The Effect Of Osteopontin Expression To The Biological Behaviour In Human Colon Cancer

Colorectal cancer (CRC) is the third most common malignancy around the world. Annually, over945,000people develop CRC around the world, and around492,000patients die from the disease. In China, the incidence of colon cancer increased year by year. CRC has become one of the most common gastrointestinal malignancy.Now, surgery is the most effective way to treat colorectal cancer. In the past30years, although advanced surgical and improved diagnostic techniques were used, the change of fatality rate is very small. In newly diagnosed patients, major of them are with advanced cancer. Only75%-80%of them has indeed underwent radical surgery, the5-year survival rate is only about60%.Tumor development is a complex process, and produce a variety of molecules around a variety of different tumor cells through autocrine and paracrine mechanisms constitute the microenvironment, fine-tune the interaction with the tumor. In1979, Senger first reported that the malignant transformation of epithelial cells secrete a phosphorylated glycoprotein, osteopontin (Osteopontin, OPN), OPN is secreted glycoprotein with rich in aspartic acid and sialic acid residues, contains a variety of functional domains. Through a combination with integrins and CD44receptor, OPN-mediated cell matrix interactions and intracellular signal transduction. Osteopontin regulate normal physiological processes, including bone resorption, promote wound healing, tissue remodeling, immune response and angiogenesis, and a variety of pathophysiological conditions, such as myocardial necrosis, restenosis, atherosclerosis, and autoimmune disease. In various stages of cancer development, Osteopontin played an important role in tumor invasion, angiogenesis and metastasis. Recent reports show that osteopontin overexpression in tumor surrounding stromal cells, which show that the process it tumor stromal interactions play an important role-led to the development of cancer. Clinical studies show that high expression of osteopontin in a variety types of cancer and a variety of tumor tissues and serum. Furthermore, enhanced expression of osteopontin can increase tumor growth and metastasis, indicating that osteopontin can be used as a biomarker for diagnosis and prognosis in various cancers. OPN gene and its related factor is a potential target for cancer prevention and treatment. However, the role of OPN in colorectal cancer and its mechanism is not fully understood. We examined the expression of OPN in colorectal adenoma-carcinoma sequence to understand OPN’s role in the formation of colorectal cancer. In cultured colon cancer cell line HT-29, we use plasmid transfection or RNA interference technology to raise or lower expression of OPN, to observe the impact to the biological behavior of tumor cells, simultaneous detect the expression of multiple tumor-related factors, and possible mechanism of OPN in colorectal tumors was discussed.PART ONE The expression of osteopontin in the colorectal adenoma-carcinoma sequenceWe collected60cases of primary colorectal cancer specimens,60cases of normal mucosa samples (taken from the same tumor specimens,10cm from the tumor margin), and50cases of colorectal adenoma esected by colonoscopy. Detected the expression of OPN in colorectal adenoma carcinoma sequence. All specimens were confirmed by histopathology, no other history of cancer. Before treatment, all patients were not underwent radiotherapy and chemotherapy. Colorectal adenoma specimens contained19cases of low-grade epithelial neoplasia and31cases of high-grade epithelial neoplasia. We first extracted total RNA and protein from all the specimens, and then the expression levels of OPN mRNA and protein in different tissues were detected by RT-PCR and Western Blot method;Using immunohistochemical techniques, OPN expression in adenoma, adenocarcinoma and normal colorectal tissue were observed. RT-PCR results showed that OPN mRNA expression levels in adenomas and adenocarcinomas were significantly higher than that in the normal mucosa, and OPN mRNA levels and malignancy degree of epithelium was positively correlated.. In contrast, OPNmRNA can not be detected in almost all normal colorectal mucosa tissue.. Consistent with this, the Western Blot results show that OPN protein expression in adenomas and adenocarcinomas were significantly higher than that in normal mucosa. The immunohistochemistry results showed and OPN positive staining mainly located in cytoplasm, the vast majority of normal mucosa cells were OPN negative staining. While the adenoma specimens OPN expression positive rate was significantly higher than in normal