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Published on: April 24, 2021
Down-regulation of SENP1 expression increases apoptosis of Burkitt lymphoma cells
Bin-Bin Huang1, Qing-Mei Gao, Wei Liang
1Department of Hematology, Tongji Hospital of Tongji University, Shanghai, China.
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.
Conclusion:
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.
Insights
Down-regulation of Sentrin/SUMO-specific protease 1 (SENP1) using shRNA effectively inhibited SENP1 expression in Burkitt lymphoma cells. This inhibition promoted cancer cell apoptosis, suggesting SENP1 as a potential target for gene therapy.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Burkitt lymphoma is an aggressive non-Hodgkin lymphoma.
- Sentrin/SUMO-specific protease 1 (SENP1) plays a role in various cellular processes.
- The role of SENP1 in Burkitt lymphoma apoptosis requires further investigation.
Purpose of the Study:
- To investigate the effect of SENP1 down-regulation on human Burkitt lymphoma (Daudi) cell apoptosis.
- To explore the potential mechanisms underlying SENP1 inhibition-induced apoptosis.
Main Methods:
- Lentiviral vectors carrying short hairpin RNA (shRNA) targeting SENP1 were constructed.
- Daudi cells were transfected with shRNA targeting SENP1 (sh-SENP1-Daudi group).
- Apoptosis-related molecules (caspase-3, 8, 9) and hypoxia-inducible factor-1α (HIF-1α) expression were analyzed using qPCR and Western blot.
Main Results:
- Efficient down-regulation of SENP1 expression was achieved in Daudi cells (79.2% interference).
- SENP1 inhibition led to characteristic apoptotic morphology and increased caspase-3, 8, and 9 expression.
- While hypoxia increased HIF-1α protein, SENP1 inhibition prevented this increase.
Conclusions:
- SENP1 shRNA effectively inhibits SENP1 expression in Daudi cells.
- SENP1 inhibition promotes apoptosis in Burkitt lymphoma cells.
- SENP1 represents a promising molecular target for Burkitt lymphoma gene therapy.
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