慢病毒介导shRNA沉默LAMP-2A和TSG101表达对PC12细胞中EGFP-α-synuclein(A53T)降解的影响

Effect of lentivirus-mediated LAMP-2A and TSG101 silencing on EGFP-α-synuclein(A53T) degradation in PC12 cells

目的采用慢病毒介导的shRNA干扰技术,下调PC12细胞中溶酶体相关膜蛋白-2A(lysosome-associated membrane protein type 2A,LAMP-2A)和肿瘤易感基因101(tumor susceptibility gene101,TSG101)的表达,并研究其对EGFP-α-synuclein(A53T)降解的影响。方法以LAMP-2A和TSG101为靶基因,合成寡核苷酸,退火形成双链DNA,与经Bam HⅠ、EcoRⅠ双酶切的p HBLV-U6-Puro载体连接获得重组慢病毒载体;将其与病毒包装辅助质粒共感染293T细胞,收集并浓缩上清液获得重组病毒,测定病毒滴度;病毒颗粒转染PC12细胞,经嘌呤酶素筛选获得稳定感染细胞株;Western blot检测LAMP-2A和TSG101蛋白的表达;分别采用LAMP-2A shRNA2和TSG101 shRNA1下调PC12细胞中LAMP-2A和TSG101蛋白的表达,并结合巴佛洛霉素A1处理,研究其对PC12细胞中EGFP-α-synuclein(A53T)、EGFP、α-synuclein(A53T)和LC3B蛋白的影响,免疫荧光检测各组PC12细胞中EGFP-α-synuclein(A53T)和LC3B蛋白的表达。结果成功构建干扰LAMP-2A和TSG101表达的慢病毒载体,并在293T细胞中包装获得病毒。重组病毒感染PC12细胞后,Western blot检测结果显示LAMP-2A shRNA2和TSG101 shRNA1干扰效果最好;下调LAMP-2A和TSG101蛋白表达均能够抑制细胞中EGFP-α-synuclein(A53T)、EGFP和α-synuclein(A53T)蛋白的降解(P<0.01),并提高LC3-Ⅱ蛋白的含量(P<0.01)。结论慢病毒介导的shRNA能有效地沉默PC12细胞中LAMP-2A和TSG101的表达,抑制细胞中EGFP-α-synuclein(A53T)的降解。

Objective To suppress the gene expression of lysosome-associated membrane protein type2A（ LAMP-2A） and tumor susceptibility gene 101（ TSG101） by lentiviruses,and observe its impact on EGFP-α-synuclein（ A53T） degradation in PC12 cells. Methods Synthesized oligonucleotides were cloned into the lentiviral vectors p HBLV-U6-Puro which was digested with Bam HⅠ and EcoRⅠ. The recombinant vectors were verified by sequencing,and transferred into 293 T package cells together with helper vectors.Recombinant lentiviruses were extracted from cell cultures to transfect PC12 cells. Stably transfected cells were screened with puromycin,and the expression of LAMP-2A and TSG101 was determined by Western blotting.The expression of LAMP-2A and TSG101 was suppressed by lentiviruses LAMP-2A shRNA2 and TSG101shRNA1,respectively,and after treatment with bafilomycin A1,the expression of enhanced green fluorescent protein（ EGFP）-α-synuclein（ A53T）,EGFP,α-synuclein（ A53T） and LC3 B protein in PC12 cells was observed. The expression of EGFP-α-synuclein（ A53T） and LC3 B protein were examined by immunofluorescence assay. Results Recombinant vectors were successfully constructed, and sufficient lentiviruses were obtained from the package cells. The recombinant lentiviruses could efficiently transfect PC12 cells,and 2 clones transfected with lentiviruses LAMP-2A shRNA2 and TSG101 shRNA1,respectively,were screened. The results of Western blotting showed that LAMP-2A and TSG101 expression was efficiently suppressed in PC12 cells. The degradation of EGFP-α-synuclein（ A53T）,EGFP and α-synuclein（ A53T） was significantly suppressed（ P〈0. 01）,and the expression of LC3-Ⅱwas increased（ P〈0. 01）. Conclusion RNAi technology based on lentivirus can efficiently suppress the gene expression of LAMP-2A and TSG101,and inhibit the degradation of EGFP-α-synuclein（ A53T） in PC12 cells.