The Construction Of Expression Vector Of Anti-sense Cyclin D1 RNA Induced By Retinoic Acid, And Its Effect On The Leukemia Cells

As an activity metabolite of vitamin A, retinoic acid (RA) plays an important role in normal cell growth and tissue development. All of these functions are mediated through its specific nuclear receptors, i.e., RARs and RXRs. These activated nuclear receptors, in turn, bind to specific DNA sequences called as retinoic acid response element (RARE) that are located in the regulatory portions of genes and thus modulate gene activity. Many experiments and clinical investigations found that RA could resist proliferation,induce differentiation and start apoptosis . Now all trans retinoic acid (ATRA) and 13-cis retinoic acid (13-cRA) have been successfully used in the clinical therapy of many cancers such as leukemia,carcinoma of kidney,mammary cancer and so on. But now there are occasionally early tolerance to RA, relapse of the disease and retinoic acid syndrome which limit the application of RA during the period of RA treatment. So it is necessary to take optimal strategies to overcome the resistance to RA and decrease ill effects in RA treatment.Now researchers think that cell cycles play an important role during the occurrenc and development of tumor. Cyclin D1 forms compound with CDK4/CDK6 in the G1 phase and hyperphosphorylate pRb to drive cell proliferation. Unregulation of cyclin D1 expression can cause disturbance of cell cycle and lead to occurrenc of tumor. Abnormalities of cyclin D1,such as CCND1 amplification,chromosomal translocation and cyclin D1 gene polymorphism , are found in many cancers . Cyclin D1 can be used as an important prognosis factor to metastasis or prognosis of malignant tumor. Anti-sense RNA is a micromolecular transcript which can bind target RNA by base complementation and restrain its function. As one of the anti-sense nucleic acid method, anti-sense RNA technique is a gene therapy method by selectively blocking target gene expression and have been developed in the field of tumor research and therapy. Many oncogenes become effective target because conditioned activation of protooncogene is a basic cause of tumor occurrence. Using DNA recombination technique, we constructed an eukaryotic expression vector(pCI-neo/RARE3-TK/AScyclinD1) containing anti-sense cyclinD1 RNA and observed anti-sense cyclin D1 is controlled in the dependence of RA by immunohistochemistry. Meanwhile with MTT assay,NBT assay,transmission electron microscope observation ,Fluorescent antibody detection,RT-PCR and western blotting ,we studied the effections to HL-60 cells by RA and anti-sense cyclin D1 induced by RA and tried to infer the likely molecular mechanismsThe results and conclusions are summarized as follows:1. The eukaryotic expression vector (pCI-neo/RARE3-TK/AScyclin D1) containing anti-sense cyclin D1 was constructed and transfected to HL-60 cells. After selected by G418, transfected cells were treated with RA, as compared with nontransfected ones treated with RA at the same time. We observed cyclin D1 expression were lower in transfected cells than nontransfected . This result showed that RA can induce the expression of anti-sense cyclin D1 in transfected cells 2. After treated by RA, we observed that RA could suppress cell proliferation and induce cell differentiation of pCI-neo/RARE3-TK/AScyclin D1 transfected cells and untransfected cells. These changes were more obvious in transfected cells than untransfected cells. Our study demonstrated that RA could induce the expression of anti-sense cyclin D1 to evoke correlative effect and also showed that RA and the product of anti-sense cyclin D1 induced by RA could cooperate in inhibition of cell growth and inducing cell differentiation. 3. After pCI-neo/RARE3-TK/AScyclinD1 transfected cells and untransfected cells were treated by RA, we found that the expression of Rb mRNA was decreased, the level of CDK4 mRNA and protein expression were also decreased and p16 protein expression was increased. These changes were more obvious in transfected cells than untransfected cells. Results suggested that the changes of them may...