即干扰效率为73%。荧光定量PCR结果显示,挑取的各个单克隆均有不同程度PRL-3基因的表达,通过公式计算,发现克隆4的干扰效率最高,表达量仅为对照的27%,即干扰效率为73%,其次是克隆1,表达量仅为对照的30%,即干扰效率为70%。通过荧光定量PCR初步得到4个可能符合实验预期目的克隆株。
Western Blot进一步检测这4个克隆株PRL-3及CDH22蛋白表达水平。综合考虑荧光定量PCR、Western Blot的结果,证实PRL-3及CDH22稳定表达敲低的细胞克隆为克隆1,命名为SW480/PRL-3-/CDH22-。
4.PRL-3和CDH22双基因表达抑制的结直肠癌细胞的体外生物学特性的改变
细胞蜡块HE染色显示:SW480/PRL-3-较SW480/PRL-3-/CDH22-、SW480、SW480/Mock细胞胞浆丰富,细胞连接紧密,SW480/PRL-3-/CDH22-、SW480、SW480/Mock3组细胞之间差异不显著。应用MTT法,我们检测SW480、SW480/Mock、SW480/PRL-3-、SW480/PRL-3-/CDH22-细胞体外增殖能力,经析因方差分析,四组差异具有显著性(F=952.067,P=0.000);经LSD法多重比较,结果表明,SW480细胞增殖速度最快,SW480/Mock、SW480/PRL-3-/CDH22-细胞次之,而SW480/PRL-3-细胞的增殖速度最慢。应用流式细胞术分析细胞周期变化,SW480、SW480/Mock、SW480/PRL-3-/CDH22-及SW480/PRL-3-四种细胞的S期细胞平均比例分别为46.6%、38.3%、30.9%和20.8%,统计分析结果显示四种细胞的S期差异有统计学意义(F=42.994,P=0.000)。SW480/PRL-3-细胞大部分阻滞在G1期,S期所占比例明显减少。
5.PRL-3和CDH22双基因表达抑制的结直肠癌细胞及对照组细胞株β-catenin/Wnt信号转导通路相关蛋白表达情况
细胞免疫化学实验显示:
1) SW480及SW480/Mock细胞E-cadherin在胞膜表达呈不同程度缺失;SW480/PRL-3-/CDH22-细胞胞膜呈间断性或异位表达,但并未出现明显的膜表达缺失;SW480/PRL-3-细胞几乎所有细胞E-cadherin成明显的胞膜着色。
2) SW480及SW480/Mock细胞β-catenin膜表达缺失明显;SW480/PRL-3-/CDH22-细胞β-catenin从细胞浆转移到细胞核膜及核周区域;SW480/PRL-3-细胞β- catenin重新定位于细胞膜。
3) CyclinD1阳性着色定位于细胞核。SW480及SW480/Mock细胞约85%的细胞CyclinD1呈核阳性表达,SW480/PRL-3-/CDH22-细胞约50%细胞呈核阳性表达,SW480/PRL-3-细胞约30%的细胞呈核阳性表达。
4) 4组细胞株CK均呈细胞膜阳性表达,无显著差异。
5) 4组细胞株Vimentin均未见阳性表达。
6) Gsk-3β在4组细胞中表现为胞浆表达。其表达强度依次为:SW480/PRL-3-/CDH22-、SW480/PRL-3-、SW480/mock、SW480。
结论
1.转染一种基因慢病毒干扰载体的细胞株可再次接受靶向另一基因干扰序列的慢病毒颗粒的转染,为研究慢病毒二次感染细胞并稳定干扰各靶基因的表达提供有实用价值的实验方法。
2.成功建立PRL-3和CDH22双基因表达抑制的结直肠癌细胞模型。
3.PRL-3、CDH22表达呈现不同水平时,Wnt/β-catenin信号转导通路相关蛋白表达强度及部位发生改变。单独下调PRL-3基因,CDH22表达上调,细胞株细胞膜E-cadherin恢复连续线性表达,细胞质或细胞核中的β-catenin重新定位于细胞膜,细胞核cyclinD1低表达。细胞之间连接变紧密,细胞增殖相对减慢,细胞阻滞在G1期,S期所占比例明显减少。
4.PRL-3基因可通过下调CDH22表达而诱导β-Catenin向细胞核聚集,从而激活Wnt信号靶基因cyclinD1的表达,导致结直肠癌细胞增殖加快。
BACKGROUND & OBJECTIVE
Colorectal cancer(CRC) is a common malignant tumor which severely threatens human health.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 cancer patients.
