Construction Of UCH-L3Gene Eukaryotic Expression Plasmid And Research On Its Impact On Rapamycin Induced Autophagy Of NCI-H460Cells

Objective:The aim of this study is to construct UCH-L3gene eukaryotic expression plasmid and to further explore the impact of UCH-L3gene on rapamycin induced autophagy of NCI-H460cells by measuring UCH-L3and LC3-II protein expression changes of these groups.Method:1.The construction of UCH-L3gene eukaryotic expression plasmid: NCI-H460cells were cultured. UCH-L3gene was amplified by RT-PCR and cloned into eukaryotic expression plasmid pcDNA3.1(+)-EGFP through double enzyme digestion and gene cloning to construct UCH-L3gene eukaryotic expression plasmid which was then confirmed by double enzyme digestion and gene sequencing.2.The experiment was divided into three groups:untransfected group, pcDNA3.1(+)-UCH-L3-EGFP group and pcDNA3.1(+)-EGFP group.NCI-H460cells were transfected with the recombinant plasmid by cationic liposome transfection.the proportion of EGFP positive cells was counted under a fluorescence microscope48hours after transfection to assess transfection efficiency.The UCH-L3gene mRNA expression of each group was detected by real-time quantitative PCR method after24hours transfection.3.Determination of the expression levels of UCH-L3and LC3-Ⅱ protein by western blot method:the experiment was divided into six groups:untransfected group, pcDNA3.1(+)-UCH-L3-EGFP group and pcDNA3.1(+)-EGFP group,each group was dividied into two groups,treated with rapamycin group and untreated control group.Cells of each group which was treated according to the experimental division above were collected,and the total proteins were extracted to measuring the expression levels of UCH-L3and LC3-II by western blot method.Results:1.The construction of UCH-L3gene eukaryotic expression plasmid:it was confirmed that pcDNA3.1(+)-UCH-L3-EGFP eukaryotic expression plasmid was constructed successfully by double digestion and gene sequencing methods.2.48hours after transfection,green fluorescent protein positive cells were visible under fluorescence microscope, which proportion account for the total number of cells was approximately50to60%. The results of Real-time quantitative PCR of the three groups showed that UCH-L3mRNA expression levels of pcDNA3.1(+)-UCH-L3-EGFP group were much higher than untransfected group and pcDNA3.1(+)-EGFP group,the difference was statistically significance(P<0.05).The UCH-L3mRNA expression levels of the untransfected group and pcDNA3.1(+)-EGFP group have no statistically significant (P>0.05).3.Determination of the expression levels of UCH-L3and LC3-II protein by western blot method:the experimental results showed that the LC3-II protein expression level of each group with drugs was higher than which of the same group without drugs and the UCH-L3level of each group with drugs was increased compared with which of the same group without drugs,the difference was statistically significant(P<0.05);the UCH-L3protein expression level of pcDNA3.1(+)-UCH-L3-EGFP group was higher than untransfected group and pcDNA3.1(+)-EGFP group in the groups with drugs or the groups without drugs,the difference was statistically significant(P<0.05).UCH-L3protein expression levels between untransfected group and pcDNA3.1(+)-EGFP group have no statistically significant (P>0.05).In the groups with drugs,LC3-Ⅱ level of pcDNA3.1(+)-UCH-L3-EGFP group decreased compared with and pcDNA3.1(+)-EGFP group and untransfected group,the difference was statistically significant (P<0.05).LC3-Ⅱ protein expression levels between untransfected group and pcDNA3.1(+)-EGFP group have no statistically significant (P>0.05)Conclusion:l.pcDNA3.1(+)-UCH-L3was successfully constructed.2.Rapamycin could induce NCI-H460cells to autophagy and UCH-L3 expressing decrease.3.UCH-L3may have an inhibition impact on autophagy of NCI-H460cells induced by rapamycin,but its specific mechanism remained unclear and needed further study.