Down-regulation of SENP 1 Expression Increases Apoptosis of Burkitt Lymphoma Cells

Burkitt lymphoma (BL) is a highly malignant B-cell non-Hodgkin’s lymphoma (NHL) (Yeh et al., 2000). Currently, chemotherapy is still a major strategy in the treatment of BL. However, the chemotherapeutics not only kill the cancer cells but severely damage the normal cells and tissues. Thus, some severe side effects are frequently found during the chemotherapy. Sentrin/SUMO-specific protease 1 (SENP1) is a member of SUMO specific protease family (SENPs) and widely distributed in the nuclei. SENP1 can regulate multiple molecules related to the biological characteristics of cancers via desumoylation. Among these molecules, hypoxia induced factor-1α (HIF-1α) which is widely considered to be one of the key regulators in cancer cells is an important one regulated by SENP1 (Yeh et al., 2000; Cheng et al., 2007; Song et al., 2011). In the present study, RNA interference was


Introduction
Burkitt lymphoma (BL) is a highly malignant B-cell non-Hodgkin's lymphoma (NHL) (Yeh et al., 2000).Currently, chemotherapy is still a major strategy in the treatment of BL.However, the chemotherapeutics not only kill the cancer cells but severely damage the normal cells and tissues.Thus, some severe side effects are frequently found during the chemotherapy.Sentrin/SUMO-specific protease 1 (SENP1) is a member of SUMO specific protease family (SENPs) and widely distributed in the nuclei.SENP1 can regulate multiple molecules related to the biological characteristics of cancers via desumoylation.Among these molecules, hypoxia induced factor-1α (HIF-1α) which is widely considered to be one of the key regulators in cancer cells is an important one regulated by SENP1 (Yeh et al., 2000;Cheng et al., 2007;Song et al., 2011).In the present study, RNA interference was

Down-regulation of SENP1 Expression Increases Apoptosis of Burkitt Lymphoma Cells
Bin-Bin Huang & , Qing-Mei Gao & , Wei Liang, Bing Xiu, Wen-Jun Zhang, Ai-Bin Liang* employed to down-regulate SENP1 expression in Burkitt lymphoma cells (Daudi cells) and then the apoptosis was observed.Moreover, the potential role of HIF-1α in this effect was also explored.Our findings may confirm SENP1 as a new target in the gene therapy of Burkitt lymphoma.

Cell culture
Daudi cells were purchased from Cell Institute of Chinese Academy of Sciences.Lenviral packaging 293T cells were maintained in DMEM containing 10% FBS.Human Burkitt lymphoma cells (Daudi cells) were grown in RPMI 1640 containing 10% FBS.Cells were cultured at 37 ℃ in a humidified environment with 5% CO 2 and those in logarithmic growth phase were used for the following experiments.

Construction of lentiviral vectors expressing SENP1 shRNA, transfection and screening of Daudi cells
According to the principles for the design of RNA interference sequence, a total of 3 sequences targeting SENP1 gene (NM_014554) were designed: SH1 (5'-TTCCTTTACACCTGTCTCG-3'), SH2 (5'-CACCACTCTGTTCCACATC3'), and SH3 (5'-TCAGGCAGTGAAACGTTGG -3').The sh1+sh2+sh3 packaging lentiviral vectors were prepared and the lentiviral packaging plasmids were used to transfect 293T cells.The viral solution was prepared and named pRNAT/shSENP1-Daudi group.At the same time, shRNA targeting scrambled sequence (general sequence: 5'-TTCTCCGAACGTGTCACGT-3') was designed and served as negative control group (pRNAT/NC).The lentivirus was labeled with green fluorescent protein (GFP).Following dilution, flow cytometry was performed to measure the proportion of GFP positive lentivirus to determine the viral titer.The lentivirus in the pRNAT/ sh-SENP1-Daudi group and pRNAT/NC was used to transfect Daudi cells at appropriate titer and named sh-SENP1-Daudi and sh-NC-Daudi (negative control) groups, respectively.In addition, Daudi cells without transfection served as a blank control group.At 24 and 48 h after transfection, the GFP expression was observed in different groups.Cells with GFP positive rate of > 50% were used in the following experiments.Moreover, at 48 h after transfection, flow cytometry was performed to screen GFP positive Daudi cells for further culture and experiments (Wang et al., 2006).