The tumor is caused by accumulated gene mutations of cells and is regulated by genetic networks resulted from gene mutations,which are the molecular basis leading to various phenotypic diversity of tumor malignancy.Functional study on these genes is becoming the essential strategy to know the mechanisms of tumor.Therefore, screening of specific genes for metastasis of CRC and elucidating the molecular mechanism of metastasis will provide us the possible new targets for treatment of CRC.
PRL-3(Phosphatase of Regenerating Liver-3 ) belongs to a subclass of protein tyrosine phosphatases(PTPs) family,which is now considered as a key gene associated with metastatic colorectal carcinomas(CRCs).However,the substrates of PRL-3,the role of PRL-3 and its signal pathways in CRC have not been well-identified.
In our previous studies,CDH22 was found as a candidate of PRL-3-interacting proteins by yeast two-hybrid screening systems.Interaction between PRL-3 and CDH22 was proved by GST-pull down assay,co-immunoprecipitation assay and co-localization analysis.Both CDH22(PB-cadherin) and E-cadherin belong to the classical subgroups of cadherin family.Both of them have five extracellular domains, one transmembrane domain and an intracellular region in structure.The intracellular domain of cadherin family has an important role in the regulation of cadherin related signaling pathways of colorectal cancer.It has been found that many members of the cadherin family are correlated with the occurrence and development of tumor.
We found previously that PRL-3 induces epithelial mesenchymal transition by down-regulation of E-cadherin,CDH22 and up-regulation of snail expression.And we also observed that PRL-3 can activate Wnt pathway by leading to nuclear accumulation ofβ-Catenin.A further study how PRL-3 affects the accumulation ofβ-Catenin is needed.As we found,there is a direct interaction between PRL-3 and CDH22,the possible roles of PRL-3 and CDH22 in related signaling pathway are observed in our study.
Recent studies found thatβ-catenin and related genes play the important roles in colorectal tumor.In normal mature cell,β-catenin is composed as the main structural components of intercellular adhesion and forms the E-cadherin-β-catenin complex to maintain the polarity and integrity of normal epithelium.Most parts ofβ-catenin are combined with intracellular domain of E-cadherin in the cytoplasm.Meanwhile,few parts ofβ-catenin are combined with APC,GSK-3βand Axin proteins to form multi-protein complex,which degradesβ-catenin through phosphorylation and dephosphorylation.Therefore,there remains only very few freeβ-catenin in the cytoplasm,it is very hard for the freeβ-catenin to accumulate in the nucleus and result in activation of Wnt signaling pathway.Wnt signal pathway is an evolutionary conservative signal pathway that acts in the development of embryos and many kinds of tumors.Freeβ-catenin accumulates in cytoplasm,combines with Tcf-4 to form a complex,enters into the cell nucleus,activates transcription of the target genes (C-myc,cyclinD1,gastri,et al.) and initiates the program of tumors.Dysfunction ofβ-catenin as well as abnormal activation of Wnt signaling pathway are closely related to the development and progression of CRC.
In this study,we established a subclone of colorectal cancer cells with knockdown of both PRL-3 and CDH22 expression using lentiviral RNAi technique. Morphological changes and biological characteristics of cells were compared within the subclone and its parent subclone.We focused on changes ofβ-catenin/Wnt signal pathway regulated by PRL-3,CDH22 and their interaction.Our study helps to further understand the molecular mechanisms of PRL-3 in tumor invasion and metastasis of CRC.
METHODS
1.A recombinant lentivirus targeting against CDH22 was produced and titered.
2.Second infection of SW480/PRL-3-cells with lentivirus targeting against CDH22. Fivex10~4 SW480/ PRL-3 cells per well were plated in a 24-well plate and incubated at 37℃.When the cells were grown to 60-70%confluence,recombinant lentivirus targeting against CDH22 with MOI at 40 was added.Then,the medium containing virus was removed 28 hours later.
3.Single cell derived clone was established by limiting dilution method.
4.Real-time quantitative PCR and Western blot were used to measure mRNA and protein expression levels of PRL-3 and CDH22 in each clone.Two clones with downregulation of both PRL-3 and CDH22 in mRNA and protein expression level were identified and established.
