骨桥蛋白与癌胚抗原相关粘附分子5在舌癌组织中的表达及相关性研究
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摘要
恶性肿瘤是经济日益发展的今天导致死亡的重要病因之一,目前全世界有超过2000万肿瘤患者,并以每年超过1000万肿瘤病人的速度新增,每年有600万患者因肿瘤死亡。发展中国家肿瘤患者占全世界的半数以上。在高收入国家因恶性肿瘤导致的死亡占总死亡率的20%,在贫困国家仅占10%。在发达国家及发展中国家肿瘤都是对公众健康事业的极大威胁。中国现已成为世界第二癌症高发国。因为酒精、烟草等因素的不良刺激,口腔癌高发,位于恶性肿瘤发病率的第八位[34]。在中国,口腔癌也是常见的头颈部肿瘤[5-8]。在口腔癌中,最常见的就是口腔舌鳞状细胞癌(oral squamous cell carcinoma, OTSCC)。由于舌是一个活动的器官,且具有丰富的血运和淋巴引流, OTSCC极易发生侵袭转移,临床进展和预后较其他的口腔癌差。在过去的数十年,OTSCC的发病率逐年上升,而治疗的临床疗效始终不佳,易发生肿瘤的早期侵袭和转移。
骨桥蛋白(Osteopontin, OPN)是一种广泛分布的多种功能分泌型钙结合磷酸化糖蛋白,对细胞的生长起着重要作用。它在许多组织和体液中存在,是重要的粘附和信号转导分子,在细胞粘附、骨转化、信号转导、血管重构、细胞免疫中都起着重要作用。最近的研究显示,OPN在多种肿瘤中均高表达,如乳腺癌、肺癌、胃癌、间皮瘤。推测OPN与许多细胞表面的受体蛋白结合后在肿瘤的恶性转化、发展、侵袭和转移的病理生理过程中发挥重要作用。有学者认为OPN可作为肿瘤基因治疗的靶点之一。
癌胚抗原相关细胞粘附分子-5(Carcinoembryonic Antigen-related Cell Adhesion Molecule5, CEACAM5)的域结构是N-A1-B1-A2-B2-A3-B3,它是人类组织分化和肿瘤细胞分化和增殖的重要调节分子。然而目前对CEACAM5的研究较少,它在OTSCC中的作用尚无研究,它和OPN在肿瘤中的相关性更无从得知。本研究拟以人OTSCC组织和舌鳞癌细胞系Tca8113细胞为研究对象,采用多种技术方法,明确OPN和CEACAM5在OTSCC中的表达,及是否存在协同作用,同时利用siRNA干扰技术沉默OPN和CEACAM5基因,探讨其对OTSCC增殖及侵袭特性的影响,将对制订更为合理的肿瘤基因治疗策略提供新的思路与有效靶点。
目的：
1.通过对人OTSCC组织及正常舌组织的免疫组化、免疫组化双标记及Real-time PCR等方法,观察OPN CEACAM5在舌癌及正常舌组织中表达情况的相关性,研究OPN与CEACAM5在组织的关系及相互作用。
2.通过免疫组化、免疫共沉淀、侵袭实验及siRNA干扰等方法,观察OPN与CEACAM5在人舌癌细胞株Tca8113及人口腔粘膜角质形成细胞(Human Oral Keratinocytes, HOK)(?)的表达情况及相关性,探讨OPN与CEACAM5在细胞中的关系及相互作用。
研究方法：
1.以OTSCC为研究对象,检测OTSCC组织中OPN与CEACAM5的表达及相互关系。
我们于2008-2010年在山东大学齐鲁医院口腔科共收集OTSCC80例及40例正常舌组织,正常舌组织的来源为与舌癌临近的正常舌组织。临床资料包括年龄、性别、肿瘤大小、肿瘤分期及肿瘤分化程度。应用免疫组化及免疫组化双标记方法检测OPN与CEACAM5在OTSCC及正常舌组织中的表达及定位,分析OPN与CEACAM5在OTSCC和正常舌组织中表达异同及其相关性,并进一步分析蛋白表达的不同与肿瘤的侵袭及分化的关系。
2.以OTSCC组织及正常组织为研究对象,进一步检测OPN与CEACAM5在OTSCC与正常舌组织中表达的差异。
应用Real-time PCR方法进一步检测舌癌组织及正常舌组织中OPN及CEACAM5的表达情况。取小量舌癌及正常舌组织块,应用Trizol方法提取组织中的总RNA,在一定的反应体系中逆转录成cDNA, Real-time PCR方法检测舌癌组织及正常舌组织块中OPN与CEACAM5在mRNA水平的表达情况。
3.人舌鳞状细胞癌细胞系Tca8113的培养及HOK的培养及生物学特性。
口腔舌鳞状细胞癌细胞系Tca8113在37℃,5%CO2饱和湿度条件下,培养于含15%胎牛血清,100IU/ml青霉素、100ug/ml链霉素的1640培养液中。
HOK细胞的原代培养：无菌条件下收集人正常粘膜组织,放置于含OKM的培养皿中,用含三抗的(青霉素100mg/l,链霉素100mg/L,两性霉素B3mg/L)的D-Hanks液洗净血迹,剪成碎块,大小约5mm*5mm,去除粘膜下组织,0.25%Dispase (Sigma)4℃消化14小时,将粘膜层与粘膜下层用眼科剪分开,粘膜层用37℃0.25%胰蛋白酶(Gibco)振荡消化5分钟,收集上清,加入OKM培养基终止消化,制成细胞悬液置于OKM培养液中培养。
用相差显微镜每天进行细胞形态学观察,同时绘制细胞生长曲线。
4.以Tca8113细胞及HOK细胞为研究对象,在基因水平检测0PN与CEACAM5在两种细胞中的表达情况。
收集生长旺盛的Tca8113细胞及HOK细胞。Trizol方法提取细胞总RN A,在一定的反应体系中逆转录成cDN A, Real-time PCR方法检测OPN与CEACAM5两种蛋白分子在mRNA水平的表达情况。
5.以Tca8113细胞及HOK细胞为研究对象,应用免疫共沉淀技术,检测OPN及CEACAM两种分子分别在Tca8113细胞及HOK细胞中的表达及相互作用。
6.以Tca8113细胞为研究对象,利用RNAi技术干扰0PN基因及CEACAM5基因的表达。
用OPN-siRNA及CEACAM5-siRNA分别及共同对Tca8113细胞中OPN及CEACAM5基因进行沉默,利用Real-timePCR及Western blot检测干扰效率,利用MTT法检测OPN及CEACAM5基因沉默后对Tca8113(?)(?)胞增殖能力的影响。
7.利用细胞侵袭实验检测OPN及CEACAM5两种分子对Tca8113细胞侵袭力的影响。
于干扰后第3天取不同组别的Tca8113细胞,调整细胞悬液浓度为2*106,将100ul的细胞悬液加入Transwell小室中,评价两种蛋白对Tca8113细胞侵袭力的影响。
结果：
1.在舌癌组织及正常舌组织中均有CEACAM5及OPN两种蛋白的表达,但是表达方式不同。在正常舌组织中,OPN蛋白主要表达在细胞浆中,而CEACAM5则表达于细胞膜上；在舌癌组织中,两种蛋白分子的表达及表达强度均强于正常舌组织,而且这两种蛋白分子均表达于细胞浆中。通过对正常舌组织和舌鳞状细胞癌两种组织的免疫组化双标记发现,在正常舌组织中,没有发现OPN和CEACAM5两种蛋白分子的共表达；在舌鳞状细胞癌组织中,可以发现OPN和CEACAM5共同表达于细胞浆中,而且这种共表达率及表达强度与肿瘤的分化侵袭程度相关。
2.通过Real-timePCR发现,在舌癌组织中,OPN及CEACAM5的表达均明显强于正常舌组织,这一结果与免疫组化结果相吻合。
3.倒置显微镜下观察发现,Tca8113细胞均呈贴壁生长,形成单层结构。细胞呈多角形及梭形,轮廓清晰,细胞间结构紧密,生长旺盛。原代培养的HOK细胞,刚获取的细胞形态多为多角形扁平细胞及球形细胞。接种24-48小时后贴壁生长,细胞为圆形,随着生长,细胞伸出短小伪足,细胞呈扁平多角形,折光性低,形成多个小克隆。培养的细胞具有扁平不规则多角形,核圆,清晰,细胞质丰富、均匀。接种5天后细胞增殖加速,数量增多,克隆增大,大约2-3周细胞长满培养瓶。传代细胞24h左右可贴壁,第2-3代细胞形态与原代相似,从第四代起,细胞增殖速度减慢。
4.分别收集生长旺盛的Tca8113及HOK细胞,分别提取总RNA.利用Real-timePCR方法检测OPN基因mRNA及CEACAM5基因mRNA的表达。结果表明在Tca8113细胞中OPN基因nRNA及CEACAM5基因mRNA的表达较HOK细胞强。
5.收集生长旺盛的Tca8113及HOK细胞,分别提取总蛋白,利用western blot检测OPN蛋白及CEACAM5蛋白的表达。结果表明在Tca8113及HOK细胞中,均可表达OPN及CEACAM5分子,且在Tca8113细胞中的表达较HOK细胞强；免疫共沉淀结果显示Tca8113细胞中OPN及CEACAM5可以相互结合,而在HOK细胞中则无相关性。
6.利用siRNA干扰Tca8113细胞中OPN及CEACAM5的表达,检测干扰效率,应用细胞侵袭实验分析单独干扰OPN、CEACAM5及联合干扰时对Tca8113细胞的作用,结果表明单独干扰OPN及CEACAM5均能减弱Tca8113细胞的侵袭力,联合干扰时Tca8113细胞侵袭力减弱更为明显。
结论：
1.在正常舌组织中CEACAM5表达在细胞膜,而在舌癌组织中该分子则表达在细胞浆中,CEACAM分子这种表达位置的变化可能在肿瘤的分化转移中起着重要的作用。
2.OPN及CEACAM5两种分子均在在舌癌中表达,并且均表达在细胞浆中,在舌癌组织中两种分子可能作为一个功能复合体起作用,而且共表达的强弱与肿瘤的分化程度及侵袭力呈正相关。
3.应用基因干扰技术,在Tca8113细胞验证了OPN与CEACAM5可能作为一个功能复合体在肿瘤的增殖和侵袭中起着重要作用,可能作为未来基因治疗的一个靶点。
Background
Cancer is one of the most common cause of mortality and morbidity nowadays. More than10million new patients and over6million deaths each year all over the world. More than20million people worldwide live with a diagnosis of malignant tumor,and over half all cancer patients live in developing countries.Cancer is responsible for more than twenty percent of all death cases in high income countries and about ten percent in low income countries. Cancer is one of the major threats to the public health in the developed world and increasingly in the developing world. While China has become the world's second largest high cancer incidence country.Due to tobacco and alcohol or other risk factor, the occurrence of oral cancer is the eighth most common cancer all over the world and it is significant component of the globe burden of cancer.In China.due to betel quid chewing, tobacco and alcohol or other similar stimulating factor, oral cancer is very common in head and neck malignancy. Cancer of the tongue is a malignant tumor that develops on the tongue or at the base of the tongue at the back of the throat.,and in tongue cancer filed,the most common is oral squamous cell carcinoma (OTSCC).OTSCC is increasingly regarded as a biologically different entity and show variations in clinical progression and prognosis compared to cancer affecting other oral sites. Because the tongue is a move organ,it has rich lymphatic network.it is more aggressive and generally associated with a higher rate of metastasis.Over the past three decades,the incidence of OTSCC has increased in China,the most important reasons is its unusual histologic makeup caused failure in clinical treatment are invasion,metastasis and resistance.
