Objective      To suppress the gene expression of lysosome-associated membrane protein type 2A (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 pHBLV-U6-Puro which was digested with BamHⅠ and EcoRⅠ. The recombinant vectors were verified by sequencing

and transferred into 293T 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 TSG101 shRNA1

respectively

and after treatment with bafilomycin A1

the expression of enhanced green fluorescent protein (EGFP)-α-synuclein(A53T)

EGFP

α-synuclein(A53T) and LC3B protein in PC12 cells was observed. The expression of EGFP-α-synuclein(A53T) and LC3B 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 (P0.01)

and the expression of LC3-Ⅱwas increased (P0.01).       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.