5.Morphological changes and biological characteristics of SW480,SW480/Mock, SW480/PRL-3-/CDH22-and SW480/PRL-3-cells were analyzed using HE staining, flow cytometry and MIT assay.
6.Protein expressions of genes involved inβ-catenin/Wnt signal pathway of SW480,SW480/Mock,SW480/PRL-3-/CDH22-and SW480/PRL-3-cells were detected using immuocytochemistry assay.
RESULTS
The main results are as follows:
1.The titer of the recombinant lentivirus targeting against CDH22 was about 8×10~5 U/ml.
2.Seventeen single cell derived cell clones were selected and established.
3.A subclone of colorectal cancer cells with knockdown of PRL-3 and CDH22 expression was established.
Real-time reverse transcription polymerase chain reaction(PCR) assays showed that two clones were selected as CDH22 stable knockdown subclones,Expression level of CDH22 mRNA in clone2 decreased to about 25%of the level in Mock group. In clone 1,expression level of CDH22 mRNA is equivalent to 27%of the level in Mock group.Western Blot experiment was performed to detect expression levels of PRL-3 and CDH22 protein in these cell clones.Real time PCR and Western Blot results showed that both PRL-3 and CDH22 in clone 1 were knocked down.Clone 1 designated as SW480/PRL-3-/CDH22-.
4.Morphological changes and biological characteristics of SW480,SW480/Mock, SW480/PRL-3-/CDH22-and SW480/PRL-3-cells
We observed that most of SW480 and SW480/Mock cells were larger and exhibited an epithelial-type appearance.Some of SW480 and SW480/Mock cells were smaller and displayed a rounder,fibroblast or spindle-shaped morphology.After PRL-3 was knocked-down,the cells grew like paving stones.The morphology of SW480/PRL-3-/CDH22-was closer to that of SW480 cells.
In vitro proliferation of SW480/PRL-3-cells decreased significantly compared with that of SW480 and SW480/MOCK cells as determined by in vitro MTT assay (F=952.067,P=0.000).However,in vitro proliferation of SW480/PRL-3-/CDH22-increased to some extent compared with that of SW480/PRL-3-cells.Flow cytometry analysis indicated that most of SW480/PRL-3-cells or SW480/PRL-3-/CDH22-were blocked in G1 phase of the cell cycle.Average ratios of SW480,SW480/mock, SW480/PRL-3-/CDH22-and SW480/PRL-3-cells in S phase were 46.6%,38.3%, 30.9%and 20.8%respectively(F=42.994,P=0.000).
5.Protein expressions of genes involved inβ-catenin/Wnt signal pathway
1) E-cadherin expression
Loss of membrane expression of E-cadherin was observed in SW480 and SW480/Mock cells.Membrane expression of E-cadherin recovered in SW480/PRL-3-cells.In SW480/PRL-3-/CDH22-cells,membrane expression of E-cadherin was partially missing.Intracellular accumulation of E-cadherin was observed in parts of SW480/PRL-3-/CDH22-cells.
2)β-catenin expression
Loss of membrane expression ofβ-catenin was observed in SW480 and SW480/Mock cells.Membrane expression ofβ-catenin recovered in SW480/PRL-3-cells.In SW480/PRL-3-/CDH22-cells,intracellular or perinuclear accumulation ofβ-catenin was observed in parts of SW480/PRL-3-/CDH22-cells.
3) cyclinD1 expression
CyclinD1 expression is located in the cellular nucleus.In SW480 and SW480/Mock cells,85%of cells showed nuclear positive staining of cyclinD1.In SW480/PRL-3-cells,only about 30%of cells showed nuclear positive staining of cyclinD1.The positive cell percentage increased to about 50%in SW480/PRL-3-/CDH22-.
4) CK expression
Membrane expressions of CK protein were observed in SW480,SW480/Mock, SW480/PRL-3-/CDH22-and SW480/PRL-3-cells.
5) Vimentin expression
No positive expressions of Vimentin were detected in SW480,SW480/Mock, SW480/PRL-3-/CDH22-and SW480/PRL-3-cells.
6) GSK-3βexpression
GSK-3βexpression is located in the cytoplasm.Moderate staining of GSK-3βwas found in SW480 and SW480/Mock cells.The positive cell percentage of GSK-3βexpression increased significantly compared with that in SW480/ Mock and SW480.