Osteopontin(OPN) which is an acidic non-collagenousglycoprotein is distributed in the extracellular matrix that plays a pivotal role in the establishment of micro-environmentfor cell growth. OPN is a very important adhesion and signal transduction moleculelt, and there are several biological functions in cell adhesion, mineralization of bone tissue, signal transduction, vascular remodeling and cell-mediated immunity and it is widely distributed invarious body fluids and tissues, including urine, blood, uterus, white blood cells, placenta, smoothmuscle, kidney, and some tumor tissues. Recently people have found that, OPN is overexpressed in many kinds of cancers, such as breast cancer,lung cancer,stomach cancer,mesothelioma,melanoma,ovarian cancer,colorectal cancer and stomach cancer and so on.The fact is that interacting with several cell surface receptors which are ubiquitously expressed makes OPN an active player participant in many pathological and physiological processes,mainly involving the malignant transformation.invasion,metastasis and development and so on in many kinds of cancers.Someone even advocated that OPN can be seen as a target for molecular targeted therapies of many types of cancers.
CEACAM5has the N-A1-B1-A2-B2-A3-B3domain structure and is one of important mediators in remodeling of diverse human tissues, and modulators of tumor cell proliferation and differentiation. Nowadays,there are less research on CEACAM5. Its biological function is not that clear. There is no research on the role of it in OTSCC.The correlation between OPN and CEACAM5in OTSCC is not clearly. This study is intended to research their expression, and whether there are synergies in OTSCC tissue and Tca8113cell by many kinds of technical methods,At the same time, we use siRNA interference technology to silence OPN and CEACAM5genes, to explore the OTSCC proliferation, apoptosis and invasion characteristics influence.We hope the two molecules will be a new thinking and effective targets to develop a more reasonable tumor gene therapy strategies.
Objective
1. In order to investigate the expression and co-localization of OPN and CEACAM5in oral tougue squamous cell carcinoma (OTSCC) and analyze the relationship between OPN and CEACAM5.then to discuss the effect of overexpression of OPN and CEACAM5in OTSCC clinicopathologic parameters.
2. To determine the expression patterns of CEACAM5and OPN on tougue squamous cell carcinoma cell line-Tca8113and human oral keratinocytes(HOK),and investigate the effect of OPN and CEACAM5on cell growth,invasion in Tca8113cells and HOK,then discuss the effect of the two molecule on metatasis and invade of OTSCC.
Methods:
1. Evaluation of OPN and CEACAM5expression in OTSCC tissue. One hundred and twenty cases including eighty OTSCC tissues and forty normal tongue tissues were collected from Qilu hospital of Shandong University from2008to2010.The clinical data included age,sex of the patents and size, stage and histologicalclassification of tumor. The expression were assessed based on the immunohistochemical and immunohistochemical double staining technique examination in paraffin-embedded tissue sections.The correlation between the expression of OPN and CEACAM5was investigated.The correlation between the expression of the two molecules and various clinicopathologic parameters was examinated.
2. The level of messenger RNA of OPN with CEACAM5in OTSCC and normal fresh frigorific samples were tested by real-time PCR. Messenger RNA content were quantitatively determined by image analyse system.The relationships between OTSCC tissues and normal tissues of mRNA of OPN and CEACAM5levels were evaluated,and the relationship with clinical and pathologic parameters was also analyzed.
3. Cuture of Human Oral Keratinocytes(HOK)and Tca8113cell to study the expression of OPN and CEACAM5in vitro.
Tca8113cells were cultured in1640Medium containing15%fetal bovine serum,100mg/l penicillin, and100mg/l streptomycin, at37℃in5%CO2atmosphere and the steps according to the manufacturer's instructions.
HOK cells in primary culture.Collected normal mucosa under sterile conditions, then placed in a petri dish containing OKM. Used D-Hanks containing three antibiotics (penicillin100mg/l. streptomycin100mg/l, amphotericin B3mg/L) to wash blood.then cut the tissue into pieces, about the size of5mm*5mm, removed of the mucosa tissue, then put into0.25%Dispase (Sigma)4℃digest14hours. The surface epithelial and subepithelial layer shear separated by ophthalmic scissors. Epithelial surface shock digested with37°C of0.25%trypsin (Gibco) for5minutes, and collected the supernatant, Added OKN in to terminate the digestion. Placed cell suspension in the culture medium. Used phase contrast microscopy to observe cell morphology and draw cell growth curve.
4. Use Tca8113cells and HOK cells as the research object to detect OPN and CEACAM5expression at the gene level.
Use vigorous growth Tca8113cells and HOK cells. Total RNA was extracted with Trizol reagent. Detected OPN and CEACAM5molecules expression at the mRNA level situation, by real-time PCR method.
5. Western blot and immune coprecipitation technique were used to detect interaction of OPN and CEACAM5in Tca8113cells and HOK cell.
6. Utilize RNA interference to silence gene expression of OPN and CEACAM5in Tca8113cells.
SiRNA of OPN and CEACAM5, respectively and jointly carried on of Tca8113cells to silence gene expression. Detected the efficiency by the Real-timePCR and Western blot. Then use MTT to assay Tca8113cell proliferative capacity the OPN and CEACAM5after gene silencing.
7. By cell invasion experiment to detect cell invasion force to detect the roles of OPN and CEACAM5in Tca8113cells. In three days after interference, adjust the concentration of cell suspension of Tca8113cells in different groups to2*106.then put100ul cell suspension into the Transwell chamber to evaluation two proteins's role in Tca8113cell of invasion force.
Result:
1. OPN and CEACAM5were both expressed In the OTSCC normal tongue tissue while the positions of expression were different. In normal tongue, OPN protein mainly expressed in the cytoplasm, and CEACAM5was presented in cell membrane. In OTSCC. the two kinds of protein molecule were both presented in cytoplasm and expression were stronger than normal tongue tissue. The use of double-labeling immunohistochemistry confirmed OPN and CEACAM5were co-expressed and co-expression and expression intensity were degree of tumor differentiation related.
2. By real-time PCR,we found in OTSCC tissue, the expression of OPN and CEACAM5strips were stronger than normal, while this result coincided with the results of immunohistochemistry.
3. Observed under the inverted microscope, Tca8113cells showed adherent growth, to form a single-layer structure. The cells were polygonal and fusiform outline a clear, close cell structure, vigorous growth. Primary cultured HOK cell which were just get polygonal, adherented growth in24-48hours after inoculation,, Along with the growth, cell stretch out short pseudopodia, the cells are flat polygon, and refraction sex low and formed multiple small clones, form multiple small cloning. The cultured cells were flat irregular polygon, they had round and clear nuclear, and abundant but well-distributed cytoplasm. After5days, the cells proliferated accelerated, in2-3weeks cells covered flasks. Passage cells adherented around24h.The2-3generation cells'morphology was similar to the primary. Since the fourth-generation, cell proliferation slowed.
4.Vigorous growth Tca8113and HOK cells were collected, were extracted total RNA. Detected the OPN gene mRNA and CEACAM5of gene mRNA by RT-PCR method. The results showed that the level of OPN gene mRNA and CEACAM5of mRNA was higher in Tca8113cells compared with HOK cells.
5. Vigorous growth Tca8113and HOK cells were collected, were extracted total protein. Detected the OPN and CEACAM5expression by western blot method. The results showed that in the Tca8113and HOK cells both expressed OPN and CEACAM5molecules, and level of expression was higher in Tca8113cells compared with HOK cells. The coimmunoprecipitation results of and HOK cells showed the OPN and CEACAM5combined with each other in Tca8113cells, while in HOK cells no correlation.
6. Using siRNA interference the expression of OPN and CEACAM5in Tca8113cells, and detected the efficiency of interference. Applicated cell invade experiment analyzed separately interference OPN. CEACAM5and combined interference in Tca8113cells. The results showed that interference OPN and CEACAM5respectively can both weaken the Tca8113cells aggressivity, while combined interference of the two molecule weakened more obviously.
Conclusion
1. CEACAM5expressed in the cell membrane in normal tongue tissue, while in OTSCC tissue was expressed in the cytoplasm. CEACAM5molecule such expression change of the position may be provided in the differentiation and metastasis of the tumor.
2. The OPN and CEACAM5molecules were both expressed in the cytoplasm in OTSCC tissue. The two molecules may function as a function of complex, and was positively correlated co-expression of the strength and the degree of tumor differentiation and invasiveness.
3. The use of RNA interference technology, we confirmed OPN and CEACAM5play a role in proliferation and metastasis of Tca8113cells. So the study of OPN and CEACAM5maybe a new target in searching drug treatment of OTSCC.
