Ultrasound Destruction LHRHa-targeted Microbubble Mediating Survivin Gene Silence And Inducing Apoptosis Of Ovarian Cancer A2780/DDP Cells

Objectives1.To investigate the reliability of encapsulating pshRNA-survivin genewith LHRHa-targeted lipid microbubbles by electrostatic adsorption and itstargeting ability in vitro.2.To screening the optimal parameters for ultrasound destructionmicrobubbles mediating gene transfecion.3.To investigate the effects of ultrasound destruction LHRHa-targetinglipid microbubbles mediated survivin silence on the proliferation andapoptosis of A2780/DDP ovarian cancer cells.Methods1.The LHRHa-targeted lipid microbubbles was prepared viabiotin-avid linkage.The pshRNA-survivin gene was encapsulated toLHRHa-targeted lipid microbubbles by adding Polylysine into the mixture.The LHRHa-targeted lipid microbubbles coated with LHRHa was detectedby immunofluorescent staining. The encapsulation efficiency was detected by spectrophotometer. The targeting ability of themicrobubbles wasobserved by optical microscopy.2.The optimal parameterof ultrasound intensity and exposure time aswell as the concentration of microbubbles and plasmid concentration forgene transfection were assayed by MTT.3.The A2780/DDP cells were divided into7groups:blank controlgroup,Plasmid group,Plasmid+microbubble group, Plasmid+LHRHa-targeted microbubble group, Plasmid+ultrasound group, plasmid+microbubble+ultrasound group and Plasmid+LHRHa-targetedmicrobubble+ultrasound group.The cell proliferation in each group wasassayed by MTT24h,48h and72h after treatment.The apoptosis rate ofeach group was detected via FCM,the expression of survivin mRNA wasdetected by Q-PCR.And the expression of surviving, caspase-9andcaspase-3protein were assayed by western blot48h after treatment.Result1.LHRHa targeted lipid microbubbles were positive inimmunofluorescent staining assay. The gene entrapment efficiencyofLHRHa targeted pshRNA-survivin loaded lipid microbubbles was（22.0±4.16）%. In vitro research of the targeting ability indicated that theLHRHa targeted pshRNA-survivin lipid mocrobubblescould adhere to theA2780/DDP cells specifically, whereas there was no adherence of untargeted microbubbles to the A2780/DDP cells.2.The inhibition rate of cell proliferationwas related to the ultrasoundintensity and exposure time as well as the concentration of mocrobubblesand plasmid. When ultrasound parameter fixed to0.5W/cm2,30s, the rate ofmicrobubbles to cell was10:1, plasmid1.6μg，which has smaller effect onthe cell proliferation.It is an optimal parameter for the further genetransfection.3.All the treatment had significant effects on A2780/DDP cell proliferationand apoptosis. However, among all the treated groups, the effects ofPlasmid+LHRHa-targeted microbubble+ultrasound group was thestrongest(P＜0.05). Apoptosis bodies were observed by transmissionelectron microscope in this group also. The Q-PCR had demonstrated thatthe expression of survivin mRNA and survivin protein was decreased in allthe treated group compared with control group(p<0.05).However, theexpression of survivin mRNA and survivin protein in Plasmid+LHRHa-targeted microbubble+ultrasound groupwas0.36±0.036、0.05±0.02,respectively,which was significantly lower than othergroups(p<0.05).The results of western blot showed that the expression ofcaspase-9and caspase-3protein in Plasmid+LHRHa-targeted microbubble+ultrasound group was0.95±0.09、2.60±0.21，respectively,which wassignificantly higher than other groups(p<0.05). Conclusion1.The LHRHa peptide was successfully linked to the lipidmicrobubble surface through biotin-avidin linkage, pshRNA-survivingene can be encapsulated on the LHRHa–targeted microbubbles viaelectrostatic adsorption. This gene loaded LHRHa–targeted microbubblecould bind to human ovarian cancer A2780/DDP cells specially andeffectively in vitro.2.Ultrasound destruction LHRHa-targeted microbubble mediatingpshRNA-survivin transfection could silence survivin gene expression andinduce apoptosis of ovarian cancer A2780/DDP cells effectively.