Study On The Effects Of RNAi Targeting COX-2Gene On Biological Behaviors Of Ovarian Cancer Cells And Serum MiR-92Expression In Patients With Epithelial Ovarian Carcinoma

Objective:To study the effects of plasmid mediated-RNAi targeting COX-2gene on biological behaviors of human ovarian cancer SKOV3cells invitro and vivo.To investigate the role of COX-2in carcinogenesis anddevelopment of ovarian cancer and provide the theroretical basis fortreatment of ovarian cancer.Methods:1. A shRNA sequence was synthesised designed for the COX-2gene.By annealing and digesting, shRNA double-stranded template wasinserted into pGPH1/GFP/Neo siRNA Expression Vector. Aftertransformation of E. coli competent cells, screening of positive coloniesand amplifying operation, the plasmid was extracted for enzyme digestionand DNA sequencing. Then the correct sequencing recombinant vectorplasmids were transfected into SKOV3cells instantaneously andpermanently. The expressions of COX-2mRNA and protein weredetected by RT-PCR and Western blot respectively.2. To study the effects of RNAi targeting COX-2gene on biologicalbehaviors of human ovarian cancer SKOV3cells in vitro,MTT assay andcolony formation test were used to detect ability of cell proliferation.Flow cytometry was used to detect cell phase changes in the cell cycle.Transwell assay was used to detect cell invasion.3. The SKOV3cells with recombinant vector plasmids and control cells were injected into nude mices, subcutaneous tumor models werebuilted. Differences of Tumor cells emergence time, volume and weightwere detected, and the impact of COX-2silence on the growth of theimplanted subcutaneously tumor in nude mice was analysised.Results:1. The pGPU6-COX-2-shRNA expression vectors were successfullyconstructed.2. Recombinant vector plasmid were transfected into SKOV3cellsinstantaneously and permanently.Stable transfected recombinant plasmidSKOV3cells were established.3. Following the transfection of the pGPU6-COX-2-shRNAPlasmid，RNAi suppressed the mRNA and protein expression of COX-2efficently and stably.4. Silencing COX-2expression of significantly inhibited the abilityof proliferation and invasion of SKOV3cells, and led to cell G1arrested.5. Compared with the control group, Tumor formation time in theinterference group was significantly prolonged, and the tumor volumeand tumor weight were significantly decreaced.Conclusion:Plasmid-mediated RNAi can effectively silence COX-2expressionin the ovarian cancer SKOV3cells. The COX-2plays an importantregulatory role in the proliferation, cell cycle regulation and invasion ofovarian cancer. RNAi targeting COX-2gene on ovarian cancer may be anuseful treatment. Objective:Ovarian cancer, especially epithelial ovarian cancer (EOC), whichaccounts for90%of ovarian cancer, continues to be the leading cause ofdeath among gynecological malignancies.Due to the asymptomaticnature of early stages of this disease, more than two-thirds of the casesare not diagnosed until the disease has spread beyond theovaries.Therefore, early detection of the disease is essential to improvethe survival of EOC patients, which can be facilitated by identifyingbiomarkers that enable early detection. Although tumor markers greatlyimprove diagnosis, the invasive, unpleasant, and inconvenient nature ofcurrent diagnostic procedures limits their develpopment and application.Thus, the development of novel non-invasive biomarkers of EOC isurgently needed for early tumor detection.MicroRNAs (miRNAs) are a recently discovered class of small(18–24nucleotides) non-coding RNAs that regulate variety of cellularprocesses, such as cell differentiation, cell cycling, and apoptosis.Several studies desmonstrate that the profiles of miRNA expressiondiffer between normal and tumor tissues.Several studies have reported the significance of circulatingmicroRNA(miRNA) as a biochemical marker of cancer. However, there are little reports that have demonstrated the significance of miRNAs inthe serum of patients with ovarian epithelial carcinomas(EOC).Therefore, the aims of this study were to clarify the status ofmicroRNA-92(miR-92) in serum EOC patients and to investigate therelationship between miR-92and the clinical feacture of EOCMethods:The miR-92levels in serum samples from50EOC patients and50healthy controls were measured by using quantitative reversetranscription-polymerase chain reaction(qRT-PCR). In addition, Clinicalparameters of EOC were assessed to determine associations with serummiR-92concentrations.Results:As a maker of EOC, serum miR-92is perfect with good sensitivityand specificity.concentration of miR-92in EOC patients wassignificantly higher than that in healthy controls (P<0.05). Theexpression of miR-92was correlated to regional lymph nodes andprogression of the clinical stages(P<0.05), and no significant associationwith age.Conclusion:These findings imply that concentration of miR-92in EOC patientswas significantly higher and miR-92may play an important regulatoryrole in carcinogenesis of EOC.The detection of miR-92in serum might serve as a novel non-invasive tumor biomarker in early diagnosis andassessment of prognosis of ovarian epithelial carcinomas.