骨桥蛋白(Osteopontin, OPN)是一种广泛分布的多种功能分泌型钙结合磷酸化糖蛋白,对细胞的生长起着重要作用。它在许多组织和体液中存在,是重要的粘附和信号转导分子,在细胞粘附、骨转化、信号转导、血管重构、细胞免疫中都起着重要作用。最近的研究显示,OPN在多种肿瘤中均高表达,如乳腺癌、肺癌、胃癌、间皮瘤。推测OPN与许多细胞表面的受体蛋白结合后在肿瘤的恶性转化、发展、侵袭和转移的病理生理过程中发挥重要作用。有学者认为OPN可作为肿瘤基因治疗的靶点之一。
癌胚抗原相关细胞粘附分子-5(Carcinoembryonic Antigen-related Cell Adhesion Molecule5, CEACAM5)的域结构是N-A1-B1-A2-B2-A3-B3,它是人类组织分化和肿瘤细胞分化和增殖的重要调节分子。然而目前对CEACAM5的研究较少,它在OTSCC中的作用尚无研究,它和OPN在肿瘤中的相关性更无从得知。本研究拟以人OTSCC组织和舌鳞癌细胞系Tca8113细胞为研究对象,采用多种技术方法,明确OPN和CEACAM5在OTSCC中的表达,及是否存在协同作用,同时利用siRNA干扰技术沉默OPN和CEACAM5基因,探讨其对OTSCC增殖及侵袭特性的影响,将对制订更为合理的肿瘤基因治疗策略提供新的思路与有效靶点。
目的：
1.通过对人OTSCC组织及正常舌组织的免疫组化、免疫组化双标记及Real-time PCR等方法,观察OPN CEACAM5在舌癌及正常舌组织中表达情况的相关性,研究OPN与CEACAM5在组织的关系及相互作用。
2.通过免疫组化、免疫共沉淀、侵袭实验及siRNA干扰等方法,观察OPN与CEACAM5在人舌癌细胞株Tca8113及人口腔粘膜角质形成细胞(Human Oral Keratinocytes, HOK)(?)的表达情况及相关性,探讨OPN与CEACAM5在细胞中的关系及相互作用。
研究方法：
1.以OTSCC为研究对象,检测OTSCC组织中OPN与CEACAM5的表达及相互关系。
我们于2008-2010年在山东大学齐鲁医院口腔科共收集OTSCC80例及40例正常舌组织,正常舌组织的来源为与舌癌临近的正常舌组织。临床资料包括年龄、性别、肿瘤大小、肿瘤分期及肿瘤分化程度。应用免疫组化及免疫组化双标记方法检测OPN与CEACAM5在OTSCC及正常舌组织中的表达及定位,分析OPN与CEACAM5在OTSCC和正常舌组织中表达异同及其相关性,并进一步分析蛋白表达的不同与肿瘤的侵袭及分化的关系。
2.以OTSCC组织及正常组织为研究对象,进一步检测OPN与CEACAM5在OTSCC与正常舌组织中表达的差异。
应用Real-time PCR方法进一步检测舌癌组织及正常舌组织中OPN及CEACAM5的表达情况。取小量舌癌及正常舌组织块,应用Trizol方法提取组织中的总RNA,在一定的反应体系中逆转录成cDNA, Real-time PCR方法检测舌癌组织及正常舌组织块中OPN与CEACAM5在mRNA水平的表达情况。
3.人舌鳞状细胞癌细胞系Tca8113的培养及HOK的培养及生物学特性。
口腔舌鳞状细胞癌细胞系Tca8113在37℃,5%CO2饱和湿度条件下,培养于含15%胎牛血清,100IU/ml青霉素、100ug/ml链霉素的1640培养液中。
HOK细胞的原代培养：无菌条件下收集人正常粘膜组织,放置于含OKM的培养皿中,用含三抗的(青霉素100mg/l,链霉素100mg/L,两性霉素B3mg/L)的D-Hanks液洗净血迹,剪成碎块,大小约5mm*5mm,去除粘膜下组织,0.25%Dispase (Sigma)4℃消化14小时,将粘膜层与粘膜下层用眼科剪分开,粘膜层用37℃0.25%胰蛋白酶(Gibco)振荡消化5分钟,收集上清,加入OKM培养基终止消化,制成细胞悬液置于OKM培养液中培养。
用相差显微镜每天进行细胞形态学观察,同时绘制细胞生长曲线。
4.以Tca8113细胞及HOK细胞为研究对象,在基因水平检测0PN与CEACAM5在两种细胞中的表达情况。
收集生长旺盛的Tca8113细胞及HOK细胞。Trizol方法提取细胞总RN A,在一定的反应体系中逆转录成cDN A, Real-time PCR方法检测OPN与CEACAM5两种蛋白分子在mRNA水平的表达情况。
5.以Tca8113细胞及HOK细胞为研究对象,应用免疫共沉淀技术,检测OPN及CEACAM两种分子分别在Tca8113细胞及HOK细胞中的表达及相互作用。
6.以Tca8113细胞为研究对象,利用RNAi技术干扰0PN基因及CEACAM5基因的表达。
用OPN-siRNA及CEACAM5-siRNA分别及共同对Tca8113细胞中OPN及CEACAM5基因进行沉默,利用Real-timePCR及Western blot检测干扰效率,利用MTT法检测OPN及CEACAM5基因沉默后对Tca8113(?)(?)胞增殖能力的影响。
7.利用细胞侵袭实验检测OPN及CEACAM5两种分子对Tca8113细胞侵袭力的影响。
于干扰后第3天取不同组别的Tca8113细胞,调整细胞悬液浓度为2*106,将100ul的细胞悬液加入Transwell小室中,评价两种蛋白对Tca8113细胞侵袭力的影响。
结果：
1.在舌癌组织及正常舌组织中均有CEACAM5及OPN两种蛋白的表达,但是表达方式不同。在正常舌组织中,OPN蛋白主要表达在细胞浆中,而CEACAM5则表达于细胞膜上；在舌癌组织中,两种蛋白分子的表达及表达强度均强于正常舌组织,而且这两种蛋白分子均表达于细胞浆中。通过对正常舌组织和舌鳞状细胞癌两种组织的免疫组化双标记发现,在正常舌组织中,没有发现OPN和CEACAM5两种蛋白分子的共表达；在舌鳞状细胞癌组织中,可以发现OPN和CEACAM5共同表达于细胞浆中,而且这种共表达率及表达强度与肿瘤的分化侵袭程度相关。
2.通过Real-timePCR发现,在舌癌组织中,OPN及CEACAM5的表达均明显强于正常舌组织,这一结果与免疫组化结果相吻合。
3.倒置显微镜下观察发现,Tca8113细胞均呈贴壁生长,形成单层结构。细胞呈多角形及梭形,轮廓清晰,细胞间结构紧密,生长旺盛。原代培养的HOK细胞,刚获取的细胞形态多为多角形扁平细胞及球形细胞。接种24-48小时后贴壁生长,细胞为圆形,随着生长,细胞伸出短小伪足,细胞呈扁平多角形,折光性低,形成多个小克隆。培养的细胞具有扁平不规则多角形,核圆,清晰,细胞质丰富、均匀。接种5天后细胞增殖加速,数量增多,克隆增大,大约2-3周细胞长满培养瓶。传代细胞24h左右可贴壁,第2-3代细胞形态与原代相似,从第四代起,细胞增殖速度减慢。
4.分别收集生长旺盛的Tca8113及HOK细胞,分别提取总RNA.利用Real-timePCR方法检测OPN基因mRNA及CEACAM5基因mRNA的表达。结果表明在Tca8113细胞中OPN基因nRNA及CEACAM5基因mRNA的表达较HOK细胞强。
5.收集生长旺盛的Tca8113及HOK细胞,分别提取总蛋白,利用western blot检测OPN蛋白及CEACAM5蛋白的表达。结果表明在Tca8113及HOK细胞中,均可表达OPN及CEACAM5分子,且在Tca8113细胞中的表达较HOK细胞强；免疫共沉淀结果显示Tca8113细胞中OPN及CEACAM5可以相互结合,而在HOK细胞中则无相关性。
6.利用siRNA干扰Tca8113细胞中OPN及CEACAM5的表达,检测干扰效率,应用细胞侵袭实验分析单独干扰OPN、CEACAM5及联合干扰时对Tca8113细胞的作用,结果表明单独干扰OPN及CEACAM5均能减弱Tca8113细胞的侵袭力,联合干扰时Tca8113细胞侵袭力减弱更为明显。
结论：
1.在正常舌组织中CEACAM5表达在细胞膜,而在舌癌组织中该分子则表达在细胞浆中,CEACAM分子这种表达位置的变化可能在肿瘤的分化转移中起着重要的作用。
2.OPN及CEACAM5两种分子均在在舌癌中表达,并且均表达在细胞浆中,在舌癌组织中两种分子可能作为一个功能复合体起作用,而且共表达的强弱与肿瘤的分化程度及侵袭力呈正相关。
3.应用基因干扰技术,在Tca8113细胞验证了OPN与CEACAM5可能作为一个功能复合体在肿瘤的增殖和侵袭中起着重要作用,可能作为未来基因治疗的一个靶点。
Background
Cancer is one of the most common cause of mortality and morbidity nowadays. More than10million new patients and over6million deaths each year all over the world. More than20million people worldwide live with a diagnosis of malignant tumor,and over half all cancer patients live in developing countries.Cancer is responsible for more than twenty percent of all death cases in high income countries and about ten percent in low income countries. Cancer is one of the major threats to the public health in the developed world and increasingly in the developing world. While China has become the world's second largest high cancer incidence country.Due to tobacco and alcohol or other risk factor, the occurrence of oral cancer is the eighth most common cancer all over the world and it is significant component of the globe burden of cancer.In China.due to betel quid chewing, tobacco and alcohol or other similar stimulating factor, oral cancer is very common in head and neck malignancy. Cancer of the tongue is a malignant tumor that develops on the tongue or at the base of the tongue at the back of the throat.,and in tongue cancer filed,the most common is oral squamous cell carcinoma (OTSCC).OTSCC is increasingly regarded as a biologically different entity and show variations in clinical progression and prognosis compared to cancer affecting other oral sites. Because the tongue is a move organ,it has rich lymphatic network.it is more aggressive and generally associated with a higher rate of metastasis.Over the past three decades,the incidence of OTSCC has increased in China,the most important reasons is its unusual histologic makeup caused failure in clinical treatment are invasion,metastasis and resistance.
Osteopontin(OPN) which is an acidic non-collagenousglycoprotein is distributed in the extracellular matrix that plays a pivotal role in the establishment of micro-environmentfor cell growth. OPN is a very important adhesion and signal transduction moleculelt, and there are several biological functions in cell adhesion, mineralization of bone tissue, signal transduction, vascular remodeling and cell-mediated immunity and it is widely distributed invarious body fluids and tissues, including urine, blood, uterus, white blood cells, placenta, smoothmuscle, kidney, and some tumor tissues. Recently people have found that, OPN is overexpressed in many kinds of cancers, such as breast cancer,lung cancer,stomach cancer,mesothelioma,melanoma,ovarian cancer,colorectal cancer and stomach cancer and so on.The fact is that interacting with several cell surface receptors which are ubiquitously expressed makes OPN an active player participant in many pathological and physiological processes,mainly involving the malignant transformation.invasion,metastasis and development and so on in many kinds of cancers.Someone even advocated that OPN can be seen as a target for molecular targeted therapies of many types of cancers.
CEACAM5has the N-A1-B1-A2-B2-A3-B3domain structure and is one of important mediators in remodeling of diverse human tissues, and modulators of tumor cell proliferation and differentiation. Nowadays,there are less research on CEACAM5. Its biological function is not that clear. There is no research on the role of it in OTSCC.The correlation between OPN and CEACAM5in OTSCC is not clearly. This study is intended to research their expression, and whether there are synergies in OTSCC tissue and Tca8113cell by many kinds of technical methods,At the same time, we use siRNA interference technology to silence OPN and CEACAM5genes, to explore the OTSCC proliferation, apoptosis and invasion characteristics influence.We hope the two molecules will be a new thinking and effective targets to develop a more reasonable tumor gene therapy strategies.
Objective
1. In order to investigate the expression and co-localization of OPN and CEACAM5in oral tougue squamous cell carcinoma (OTSCC) and analyze the relationship between OPN and CEACAM5.then to discuss the effect of overexpression of OPN and CEACAM5in OTSCC clinicopathologic parameters.
2. To determine the expression patterns of CEACAM5and OPN on tougue squamous cell carcinoma cell line-Tca8113and human oral keratinocytes(HOK),and investigate the effect of OPN and CEACAM5on cell growth,invasion in Tca8113cells and HOK,then discuss the effect of the two molecule on metatasis and invade of OTSCC.
Methods:
1. Evaluation of OPN and CEACAM5expression in OTSCC tissue. One hundred and twenty cases including eighty OTSCC tissues and forty normal tongue tissues were collected from Qilu hospital of Shandong University from2008to2010.The clinical data included age,sex of the patents and size, stage and histologicalclassification of tumor. The expression were assessed based on the immunohistochemical and immunohistochemical double staining technique examination in paraffin-embedded tissue sections.The correlation between the expression of OPN and CEACAM5was investigated.The correlation between the expression of the two molecules and various clinicopathologic parameters was examinated.
2. The level of messenger RNA of OPN with CEACAM5in OTSCC and normal fresh frigorific samples were tested by real-time PCR. Messenger RNA content were quantitatively determined by image analyse system.The relationships between OTSCC tissues and normal tissues of mRNA of OPN and CEACAM5levels were evaluated,and the relationship with clinical and pathologic parameters was also analyzed.
3. Cuture of Human Oral Keratinocytes(HOK)and Tca8113cell to study the expression of OPN and CEACAM5in vitro.
Tca8113cells were cultured in1640Medium containing15%fetal bovine serum,100mg/l penicillin, and100mg/l streptomycin, at37℃in5%CO2atmosphere and the steps according to the manufacturer's instructions.
HOK cells in primary culture.Collected normal mucosa under sterile conditions, then placed in a petri dish containing OKM. Used D-Hanks containing three antibiotics (penicillin100mg/l. streptomycin100mg/l, amphotericin B3mg/L) to wash blood.then cut the tissue into pieces, about the size of5mm*5mm, removed of the mucosa tissue, then put into0.25%Dispase (Sigma)4℃digest14hours. The surface epithelial and subepithelial layer shear separated by ophthalmic scissors. Epithelial surface shock digested with37°C of0.25%trypsin (Gibco) for5minutes, and collected the supernatant, Added OKN in to terminate the digestion. Placed cell suspension in the culture medium. Used phase contrast microscopy to observe cell morphology and draw cell growth curve.
4. Use Tca8113cells and HOK cells as the research object to detect OPN and CEACAM5expression at the gene level.
Use vigorous growth Tca8113cells and HOK cells. Total RNA was extracted with Trizol reagent. Detected OPN and CEACAM5molecules expression at the mRNA level situation, by real-time PCR method.
5. Western blot and immune coprecipitation technique were used to detect interaction of OPN and CEACAM5in Tca8113cells and HOK cell.
6. Utilize RNA interference to silence gene expression of OPN and CEACAM5in Tca8113cells.
SiRNA of OPN and CEACAM5, respectively and jointly carried on of Tca8113cells to silence gene expression. Detected the efficiency by the Real-timePCR and Western blot. Then use MTT to assay Tca8113cell proliferative capacity the OPN and CEACAM5after gene silencing.