CONCLUSION
1.RNAi lentivirus targeting against another gene can be used to establish double-gene knockdown cell line after the primary gene of the cell line was knocked down.
2.A subclone of colorectal cancer cell line with knockdown of PRL-3 and CDH22 is established using lentiviral RNAi technique.
3.In vitro proliferation of SW480 decreases significantly after PRL-3 is knocked down.Most of SW480 cells are blocked in G1 phase of the cell cycle after PRL-3 is knocked down.
4.Interaction between PRL-3 and CDH22 contributes to activation of Wnt/β-catenin signal pathway by affecting nuclear accumulation ofβ-catenin,which results in transcription of cyclinD1..
引文
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9.Rouleau C,Roy A,St Martin T,et al.Protein tyrosine phosphatase PRL-3 in malignant cells and endothelial cells:expression and function[J].Mol Cancer Ther,2006,5(2):219-229.
10.Wang Y,Li ZF,He J,et al.Expression of the human phosphatases of regenerating liver (PRLs) in colonic adenocarcinoma and its correlation with lymph node metastasis[J].Int J Colorectal Dis,2007,22(10):1179-1841
11.Wu X,Zeng H,Zhang X,et al.Phosphatase of regenerating liver-3 promotes motility and metastasis of mouse melanoma cells[J].Am J Pathol,2004,164(6):2039-2054.
12.吴玫,龙飞,李姝玉.PRL23在乳腺癌中的表达及意义[J].中国组织化学与细胞化学杂志,2006,15(4):410-415.
13. Bardelli A, Saha S, Sager JA , et al . PRL-3 expression in metastatic cancers[J]. Clin Cancer Res ,2003 ,9 (15):5607 - 5615
14. Peng L, Ning J, Meng L , et al. The association of the expression level of protein tyrosine phosphatase PRL-3 protein with liver metastasis and prognosis of patients with colorectal cancer[J ]. J Cancer Res Clin Oncol ,2004 ,130 (9) :521 -526.
15. Kato H, Semba S, Miskad UA, et al. High expression of PRL-3 promotes cancer cell motility and liver metastasis in human colorectal cancer :a predictive molecular marker of metachronous liver and lung metastases[J ]. Clin Cancer Res ,2004 ,10(21) :7318 - 7 328.
16. Wallin AR, Svanvik J, Adell G, et al. Expression of PRL proteins at invasive margin of rectal cancers in relation to preoperative radio therapy[J]. Int J Radiat Oncol Biol Phys ,2006 ,65(2):452 - 458.
17. Peng L, Li Y, Meng L et al. Preparation and characterization of monoclonal antibody against protein tyrosine phosphatase PRL-3[J]. Hybrid Hybridomics 2004;23(1):23-27.
18. Diamond RH, Cressman DE, Laz TM et al. PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth[J]. Mol Cell Biol 1994;14(6):3752-3762.
19. Cates CA, Michael RL, Stayrook KR et al. Prenylation of oncogenic human PTP(CAAX) protein tyrosine phosphatases[J]. Cancer Lett 1996;110(1-2):49-55.
20. Zeng Q, Si X, Horstmann H et al. Prenylation-dependent association of protein-tyrosine phosphatases PRL-1, -2, and -3 with the plasma membrane and the early endosome.[J].Biol Chem 2000;275(28):21444-21452.
21. Waterhouse PM, Wang MB, Lough T. Gene silencing as an adaptive defence against viruses[J]. Nature 2001 ;411 (6839):834-842.
22. Kozlov G, Cheng J, Ziomek E et al. Structural insights into molecular function of the metastasis-associated phosphatase PRL-3[ J ].Biol Chem 2004;279(12):11882-11889.
23. Wu X, Zeng H, Zhang X, et al. Phosphatase of regenerating liver -3 promotes motility and metastasis of mouse melanoma cells[J ] . Am J Pathol ,2004 ,164(6) :2 039 - 2 054.
24. Peng L , Jin G, WangL , et al . Identification of integrin alphal as an interacting protein of protein Tyrosine phosphatase PRL-3[J]. Bio-chem Biophys Res Commun, 2006, 342(1): 179-183.
