Vector Construction And Silencing Effect Of Edg4 Gene Targeted Small Interfering RNA

Background and aimsOvarian carcinoma is difficult to cure due to its high mortality and great hazard. Because of located in the deep pelvic bottom without early signs and symptoms, about 75-80% of all patients are final diagnosed to be advanced stage, which leads to difficult to diagnose and treat the ovarian cancer in clinic. Lysophosphatidic acid (LPA), deriving from grease of cellular membrane, is present at elevated concentrations in the ascites and plasma of ovarian cancer patients. It is produced and released from ovarian cancer cells constitutively or after stimulation. LPA can increase ovarian caner cell proliferation, survival, protease production and activation, invasion, production of cytokines, and resistance to chemotherapeutic agents. It is thought that LPA has a close relation with occurrence, development, invasion, metastasis of carcinoma. It is very important to carcinogenesis and development of ovarian cancer to decrease extracellular LPA concentration by modifying its metabolism, blocking LPA receptors, or interfering with the LPA signal transduction pathway. Blocking a series of LPA cascade reaction will possibly become a new method to ovarian cancer therapy.LPA(1-acyl-2-lyso-sn-glycero-3-phosphate) is the simplest phospholipids and mediates mult- iple functions ranging from growth promotion and increased cell cycle progression to cell survival. The effections of LPA signaling are determined by the LPA receptors expressed on the cell surfaces. The LPA receptors belong to members of the Edg family of G protein-coupl-ed receptors, which have been proposed to mediate LPA signaling transduction pathway for multiple biological effection. Edg2 expresses stably in the normal or immortalized ovarian cells. Edg4 and Edg7 yet overexpress in the ovarian epithelial cells, low expression or non-expession in the normal and immortalized ovarian cells, which suggests that high expression of Edg4 and Edg7 maybe relate to the effection of LPA in the ovarian cancer.RNAi which develops in recent years open up a new door for molecule biological therapy of tumor. Compared with traditional gene silencing, it has effect and lasting a long period of time. At present, the technique has taken on an attracting perspective in gene therapy of tumor. So this experiment design and construct expression vector of siRNA to aim directly at Edg4, transfect it into human ovarian cancer cell line SKOV3, observe its silencing establyMethodsAccording to the Edg4 sequence GenBank has been adopted, we used the siRNA design and analysis software which were provided by following Web address (http://www. ambion.com/techlib/misc/siRNA_design.html) to design the target oligonucleotides. Then the oligonucleotides of target siRNAs were indexed through National Center for Biotechnology Information (http://www.ncbi.nlm.nih. gov/Blast) to compare their homologization, in order to identify the best siRNA oligonucleotides of Edg4. Afterwards, the siRNA oligonucleotides of Edg4 which have palindrome and loop structure were synthesized. The DNA was cloned into pGEM-T Easy vector. Then,α-complementation test and T7/SP6 PCR were used to screen the positive clones(pGEM-T-siEdg4-1 and pGEM-T-siEdg4-2); After this, the target DNA was cut down from pGEM-T-siEdg4-1 and pGEM-T-siEdg4-2 by BamHI and XhoI restriction enzyme, and linked with siRNA express vector(pRNAT-U6.1);The positive ones(pRNAT-U6.1-siEdg4-l and pRNAT-U6.1-siEdg4-2) were selected by PCR. Then the target DNA was sequenced. Eventually, the siRNA express vector pRNAT-U6.1-siEdg4-l and pRNAT-U6.1-siEdg4-2 were transfected into human ovarian cancer cell line (SKOV3). The expression level of Edg4 mRNA was detected by real-time PCR.Result1. The 20 target siRNA oligonucleotides were obtained. After BLAST in NCBI, two target siRNA sequences with 19 bases were ensured.SE1:GACCATCGGCTTCTTCTAT (212-230);SE2:GACCAATCTGCTGGTCATA (323-341) .2. After the hairpin DNA of siRNA annealing, the results of agar gel electrophoresis analysis showed that there was an obvious stripe whose molecular weight was similar to the expected.3. The annealing products was recombined with pGEM-T Easy, which was transformed into JM109.After screened and identified,the positive clone (pGEM-T-siEdg4-l and pGEM-T-siEdg4-2) was obtained.4. After sub-clone, the pRNAT-U6.1-siEdg4-l and pRNAT-U6.1-siEdg4-2 were obtained andselected by PCR.5.After sequenced, the DNA sequences inserted the recombination pRNAT-U6.1-siEdg4-l and pRNAT-U6.1-siEdg4-2 were conformed completely with the design.6. A great quantity of GFP can be observed in transfected SKOV3 cell by fluorescence microscope 48 hours later. And cell clones resistanting to G418 were selected.7. The mRNA level of Edg4 in experimental group transfected by pRNAT-U6.1-siEdg4-l and pRNAT-U6.1-siEdg4-2 was obviously reduced. The mRNA level of Edg4 was still high in empty vector control group and untransfected group.Conclusion1. The siRNA oligonucleotides hairpin target Edg4 gene were design.2. The express vector pRNAT-U6.1-siEdg4 and pRNAT-U6.1-siEdg4-2 were constructed successfully.3. The express of Edg4 mRNA in ovarian cancer cell line (SKOV3) was obviously silenced after pRNAT-U6.1-siEdg4 and pRNAT-U6.1-siEdg4-2 transfected.