7. By cell invasion experiment to detect cell invasion force to detect the roles of OPN and CEACAM5in Tca8113cells. In three days after interference, adjust the concentration of cell suspension of Tca8113cells in different groups to2*106.then put100ul cell suspension into the Transwell chamber to evaluation two proteins's role in Tca8113cell of invasion force.
Result:
1. OPN and CEACAM5were both expressed In the OTSCC normal tongue tissue while the positions of expression were different. In normal tongue, OPN protein mainly expressed in the cytoplasm, and CEACAM5was presented in cell membrane. In OTSCC. the two kinds of protein molecule were both presented in cytoplasm and expression were stronger than normal tongue tissue. The use of double-labeling immunohistochemistry confirmed OPN and CEACAM5were co-expressed and co-expression and expression intensity were degree of tumor differentiation related.
2. By real-time PCR,we found in OTSCC tissue, the expression of OPN and CEACAM5strips were stronger than normal, while this result coincided with the results of immunohistochemistry.
3. Observed under the inverted microscope, Tca8113cells showed adherent growth, to form a single-layer structure. The cells were polygonal and fusiform outline a clear, close cell structure, vigorous growth. Primary cultured HOK cell which were just get polygonal, adherented growth in24-48hours after inoculation,, Along with the growth, cell stretch out short pseudopodia, the cells are flat polygon, and refraction sex low and formed multiple small clones, form multiple small cloning. The cultured cells were flat irregular polygon, they had round and clear nuclear, and abundant but well-distributed cytoplasm. After5days, the cells proliferated accelerated, in2-3weeks cells covered flasks. Passage cells adherented around24h.The2-3generation cells'morphology was similar to the primary. Since the fourth-generation, cell proliferation slowed.
4.Vigorous growth Tca8113and HOK cells were collected, were extracted total RNA. Detected the OPN gene mRNA and CEACAM5of gene mRNA by RT-PCR method. The results showed that the level of OPN gene mRNA and CEACAM5of mRNA was higher in Tca8113cells compared with HOK cells.
5. Vigorous growth Tca8113and HOK cells were collected, were extracted total protein. Detected the OPN and CEACAM5expression by western blot method. The results showed that in the Tca8113and HOK cells both expressed OPN and CEACAM5molecules, and level of expression was higher in Tca8113cells compared with HOK cells. The coimmunoprecipitation results of and HOK cells showed the OPN and CEACAM5combined with each other in Tca8113cells, while in HOK cells no correlation.
6. Using siRNA interference the expression of OPN and CEACAM5in Tca8113cells, and detected the efficiency of interference. Applicated cell invade experiment analyzed separately interference OPN. CEACAM5and combined interference in Tca8113cells. The results showed that interference OPN and CEACAM5respectively can both weaken the Tca8113cells aggressivity, while combined interference of the two molecule weakened more obviously.
Conclusion
1. CEACAM5expressed in the cell membrane in normal tongue tissue, while in OTSCC tissue was expressed in the cytoplasm. CEACAM5molecule such expression change of the position may be provided in the differentiation and metastasis of the tumor.
2. The OPN and CEACAM5molecules were both expressed in the cytoplasm in OTSCC tissue. The two molecules may function as a function of complex, and was positively correlated co-expression of the strength and the degree of tumor differentiation and invasiveness.
3. The use of RNA interference technology, we confirmed OPN and CEACAM5play a role in proliferation and metastasis of Tca8113cells. So the study of OPN and CEACAM5maybe a new target in searching drug treatment of OTSCC.
引文
[1]World health organization.The world report 2004:Changing History.Geneva;WHO,2004.
[2]B.W. Stewart. P. Kleihues (Eds.), World cancer report, WHO International Agency for Research on Cancer. Lyon (2003)
[3]Petersen PE. The world oral health report 2003:continuous improvement of oral health in the 21st century-the approach of the WHO global oral health programme. Community Dent Oral Epidemiol 2003; 31(Supp.1):3-24.
[4]P.E. Petersen. D. Bourgeois, D. Bratthall, H. Ogawa.Oral health information systems-towards measuring progress in oral health promotion and disease prevention Bull World Health Organ,83 (2005), pp.686-693
[5]J.H.C Ho An epidemiologic and clinical study of nasopharyngeal carcinoma Int J Radiat Oncol Biol Phys.4 (1978). pp.183-198
[6]D.P Huang. J.H.C Ho. W.K Chan. W.H Lau. M Lui Cytogenetics of undifferentiated nasopharyngeal carcinoma xenografts from southern Chinese Int J Cancer,43 (1989). pp.936-939
[7]M.L Lung, W.P Lam. J Sham et al. Detection and prevalence of the "f" variant of Epstein-Barr virus in Southern China Virology,185 (1) (1991), pp.67-71.
[8]P.M.L Teo, W.Y Lee. P Yu The prognostic significance of parapharyngeal tumour involvement in nasopharyngeal carcinoma Radiother Oncol,39 (1996), pp.209-221.
[9]Regezi JA, Sciubba JJ. Jordan RCK. Oral pathology:clinical, pathologic correlations.5th ed. St Louis (MO):Saunders Elsevier:2008.
[10]Rautava J, Luukkaa M, Heikinheimo K. Alin J, Grenman R, Happonen RP. Squamous cell carcinomas arising from different types of oral epithelia differ in their tumor and patient characteristics and survival. Oral Oncol 2007; 43(9): 911-9.
[11]Sathyan KM, Sailasree R, Jayasurya R, Lakshminarayanan K, Abraham T.Nalinakumari KR, et al. Carcinoma of tongue and the buccal mucosa represent different biological subentities of the oral carcinoma. J Cancer Res Clin Oncol 2006; 132 (9):601-9.
[12]Rusthoven K, Ballonoff A. Raben D, Chen C. Poor prognosis in patients withstage Ⅰ and Ⅱ oral tongue squamous cell carcinoma. Cancer 2008; 112 (2): 345-51.
[13]Lim MS. RE:correlational of oral tongue cancer inversion with matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) expression, by Kim S-H, Cho NH, Kim K. et al.. J Surg Oncol 2006; 93(4):253-4
[14]Leipzig B, Cummings CW, Chung CT, Johnson JT, Sagerman RH. Carcinoma of the anterior tongue. Ann Otol Rhinol Laryngol1982; 91(1 Pt 1):94-7.
[15]Byers RM, El-Naggar AK, Lee YY, Rao B, Fornage B, Terry NHA, et al. Can we detect or predict the presence of occult nodal metastases in patients with squamous carcinoma of the oral tongue?Head Neck 1998; 20(2):138-44.
[16]Huang SF, Kang CJ. Lin CY, Fan KH, Yen TC, Wang HM, et al. Neck treatment of patients with early stage oral tongue cancer:comparison between observation, supraomohyoid dissection, and extended dissection. Cancer 2008; 112 (5):1066-75.
[17]D'Cruz AK, Siddachari RC, Walvekar RR, Pantvaidya GH, Chaukar DA, Deshpande MS. et al. Elective neck dissection for the management of the NO neck in early cancer of the oral tongue:need for a randomized controlled trial. Head Neck 2009; 31(5):618-24.
[18]Sodek J.Batista Da,Sliva AP.et al.Osteopotin and mucosal protection.J Dent Res.2006.85:404-15.
[19]Denhardt DT.Giachelli CM.Rittling SR.Role of osteopontin in cellular signaling and toxicant injury.Annu Rev Pharmacol Toxicol,2001b,41:723-49.
[20]Sodek J.Ganss B,Mckee MD.Osteopontin.Crit Rev Oral Biol Med.2000.11:279-303.
[21]Patanin N. Jouhra F.Jiang W.Mokbel K.Osteopontin expression profiles predict pathological and clinical outcome in breast cancer.Anticancer Res.2008 Nov-Dec:28(6B):4105-10.
[22]Ashkar S,Weber GF,Panoustsakopoulou V,et al.Eta-1(Osteopontin):an early component of type-1 (cell-mediated) immunity.Science,2000,287:860-4.
[23]Higashiyama M.Ito T.Tanaka E.et al.Prognostic significance of osteopontin expression in human gastric carcinoma.Anna Surg Oncol,2007.14(12):3419-27.
[24]Zhang X.Tsukamoto T,Mizoshita T,et al.Expression of osteopontin and CDX2:indications of phenotypes and prognosis in advanced gastric cancer.Oncol Rep.2009.21(3):609-13.
[25]Weber GF.The metastasis gene osteopontin:a candidate targe for cancer therapy.Biochim Biophys Acta,2001.28.1552(2):61-85.
[26]Johnston NI.Gunasekharan VK.Ravindranath A.et al.Osteopontin as a targe for cancer therapy.Front Biosci,08.13:4361-72.
[27]S. Hammarstrom.The carcinoembryonic antigen (CEA) family:structures, suggested functions and expression in normal and malignant tissues Semin Cancer Biol,9 (1999), pp.67-81.
[28]World Health Organization. The World Oral Health Report 2003. Geneva:World Health Organization; 2003.
[29]Rautava J, Luukkaa M, Heikinheimo K, Alin J. Grenman R, Happonen RP. Squamous cell carcinomas arising from different types of oral epithelia differ in their tumor and patient characteristics and survival. Oral Oncol 2007;43(9): 911-9.
[30]Sathyan KM, Sailasree R. Jayasurya R, Lakshminarayanan K, Abraham T, Nalinakumari KR, et al. Carcinoma of tongue and the buccal mucosa represent different biological subentities of the oral carcinoma. J Cancer Res Clin Oncol 2006;132(9):601-9.
[31]Rusthoven K, Ballonoff A. Raben D, Chen C. Poor prognosis in patients with stage I and II oral tongue squamous cell carcinoma. Cancer 2008;112(2):345-51.
[32]Regezi JA, Sciubba JJ, Jordan RCK. Oral pathology:clinical, pathologic correlations.5th ed. St Louis (MO):Saunders Elsevier; 2008.
[33]Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100(1):57-70.
[34]Lim MS. RE:correlational of oral tongue cancer inversion with matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) expression, by Kim S-H, Cho NH, Kim K, et al.. J Surg Oncol 2006;93(4):253-4.
[35]Sano D, Myers JN. Metastasis of squamous cell carcinoma of the oral tongue. Cancer Metastasis Rev 2007;26(3-4):645-62.
[36]Leipzig B, Cummings CW, Chung CT, Johnson JT, Sagerman RH. Carcinoma of the anterior tongue. Ann Otol Rhinol Laryngol 1982;91(1 Pt 1):94-7.
[37]Byers RM, El-Naggar AK, Lee YY, Rao B, Fornage B, Terry NHA, et al. Can we detect or predict the presence of occult nodal metastases in patients with squamous carcinoma of the oral tongue? Head Neck 1998:20(2):138-44.
[38]Huang SF, Kang CJ, Lin CY, Fan KH, Yen TC, Wang HM, et al. Neck treatment of patients with early stage oral tongue cancer:comparison between observation. supraomohyoid dissection, and extended dissection. Cancer 2008;112(5):1066-75.
[39]D'Cruz AK, Siddachari RC, Walvekar RR, Pantvaidya GH, Chaukar DA. Deshpande MS, et al. Elective neck dissection for the management of the NO neck in early cancer of the oral tongue:need for a randomized controlled trial. Head Neck 2009:31(5):618-24.
[40]Haddadin KJ. Soutar DS, Oliver RJ. Webster MH. Robertson AG. MacDonald DG. Improved survival for patients with clinically T1/T2. NO tongue tumors undergoing a prophylactic neck dissection. Head Neck 1999;21 (6):517-25.
