Screening Of Antisense Oligonucleotides,SiRNA Targeting The Messenger RNA Of Endothelial Vascular Factor Feceptor-2 And Study Of Their Anti-Tumor Function

Objective: The completion of human gene sequencing and the progress in function genomics and proteomics results in the rapid identification of target gene involved in neoplastic transformation and tumor growth, which provide the premise for tumor gene therpapy. Antisense oligonucleotides and the recently developed RNA interference are the useful tool for gene therapy and the study of gene function. But it has been found that not all antisense molecule can effectively hybridize with taget gene and block the gene expression, due to the existing of second structure of target gene mRNA. Vascular endothelial growth factor receptor-2/ KDR (VEGFR2, also named as kinase-insert domain containing receptor: KDR ) is diffusively expressed in endothelial cells, partially in some tumor cells, and appears to play a pivotal role on promoting the tumor vascularization, tumor growth and metastasis. VEGF plays its role throuth paracrine or autocrine mechanism:(1) The paracrine stimulation of angiogenesis : solid tumor cells secret VEGF, which binds to KDR on vascular endothelial cells and activates the signaling pathway, resulting in the endothelial cell proliferation , neovascularization and the increase of vascular permeability. (2) The autocrine stimulatory effect on tumor proliferation: VEGF is an autocrine growth factor for some KDR positive human tumors, such as breast cancer. The exsting VEGF-KDR autocrine loops promote tumor cell proliferation directly. Inhibition of KDR expression have the potential to inhibit tumor growth on two levels: the level of the anigogenesis and the level of tumor cell. KDR is the suitable target molecule for gene therapy. In this study,from the full length of KDR mRNA,we try to select the antisense oligonucleotides which could hybridize with KDR mRNA in an effective and specific way,using the method of combination of computer prediction design or oligonucleotide library hybridization with oligonucleotide microarrays hybridization,respectively. According to the target sites of antisense oligonucleotides selected, DNA vector-based pPUR/U6/KDR-siRNAs for intracellular transcription KDR specific short hairpin siRNA were designed and constructed. In cultured MCF-7 cell line,the anti-cancer effects of asONs and siRNA were explored,and some signaling proteins expression changes after the inhibition of KDR protein expression were detected . At last,the anti-tumor activity of antisense oligonucleotide in vivo was investigated in the breast cancer nude micemodel. With these data, we tried to provide the experimental support for the possible clinical use of antisense oligonucleotides in the future. Methods: 1.Cloning VEGFR2/KDR gene:The VEGFR2/KDR gene was divided into KDR1(20-2823) and KDR2 (2711-5330) gene fragments to screen the mRNA antisense oligonucleotides accessible sites. Total RNA was isolated from A375 metastatic melanoma cells,and KDR1 and KDR2 gene fragments were got from RT-PCR .The PCR products were ligated with pGEMR-T vector and were named as pGEMR-T- KDR1, pGEMR-T- KDR2, respectively. After sequencing analysis, the plasmids with the inserted fragments (direction:5'~3') being the downstream of T7 promoter were selected for the following tests. 2. Screening the VEGFR2 mRNA accessible sites by combining computer prediction design with oligonucleotide microarrays:The minimal free energy second structures of KDR1 mRNA and KDR2 mRNA were analyzed with Mfold web server ,respectively. Antisense oligonucleotides probes targeting to local loose structure of RNA, such as hairpin or stem-loop etc, were designed. Oligonucleotide microarrays were fabricated by covalently binding the antisense oligonucleotides probes to aldehyde-coated glass slides. The recombinant plasmids pGEMR-T- KDR1, pGEMR-T- KDR2 were lined with Nde I, and internally radiolabelled KDR1 mRNA, KDR2 mRNA were in vitro transcribed by T7 RNApolymerase in the presence of [α-32P] UTP, respectively. Microarrays were hybridized in a closed chamber, and the hybridization intensity was analyzed using computer program ImageQuant 5.2...