Detection of transfection efficiency
Real-time fluorescence quantitative PCR and western blot assay were employed to measure the mRNA and protein expressions of SENP1 before and after transfection.At 48 h after transfection, 1×10 7 cells were collected and total RNA was extracted with TRIzol reagent.Then, 1 μl of dNTP, 1 μl of MMLV, 5 μl 5×buffer, 1 μl of Olig(dt), 0.5 μl of RNasin and 1 μl of RNA were mixed followed by addition of water to 20 μl.The mixture was incubated at 42 ℃ for 50 min followed by denaturation at 95 ℃ for 5 min and subsequent reverse transcription.Then, 0.8 μl of cDNA, 10 μl of Syber green PremixII, 0.4 μl of Primer 1 (10 μM), 0.4 μl of Primer 2 (10 μM) and water were mixed at a final volume of 20 μl.PCR was performed in Roche thermal cycler.The PCR conditions were: pre-denaturation at 95℃ for 1 min, 40 cycles of 95℃ for 10 s, 60℃ for 10 s and 72℃ for 20 s. β-actin served as an internal reference and 2 -rrCT method was employed to determine the relative expression of target genes.Cell culture was identical to those above, and cells were collected 48 h later.Total protein was extracted with a kit and subjected to SDS-PAGE.Then, the proteins were transferred onto PVDF membrane which was treated with SENP1 or β-actin antibodies (1:500) and horseradish peroxidase conjugated IgG.Following visualization, bands were scanned into a computer followed by analysis of optical density.Experiment was performed three times.

RNA interference of SENP-1 expression on apoptosis of Daudi cells
At 24 h, 48 h and 72 h after transfection, total RNA was extracted with TRizol reagent and reverse transcribed into cDNA.Real-time fluorescence quantitative PCR was done to measure the mRNA expression of caspase-3, 8 and 9 in cells of sh-SENP1-Daudi group.At the same time points, total proteins were extracted and western blot assay was employed to measure the protein expression of caspase-3, 8 and 9 in cells of sh-SENP1-Daudi group.The above experiments were done three times.

RNA interference of SENP-1 expression on HIF-1α expression in Daudi cells
CoCl 2 (100 mmol/L) was used to mimic the hypoxic environment and treat cells in the blank control group, sh-NC-Daudi group and sh-SENP1-Daudi group for 24 h.Real-time quantitative PCR and western blot assay were performed to measure the mRNA and protein expression of HIF-1α.

Statistical analysis
Statistical analysis was performed by using SPSS 15.0 Software.The comparisons among different group were done with Chi-square test.P < 0.05 was considered significantly different.

Daudi cells experiencing transfection
Peripheral blood lymphcytes (PBL) were separated by Percoll density gradient centrifugation in 10 healthy volunteers.Fluorescence quantitative PCR was performed to measure the mRNA expression of SENP-1 in Daudi cells.Results showed the mRNA expression of Daudi in Daudi cells was 27.83±6.64higher than that in normal cells (P<0.05).The NC-shRNA and SENP1-shRNA lentiviral vectors were used to transfect Daudi cells at MOI of 15 for 48 h followed by laser scanning confocal microscopy.Results showed the transfection efficiency was > 50% and these cells can be used for further experiments (Figure 1).

Detection of interference efficiency
At 48 h after transfection, flow cytometry was performed to screen the GFP positive cells (Figure 2A).Quantitative PCR showed, when compared with sh-NC-  Daudi cells undergoing sh-SENP1 transfection at 48 h and 72 h after transfection.At 24 h, 48 h and 72 h after transfection, the mRNA expression of caspase-3, 8 and 9 was increased by 1.679±0.036and 1.788±0.032,2.184±0.034and 2.369±0.040and 1.700±0.025and 1.793±0.037,respectively, when compared with that in the blank control group.B: Protein expression of caspase-3, 8 and 9 in Daudi cells undergoing sh-SENP1 transfection at 48 h and 72 h after transfection Daudi group and blank control group, the inhibition rate of SENP1 mRNA expression was 79.17±0.026% in the sh-SENP1-Daudi group.Western blot assay revealed the protein expression of SENP1 in the sh-SENP1-Daudi group was markedly reduced as compared to the remaining two groups but there was no significant difference in the protein expression of SENP1 between sh-NC-Daudi group and blank control group (Figure 2B, 2C).