25. Polte TR, Hanks SK. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas[J ]. Proc Natl Acad Sci USA ,1995 ,92(23) :10678 - 10682
26. Honda H, Oda H, Nakamoto T, et al. Cardiovascular anomaly impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas[J]. Nat Genet ,1998 ,19 (4):361 -365.
27. Liang F, Liang J, Wang WQ, et al. PRL-3 promotes cell invasion and proliferation by down regulation of Csk leading to Src activation [J ]. J Biol Chem ,2007 ,282 (8):5413 - 5 419.
28. Fiordalisi JJ, Keller PJ, Cox AD. PRL tyrosine phosphatases regulate rho family GTPases to promote invasion and motility[J ]. Cancer Res , 2006 ,66(6) :3153 - 3161..
29. Wang H, Quah SY, Dong JM, et al . PRL-3 down regulates PTEN expression and signals through P13K to promote epithelial-mesenchyma transition[J]. Cancer Res, 2007, 67(7) 2922-2926.
30. TomitaK, vanBokhovenA, van LeendersGJ, et al. Cadherin switching in human prostate cancer progression [ J]. Cancer Res, 2000,60:3650-3654.
31. Munoz - GuerraMF, Marazuela EG, Fernandez - ContrerasME, et al. P - cadherin exp ression reduced in squamous cell carcinoma of the oral cavity: an indicatior of poor p rognosis [ J ]. Cancer, 2005,103 (5) :960-969.
32. Taniuchi K, Nakagawa H, HosokawaM, et al. Overexp ressed P - cadherin /CDH3 promotesmotility of pancreatic cancer cells by interacting with p120ctn and activating rho -family GTPases [ J ]. Cancer Res,2005, 65 (8): 3092-3099
33. Shimazui T,Qosterwijk E,Akaza H et al.Expression of cadherin-6 as a novel diagnostic tool to predict prognosis of patients with E-cadherin-absent renal cell carcinoma.Clin Cancer Res1998;4(10);2419-2424.
34. Bauer R, Bosserhoff AK. Functional implication of truncated P-cadherin expression in malignant melanoma [ J]. Exp Mol Pathol,2006, 81: 224-230.
35. Bonilla C, Mason T, Long L, et al. E-cadherin polymorphisms and haplotypes influence risk for prostate cancer[ J]. Prostate, 2006, 66:546-556.
36. Charalabopoulos K, GogaliA, Dalavaga Y, et al. The clinical significance of soluble E - cadherin in nonsmall cell lung cancer [ J ]. Exp Oncol, 2006,28 (1): 83-85.
37. Muller T, Bain G, Wang X, et al . Regulation of ep -ithelial cell migration and tumor formation by beta - catenin signaling [ J ]. Exp Cell Res, 2002, 280: 119-133.
1.Elbashir SM,Harborth J.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J].Nature,2001,411,(6836):494.
2.Meng-TsaiWu,Wen -Hui Tsai,Wen -Tsan Chang et al.Simple and efficient DNA vector based RNAi systems in mammalian cells[J].Biochem Biophys Res Commun,2005,330(1):53-59.
3.Imamur T,Yokosuk O,OmatM,et al.Proteomic analysis of the TGF -b signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad 4 expression[J].Biochem Biophys Res Commun,2004,318(1):289-296.
4.Takeshita F,Ochiya T.Therapeutic potential of RNA interference against cancer[J].Cancer Sci,2006,97(8):689-696.
5.Cabot RA,Kuhholzer B,Chan AW,et al.Transgenic pigs pro-duced using in vitro matured occytes infected with a retroviral vector[J].Anim Biotechnol,2001,12(2):205-214
1. Kozlov G, Cheng J, Ziomek E, et al. Structural insights into molecular function of the metastasis-associated phosphatase PRL-3[J].J Biol Chem ,2004,279:11882-9
2. Kim KA, Song JS, Jee J, et al. Stru cture of human PRL-3, the phosphatase associated with cancer metastasis[J]. FEBS Lett ,2004,565:181-7..
