Effects Of Ultrasound Destructing Lhrha-targeted Microbubble Mediated Survivin Targeted Silencing On The Migration And Metastasis Of Ovarian Cancer A2780/DDP Cells

PARTⅠ Expression of Survivin in ovarian cancer cell linesObjective: To detect the expression of Survivin protein in severalovarian cancer cell lines.Methods: Three ovarian cancer cell lines HO8910, SKOV3andA2780/DDP were cultured in vitro. The expression level of survivin proteinwas determined by immunofluorescence staining and Western-blot.Results:①immunofluorescence staining: The Fluorescent labeling rateof HO8910, SKOV3and A2780/DDP cell lines were75.34%±2.52%,74.77%±2.65%and81.04%±2.12%, respectively. The Fluorescentlabeling rate of A2780/DDP cell lines were significantly higher thanHO8910and SKOV3cell lines(P<0.05).②Western-blot: The ratio ofsurvivin/β-action in HO8910, SKOV3and A2780/DDP cell lines were0.624±0.028,0.638±0.029and0.702±0.017,respectively. The ratio ofsurvivin/β-action in A2780/DDP cell lines were significantly higher thanHO8910and SKOV3cell lines(P<0.05).Conclusion: Compared with ovarian cancer HO8910and SKOV3cellline, A2780/DDP cell line has higher expression of Survivin protein. It is a suitable ovarian cancer cell line for studying the function of survivin geneas well as targeted silence.PARTⅡ Recombinant plasmid pshRNA-Survivin transferedinto ovarian cancer A2780/DDP cells by the ultrasound mediatedLHRHa-targeted microbubbles destruction and the influence ofthe invasion and metastasis after treatment.objective: To transfect recombinant plasmid pshRNA-Survivin usingultrasound targeted microbubble destruction(UTMD) techniques andinvestigate the effects of RNA interference on ovarian cancer cell lineA2780/DDP in silencing survivin gene and inhibiting invasion andmetastasis.Methods: Recombinant expression plasmid of short hairpinRNA(shRNA) targeting survivin gene named pshRNA-Survivin wasconstructed. the A2780/DDP cells were randomly divided into7groups:control group(C), Plasmid group(P), Plasmid+microbubble group(P+Mb),Plasmid+LHRHa-targeted microbubble group(P+TMb), Plasmid+ultrasound group(P+US), plasmid+microbubble+ultrasound group(P+Mb+US) and Plasmid+LHRHa-targeted microbubble+ultrasoundgroup (P+TMb+US), the expression level of survivin protein wasquantitatively determined by western-blot. the adhesion of A2780/DDPcells to artifical basement membrane Matrigel was measured by MTT assay.The invasion and metastasis were evaluated by transwell chambers attachedwith polycarbonate filters.The expression of MMP-2、E-cadherin proteinwas quantitatively measured by western-blot.Results:①Compared with the control group, the expression of survivinprotein in P+US,P+Mb+US and P+TMb+US groups were significantlyinhibited according to results of western-blot (p<0.05). The invasive and metastatic cell numbers in those groups were significantly decreasedalso(p<0.05). The result of western-blot showed a down-regulationMMP-2expression (p<0.05) and an up-regulation E-cadherin expression inthose three groups(p<0.05); The expression level of survivin protein, theinvasive and metastatic cell numbers, the expression of MMP-2inP+TMb+US groups were significantly lower than that in P+US andP+Mb+US groups(p<0.05).However, the expression level of E-cadherinprotein in P+TMb+US group was significantly higher than that in P+USand P+Mb+US groups(p<0.05).②Compared with the control group，theadhesion force in P+Mb+US and P+TMb+US groups were significantlydecreased (p<0.05); the adhesion force in P+TMb+US group wassignificantly lower than that in P+Mb+US group also(p<0.05).Conclusion: UTMD mediating RNA interference could silencesurvivin gene and suppress the abilities of adhesion,invasion and metastasisof ovary cancer cells，which might become an effective adjuvant therapyfor ovarian cancer in the future.