SENP1-shRNA transfection on morphology of Daudi cells
At 48 h after transfection, flow cytometry was performed to screen GFP positive cells (Daudi cells without transfection did not experience screening).Following fixation, Giemsa staining of these cells were done and the morphology was observed under a light microscope (100×).Results showed cells in the sh-SENP1-Daudi were shrunk, had irregular edge in the membrane, pyknosis and nuclear fragmentation and apoptotic bodies were also noted in these cells.In the blank control group and Sh-NC-Daudi group, cells had clear edge in the membrane and no or extremely few apoptotic bodies (Figure 3)

Effect of SENP1 down-regulation on mRNA and protein expressions of caspase-3, 8 and 9 in Daudi cells
At 24 h, 48 h and 72 h after transfection, FCM was done to screen GFP positive cells (data were not shown).
Quantitative PCR and Western blot assay showed the mRNA and protein expressions of caspase-3, 8 and 9 increased with the prolongation of transfection (P<0.05)(Figure 4A and 4B).

Effect of RNA interference of SENP1 on mRNA and protein expression of HIF-1α in Daudi cells
CoCl 2 at 100 mmol/L was used to mimic hypoxic environment and treat cells in blank control group, Sh-NC-Daudi group and group for 24 h.Fluorescence quantitative PCR and Western blot assay were employed to measure the mRNA and protein expression HIF-1α at 0 h and 24 h after CoCl 2 treatment.Results showed the mRNA expression of HIF-1α remained unchaged in three groups (P>0.05)(Figure 5A).However, after CoCl 2 treatment for 24 h, the protein expression of HIF-1α in the blank control group and Sh-NC-Daudi group was markedly increased but that in the Sh-SENP-1-Daudi group remained unchanged (Figure 5B).