3. Li Z, Zhan W, Wang Z, et al. Inhibition of PRL-3 gene expression in gastric cancer cell line SGC7901 viamicro RNA suppressed reduces peritoneal metastasis[J].Biochem Biophys Res Commun ,2006,348:229-237
4. Saha S, Bardelli A, Buckhaults P, et al. A phosphatase associated with metastasis of colorectal cancer[J]. Science ,2001,294 (5545): 1343-6
5. Bardelli A, Saha S, Sager JA, et al. PRL-3 expression in metastatic cancers[J].Clin Cancer Res ,2003,9:5607-15.
6. Zeng Q, Dong JM, Guo K, et al. PRL-3 and PRL-1 promote cell migration, invasion, and metastasis[J].Cancer Res ,2003, 63:2716-22.
7. Wu X, Zeng H, Zhang X, et al. Phosphatase of regenerating liver-3 promotes motility and metastasis of mouse melanoma cells[J]. Am J Pathhol ,2004,64:2039-54.
8. Guo K, Li J, Tang JP, et al. Catalytic Domain of PRL-3 plays an Essential Role in Tumor Metastasis: Formation of PRL-3 Tumors Inside the Blood Vessels[J].Cancer Biol Ther, 2004, In press .
9. Russo A , Bazan V , Migliavacca M , et al. DNA aneuploidy and high proliferative activity but not K-ras-2 mutations as independent predictors of clinical outcome in operable gast ric carcinoma : result s of a 5-year Gruppo Oncologico dell' Italia Meridonale (GDIM) prospective study[J]. Cancer ,2001 , 92 (2): 294-302.
1. Gregorieff A,Clevers H. Wnt signaling in the intestinal epithelium:from endoderm to cancer[J]. Genes Dev,2005,19 (8): 877- 890.
2. Moon RT, Bowerman B, Boutros M, et al. The Promise and perils of Wnt signaling through bata-catenin[J]. Science, 2002, 296(5573): 1644- 1646.
3. .Huber AH, Stewart DB,Laurents DV et,al. The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin.A possible mechanism for regulating cadherin turnover,J Biol Chem 2001,276(15):12301-12309.
2.Zeng Q,Si X,Horstmann H,et al.Prenylation dependent association of protein tyrosine phosphatases PRL-1,-2,and-3 with the plasma membrane and the early endosome[J].J Biol Chem,2000,275(28):21444-21 452.
3.Zeng Q,Hong W,Tan YH,Mouse PRL-2 and PRL-3,two potentially prenylated protein tyrosine phosphatases homologous to PRL-1[J].Biochem Biophys Res Commun,1998,244(2):421-427.
4.Matter WF,Estridge T,Zhang C,et al.Role of PRL-3,a human muscle specific tyrosine phosphatase,in angiotensin Ⅱ signaling[J].Biochem Biophys Res Commun,2001,283(5):1061-1068.
5.Guo K,Li J,Wang H,et al.PRL-3 initiates tumor angiogenesis by recruiting endothelial cells in vitro and in vivo[J].Cancer Res,2006,66(19):9625-9635.
6.Miskad UA,Semba S,Kato H,et al.Expression of PRL-3 phosphatase in human gastric carcinomas:close correlation with invasion and metastasis[J].Pathobiology,2004,71(4):176-184.
7.Polato F,Codegoni A,Fruscio R,et al.PRL-3 phosphatase is implicated in ovarian cancer growth[J].Clin Cancer Res,2005,11(19 Pt 1):6835-6839
8.Radke I,Gotte M,Kersting C,et al.Expression and prognostic impact of the protein tyrosine phosphatases PRL-1,PRL-2,and PRL-3 in breast cancer[J].Br J Cancer,2006,95(3):347-354
9.Rouleau C,Roy A,St Martin T,et al.Protein tyrosine phosphatase PRL-3 in malignant cells and endothelial cells:expression and function[J].Mol Cancer Ther,2006,5(2):219-229.
10.Wang Y,Li ZF,He J,et al.Expression of the human phosphatases of regenerating liver (PRLs) in colonic adenocarcinoma and its correlation with lymph node metastasis[J].Int J Colorectal Dis,2007,22(10):1179-1841
11.Wu X,Zeng H,Zhang X,et al.Phosphatase of regenerating liver-3 promotes motility and metastasis of mouse melanoma cells[J].Am J Pathol,2004,164(6):2039-2054.
12.吴玫,龙飞,李姝玉.PRL23在乳腺癌中的表达及意义[J].中国组织化学与细胞化学杂志,2006,15(4):410-415.
