Dissertation > Excellent graduate degree dissertation topics show

Vector Construction and Silencing Effect of Edg4 Gene Targeted Small Interfering RNA

Author: ZhouWei
Tutor: LiLiuXia；QiaoYuHuan
School: Zhengzhou University
Course: Obstetrics and Gynaecology
Keywords: Edg4 RNA interference siRNA vector Ovarian Cancer Quantitative PCR
CLC: R346
Type: Master's thesis
Year: 2007
Downloads: 79
Quote: 0
Read: Download Dissertation

Abstract

Background and purpose of ovarian cancer is a common gynecological malignancies, clinically refractory tumors due to the location secluded pelvic floor, and the lack of early symptoms and signs, 75% -80% of patients diagnosed at advanced stage cause difficulties in the diagnosis and treatment of ovarian cancer. Therefore, the ovarian cancer mortality rate is the highest, the most harmful gynecological malignancies. Lysophosphatidic acid (lysophosphatidic acid, LPA) is a membrane lipid derivatives Recent studies have found that patients with ovarian cancer in plasma and ascites LPA levels were significantly increased, in vitro experiments show lower levels of LPA can inhibit ovarian tumor cell proliferation and promote its apoptosis. LPA promotes ovarian cancer cell proliferation, survival, and activation of protease, to promote the generation of angiogenic factors such as cytokines and tumor occurrence, development, invasion, metastasis are closely related. Thus affecting the generation of LPA metabolism, receptor expression and signal transduction pathways of the development of ovarian cancer is important, it is possible to become a new way of ovarian cancer treatment. Lysophosphatidic acid i.e. 1 - acyl glycerol 3 - phosphate (1-acyl-sn-glycerol-3-phosphate), and its basic structure contains a phosphoric acid group, the three parts of the glycerol backbone, and the long chain fatty acids. LPA is a lipid, small molecular substances, is a membrane source of biologically active lipid neurotransmitter, a signal transduction pathway between cells, as an intermediate product of the lipid metabolism. LPA mainly through its receptors play a variety of biological effects that many G protein receptor-mediated signal transduction pathways. Currently known human LPA receptor, there are three, namely endothelial differentiation gene protein (of endothelial differentiation gene Edg) Edg2 Edg4 Edg7. The where Edg2 in normal ovarian cells immortalized ovarian cells stably expressing the Edg4 and Edg7 overexpression in ovarian cancer cells, immortalized ovarian cells do not express or low expression in normal ovarian cells. Therefore, Edg4, and Edg7 expression may be related to the LPA on the role of tumor. RNA interference, the rise in recent years has opened up new avenues for the molecular biology of the tumor treatment. Compared to traditional gene silencing technology, RNA interference has the effect of long duration and other advantages, this technology in cancer gene therapy shows tantalizing prospect. This study was to construct the recombinant vector for the LPA receptors Edg4 the siRNA, double-stranded hairpin siRNA corresponding DNA sequence cloned into the vector transfected SKOV3 cells, intracellular expression Edg4 targeted siRNA molecules specific silencing Gene Edg4, thereby blocking the role of LPA, inhibit the growth of ovarian cancer cells, looking for new methods and targets for gene therapy of ovarian cancer. Method based on in GenBank Edg4 gene sequence (NM ₀ 04720), design, follow http://www.ambion.com/techlib/misc/siRNA-design.html principle of candidate sequences by the United States The retrieved Center for Biotechnology Information (National Center for Biotechnology Information) online homologous sequences http://www.ncbi.nlm.nih.gov/Blast select OK siRNA target sequence of 19 nucleotides. Separately synthesized targeting Edg4 siRNA sequence, annealed into double-stranded DNA, and cloned into pGEM-T Easy vector using α complementary and T7/SP6 PCR amplification Screening and identification of recombinants the pGEM-T-siEdg4-1 and pGEM-T- enzyme digested recombinant siEdg4-2; cut with BamH Ⅰ and Xho Ⅰ restriction and siRNA the expression the carrier pRNAT-U6.1; will be the double stick Edg4 hairpin siRNA fragment was subcloned into the siRNA expression vector pRNAT-U6.1; utilize The universal primers Screening and identification the recombinant pRNAT-U6.1-siEdg4-1 and pRNAT-U6.1-siEdg4-2; insertion sequence DNA sequence analysis. SiRNA expression vector pRNAT-U6.1-siEdg4-1 is pRNAT-U6.1-siEdg4-2 transfected into human ovarian cancer cell line SKOV3, SKOV3 cells transfected using fluorescence microscopy; using quantitative PCR The detected cells Edg4 mRNA expression levels. Results 1. Homologous sequences retrieved 20 Edg4 siRNA candidate target segment of these candidate sequences by the U.S. Center for Biotechnology Information Online finalized 212-230 bits (GACCATCGGCTTCTTCTAT) and 323-341 (GACCAATCTGCTGGTCATA) siRNA target sequence. 2. siRNA the card issuer DNA annealing, the the electrophoresis visible bright bands, located approximately 70bp at exactly the same and Design. 3. After the connection of the the annealing product and the pGEM-T Easy, transforming JM109, Screening and identification obtained the recombinants the pGEM-T-siEdg4-1 and pGEM-T-siEdg4-2 4. After subcloning, were identified using universal primers recombinant plasmid obtained pRNAT-U6.1-siEdg4-1 and pRNAT-U6.1-siEdg4-2 5. Recombinant the sub pRNAT-U6.1-siEdg4-1 and pRNAT-U6.1-siEdg4 2 insert DNA sequencing results with the design exactly. 6. 48 hours after transfection with a fluorescent microscope ovarian cancer can see a large number of GFP expression by G418 selection of transfected cells get their resistant cells communities. 7. Transfection Edg4 targeting siRNA (pRNAT-U6.1-siEdg4-1 ovarian cancer cells, pRNAT-U6.1-siEdg4-2) in the experimental group Edg4 mRNA expression levels significantly lower. The two control groups (control group and empty vector control group) there is a higher level of Edg4 mRNA expression in ovarian cancer cell line SKOV3 cells. Conclusion 1. Design for Edg4 hairpin siRNA oligonucleotide nucleotide fragment. 2. Successfully constructed for Edg4 of siRNA expression the carrier pRNAT-U6.1-siEdg4-1 and pRNAT-U6.1-siEdg4-2 3. the pRNAT-U6.1-siEdg4-1 and the pRNAT-U6.1-siEdg4 2 transfected ovarian cancer cell line SKOV3 can significantly reduce cell Edg4 mRNA expression.

Vector Construction and Silencing Effect of Edg4 Gene Targeted Small Interfering RNA

Abstract

Related Dissertations