[41]Sodek J, Ganss B, McKee MD. Osteopontin[J]. Crit Rev Oral Biol Med,2000, 11(3):279-303
[42]Senger DR, Wirth DF, Hynes RO. Transformed mammalian cells secrete specific proteins and phosphoproteins [J]. Cell.1979,16 (4): 885-893
[43]Gravallese EM. Osteopontin:a bridge between bone and the immune system[J]. J Clin Invest,2003.112(2):147-149
[44]Matsui Y, Iwasaki N, Kon S, et al. Accelerated development of agingassociated and instability-induced osteoarthritis in osteopontin-deficient mice[J]. Arthritis Rheum,2009,60(8):2362-2371
[45]Ashkar S. Weber GF, Panoutsakopoulou V, et al. Eta-1(Osteopon-tin): an early component of type-1 (cell-mediated) immunity [J]. Science, 2000,287(5454):860-864
[46]Sun BS, Dong QZ, Ye QH, Sun HJ. Jia HL. Zhu XQ, Liu DY, Chen J, Xue Q, Zhou HJ. Ren N, Qin LX. Lentiviral-mediated miRNA against osteopontin suppresses tumor growth and metastasis of human hepatocellular carcinoma. Hepatology.2008 Dec;48(6):1834-42.
[47]Mirza M. Shauglinessy E. Hurley JK. Vanpatten KA. Pestano GA. He B. Weber GF. Osteopontin-c is a selective marker of breast cancer. Int J Cancer.2008 Feb 15;122(4):889-97.
[48]Wong YF, Cheung TH, Tsao GS, Lo KW, Yim SF, Wang VW, Heung MM. Chan SC, Chan LK. Ho TW, Wong KW. Li C, Guo Y, Chung TK, Smith DI. Genome-wide gene expression profiling of cervical cancer in Hong Kong women by oligonucleotide microarray. Int J Cancer.2006 May 15;118(10):2461-9.
[49]Laur IE NA, Comegysm M, Carreiro MP, et al. Careinoembry-onic antigen related cell adhesion moleculel a-4L suppression of rat hepatocellular carcinomas [J]. Cancer Res,2005,65(23):11010.11017.
[50]Okegawa T, Pong RC, Li Y, et al. The role of cell adhesion molecule in cancer progression and its application in cancer therapy[J]. Acts Biochim Pol,2004,51(2):445.
[51]Tiezzi DG, Fernandez SV, Russo J, Epithelial mesenehymal transition during the neoplastic transformation of human breast epithelial cells by estrogen[J]. Oncol,2007,31(4):823.
[52]Klaile E, Muller MM, Kannicht C. Singer BB, Lucka L.CEACAM1 functionally interacts with filamin A and exerts adual role in the regulation of cell migration[J]. J Cell Science,2005,118(Pt 23),5513-5524.
[53]Ebrahimnejad A, Streichert T, Nollau P, et al. CEACAM1 enhances invasion and migration of melanocytic and melanoma cells[J]. Am J Pathol,2004,165: 1781-1787.
[54]Wirth T. Soeth E, Czubayko F, Juhl H. Inhibition of endogenous carcinoembryonic antigen (CEA) increases the apoptotic rate of colon cancer cells and inhibits metastatic tumor growth. Clin Exp Metastasis.2002;19(2):155-60.
[55]Gangopadhyay A, Lazure DA, Thomas P. Adhesion of colorectal carcinoma cells to the endothelium is mediated by cytokines from CEA stimulated Kupffer cells. Clin Exp Metastasis.1998 Nov:16(8):703-12.
[56]Price JT, Thompson EW. Mechanism of tumor invasion and metastasis: emerging targets for therapy[J]. Expert Opin Ther Targets,2002,6(2):217-233.
[57]Dai J, Peng L, Jain S, Kundu GC, et al. Osteopontin induces angiogenesis through activation of PI3K/AKT and ERK1/2 in endothelial cells. Oncogene. 2009.28(38):3412-3422.
[58]Weismann D. Briese J, Niemann J, et al. Osteopontin stimulates invasion of NCI-h295 cells but is not associated with survival in adrenocortical carcinoma. J Pathol,2009,218 (2):232-240.
[59]Shevde LA. Das S, Clark D W, et al. Osteopontin:an effector and an effect of tumor metastasis, Curr Mol Med,2009.
[60]项斌,刘兵团.人结肠癌组织中骨桥蛋白的表达研究[J].南通大学学报(医学版),2011,31(2)：141——142.
[61]庞晓云,伍建国.子宫颈癌患者中骨桥蛋白的表达及其临床意义[J].中外医学研究,2011,9(21)：86—88
[62]董晓瑜,杨静华.骨桥蛋白在卵巢上皮癌组织中的表达及其与预后的关系[J].中国综合临床,2010,26(8)：121——125
[63]Move VE, Barraclough R, West C, et al. Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducingDNA-transfected rat mammary cell lines[J]. Br J Cancer,2004,90(9):1796-1802.
[64]Yeatman TJ, Chambers AF. Osteopontin and colon cancer progression[J]. Clin Exp Metastasis,2003,20(1):85-90
[65]Wai PY, Kuo PC. The role of osteopontinintumormetastasis[J]. J SurgRes,2004, 121(2):228-241.
[66]Muramatsu T, Shima K, Ohta K, et al. Inhibition of osteopontin expression and function in oral cancer cell lines by antisense oligonucleotides[J]. Cancer Lett,2005, 217(1):87-95.
[67]Ito T,Hashimoto Y, Tanaka E, et al. An inducible short-hairpin RNA vector against osteopontin reduces metastatic potential of human esophageal squamous cell carcinoma in vitro and in vivo [J].Clin Cancer Res,2006,12(4):1308-1316.
[68]Allan AL, George R, Vantyghem SA, et al. Role of the integrinbinding protein osteopontin in lymphatic metastasis of breast cancer[J]. Am J Pathol,2006,169(1): 233-246.
[69]商冠宁,吕刚.骨桥蛋白在骨肉瘤中表达及其与侵袭转移关系[J].现代肿瘤医学,2010,18(11)：2251一2253
[70]Sakaguchi H. Fujimoto J. Clinical implications of osteopontin in metastatic lesions of uterine cervical cancers[J]. Cancer Lett,2007,247(1):98-102
[71]付维,范尔钟,杨征等.骨桥蛋白在喉癌组织中的表达[J].中国耳鼻喉头颈外科,2008,48(6)：1834-1842.
[72]孙永梅,刘韶平,马喆等.骨桥蛋白在乳头状甲状腺癌中的表达及意义.中国现代普通外科进展,2009,12(1)：24-28.
[73]Wong YF, Cheung TH, Tsao GS, et al.Genome-wide gene expression profiling of cervical cancer in Hong Kong women by ologonucleotide microarray. Int J Cancer, 2006 118(10):2461-2469.
[74]Matsuzaki H Shima K'MuramatsuT, etal. Osteopontinasbiomarker in early invasion by squam ous cell carcinoma in tongue [J]. J Oral Pathol Med,2007,36(1): 30-4.
75 Ogbureke K U, Nikitakis NG, Warburton G, et al. Up-regulation of SIBLING proteins and correlation wim cognate MNIP expression in oral cancer[J]. Oral Oncol, 2007,43(9):920-32.
[76]吕晓智,殷学民,冯元勇,段丽琼,陈万涛,张陈平.骨桥蛋白mRNA在口腔鳞癌与正常黏膜中的表达及临床意义[J].南方医科大学学报,2008；28(7)：1165-7.
[77]Snitcovsky I, Leitao GM, Pasini FS, et al. Plasma osteopontin levels in patients with head and neck cancer undergoing chemoradiotherapy. Arch Otolaryngol Head Neck Surg,2009.135(8):807-811.
[78]Gold P, Freedman SC. Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and ahsorption techniques. J Exp Med,1965,121:439
[79]Anonymous. Carcinoembryonic antigen:it s role as a marker in the management of cancer. Summary of an NIH consensus statement [J]. Lancet.1981.282:373-375
[80]Grem J. The prognosti importance of tumormarkers in adenocarcinomas of the gastrointestinal tract [J]. Curr Opinion Oncol.1997.9:380-387
[81]黄传涌,卢贤立。血清CA19-9、CEA检测消化道恶性肿瘤的诊断价值[J]. 医学理论与实践,2000,13(1)：23
[82]王仁华,马思平,王辉等.血清CA19-9,CEA、CA50在大肠癌中的作用[J].实用肿瘤学杂志,1999,13(2)：128-9.
[83]Ohta K, Yamaguchi Y.Shimizu K,et al. Novel system for generating cytotoxic effector lymphocytes using carcinoembryonic antigen(CEA) peptide and cultured dendritic cell. Anticancer res 2002.22(5):2597-606
[84]Greiner JW.Guadagni F, Goldstein D, et al.Intraperitoneal administration of inglycoprotein-72 and carcinoembryonic antigen on malignant ascites cells.J Clin Oncol 1992,10(5):735-46
[85]Grotowski M. Antigens(CEA and CA.19-9)in diagnosis and prognosis colorectal cancer. Pol Merkuriusz Lek.2002,12(67):77-80
[86]张忻平,尉承泽.大肠癌患者抑郁和焦虑情绪的临床特征[J].中国临床康复,2004,8(3)：420-1
[87]段宝玲,孙巧惠,边冬梅等.胃肠道肿瘤患者术后抑郁和焦虑的临场特征[J].中国临床康复,2003,7(30)：4156.
[88]Anonymous. Clinical practice guidelines for the use of tumor markers in breast and colorectal cancer[J]. J Clin Oncol,1996,14:2843-2877
[89]郭俊明,罗超权.癌胚抗原阳性肿瘤基因治疗的研究进展[J].中国肿瘤生物治疗杂志,19996(2)：152-154.
[90]Thies A et al. J Clin Oncol,2002,20(10):2530-2536
[91]Gangopadhyay A et al. Clin Exp Metastasis,1998,16(8):703-712
[92]Hammarstrom S et al. Semin Caner Bilo,1999.9(2):67-81
[93]Wirth T et al. Clin Exp Metastasis,2002,19(2):155-160
[94]Thompson J et al. Cancer Res.1997,57(9):1776-1784
[95]Bedi A et al. Cancer Res,1995,55(9):1811-1816
[96]Beauchemin N. Kunath T. Robitaille J. Chow B, Turbide C. Daniels E. et al.Association of biliary glycoprotein with protein tyrosine phosphatase SHP-1 in malignant colon epithelial cells.Oncogene 1997; 14:783-90.
[97]Lutz Riethdorf L. Lisboa BW, Henkel U, Naumann M, Waqener C, et al. Differential expression of CD66a (BGP), a cell adhesion molecule of the carcinoembryonic antigen family, in benign, premalignant, and malignant lesions of the human mammary gland.J Histochem Cytochem 1997; 45:957-63.
[98]Giachelli CM, Steitz S. Osteopontin:a versatile regulator of inflammation and biomineralization.Matrix Biol 2000; 19:615-22.
[99]Senger DR. Ledbetter SR, Claffey KP, Papadopoulos-Sergiou A. Perruzzi CA, Detmar M.Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the av B3 integrin, osteopontin, and thrombin.Am J Pathol 1996; 149:293-305.
[100]Denhardt DT, Guo X. Osteopontin.a protein with diverse functions.FASEB J 1993; 7:1475-82.
[101]Shijubo N. Uede T, Kon S, Nagata M, Abe S.Vascular endothelial growth factor and osteopontin in tumor biology.Crit Rev Oncog 2000; 11:135-46.
[102]Morimoto J, Inobe M, Kimura C, Kon S. Diao H, Aoki M, et al.Osteopontin affects the persistence of β-glucan-induced hepatic granuloma formation and tissue injury through two distinct mechanisms.Int Immunol 2004; 16:477-88.
[103]Coppola D, Szabo M, Boulware D, Muraca P, Alsarraj M, Chambers AF, et al.Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies.Clin Cancer Res 2004; 10:184-90.
[104]Chien CY. Su CY, Chuang HC, Fang FM. Huang HY, Chen CM, et al.Clinical significance of osteopontin expression in T1 and T2 tongue cancers.Head Neck 2008; 30:776-81.