Discussion
BL is a highly malignant tumor and usually progresses rapidly.Patients with BL have high mortality.The prevalence of BL is about 40% in Children with NHL and 2% in adults with NHL (Shen et al., 2003).With the development in molecular biology, immunohistochemistry and cytogenetics, most patients with BL may recover after high dose chemotherapy.However, after repeated chemotherapy, cancer cells may combat with the chemotherapeutics induced apoptosis and thus, a fraction of patients are non-responsive treatment or develop recurrence.Thus, to find the factors related to cancer cells apoptosis and to explore these factors as therapeutic target may provide new strategy for the treatment of BL.
SENPs mediated desumoylation has been a hot topic in the studies on post-translational modification (Yeh et al., 2000).In recent years, abnormal SENP1 expression has been found in some cancers.For example, in human prostate cancer and thyroid oncocytoma, the protein expression of SENP1 was found to be up-regulated and the SENP1 expression was related to the invasiveness of cancer cells (Shalizi et al., 2006;Bawa-Khalfe et al., 2007).Over-expression of SENP1 was found to induce canceration of prostate epithelial cells in mice, and silencing LNCaP SENP1 expression by RNA interference in prostate cancer cell line was demonstrated to increase the spontaneous apoptosis rate and the sensitivity of prostate cancer cells to chemotherapeutics.
SENP1 can regulate multiple factors related to the biological behaviors of cancer cells among which HIF-1α is an important one.HIF-1 is a widely distributed transcriptional factor in mammalians and humans in the presence of hypoxia.HIF-1 is the heterodimer of α unit and β unit.HIF-1α is a unique subunit regulated by oxygen and oxygen determines the HIF1 activity.The Lysine residues at 391 and 477 are the site modified by SUMO-1.Following binding to target genes, HIF1 can increase the transcription of down-stream genes (vascular endothelial growth factor and multidrug resistance gene) via transcription and post-transcriptional regulation.This may alter the signal transduction of cell survival and apoptosis which plays an important role in the adaptation to hypoxia and invasion and metastasis of cancers (Yates et al., 2008).SENP1 can regulate the stability of HIF-1α to exert a series of biological effects involving in survival and immune escaping of cancer cells (Yates et al., 2008).Currently, there is controversy on the effect of SUMO on HIF-1α stability.Bae et al. (2004) found that SUMO-1 can compete with ubiquitin to bind to the same sites, which increases the HIF-1α stability.However, Cheng et al. (2007) found the HIF-1α stability reduced in mice with SENP1 deficiency, which suggests that the desumoylation of HIF-1α by SENP1 is essential for the HIF-1α stability.Thus, in the occurrence and development of cancers, the relationship between SENP1 and HIF-1α is required to be further elucidated.This elicits the interest on the role of SENP1 in the occurrence and development of cancers.
In the present study, we for the first time confirmed that the SENP1 expression in Daudi cells was higher than that in normal peripheral blood lymphocytes.This suggests SENP1 is closely related to the Burkit lymphoma cells.Then, RNA interference was employed to down-regulate SENP1 expression which was demonstrated by Western blot assay.Microscopy showed Daudi cells with SENP1 down-regulation had characteristics of apoptosis such as cell shrinkage, nuclear fragmentation and apoptotic bodies.In addition, the classic apoptotic factors caspase-3, 8 and 9 were up-regulated at mRNA and protein levels.This suggests SENP1 down-regulation increases the apoptosis of Daudi cells.Whether this process has involvement of HIF-1α stability is still unclear (Chavez et al., 2002; Jean,  doi.org/10.7314/APJCP.2012.13.5.2045Down-regulation of SENP1 Expression Increases Apoptosis of Burkitt Lymphoma Cells 2004;Resar et al., 2005;Zhou et al., 2011).In this study, simulated hypoxia was employed and results showed the mRNA expression of HIF-1α was comparable in normal Daudi cells and those with SENP1 down-regulation, but the protein expression of HIF-1α was markedly different between them.This indicates SENP1 can regulate the protein expression of HIF-1α.The hypoxia the balance between generation and degradation of HIF-1α protein, and increase the protein expression of HIF-1α.In Daudi cells with SENP1 down-regulation, the HIF-1α expression remained unchanged.Thus, we speculated that SENP1 down-regulation increases the degradation of HIF-1α.On the basis of the role of HIF-1α in cancer cells and our findings, we postulated that the SENP1 down-regulation in Daudi cells down-regulate HIF-1α protein expression, which then affected the transcription and post-transcriptional regulation of downstream factors including apoptosis related factors.This makes cancer cell unable to adapt to the hypoxic environment in the cancer resulting in apoptosis of cancer cells (Barliya et al., 2011;Du et al., 2011;El et al., 2011;Freise et al., 2011;Sapra et al., 2011;Liao et al., 2012).
Taken together, down-regulation of SENP1 expression in Daudi cells can reduce the stability of HIF-1α protein and induce the apoptosis of Daudi cells.Thus, gene therapy targeting SENP1 may become an adjunctive therapy of Burkitt lymphoma.

Figure 1 .Figure 3 .
Figure 1.Microscopy of Daudi Cells after Transfection with Respective Vectors.Lentiviral vectors in the pRNAT/ sh-SENP1-Daudi group and pRNAT/NC group were used to transfect Daudi cells at appropriate titer and named sh-SENP1-Daudi group and sh-NC-Daudi group (negative control group), respectively.At the same time, Daudi cells without transfection served as a blank control group.At 48 h after transfection, the GFP expression was observed in sh-SENP1-Daudi group and sh-NC-Daudi group

Figure 4 .
Figure 4. Effect of SENP1 Down-regulation on mRNA and Protein Expressions of Caspase-3, 8 and 9 in Daudi Cells.A: mRNA expression of caspase-3, 8 and 9 in

Figure 5 .
Figure 5.Effect of RNA Interference of SENP1 on mRNA and Protein Expression of HIF-1α in Daudi Cells.A: mRNA expression of HIF-1α following hypoxia treatment.Cells in the blank control group, sh-NC-Daudi group and sh-SENP1-Daudi group were treated with CoCl2 at 100 mmol/L for 24 h.Results showed the mRNA expression of HIF-1α was 1.076±0.110,1.054±0.120and 1.072±0.131 in the blank control group, sh-NC-Daudi group and sh-SENP1-Daudi group, respectively, showing no marked difference (P>0.05).B: Protein expression of HIF-1α following hypoxia treatment (Western blot)