| Colorectal cancer (CRC) is one of the most common cancers in clinic, its incidence rate has raised to the third in large cities in China, and it is the third leading cause of cancer death. In despite of improvements treatment, the therapeutic outcome of CRC remains the same level as it was in 1970s and the overall five-year survival is about 50% to 55%. Local recurrences and distant metastasis are main reasons of therapy failure. The liver is the most common organ of distant metastasis of CRC, with extraordinary high incidence. At the time of initial diagnosis, 20%-40% of patients with CRC exist liver metastasis. In autopsies of patients who died of CRC, 36%-81% of autopsies revealed liver metastasis. It is one of the important factors for treatment of CRC.The formation of liver metastasis is related with biological characteristics of CRC cells, immune status of human body and microenvironment of the liver. The process can be expressed as: CRC primary lesion - portal vein - liver - hepatic veins - pulmonary circulation - systemic circulation ( or into bile). Each link of it is regulated by different genes, with synergistic or antagonistic effect each other. Only each condition is matched , the liver metastasis in CRC will be formed.Some of the molecules with relation to CRC liver metastasis include CEA, CD44, integrin,MMPs/TIMPs, SleX, nm23, DPC4, maspin, and so on. But the details are still unclear. Besides common mechanisms of other malignant tumors in digestive tract, liver metastasis in CRC may have some specific aspects.Complete understanding of the exact molecular mechanism of CRC liver metastasis, which may provide basis for efficient intervention of CRC liver metastasis in clinical treatment, is the hot spot at present, and also the main objective of this study.The affymatrix gene chips were used to measure the differential expression of more than 12,000 genes and ESTs in 21 pairs CRC and liver metastatic tissues. Osteopontin (OPN) was found to show much higher expression in liver metastasis tissues of CRC.OPN is a phosphorylated glycoprotein, rich in serine residue. The ROD (Arg-Gly-Asp ) domain in it is an important motif for cell adhesion. OPN is produced by osteoclasts, macrophages, T-cells, kidneys ,and vascular smooth muscle cells. It contributes to macrophage homing and cellular immunity. The importance of OPN in bone mineralization, kidney stone formation, cardiovascular disease and bacterial infection was widely reported. The function of OPN in tumorigenesis and metastasis is catching the sight of scientists. The mechanism of OPN in CRC liver metastasis is far from clear and the correlative reports are lacking.In this paper, we cloned the cDNA of OPN and carried out more detailed study of the possible role of OPN in CRC liver metastasis. Part I Differential expression of OPN mRNA in cancer and adjacent tissuesOPN mRNA expression level was detected in CRC tissue (including its adjacent normal mucosa and liver metastatic tissue), gastric cancer, breast cancer, hepatocarcinoma, peripheral blood, and several CRC cell lines by RT-PCR and real-time fluorescent quantitative PCR. The results show that OPN mRNA is the highest in liver metastatic tissue, moderate in CRC tissue, and the lowest in adjacent normal mucosa. OPN mRNA has a elevating tendency with the Dukes' stage ascending, but is not related with pathologic grade; OPN mRNA expression is both higher in gastric cancer and hepatocarcinoma than in their adjacent tissue; but the differences is not apparent in paired breast cancer; Differential expression of OPN mRNA is detected in peripheral blood of CRC patients. OPN mRNA expression is positive in Colo 205 cell line and is negative in SW480. Part II Construction of sense- and antisense-OPN eukaryotic expression plasmidsTo prepare RNA probe for in situ hybridization and to prepare cell transfection, pGEM-T Easy -OPN (antisense 201bp) and pGEM-T Easy -OPN (ORF) were cloned. pcDNA 3.1 (+)-OPN (antisense 201bp) and pcDNA 3.1 (+)-OPN (ORF) eukaryotic expressi...
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