[105]Chiu YW, Tu HF, Wang IK, Wu CH, Chang KW, Liu TY, et al.The implication of osteopontin (OPN) expression and genetic polymorphisms of OPN promoter in oral carcinogenesis.Oral Oncol 2010; 46:302-6.
[106]Briese J, Oberndorfer M, Patschenik C, Schulte HM, Makriqiannakis A, Loning T, et al.Osteopontin Is Colocalized with the Adhesion Molecule CEACAM1 in the ExtravillousTrophoblast of the Human Placenta and Enhances Invasion of CEACAM1-Expressing Placental Cells.J Clin Endocrinol Metab 2005; 90:5407-13
[1]Chaudary N, Hill RP. Increased expression of metastasis-related genes in hypoxic cells sorted from cervical and lymph nodal xenograft tumors[J]. Lab Invest,2009,89(5):587-596
[2]Anborgh PH, Wilson SM, Tuck AB, et al. New dual monoclonal ELISA for measuring plasma osteopontin as a biomarker associated with survival in prostate cancer:clinical validation and comparison of ml. Htiple ELISAs[J]. Clin Chem,2009,55(5):895-903
[3]Suzuki M, Mose E, Galloy C, et al.Osteopontin gene expression determines spontaneous metastatic performance of orthotopic human breast cancer xenografts.Am J Pathol,2007,171 (2):682-692.
[4]Li Destri G, Curreri R. Lanteri R. Gagliano G, Rodolico M. Di Cataldo A. Puleo S. Biliary carcinoembryonic antigen in the diagnosis of occult hepatic metastases from colorectal cancer.J Surg Oncol.2002 Sep;81(1):8-11.
[5]Gangopadhyay A, Lazure DA, Thomas P. Adhesion of colorectal carcinoma cells to the endothelium is mediated by cytokines from CEA stimulated Kupffer cells. Clin Exp Metastasis.1998 Nov;16(8):703-12.
[6]Steller H:Mechanisms and genes of cellular suicide.Science 1995, 267:1445-1449.
[7]Thompson CB:Apoptosis in the pathogenesis and treatment of disease.Science 1995.267:1456-1462.
[8]Liaw L, Skinner MP, Raines EW, Ross R. Cheresh DA, Schwartz SM, et al. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro.J Clin Invest 1995; 95:713-24.
[9]Daiter E. Omigbodun A. Wang S. Walinsky D. Strauss JF Ⅲ. Hoyer JR., et al.Cell differentiation and endogenous cyclic adenosine 3.5,-monophosphate regulate osteopontin expression in human trophoblasts.Endocrinology 1996; 137:1785-90.
[10]Yue TL. McKenna PJ. Ohlstein EH, Farach-Carson MC, Butler WT, Johanson K. et al.Osteopontin-stimulated vascular smooth muscle cell migration is mediated by beta 3 integrin.Exp Cell Res 1994; 214:459-64.
[11]Kurupatip P, Tank S, Kumarasinghe G, et al. Inhibition of gene expression and growth by antisense peptide nucleic acids in a muhiresistant beta lactamase producing Klebsiella pneumonia strain[J]. Antimicrob Agents Chemother,2007,51(3): 8052-8111.
[12]Huang X W, Pan J, An X Y, et al. Inhibition of bacterial translation and growth by peptidenucleic acidstargetedtodomainll of23S rRNA[J]. J Pept Sci,2007, 13(4).2202-2261.
[13]Ferber D. A new way to combat therapy side effects [J]. Science,1999,285: 1651-1653.
[14]Ahlquist P. RNA-dependent RNA polymerases and RNA interference (RNAi) [J]. Cell Cycle,2003,2:22-24.
[15]Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature,1998,391(6669): 806-811.
[16]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans[J]. Nature,1998,391(6669): 806-811.
[17]Elbashir SM, Harbonh J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature,2001,411(6836): 494-498.
[18]Hannon GJ. RNA interference [J]. Nature,2002,418(6894):244
[19]Zamore PD, Tuschl T, Sharp PA, et al. RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell,2000 101 (1):25
[20]Zamore PD. RNA interference:listening to the sound of silence. Nat Struct Biol, 2001,8(9):746
[21]Bmmmelkamp TR, Bemards R, Agam i R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J]. Cancer cell,2002,2(3):243-247.
[22]Grzino F, Arduini F, Ruzzoa A, et al. Combined analysis of Ecadherin gene(CDH1) promoter hypermethlation and E-cad-herin protein expression in patients with gastric can cer:implication for treatment with demethylating drugs[J]. Ann Oncol,2004,15(3):489-492.
[23]Ming J,Milner J.Selective silencing of viral gene expression in HPV-Positive human cervical carcinoma ceils treated with siRNA, a P rimer of RNA interference [J]. Oncogene,2002,21:6041-6048.
[24]Spankuch-Schmitt B.Bereiter-Hahn J, Kaufmann M.et al. Effect of RNA silencing of polo-like kinase-1(PLK1) on apoptosis and spindle formation in human cancer cell[J]s. J Natl Cancer Inst,2002,94(24):1863
[25]Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference [J]. Cancer Cell,2002,2(3):243
[26]Sbankar P, Manjunath N, Lieberman J. The prospect of silencing disease using RNA interference[J]. JAMA,2005,293(11):1367-1373
[27]Coppola D, Szabo M, Boulware D et al.Correlation ofesteopontin protein expression and pathological stage across a wide variety of tumor histologies.Clin Cancer Res,2004.10:184-190.
[28]Adwan H, Bauerle TJ.Berger MR.Downregulation of esteopontin and bone sialoprotein Ⅱ is related to reduced colony formation and metastasis formation of MDA-MB-231 human breast cancer cells. Cancer Gene Ther.2004,11:109-120.
[29]薛松,汪水义,尹邦良,等.骨桥蛋白小干扰性RNA抑制大隐静脉平滑肌细胞增殖和迁移能力的研究.中华实验外科杂志,2007,24：990-992.
[30]柳昊,陈安民,郭风劲,蒋林,胡伟华,解杰.体外RNA干扰抑制骨桥蛋白 表达对肾癌细胞生长和侵袭的影响.中华实验外科杂志2011,28：857-860
[31]Jen KY.Gewirtz AM.Suppression of gene epression by targeted disruption of messenger RNA; available options and current stratefies[J]. Stem Cells.2000.18(5):307-319
[32]Shevde LA, Samant RS, Paik JC.et al. Osteopontin knockdown suppresses tumourigennicity of human metastatic breast carcinoma MDA-MB-435[J]. Clin Exp Metastasis,2006,23(2):123-133
[33]Guadagni F'Kantor J,Aloe S, et al.Detection of blood-borne cells in colorectal cancer patients by nested reverse, transcripdon-polymerase chain reaction for cardnocmbryonic antigen messenger RNA:longitudinal analyses and demonstration of its potential importance as an adjunct to multiple serum markers[J].Cancer Res, 2001,61(6):2523-2532
[34]Sienel W et al. Elevated expression of carcinoembryonic antigen-related cell adhesion molecule 1 promotes progression of non-small cell lung cancer.Clin Cancer Res.2003.9:2260-2266.
[35]Thies A et al. CEACAM1 expression in cutaneous malignant melanoma predicts the development of metastatic disease.J Clin Oncol,2002.20(10):2530-2536
[36]Gangopadhyay A, Lazure DA, Thomas P. Adhesion of colorectal carcinoma cells to the endothelium is mediated by cytokines from CEA stimulated Kupffer cells. Clin Exp Metastasis.1998 Nov;16(8):703-12.
[37]Wirth T et al.[J]. Inhibition of endogenous carcinoembryonic antigen (CEA) increases the apoptotic rate of colon cancer cells and inhibits metastatic tumor growth. Exp Metastasis,2002,19(2):155-160
[2]B.W. Stewart. P. Kleihues (Eds.), World cancer report, WHO International Agency for Research on Cancer. Lyon (2003)
[3]Petersen PE. The world oral health report 2003:continuous improvement of oral health in the 21st century-the approach of the WHO global oral health programme. Community Dent Oral Epidemiol 2003; 31(Supp.1):3-24.
[4]P.E. Petersen. D. Bourgeois, D. Bratthall, H. Ogawa.Oral health information systems-towards measuring progress in oral health promotion and disease prevention Bull World Health Organ,83 (2005), pp.686-693
[5]J.H.C Ho An epidemiologic and clinical study of nasopharyngeal carcinoma Int J Radiat Oncol Biol Phys.4 (1978). pp.183-198
[6]D.P Huang. J.H.C Ho. W.K Chan. W.H Lau. M Lui Cytogenetics of undifferentiated nasopharyngeal carcinoma xenografts from southern Chinese Int J Cancer,43 (1989). pp.936-939
[7]M.L Lung, W.P Lam. J Sham et al. Detection and prevalence of the "f" variant of Epstein-Barr virus in Southern China Virology,185 (1) (1991), pp.67-71.
[8]P.M.L Teo, W.Y Lee. P Yu The prognostic significance of parapharyngeal tumour involvement in nasopharyngeal carcinoma Radiother Oncol,39 (1996), pp.209-221.
[9]Regezi JA, Sciubba JJ. Jordan RCK. Oral pathology:clinical, pathologic correlations.5th ed. St Louis (MO):Saunders Elsevier:2008.
[10]Rautava J, Luukkaa M, Heikinheimo K. Alin J, Grenman R, Happonen RP. Squamous cell carcinomas arising from different types of oral epithelia differ in their tumor and patient characteristics and survival. Oral Oncol 2007; 43(9): 911-9.
[11]Sathyan KM, Sailasree R, Jayasurya R, Lakshminarayanan K, Abraham T.Nalinakumari KR, et al. Carcinoma of tongue and the buccal mucosa represent different biological subentities of the oral carcinoma. J Cancer Res Clin Oncol 2006; 132 (9):601-9.
[12]Rusthoven K, Ballonoff A. Raben D, Chen C. Poor prognosis in patients withstage Ⅰ and Ⅱ oral tongue squamous cell carcinoma. Cancer 2008; 112 (2): 345-51.
[13]Lim MS. RE:correlational of oral tongue cancer inversion with matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) expression, by Kim S-H, Cho NH, Kim K. et al.. J Surg Oncol 2006; 93(4):253-4
[14]Leipzig B, Cummings CW, Chung CT, Johnson JT, Sagerman RH. Carcinoma of the anterior tongue. Ann Otol Rhinol Laryngol1982; 91(1 Pt 1):94-7.
[15]Byers RM, El-Naggar AK, Lee YY, Rao B, Fornage B, Terry NHA, et al. Can we detect or predict the presence of occult nodal metastases in patients with squamous carcinoma of the oral tongue?Head Neck 1998; 20(2):138-44.
[16]Huang SF, Kang CJ. Lin CY, Fan KH, Yen TC, Wang HM, et al. Neck treatment of patients with early stage oral tongue cancer:comparison between observation, supraomohyoid dissection, and extended dissection. Cancer 2008; 112 (5):1066-75.
[17]D'Cruz AK, Siddachari RC, Walvekar RR, Pantvaidya GH, Chaukar DA, Deshpande MS. et al. Elective neck dissection for the management of the NO neck in early cancer of the oral tongue:need for a randomized controlled trial. Head Neck 2009; 31(5):618-24.
[18]Sodek J.Batista Da,Sliva AP.et al.Osteopotin and mucosal protection.J Dent Res.2006.85:404-15.
[19]Denhardt DT.Giachelli CM.Rittling SR.Role of osteopontin in cellular signaling and toxicant injury.Annu Rev Pharmacol Toxicol,2001b,41:723-49.
[20]Sodek J.Ganss B,Mckee MD.Osteopontin.Crit Rev Oral Biol Med.2000.11:279-303.
[21]Patanin N. Jouhra F.Jiang W.Mokbel K.Osteopontin expression profiles predict pathological and clinical outcome in breast cancer.Anticancer Res.2008 Nov-Dec:28(6B):4105-10.
