Experimental Study Of Interfering RNA Targeting H-ras Gene Combined With Energy Controllable Steep Pulses Treating Ovarian Carcinoma

Steep pulsed electric fields(SPEFs) is a new low-invasive or mini-invasive physical approach in treatment neoplasm, which causes irreversible electrical breakdown(IREB) on the tumor cell membrane and results in cell death. Killing the survival tumor cells suppletively in marginal tissue after ECSP is a key question that influences the effect of energy-controllable steep pulse(ECSP) therapy. Tumor is a genome functional disease with characteristics of activation of cancer gene and nonactivation of tumor suppressor gene, which limits the availability of traditional therapy. With the development of molecularbiology ,immunology and virology , gene therapy could become a new method to effect a radical cure for cancer following surgical operation,chemotherapy and radiotherapy. Ras gene is a kind of proto-oncogene in normal cells which has a close relation with proliferation and differentiation of cells. Continuing activation of ras gene will lead to cell proliferation and cause immortal cells. Wild H-ras gene is higher expression in ovarian serous cystadenocarcinoma. We have used RNAi technology to silence H-ras expression and observe the cells proliferation, apoptosis and growth of tumor in order to ascertain the function of H-ras gene in development of ovarian carcinoma. ECSP combined with short hairpin RNA targeting H-ras gene were used to solve the remnants and relapse after ECSP. Our experiments provide a theoretical and experimental basis of combination ECSP and gene treatment for cancer. PART ONE CONSTRUCTING AND IDENTIFICATION OF THE SHORT HAIRPIN RNA RECOMBINANT PLASMID TARGETING H-RAS GENE OBJECTIVE: To construct the recombinant plasmid targeting H-ras gene coding sequence pshRNA/H-ras and analyzing nucleic acid sequence for further researching the role in the development of ovarian carcinoma. METHODS: A 21bp reverse repeated motif of H-ras gene target sequence with 4-5bp spacer were designed and synthesized. Annealing and inserted into pTZU6+1. These recombinant plasmids were transformed into JM109 strain. Then the recombinant plasmids were identified by restriction enzyme and DNA sequence analyzing. RESULTS: The recombinant plasmids targeting H-ras gene were constructed successfully. CONCLUSION: Constructing the pshRNA/H-ras recombinant plasmid helps to further study the inhibitory role of H-rasmRNA and the protein expression.PART TWO INHIBITION OF ENDOGENETIC H-RAS GENE EXPRESSION BY PSHRNA/H-RAS IN OVARIAN CANCER CELLS AND THE OVARIAN CANCER RESISTANT CELLS OBJECTIVE: To investigate the inhibition of endogenetic H-ras gene expression applying siRNA directed against H-ras in SKOV3 and SKOV3/ADM strains. METHODS: The expression level of H-ras gene in SKOV3 and SKOV3/ADM cells were measured by Immunocytochemistry. The recombinant plasmid targeting H-ras gene were transfected into SKOV3 and SKOV3/ADM with DOTAP lipofectmine and the expression of H-ras were detected by Reverse Transcripitional RCR,Western blot. RESULTS: There are higher expression of H-ras in SKOV3 and SKOV3/ADM cells. The expression of H-ras are all suppressed after transfected pshRNA/H-ras1 and pshRNA/H-ras2 respectively by RT-PCR and Western blot. CONCLUSION: These results indicated that the siRNA directed against H-ras could inhibit the expression of H-ras in SKOV3 and SKOV3/ADM cells and established a foundation to further study theunusual proliferation in cancer cells. PART THREE THE PROLIFERATIVE EFFECT ON SILENCING OF ENDOGENETIC H-RAS GENE EXPRESSION BY RNA INTERFERENCE IN SKOV3 AND SKOV3/ADM OBJECTIVE: To investigate the cells proliferation effect after transfected pshRNA/H-ras and discuss the role of H-ras gene in the development of ovarian cancer. METHODS: There are four groups in our study : normal control group, 24h , 48h and 72h experimental group after transfected pshRNA/H-ras. The influence on proliferation and apoptosis were investigated by MTT assay, cloning test, flow cytometry and AO/EB respectively. RESULTS: It shows the inhibition of cell cycle after transfected pshRNA/H-ras 24h and 48h in...