13. Bardelli A, Saha S, Sager JA , et al . PRL-3 expression in metastatic cancers[J]. Clin Cancer Res ,2003 ,9 (15):5607 - 5615
14. Peng L, Ning J, Meng L , et al. The association of the expression level of protein tyrosine phosphatase PRL-3 protein with liver metastasis and prognosis of patients with colorectal cancer[J ]. J Cancer Res Clin Oncol ,2004 ,130 (9) :521 -526.
15. Kato H, Semba S, Miskad UA, et al. High expression of PRL-3 promotes cancer cell motility and liver metastasis in human colorectal cancer :a predictive molecular marker of metachronous liver and lung metastases[J ]. Clin Cancer Res ,2004 ,10(21) :7318 - 7 328.
16. Wallin AR, Svanvik J, Adell G, et al. Expression of PRL proteins at invasive margin of rectal cancers in relation to preoperative radio therapy[J]. Int J Radiat Oncol Biol Phys ,2006 ,65(2):452 - 458.
17. Peng L, Li Y, Meng L et al. Preparation and characterization of monoclonal antibody against protein tyrosine phosphatase PRL-3[J]. Hybrid Hybridomics 2004;23(1):23-27.
18. Diamond RH, Cressman DE, Laz TM et al. PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth[J]. Mol Cell Biol 1994;14(6):3752-3762.
19. Cates CA, Michael RL, Stayrook KR et al. Prenylation of oncogenic human PTP(CAAX) protein tyrosine phosphatases[J]. Cancer Lett 1996;110(1-2):49-55.
20. Zeng Q, Si X, Horstmann H et al. Prenylation-dependent association of protein-tyrosine phosphatases PRL-1, -2, and -3 with the plasma membrane and the early endosome.[J].Biol Chem 2000;275(28):21444-21452.
21. Waterhouse PM, Wang MB, Lough T. Gene silencing as an adaptive defence against viruses[J]. Nature 2001 ;411 (6839):834-842.
22. Kozlov G, Cheng J, Ziomek E et al. Structural insights into molecular function of the metastasis-associated phosphatase PRL-3[ J ].Biol Chem 2004;279(12):11882-11889.
23. Wu X, Zeng H, Zhang X, et al. Phosphatase of regenerating liver -3 promotes motility and metastasis of mouse melanoma cells[J ] . Am J Pathol ,2004 ,164(6) :2 039 - 2 054.
24. Peng L , Jin G, WangL , et al . Identification of integrin alphal as an interacting protein of protein Tyrosine phosphatase PRL-3[J]. Bio-chem Biophys Res Commun, 2006, 342(1): 179-183.
25. Polte TR, Hanks SK. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas[J ]. Proc Natl Acad Sci USA ,1995 ,92(23) :10678 - 10682
26. Honda H, Oda H, Nakamoto T, et al. Cardiovascular anomaly impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas[J]. Nat Genet ,1998 ,19 (4):361 -365.
27. Liang F, Liang J, Wang WQ, et al. PRL-3 promotes cell invasion and proliferation by down regulation of Csk leading to Src activation [J ]. J Biol Chem ,2007 ,282 (8):5413 - 5 419.
28. Fiordalisi JJ, Keller PJ, Cox AD. PRL tyrosine phosphatases regulate rho family GTPases to promote invasion and motility[J ]. Cancer Res , 2006 ,66(6) :3153 - 3161..
29. Wang H, Quah SY, Dong JM, et al . PRL-3 down regulates PTEN expression and signals through P13K to promote epithelial-mesenchyma transition[J]. Cancer Res, 2007, 67(7) 2922-2926.
30. TomitaK, vanBokhovenA, van LeendersGJ, et al. Cadherin switching in human prostate cancer progression [ J]. Cancer Res, 2000,60:3650-3654.
31. Munoz - GuerraMF, Marazuela EG, Fernandez - ContrerasME, et al. P - cadherin exp ression reduced in squamous cell carcinoma of the oral cavity: an indicatior of poor p rognosis [ J ]. Cancer, 2005,103 (5) :960-969.
32. Taniuchi K, Nakagawa H, HosokawaM, et al. Overexp ressed P - cadherin /CDH3 promotesmotility of pancreatic cancer cells by interacting with p120ctn and activating rho -family GTPases [ J ]. Cancer Res,2005, 65 (8): 3092-3099
33. Shimazui T,Qosterwijk E,Akaza H et al.Expression of cadherin-6 as a novel diagnostic tool to predict prognosis of patients with E-cadherin-absent renal cell carcinoma.Clin Cancer Res1998;4(10);2419-2424.