[22]Ashkar S,Weber GF,Panoustsakopoulou V,et al.Eta-1(Osteopontin):an early component of type-1 (cell-mediated) immunity.Science,2000,287:860-4.
[23]Higashiyama M.Ito T.Tanaka E.et al.Prognostic significance of osteopontin expression in human gastric carcinoma.Anna Surg Oncol,2007.14(12):3419-27.
[24]Zhang X.Tsukamoto T,Mizoshita T,et al.Expression of osteopontin and CDX2:indications of phenotypes and prognosis in advanced gastric cancer.Oncol Rep.2009.21(3):609-13.
[25]Weber GF.The metastasis gene osteopontin:a candidate targe for cancer therapy.Biochim Biophys Acta,2001.28.1552(2):61-85.
[26]Johnston NI.Gunasekharan VK.Ravindranath A.et al.Osteopontin as a targe for cancer therapy.Front Biosci,08.13:4361-72.
[27]S. Hammarstrom.The carcinoembryonic antigen (CEA) family:structures, suggested functions and expression in normal and malignant tissues Semin Cancer Biol,9 (1999), pp.67-81.
[28]World Health Organization. The World Oral Health Report 2003. Geneva:World Health Organization; 2003.
[29]Rautava J, Luukkaa M, Heikinheimo K, Alin J. Grenman R, Happonen RP. Squamous cell carcinomas arising from different types of oral epithelia differ in their tumor and patient characteristics and survival. Oral Oncol 2007;43(9): 911-9.
[30]Sathyan KM, Sailasree R. Jayasurya R, Lakshminarayanan K, Abraham T, Nalinakumari KR, et al. Carcinoma of tongue and the buccal mucosa represent different biological subentities of the oral carcinoma. J Cancer Res Clin Oncol 2006;132(9):601-9.
[31]Rusthoven K, Ballonoff A. Raben D, Chen C. Poor prognosis in patients with stage I and II oral tongue squamous cell carcinoma. Cancer 2008;112(2):345-51.
[32]Regezi JA, Sciubba JJ, Jordan RCK. Oral pathology:clinical, pathologic correlations.5th ed. St Louis (MO):Saunders Elsevier; 2008.
[33]Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100(1):57-70.
[34]Lim MS. RE:correlational of oral tongue cancer inversion with matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) expression, by Kim S-H, Cho NH, Kim K, et al.. J Surg Oncol 2006;93(4):253-4.
[35]Sano D, Myers JN. Metastasis of squamous cell carcinoma of the oral tongue. Cancer Metastasis Rev 2007;26(3-4):645-62.
[36]Leipzig B, Cummings CW, Chung CT, Johnson JT, Sagerman RH. Carcinoma of the anterior tongue. Ann Otol Rhinol Laryngol 1982;91(1 Pt 1):94-7.
[37]Byers RM, El-Naggar AK, Lee YY, Rao B, Fornage B, Terry NHA, et al. Can we detect or predict the presence of occult nodal metastases in patients with squamous carcinoma of the oral tongue? Head Neck 1998:20(2):138-44.
[38]Huang SF, Kang CJ, Lin CY, Fan KH, Yen TC, Wang HM, et al. Neck treatment of patients with early stage oral tongue cancer:comparison between observation. supraomohyoid dissection, and extended dissection. Cancer 2008;112(5):1066-75.
[39]D'Cruz AK, Siddachari RC, Walvekar RR, Pantvaidya GH, Chaukar DA. Deshpande MS, et al. Elective neck dissection for the management of the NO neck in early cancer of the oral tongue:need for a randomized controlled trial. Head Neck 2009:31(5):618-24.
[40]Haddadin KJ. Soutar DS, Oliver RJ. Webster MH. Robertson AG. MacDonald DG. Improved survival for patients with clinically T1/T2. NO tongue tumors undergoing a prophylactic neck dissection. Head Neck 1999;21 (6):517-25.
[41]Sodek J, Ganss B, McKee MD. Osteopontin[J]. Crit Rev Oral Biol Med,2000, 11(3):279-303
[42]Senger DR, Wirth DF, Hynes RO. Transformed mammalian cells secrete specific proteins and phosphoproteins [J]. Cell.1979,16 (4): 885-893
[43]Gravallese EM. Osteopontin:a bridge between bone and the immune system[J]. J Clin Invest,2003.112(2):147-149
[44]Matsui Y, Iwasaki N, Kon S, et al. Accelerated development of agingassociated and instability-induced osteoarthritis in osteopontin-deficient mice[J]. Arthritis Rheum,2009,60(8):2362-2371
[45]Ashkar S. Weber GF, Panoutsakopoulou V, et al. Eta-1(Osteopon-tin): an early component of type-1 (cell-mediated) immunity [J]. Science, 2000,287(5454):860-864
[46]Sun BS, Dong QZ, Ye QH, Sun HJ. Jia HL. Zhu XQ, Liu DY, Chen J, Xue Q, Zhou HJ. Ren N, Qin LX. Lentiviral-mediated miRNA against osteopontin suppresses tumor growth and metastasis of human hepatocellular carcinoma. Hepatology.2008 Dec;48(6):1834-42.
[47]Mirza M. Shauglinessy E. Hurley JK. Vanpatten KA. Pestano GA. He B. Weber GF. Osteopontin-c is a selective marker of breast cancer. Int J Cancer.2008 Feb 15;122(4):889-97.
[48]Wong YF, Cheung TH, Tsao GS, Lo KW, Yim SF, Wang VW, Heung MM. Chan SC, Chan LK. Ho TW, Wong KW. Li C, Guo Y, Chung TK, Smith DI. Genome-wide gene expression profiling of cervical cancer in Hong Kong women by oligonucleotide microarray. Int J Cancer.2006 May 15;118(10):2461-9.
[49]Laur IE NA, Comegysm M, Carreiro MP, et al. Careinoembry-onic antigen related cell adhesion moleculel a-4L suppression of rat hepatocellular carcinomas [J]. Cancer Res,2005,65(23):11010.11017.
[50]Okegawa T, Pong RC, Li Y, et al. The role of cell adhesion molecule in cancer progression and its application in cancer therapy[J]. Acts Biochim Pol,2004,51(2):445.
[51]Tiezzi DG, Fernandez SV, Russo J, Epithelial mesenehymal transition during the neoplastic transformation of human breast epithelial cells by estrogen[J]. Oncol,2007,31(4):823.
[52]Klaile E, Muller MM, Kannicht C. Singer BB, Lucka L.CEACAM1 functionally interacts with filamin A and exerts adual role in the regulation of cell migration[J]. J Cell Science,2005,118(Pt 23),5513-5524.
[53]Ebrahimnejad A, Streichert T, Nollau P, et al. CEACAM1 enhances invasion and migration of melanocytic and melanoma cells[J]. Am J Pathol,2004,165: 1781-1787.
[54]Wirth T. Soeth E, Czubayko F, Juhl H. Inhibition of endogenous carcinoembryonic antigen (CEA) increases the apoptotic rate of colon cancer cells and inhibits metastatic tumor growth. Clin Exp Metastasis.2002;19(2):155-60.
[55]Gangopadhyay A, Lazure DA, Thomas P. Adhesion of colorectal carcinoma cells to the endothelium is mediated by cytokines from CEA stimulated Kupffer cells. Clin Exp Metastasis.1998 Nov:16(8):703-12.
[56]Price JT, Thompson EW. Mechanism of tumor invasion and metastasis: emerging targets for therapy[J]. Expert Opin Ther Targets,2002,6(2):217-233.
[57]Dai J, Peng L, Jain S, Kundu GC, et al. Osteopontin induces angiogenesis through activation of PI3K/AKT and ERK1/2 in endothelial cells. Oncogene. 2009.28(38):3412-3422.
[58]Weismann D. Briese J, Niemann J, et al. Osteopontin stimulates invasion of NCI-h295 cells but is not associated with survival in adrenocortical carcinoma. J Pathol,2009,218 (2):232-240.
[59]Shevde LA. Das S, Clark D W, et al. Osteopontin:an effector and an effect of tumor metastasis, Curr Mol Med,2009.
[60]项斌,刘兵团.人结肠癌组织中骨桥蛋白的表达研究[J].南通大学学报(医学版),2011,31(2)：141——142.
[61]庞晓云,伍建国.子宫颈癌患者中骨桥蛋白的表达及其临床意义[J].中外医学研究,2011,9(21)：86—88
[62]董晓瑜,杨静华.骨桥蛋白在卵巢上皮癌组织中的表达及其与预后的关系[J].中国综合临床,2010,26(8)：121——125
[63]Move VE, Barraclough R, West C, et al. Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducingDNA-transfected rat mammary cell lines[J]. Br J Cancer,2004,90(9):1796-1802.
[64]Yeatman TJ, Chambers AF. Osteopontin and colon cancer progression[J]. Clin Exp Metastasis,2003,20(1):85-90
[65]Wai PY, Kuo PC. The role of osteopontinintumormetastasis[J]. J SurgRes,2004, 121(2):228-241.
[66]Muramatsu T, Shima K, Ohta K, et al. Inhibition of osteopontin expression and function in oral cancer cell lines by antisense oligonucleotides[J]. Cancer Lett,2005, 217(1):87-95.
[67]Ito T,Hashimoto Y, Tanaka E, et al. An inducible short-hairpin RNA vector against osteopontin reduces metastatic potential of human esophageal squamous cell carcinoma in vitro and in vivo [J].Clin Cancer Res,2006,12(4):1308-1316.
[68]Allan AL, George R, Vantyghem SA, et al. Role of the integrinbinding protein osteopontin in lymphatic metastasis of breast cancer[J]. Am J Pathol,2006,169(1): 233-246.
[69]商冠宁,吕刚.骨桥蛋白在骨肉瘤中表达及其与侵袭转移关系[J].现代肿瘤医学,2010,18(11)：2251一2253
[70]Sakaguchi H. Fujimoto J. Clinical implications of osteopontin in metastatic lesions of uterine cervical cancers[J]. Cancer Lett,2007,247(1):98-102
[71]付维,范尔钟,杨征等.骨桥蛋白在喉癌组织中的表达[J].中国耳鼻喉头颈外科,2008,48(6)：1834-1842.
[72]孙永梅,刘韶平,马喆等.骨桥蛋白在乳头状甲状腺癌中的表达及意义.中国现代普通外科进展,2009,12(1)：24-28.
[73]Wong YF, Cheung TH, Tsao GS, et al.Genome-wide gene expression profiling of cervical cancer in Hong Kong women by ologonucleotide microarray. Int J Cancer, 2006 118(10):2461-2469.
[74]Matsuzaki H Shima K'MuramatsuT, etal. Osteopontinasbiomarker in early invasion by squam ous cell carcinoma in tongue [J]. J Oral Pathol Med,2007,36(1): 30-4.
75 Ogbureke K U, Nikitakis NG, Warburton G, et al. Up-regulation of SIBLING proteins and correlation wim cognate MNIP expression in oral cancer[J]. Oral Oncol, 2007,43(9):920-32.
[76]吕晓智,殷学民,冯元勇,段丽琼,陈万涛,张陈平.骨桥蛋白mRNA在口腔鳞癌与正常黏膜中的表达及临床意义[J].南方医科大学学报,2008；28(7)：1165-7.
[77]Snitcovsky I, Leitao GM, Pasini FS, et al. Plasma osteopontin levels in patients with head and neck cancer undergoing chemoradiotherapy. Arch Otolaryngol Head Neck Surg,2009.135(8):807-811.
