| Ribonuclease inhibitor (RI) is an acidic cytoplasmic glycoprotein with a 50 kDa estimated molecular weight. RI can inhibit the activity of ribonuclease A (RNase A) by tight combination with it (ki=4.0×10-14M). The amino acid sequence of angiogenin (Ang) is high conservative compared with RNase A. Ang is a member of RNase superfamily. Ang is produced and secreted by tumor cells and normal cells and its molecular mass is 14.4 kDa. It has a robust capacity to induce blood vessel formation in vivo and in vitro. On the basis of analysis of cDNA sequence, Ang is 35% identical with human pancreatic RNase. Ang forms more restricted complex (Ki=7.1×10-16) with RI than that formed by RI and RNase A. X-ray analysis showed that Ang had a very similia space structure. They can both form tight complex with RI. So it can inhibit the activity of Ang. Ang expresses highly and promotes angiogenesis in colon, liver tumor,etc. RI may inhibit angiogenesis through blocking on b-FGF pass way too. So anti-angiogenesis will be an efficient method in the inhibition of the growth and metastasis of cancer. The experiment demonstrated that RI could effectively block the angiogenin induced angiogenesis. RI is constructed almost entirely of leucine-rich repeats. Such repeats have been identified in more than 100 proteins that exhibit a wide range functions, including cell-cycle regulation, DNA repair, extracellular matrix interaction, and enzyme inhibition. RI was recognized as a regulating element on formation of new blood vessels in embryo developing, wound healing, and tumor occurring. RI located on the chromosome 11p15.5, nearly ras gene, 11p15.5 often has change in cancer patients. The study indicated that more than one locus at 11p15 could be involved in tumorigenesis, therefore, RI may has relation with the cell growth and differentiation,which might involve in unclear biological effects. Previous study indicated that RI extracted from human placenta could inhibit the some kinds of transplant tumor growth in the animal body, including Ca761 breast carcinoma, S-180 sarcoma, SHG44 and C6 glioma. Our previous experiments also showed that RI activity reduced in the blood of tumor patients and RI mRNA was significantly lower in the human breast than that in the normal tissue. Angiogenesis, the formation of new blood vessel, plays an important role in the growth of solid tumors. If solid tumor grows to 1-2 mm without new blood vessel formation, the tumor will degenerate. These results suggest RI might be correlated with some tumorigenesis and metastasis as one of candidate tumor suppressor genes (TSGs). However, nowadays whether RI has other tumor-suppressive effects remains unclear, its exact molecular mechanism of antitumor has not been totally ascertained. In order to understand further the function of RI and investigate the relationship between RI and tumor growth, invasion, metastasis for its exact molecular mechanism of antitumor. The study detected the effects on DNA methylation inhibitor on the expression of ribonuclease inhibitor in the cancer cell lines; established a transfection of human RI gene cDNA into murine B16 cells by the retroviral packaging cell line PA317 carrying the pLNCX-RI in vitro to observe the effects of transfected RI on cell proliferation, apoptosis in vitro and tumor growth, metastasis in vivo; analyzed the expression of RI gene using human bladder transitional cell carcinoma with different invasive potential as model to explore effects of RI on invasion and metastasis. The main work is as followings:1. Human breast carcinoma cell line MCF-7, human gastric carcinoma cell line BGC-823, human prostate carcinoma cell line DU-145 and human colon carcinoma cell line HT-29 were treated by methylation inhibitor, 5-aza-2'-deoxycytidine(5-Aza-CdR). The expression of RI gene was analyzed by RT-PCR, Western blotting, immunofluorescence and immunocytochemistry. The data showed that 5-Aza-CdR can significantly elevate the expression of RI at both mRNA and protein leves in MCF-7, BGC-823, DU-145 cell lines, but n... |
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