34. Bauer R, Bosserhoff AK. Functional implication of truncated P-cadherin expression in malignant melanoma [ J]. Exp Mol Pathol,2006, 81: 224-230.
35. Bonilla C, Mason T, Long L, et al. E-cadherin polymorphisms and haplotypes influence risk for prostate cancer[ J]. Prostate, 2006, 66:546-556.
36. Charalabopoulos K, GogaliA, Dalavaga Y, et al. The clinical significance of soluble E - cadherin in nonsmall cell lung cancer [ J ]. Exp Oncol, 2006,28 (1): 83-85.
37. Muller T, Bain G, Wang X, et al . Regulation of ep -ithelial cell migration and tumor formation by beta - catenin signaling [ J ]. Exp Cell Res, 2002, 280: 119-133.
1.Elbashir SM,Harborth J.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J].Nature,2001,411,(6836):494.
2.Meng-TsaiWu,Wen -Hui Tsai,Wen -Tsan Chang et al.Simple and efficient DNA vector based RNAi systems in mammalian cells[J].Biochem Biophys Res Commun,2005,330(1):53-59.
3.Imamur T,Yokosuk O,OmatM,et al.Proteomic analysis of the TGF -b signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad 4 expression[J].Biochem Biophys Res Commun,2004,318(1):289-296.
4.Takeshita F,Ochiya T.Therapeutic potential of RNA interference against cancer[J].Cancer Sci,2006,97(8):689-696.
5.Cabot RA,Kuhholzer B,Chan AW,et al.Transgenic pigs pro-duced using in vitro matured occytes infected with a retroviral vector[J].Anim Biotechnol,2001,12(2):205-214
1. Kozlov G, Cheng J, Ziomek E, et al. Structural insights into molecular function of the metastasis-associated phosphatase PRL-3[J].J Biol Chem ,2004,279:11882-9
2. Kim KA, Song JS, Jee J, et al. Stru cture of human PRL-3, the phosphatase associated with cancer metastasis[J]. FEBS Lett ,2004,565:181-7..
3. Li Z, Zhan W, Wang Z, et al. Inhibition of PRL-3 gene expression in gastric cancer cell line SGC7901 viamicro RNA suppressed reduces peritoneal metastasis[J].Biochem Biophys Res Commun ,2006,348:229-237
4. Saha S, Bardelli A, Buckhaults P, et al. A phosphatase associated with metastasis of colorectal cancer[J]. Science ,2001,294 (5545): 1343-6
5. Bardelli A, Saha S, Sager JA, et al. PRL-3 expression in metastatic cancers[J].Clin Cancer Res ,2003,9:5607-15.
6. Zeng Q, Dong JM, Guo K, et al. PRL-3 and PRL-1 promote cell migration, invasion, and metastasis[J].Cancer Res ,2003, 63:2716-22.
7. Wu X, Zeng H, Zhang X, et al. Phosphatase of regenerating liver-3 promotes motility and metastasis of mouse melanoma cells[J]. Am J Pathhol ,2004,64:2039-54.
8. Guo K, Li J, Tang JP, et al. Catalytic Domain of PRL-3 plays an Essential Role in Tumor Metastasis: Formation of PRL-3 Tumors Inside the Blood Vessels[J].Cancer Biol Ther, 2004, In press .
9. Russo A , Bazan V , Migliavacca M , et al. DNA aneuploidy and high proliferative activity but not K-ras-2 mutations as independent predictors of clinical outcome in operable gast ric carcinoma : result s of a 5-year Gruppo Oncologico dell' Italia Meridonale (GDIM) prospective study[J]. Cancer ,2001 , 92 (2): 294-302.
1. Gregorieff A,Clevers H. Wnt signaling in the intestinal epithelium:from endoderm to cancer[J]. Genes Dev,2005,19 (8): 877- 890.
2. Moon RT, Bowerman B, Boutros M, et al. The Promise and perils of Wnt signaling through bata-catenin[J]. Science, 2002, 296(5573): 1644- 1646.
3. .Huber AH, Stewart DB,Laurents DV et,al. The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin.A possible mechanism for regulating cadherin turnover,J Biol Chem 2001,276(15):12301-12309.
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