Inhibitory Effects And Possible Mechanism Of5-LOXsiRNA On Growth Of HepG2Cell Xenografts In Small Nude Mice

Objective To design5-lipoxygenase（5-LOX） siRNA through the technology ofRNA interference （RNAi）.detect5-LOX expression’s changes in hepatoma cellline HepG₂after being transfected by siRNA.to investigate the inhibitory effect of5-LOXsiRNA on proliferation of HepG₂cell xenografts in small nude mice and theeffect of the ERK（extracellular regulated protein kinase）1/2signal transductionpathway.and to explore the mechanism of inhibitory growth in nude mice proliferation.further to broaden the pathogenesis of hepatocellular carcinoma.provide thebasis and the target of genetherapy for liver tumors.at last,discuss the value of RNAi indisease research，treatment and prophylaxis.Methods RNAi was synthesized to interfere function of5-LOX, HepG₂cells weretransfected by5-LOX siRNA and cells were divided into three groups:1.positive interference group （transfected with siRNA interference plasmid）.2. negative control group（transfected with empty vector plasmid）.3. blank group （non-transfected plasmid）.Cells were collected at the48h after transient transfection,5-LOX’s expression were significantly decreased which detected by real·timeflorescence quantitative PCR method.The xenografts model derived from thetransfected HepG₂cells were established in nude mice. experimental nude mice wererandomly divided into three groups:1. transfected group （inoculated with HepG₂cells transfected by5-LOXsiRNA）,2.free vector transfected group （negativecontrol）,3.untransfected group （with normal HepG₂cell）. The nude mice were killed21th days later.Tumor tissues were taked out and volumes were measured.（tumor volume （V）=long diameter×short diameter2×0.5）,and tumor rate of declinewas measured. Protein levels of5-LOX,ERK1/2were determined byWestern-blot,mRNA level of5-LOX,ERK1/2was analyzed by reverse transcription-polymerase chain reaction（RT-PCR）.Results1. The numerus of5-LOX expression relative quantitative inpositive interference group is0.341obviously declined than the result of0.822innegative control and1.074in blank group（P <0.01）.2.The mean tumors volumein transfected group is significantly reduced （10.660±4.552） cm3compared with（19.576±6.081） cm3in free vector transfected group and （20.465±8.395）cm3inuntransfected group （P<0.01）. tumor rate of decline is52.09%in transfectedgroup.3. The protein expression of5-LOX, ERK1/2in transfection group were0.167±0.015,0.187±0.006,0.183±0.015,which significantly decreased, comparedwith427±0.015,0.367±0.153,0.323±0.021in free vector transfected groupand0.443±0.021,0.390±0.030,0.343±.015in untransfected group （P <0.01）.while the mRNA expression of5-LOX, ERK1/2also significant declined intransfection group（P <0.05）.Conclusion1.siRNA is one of effective gene silencing methods. The expression5-LOX mRNA in human hepatoma HepG₂cell line was significantly inhibiteddetected at the cellular level after transfected by siRNA2.Inhibition of5-LOX’s expression can significantly inhibit the hepatocellular carcinoma tumor growthin vivo experiments3. Inhibition of5-lipoxygenase （5-LOX） expression cansignificantly inhibit the expression of ERK1/2，which shows that5-LOXsiRNA may inhibits tumor proliferation via extracellular signal-regulated kinase （ERK） signaltransduction pathway4. RNAi will likely become a new approach in cancer genetherapy and will broad in application values.