[78]Gold P, Freedman SC. Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and ahsorption techniques. J Exp Med,1965,121:439
[79]Anonymous. Carcinoembryonic antigen:it s role as a marker in the management of cancer. Summary of an NIH consensus statement [J]. Lancet.1981.282:373-375
[80]Grem J. The prognosti importance of tumormarkers in adenocarcinomas of the gastrointestinal tract [J]. Curr Opinion Oncol.1997.9:380-387
[81]黄传涌,卢贤立。血清CA19-9、CEA检测消化道恶性肿瘤的诊断价值[J]. 医学理论与实践,2000,13(1)：23
[82]王仁华,马思平,王辉等.血清CA19-9,CEA、CA50在大肠癌中的作用[J].实用肿瘤学杂志,1999,13(2)：128-9.
[83]Ohta K, Yamaguchi Y.Shimizu K,et al. Novel system for generating cytotoxic effector lymphocytes using carcinoembryonic antigen(CEA) peptide and cultured dendritic cell. Anticancer res 2002.22(5):2597-606
[84]Greiner JW.Guadagni F, Goldstein D, et al.Intraperitoneal administration of inglycoprotein-72 and carcinoembryonic antigen on malignant ascites cells.J Clin Oncol 1992,10(5):735-46
[85]Grotowski M. Antigens(CEA and CA.19-9)in diagnosis and prognosis colorectal cancer. Pol Merkuriusz Lek.2002,12(67):77-80
[86]张忻平,尉承泽.大肠癌患者抑郁和焦虑情绪的临床特征[J].中国临床康复,2004,8(3)：420-1
[87]段宝玲,孙巧惠,边冬梅等.胃肠道肿瘤患者术后抑郁和焦虑的临场特征[J].中国临床康复,2003,7(30)：4156.
[88]Anonymous. Clinical practice guidelines for the use of tumor markers in breast and colorectal cancer[J]. J Clin Oncol,1996,14:2843-2877
[89]郭俊明,罗超权.癌胚抗原阳性肿瘤基因治疗的研究进展[J].中国肿瘤生物治疗杂志,19996(2)：152-154.
[90]Thies A et al. J Clin Oncol,2002,20(10):2530-2536
[91]Gangopadhyay A et al. Clin Exp Metastasis,1998,16(8):703-712
[92]Hammarstrom S et al. Semin Caner Bilo,1999.9(2):67-81
[93]Wirth T et al. Clin Exp Metastasis,2002,19(2):155-160
[94]Thompson J et al. Cancer Res.1997,57(9):1776-1784
[95]Bedi A et al. Cancer Res,1995,55(9):1811-1816
[96]Beauchemin N. Kunath T. Robitaille J. Chow B, Turbide C. Daniels E. et al.Association of biliary glycoprotein with protein tyrosine phosphatase SHP-1 in malignant colon epithelial cells.Oncogene 1997; 14:783-90.
[97]Lutz Riethdorf L. Lisboa BW, Henkel U, Naumann M, Waqener C, et al. Differential expression of CD66a (BGP), a cell adhesion molecule of the carcinoembryonic antigen family, in benign, premalignant, and malignant lesions of the human mammary gland.J Histochem Cytochem 1997; 45:957-63.
[98]Giachelli CM, Steitz S. Osteopontin:a versatile regulator of inflammation and biomineralization.Matrix Biol 2000; 19:615-22.
[99]Senger DR. Ledbetter SR, Claffey KP, Papadopoulos-Sergiou A. Perruzzi CA, Detmar M.Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the av B3 integrin, osteopontin, and thrombin.Am J Pathol 1996; 149:293-305.
[100]Denhardt DT, Guo X. Osteopontin.a protein with diverse functions.FASEB J 1993; 7:1475-82.
[101]Shijubo N. Uede T, Kon S, Nagata M, Abe S.Vascular endothelial growth factor and osteopontin in tumor biology.Crit Rev Oncog 2000; 11:135-46.
[102]Morimoto J, Inobe M, Kimura C, Kon S. Diao H, Aoki M, et al.Osteopontin affects the persistence of β-glucan-induced hepatic granuloma formation and tissue injury through two distinct mechanisms.Int Immunol 2004; 16:477-88.
[103]Coppola D, Szabo M, Boulware D, Muraca P, Alsarraj M, Chambers AF, et al.Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies.Clin Cancer Res 2004; 10:184-90.
[104]Chien CY. Su CY, Chuang HC, Fang FM. Huang HY, Chen CM, et al.Clinical significance of osteopontin expression in T1 and T2 tongue cancers.Head Neck 2008; 30:776-81.
[105]Chiu YW, Tu HF, Wang IK, Wu CH, Chang KW, Liu TY, et al.The implication of osteopontin (OPN) expression and genetic polymorphisms of OPN promoter in oral carcinogenesis.Oral Oncol 2010; 46:302-6.
[106]Briese J, Oberndorfer M, Patschenik C, Schulte HM, Makriqiannakis A, Loning T, et al.Osteopontin Is Colocalized with the Adhesion Molecule CEACAM1 in the ExtravillousTrophoblast of the Human Placenta and Enhances Invasion of CEACAM1-Expressing Placental Cells.J Clin Endocrinol Metab 2005; 90:5407-13
[1]Chaudary N, Hill RP. Increased expression of metastasis-related genes in hypoxic cells sorted from cervical and lymph nodal xenograft tumors[J]. Lab Invest,2009,89(5):587-596
[2]Anborgh PH, Wilson SM, Tuck AB, et al. New dual monoclonal ELISA for measuring plasma osteopontin as a biomarker associated with survival in prostate cancer:clinical validation and comparison of ml. Htiple ELISAs[J]. Clin Chem,2009,55(5):895-903
[3]Suzuki M, Mose E, Galloy C, et al.Osteopontin gene expression determines spontaneous metastatic performance of orthotopic human breast cancer xenografts.Am J Pathol,2007,171 (2):682-692.
[4]Li Destri G, Curreri R. Lanteri R. Gagliano G, Rodolico M. Di Cataldo A. Puleo S. Biliary carcinoembryonic antigen in the diagnosis of occult hepatic metastases from colorectal cancer.J Surg Oncol.2002 Sep;81(1):8-11.
[5]Gangopadhyay A, Lazure DA, Thomas P. Adhesion of colorectal carcinoma cells to the endothelium is mediated by cytokines from CEA stimulated Kupffer cells. Clin Exp Metastasis.1998 Nov;16(8):703-12.
[6]Steller H:Mechanisms and genes of cellular suicide.Science 1995, 267:1445-1449.
[7]Thompson CB:Apoptosis in the pathogenesis and treatment of disease.Science 1995.267:1456-1462.
[8]Liaw L, Skinner MP, Raines EW, Ross R. Cheresh DA, Schwartz SM, et al. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro.J Clin Invest 1995; 95:713-24.
[9]Daiter E. Omigbodun A. Wang S. Walinsky D. Strauss JF Ⅲ. Hoyer JR., et al.Cell differentiation and endogenous cyclic adenosine 3.5,-monophosphate regulate osteopontin expression in human trophoblasts.Endocrinology 1996; 137:1785-90.
[10]Yue TL. McKenna PJ. Ohlstein EH, Farach-Carson MC, Butler WT, Johanson K. et al.Osteopontin-stimulated vascular smooth muscle cell migration is mediated by beta 3 integrin.Exp Cell Res 1994; 214:459-64.
[11]Kurupatip P, Tank S, Kumarasinghe G, et al. Inhibition of gene expression and growth by antisense peptide nucleic acids in a muhiresistant beta lactamase producing Klebsiella pneumonia strain[J]. Antimicrob Agents Chemother,2007,51(3): 8052-8111.
[12]Huang X W, Pan J, An X Y, et al. Inhibition of bacterial translation and growth by peptidenucleic acidstargetedtodomainll of23S rRNA[J]. J Pept Sci,2007, 13(4).2202-2261.
[13]Ferber D. A new way to combat therapy side effects [J]. Science,1999,285: 1651-1653.
[14]Ahlquist P. RNA-dependent RNA polymerases and RNA interference (RNAi) [J]. Cell Cycle,2003,2:22-24.
[15]Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature,1998,391(6669): 806-811.
[16]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans[J]. Nature,1998,391(6669): 806-811.
[17]Elbashir SM, Harbonh J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature,2001,411(6836): 494-498.
[18]Hannon GJ. RNA interference [J]. Nature,2002,418(6894):244
[19]Zamore PD, Tuschl T, Sharp PA, et al. RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell,2000 101 (1):25
[20]Zamore PD. RNA interference:listening to the sound of silence. Nat Struct Biol, 2001,8(9):746
[21]Bmmmelkamp TR, Bemards R, Agam i R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J]. Cancer cell,2002,2(3):243-247.
[22]Grzino F, Arduini F, Ruzzoa A, et al. Combined analysis of Ecadherin gene(CDH1) promoter hypermethlation and E-cad-herin protein expression in patients with gastric can cer:implication for treatment with demethylating drugs[J]. Ann Oncol,2004,15(3):489-492.
[23]Ming J,Milner J.Selective silencing of viral gene expression in HPV-Positive human cervical carcinoma ceils treated with siRNA, a P rimer of RNA interference [J]. Oncogene,2002,21:6041-6048.
[24]Spankuch-Schmitt B.Bereiter-Hahn J, Kaufmann M.et al. Effect of RNA silencing of polo-like kinase-1(PLK1) on apoptosis and spindle formation in human cancer cell[J]s. J Natl Cancer Inst,2002,94(24):1863
[25]Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference [J]. Cancer Cell,2002,2(3):243
[26]Sbankar P, Manjunath N, Lieberman J. The prospect of silencing disease using RNA interference[J]. JAMA,2005,293(11):1367-1373
[27]Coppola D, Szabo M, Boulware D et al.Correlation ofesteopontin protein expression and pathological stage across a wide variety of tumor histologies.Clin Cancer Res,2004.10:184-190.
[28]Adwan H, Bauerle TJ.Berger MR.Downregulation of esteopontin and bone sialoprotein Ⅱ is related to reduced colony formation and metastasis formation of MDA-MB-231 human breast cancer cells. Cancer Gene Ther.2004,11:109-120.
[29]薛松,汪水义,尹邦良,等.骨桥蛋白小干扰性RNA抑制大隐静脉平滑肌细胞增殖和迁移能力的研究.中华实验外科杂志,2007,24：990-992.
[30]柳昊,陈安民,郭风劲,蒋林,胡伟华,解杰.体外RNA干扰抑制骨桥蛋白 表达对肾癌细胞生长和侵袭的影响.中华实验外科杂志2011,28：857-860
[31]Jen KY.Gewirtz AM.Suppression of gene epression by targeted disruption of messenger RNA; available options and current stratefies[J]. Stem Cells.2000.18(5):307-319
[32]Shevde LA, Samant RS, Paik JC.et al. Osteopontin knockdown suppresses tumourigennicity of human metastatic breast carcinoma MDA-MB-435[J]. Clin Exp Metastasis,2006,23(2):123-133
[33]Guadagni F'Kantor J,Aloe S, et al.Detection of blood-borne cells in colorectal cancer patients by nested reverse, transcripdon-polymerase chain reaction for cardnocmbryonic antigen messenger RNA:longitudinal analyses and demonstration of its potential importance as an adjunct to multiple serum markers[J].Cancer Res, 2001,61(6):2523-2532
[34]Sienel W et al. Elevated expression of carcinoembryonic antigen-related cell adhesion molecule 1 promotes progression of non-small cell lung cancer.Clin Cancer Res.2003.9:2260-2266.
[35]Thies A et al. CEACAM1 expression in cutaneous malignant melanoma predicts the development of metastatic disease.J Clin Oncol,2002.20(10):2530-2536
[36]Gangopadhyay A, Lazure DA, Thomas P. Adhesion of colorectal carcinoma cells to the endothelium is mediated by cytokines from CEA stimulated Kupffer cells. Clin Exp Metastasis.1998 Nov;16(8):703-12.
[37]Wirth T et al.[J]. Inhibition of endogenous carcinoembryonic antigen (CEA) increases the apoptotic rate of colon cancer cells and inhibits metastatic tumor growth. Exp Metastasis,2002,19(2):155-160