mucosa. With increasing degree of malignancy of the adenoma level, positive rate of OPN expression in high-grade epithelial neoplasia was higher than that in the low grade epithelial neoplasia, Positive rate of OPN expression in colorectal adenocarcinoma was higher than the adenoma. These results suggest that in the colorectal adenoma-carcinoma sequence, with the increase of atypical hyperplasia severity of epithelial cells, OPN expression level was significantly elevated, indicating that OPN may play an important role in the process of malignant transformation in colorectum.Part II The effect of OPN on the growth and migration of colon cancer cellsAlthough studies have shown that increased OPN expression in a variety of tumor tissue, but the effect and mechanism of OPN on colorectal cancer remains unclear. To study the role of OPN in colorectal tumor cells, we used human colon cancer cell line HT-29as an in vitro model to study the role of OPN in colorectal cancer. We transfected the OPN knock-down (KD) and OPN expression plasmid to the human colon cancer cell line HT-29to up and down regulate OPN expression in colon cancer cells. Western blot analysis showed the significant higher level of OPN expression in OPN expression plasmid transfected colon cancer cells. OPN expression levels in colon cancer cells transfected with the OPN-KD plasmid was significantly lower than the control group. From cell viability, proliferation, and migration and in vivo tumorigenesis, We studyed the role of OPN on colon cancer cells. First, we used MTT to assay the effect of OPN on colon cancer cell viability. The results showed that, compared with the control group, reduced expression of OPN significantly inhibit the growth of colon cancer cells. Conversely, over-expression of OPN promoted the activity of the colon cancer cells. Furthermore, we adopted the method of cell colony formation rate to evaluate the influence of OPN on long-term proliferation of colon cancer cells. The results showed that the lower OPN expression significantly reduced the rate of colon cancer cell colony formation; contrast, over-expression of OPN colon increased cancer cell colony formation. These results suggest that high expression of OPN to promote the growth and proliferation of colon cancer cells. In addition to the unlimited growth and proliferation, infiltration and invasion to normal tissue is another notable features of tumor cells. Through the cell scratch test, we examined the impact of OPN on colon cancer cell migration. The results show that the lower OPN inhibited cell migration; contrast, over-expression of OPN promoted the migration of the colon cancer cells. Finally, by in vivo nude mouse tumor experiments, we tested the impact of OPN on the colon cancer cell tumorigenesis ability. The results showed that reduced OPN suppressed the tumorigenesis of cells in vivo; contrast, over-expression of OPN promoted colon cancer cells in vivo tumorigenesis.In short, high expression of OPN promoted the growth, proliferation, migration and in vivo tumorigenesis of colon cancer cell. Conversely, lowered the OPN expression inhibited the biological activity of colon cancer cells.Part III THE effect of OPN expression tumor-associated factors in colon cancer cellsNumerous studies show that Matrix metallopeptidase9(MMP-9),vascular endothelial growth factor(VEGF),Fibroblast Growth Factor(FGF-2) and Cyclooxygenase-2play important roles in tumor angiogenesis, invasion and growth of tumor cells. We adopted the same colon cancer cell model and the same OPN regulation mode to the second part, to explore the effect of OPN expression changes on MMP-9, VEGF, FGF-2and COX-2. MMP-9, VEGF, FGF-2, COX-2protein expression levels were detected by Western blot. The results of this study show that, reduced OPN expression significantly inhibited the expression of MMP-9, VEGF, FGF-2and COX-2, while raised OPN expression appeared opposite effect. The results suggest a positive correlation between OPN expression with variety of tumor factors. The impact of OPN on colon cancer cells Is at least partially achieved by affecting MMP-9, VEGF, FGF-2, COX-2and other tumor-related factors.In summary, we have revealed gradual higher expression of OPN in colorectal adenoma-adenocarcinoma sequence. High expression of OPN promotedf colon cancer cell growth and migration, down regulaed OPN expression had contrary effect. Furthermore, we also confirmed the impact of OPN to tumor-associated factors in colon cancer cells,and may play a role in colon tumor development through this mechanism. In view the role of OPN in colorectum, OPN gene and its related factor is a potential target for cancer prevention and treatment.