Cloning, Function And Anti-tumor Study Of PNAS-4 Gene

Malignant tumor is a great threaten to human hearth and life. With the comingof 21st century, malignant tumor has become the first cause of death amongvarious human diseases. Conventional treatment ways such as radiotherapy,them-therapy and operation-therapy for tumor therapy had maken a greatprogress, but new treatment ways against tumor have to been found to refrainfrom their increasing drawbacks. With the development of molecular biologyand the deepening of knowledge of tumor molecular mechanism, Gene therapyprovides chances for healing well of tumor indeed. Since 1990s, scientists allover the world have maken many great contributions to tumor gene therapy,which caused a rising and fast development of tumor gene therapy. However,there are still lots of problems to be solved, the most urgent among them focuseson few valuable genes which can inhibit tumou growth, their complicated signalpathways and unclear protein-protein interaction.Apoptosis describes a phenomenon about gradual death of cells. The processduring which the cells die initiatively is strictly regulated by genes. Melucularoncology studies found apoptosis has close relationship with the genesis,development of tumor and effectiveness of treatment against tumor. The genesisof tumor mainly stems from the mutation of oncogenes, anti-oncogenes or otherrelative regulation genes, which destabilizates cellular genome. As a result, tumor develope and deteriorate. It is one of important strategies in the field ofmalignant tumor therapy to induce selectively cell apoptosis of tumor. Of these,the most appealing is the gene therapy strategy aimed at inducing tumor cellapoptosis.With the completion of sequencing for human genome in 2004, it proclaimedthe coming of an era of function genome aimed at cloning new genes andelucidating their functions and interaction of genes, which facilitates us to findand explore more new apoptosis-related genes for various diesease therapyincluding tumor therapy. PNAS-4 is one of new genes identified in large scalesequencing by NIH. Studies reported that PNAS-4 is a novel pro-apoptotic geneactivated during the early response to DNA damage. Its overexpression cancause death of human osteosarcoma U2OS cells via apoptosis, which make it ofa new valuable gene applied to tumor gene therapy. But so far, its detailedmechanisms have hardly been known. For this reason, this paper reported theisolation of Xenopus, mouse and human PNAS-4 and the preliminaryinvestigation on this gene in embryonic development, mouse tumor therapy andthe mechanisms by which the overexpression of this gene inhibits tumor growth.The results are as follows:1. We isolated the Xenopus PNAS-4 (xPNAS-4) gene and characterizated it bybiology informatics, xPNAS-4 protein is a cytoplasm protein that appearsaround perinuclear location. Development expression profile assay showedxPNAS-4 is a maternal inheritance gene expressing in all stages of embryos.In embryonic tissue, xPNAS-4 mainly appears in brain, heart and dorsalnerve, overexpression of xPNAS-4 by mRNA microinjection caused eyedevelopment defects, showing reduced eye size and hypopigmentation inXenopus embryos, and dissymmetric eye or one eye phenotype inzebrafish embryos. Bend chorda vertebralis can also be observed in stronglyaffected zebrafish embryos.2. We alse isolated the mouse PNAS-4 (mPNAS-4) gene. Cell proliferation activity (MTT) analysis in vitro found overexpression of mPNAS-4 caninhibit mouse LL2 and CT26 tumor cell growth. PI staining and DNA ladderassay showed this inhibitation caused from apoptosis. The anti-tumorexprements in vivo showed the overexpression of mPNAS-4 cansignificantly inhibit tumor growth and prolong life of mouse bearing tumor.3. Moreover, we detected the functional mechanisms of human PNAS-4 andfound overexpression of human PNAS-4 can result in cell cycle arrest in Sstage and induce cell apoptosis in A549 cells. Western blotting analysisshowed overexpression of PNAS-4 can cause Cyt c transposition tocytoplasm from mitochondria, up-regulation of p21,E2F1 and Bax,down-regulateion of PCNA, HSP70 and Bcl-2, appearance of activityfragment of Bid and caspase 3. But we failed to detcet p53 change in A549cells overexpressing PNAS-4. Using p53 inhibitor (Pft-a) can not rescuethe apoptosis resulted from overexpression of PNAS-4.Taken together, these findings may provide a foundation for furthermechanism study and its application of PNAS-4 gene to tumor gene therapy.