The Roles Of OPN And CEACAM5Expression In OTSCC And Correlation Research

BackgroundCancer 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.Objective1. 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.Conclusion1. 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.