Down-regulation of SENP1 expression increases apoptosis of Burkitt lymphoma cells.
OBJECTIVE
To investigate the effect of down-regulation of Sentrin/SUMO-specific protease 1 (SENP1) expression on the apoptosis of human Burkitt lymphoma cells (Daudi cells) and potential mechanisms.


METHODS
Short hairpin RNA (shRNA) targeting SENP1 was designed and synthesized and then cloned into a lentiviral vector. A lentiviral packaging plasmid was used to transfect Daudi cells (sh-SENP1-Daudi group). Daudi cells without transfection (Daudi group) and Daudi cells transfected with blank plasmid (sh-NC-Daudi group) served as control groups. Flow cytometry was performed to screen GFP positive cells and semiquantitative PCR and Western blot assays were employed to detect the inference efficiency. The morphology of cells was observed under a microscope before and after transfection. Fluorescence quantitative PCR and Western blot assays were conducted to measure the mRNA and protein expression of apoptosis related molecules (caspase-3, 8 and 9). After treatment with COCl2 for 24 h, the mRNA and protein expression of hypoxia inducible factor -1α (HIF-1α) was determined.


RESULTS
Sequencing showed the expression vectors of shRNA targeting SENP1 to be successfully constructed. Following screening of GFP positive cells by FCM, semiqualitative PCR showed the interference efficiency was 79.2±0.026%. At 48 h after transfection, the Daudi cells became shrunken, had irregular edges and presented apoptotic bodies. Western blot assay revealed increase in expression of caspase-3, 8 and 9 with prolongation of transfection (P<0.05). Following hypoxia treatment, mRNA expression of HIF-1α remained unchanged in three groups (P>0.05) but the protein expression of HIF-1α markedly increased (P<0.05). However, in the sh-SENP1-Daudi group, the protein expression of HIF-1α remained unchanged.


CONCLUSIONS
SENP1-shRNA can efficiently inhibit SENP1 expression in Daudi cells. SENP1 inhibition may promote cell apoptosis. These findings suggest that SENP1 may serve as an important target in the gene therapy of Burkitts lymphoma.
Department of Hematology, Tongji Hospital of Tongji University, Shanghai, China.
Bin-Bin
BB
Huang
Qing-Mei
QM
Gao
Wei
W
Liang
Bing
B
Xiu
Wen-Jun
WJ
Zhang
Ai-Bin
AB
Liang
D017209:Apoptosis; D015153:Blotting, Western; D002051:Burkitt Lymphoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D020169:Caspases / Q000378:metabolism; D003546:Cysteine Endopeptidases; D015536:Down-Regulation; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D010450:Endopeptidases; C404512:SENP1 protein, human; D020169:Caspases; D003546:Cysteine Endopeptidases