Analysis of the expression pattern of the BCL11B gene and its relatives in patients with T-cell acute lymphoblastic leukemia

Background In a human T-cell acute lymphoblastic leukemia (T-ALL) cell line (Molt-4), siRNA-mediated suppression of BCL11B expression was shown to inhibit proliferation and induce apoptosis, functions which may be related to genes involved in apoptosis (such as TNFSF10 and BCL2L1) and TGF-β pathways (such as SPP1and CREBBP). Methods The expression levels of the above mentioned genes and their correlation with the BCL11B gene were analyzed in patients with T-ALL using the TaqMan and SYBR Green I real-time polymerase chain reaction technique. Results Expression levels of BCL11B, BCL2L1, and CREBBP mRNA in T-ALL patients were significantly higher than those from healthy controls (P <0.05). In T-ALL patients, the BCL11B expression level was negatively correlated with the BCL2L1 expression level (rs = -0.700; P <0.05), and positively correlated with the SPP1 expression level (rs = 0.683; P <0.05). In healthy controls, the BCL11B expression level did not correlate with the TNFSF10, BCL2L1, SPP1, or CREBBP expression levels. Conclusions Over-expression of BCL11B might play a role in anti-apoptosis in T-ALL cells through up-regulation of its downstream genes BCL2L1 and CREBBP.

Background T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of newly diagnosed ALL cases in children and 20-25% of ALL cases in adults [1,2]. Overall, these are aggressive malignancies that do not respond well to chemotherapy and have a poorer prognosis than their B-cell counterparts [3]. The development of targeted therapies, including monoclonal antibodies and gene therapy, continues. Small interfering RNA (siRNA) is a promising gene-targeting agent that has shown great potential, particularly in the field of cancer treatment [4][5][6].
BCL2-like 1 (BCL2L1; Bcl-xL) is similar to Bcl-2 because it restrains the apoptosis induction of multiple stimuli, and is a key factor in the terminal step of apoptosis regulation. Studies have shown that BCL2L1 participates in various protein-protein interactions, playing a role in inhibiting apoptosis. In the endogenous apoptosis pathway, BCL2L1 of the BCL-2 family inhibits apoptosis by blocking the translocation of Bax to the mitochondrial outer membrane [14]. cAMP-response element binding protein (CREBBP) plays a critical role in embryonic development, growth control, and homeostasis by coupling chromatin remodeling to transcription factor recognition. A CREBBP gene rearrangement with chromosomal translocation has been identified in acute myeloid leukemia [15,16] and over-expression of CREBBP was found in Jurkat cells. Additionally, enhancement of apoptotic cell death occurred in the presence of CREB1 siRNA [17]. Tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10; TRAIL) is a tumor necrosis factor superfamily member, and induces apoptosis through its interaction with death receptors. BCL-2 family genes and TNFSF10 probably act together through crosstalk between the intrinsic and death receptor-mediated apoptosis pathways [18]. Secreted phosphoprotein 1 (SPP1) is also known as OPN and its abnormal activation can stimulate tumor growth, invasion, angiogenesis, and immune suppression, with wideranging effects on cell proliferation, apoptosis, differentiation, and migration [19,20].
Previous studies [21,22] showed that the inhibition of BCL11B expression by siRNA selectively inhibited proliferation and effectively induced apoptosis in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines (Jurkat, Molt-4). Additionally, global gene expression profiling revealed that BCL11B siRNA-mediated cell apoptosis may be related to BCL-2 family genes of the mitochondrial pathway, and the TRAIL (TNFSF10) gene of the death receptor signaling pathway [22], furthermore, in our previous study, the genes (SPP1 and CREBBP) of the TGF-β pathway (unpublished data). Little is known about the expression pattern of these genes in T-ALL. Thus, analyzing the expression pattern of these genes in malignant T-cells is important because BCL11B disruption and disturbed expression may contribute to the development of T-cell malignancies in humans [8]. In the present study, we further analyzed expression levels of TNFSF10, BCL2L1, SPP1, and CREBBP, and their correlation with BCL11B in male patients with T-ALL, to clarify the role of BCL11B in T-cell malignancies.
All procedures were conducted in accordance with the guidelines of the Medical Ethics committees of the health bureau of Guangdong Province, PR China.

RNA extraction and cDNA synthesis
RNA was extracted using the Trizol kit (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into the first-strand cDNA using random hexamer primers and the reverse transcriptase Superscript II Kit (Invitrogen), according to the manufacturer's instructions.

Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Quantitative detection of the BCL11B gene expression level in cDNA from PBMCs was performed using Taq-Man real-time PCR. PCR was performed as described previously [8]. To precisely determine the copy numbers of BCL11B, a duplex vector, including a fragment of the BCL11B and the b2 microglobulin (b2M) genes was constructed and used as a reference (the duplex vector was a gift from Prof. C.A. Schmidt, Ernst-Moritz-Arndt University Greifswald, Germany). Based on the DNA concentration, measured by spectrophotometry and confirmed by quantitative gel eletrophoresis, standard dilutions of the vector from 10 7 to 10 1 copies were prepared [8]. Briefly, PCR was performed in a 25-μL total volume containing 2 μL of cDNA, 25 pmol of each primer (BCL11B-f and BCL11B-b for BCL11B gene amplification; β2Mf and β2Mb for b2M gene amplification), 10 nmol of each dNTP, 1.5 U AmpliTaq Gold (Applied Biosystems, Branchburg, NJ, USA), 5 pmol of 6FAM-TAMRA probe, and PCR buffer containing 4.5 mM MgCl 2 . After an initial denaturation at 95°C for 5 min, 50 cycles consisting of 95°C for 15 s and 64°C for 1 min were performed. Primers and probes for BCL11B and b2M gene amplification were synthesized by TIB Molbiol Co. (Berlin, Germany; Table 1).
The absolute amounts of BCL11B and b2M were measured in two independent assays and BCL11B content per 100,000 b2M copies was calculated using the formula: n = 100000 × BCL11B/β2M. Expression levels of TNFSF10, BCL2L1, SPP1, CREBBP, and the reference gene b2-MG were determined by SYBR Green I real-time PCR. Briefly, PCR was performed in a 25-μL total volume containing 1 μL of cDNA, 9 μL of 2.5× SYBR Green mix (Tiangen, Beijing, PR China), and 10 μmol/L primer pairs. The following cycling conditions were used: initial denaturation at 95°C for 2 min, followed by 44 cycles at 95°C for 15 s, and 81°C (TNFSF10, SPP1, CREBBP, and b-2-MG) or 84°C (BCL2L1) for 1 min. The relative amounts of the genes of interest and the b2M reference gene were measured in two independent assays. The 2 (-ΔΔCT) method was used to present the data of the genes of interest relative to an internal control gene [23,24]. The efficiencies of real-time PCR for expression analysis of different genes were evaluated using diluted Molt-4 cDNA (1, 5 -1 , 5 -2 , 5 -3 , 5 -4 ) as templates to construct relative standard curves. Additionally, the specific amplification of PCR products was analyzed by melting curve analysis and agarose electrophoresis. Primers used in the SYBR Green I real-time PCR for all four gene amplifications were synthesized by Shanghai Biological Engineering Technology Services Co., Ltd. (Table 2).
RT-PCR for TNFSF10, BCL2L1, SPP1, and CREBBP genes was performed using the same primers as described above, and the PCR products were sent to Shanghai Invitrogen Biotechnology Co. for DNA sequence analysis.

Statistical analyses
Independent-sample t -test analysis was used for the BCL11B gene mRNA levels in different samples, while the Mann-Whitney U test and Spearman's rank correlation analyses were used for non-normally distributed data using the SPSS 13.0 statistical software. Differences were considered statistically significant at P < 0.05.

Expression of TNFSF10, BCL2L1, SPP1, and CREBBP genes in T-ALL
The high amplification efficiency of the four genes of interest (TNFSF10, BCL2L1, SPP1, and CREBBP) was consistent with that of the b2M reference gene. For example, the accurate standard curve graphs of BCL2L1 and b 2 M control gene amplification are illustrated in Figure 2A and 2B (r 2 = 0.995). The amplification efficiencies of BCL2L1 and the b2M control gene were 95.30% and 95.16%, respectively, and the melting curves are shown in Figure 2C. PCR products from the b2M control gene and genes of interest were confirmed using 2.5% gel electrophoresis ( Figure 2D, E), followed by sequence confirmation (data not shown).
In T-ALL patients, Spearman's rank correlation analyses revealed that the BCL11B expression level was negatively    correlated with the BCL2L1 relative expression level (r s = -0.700; P = 0.036; Figure 3A), and positively correlated with the SPP1 relative expression level (r s = 0.683; P = 0.042; Figure 3B). The BCL11B expression level did not exhibit an obvious correlation with TNFSF10 or CREBBP relative expression levels. No significant correlation was found between the BCL11B gene and the other four genes of interest in the healthy controls.

Discussion
Increasing numbers of translocations involving the BCL11B locus [8,10,11] or high levels of BCL11B mRNA expression in most T-ALL cases [8,12] have been reported; however, the mechanism of BCL11B-mediated oncogenesis remains unknown. To clarify the role of BCL11B in T-cell malignancies, we further analyzed the expression levels of TNFSF10, BCL2L1, SPP1, and CREBBP genes and their correlations with BCL11B in patients with T-ALL and controls. Over-expression of the BCL11B gene, as well as BCL2L1 and CREBBP mRNA, were characteristic features of T-ALL. Recent evidence has suggested that multiple mechanisms may regulate the release of mitochondrial factors, some of which depend on the action of caspases. BCL2L1 may inactivate caspase-8 by decreasing death-inducing signaling complex (DISC) formation in the plasma membrane, nucleus, and Golgi complex while diverting DISC formation to the mitochondria. The inhibitory effects of BCL2L1 on DISC formation may play a significant role in protecting endothelial cells from hypoxia/reoxygenation (H/R)-induced cell death [25]. Thus, over-expression of the BCL2L1 gene suggests that it might be related to the occurrence of T-ALL by defective regulation of apoptosis. During the process of T-ALL, over-expressed BCL2L1 is thought to suppress the activity of caspase-8; thus, as a kind of protection mechanism, the TNFSF10 gene of some patients is highly expressed, promoting caspase-8 activity in response to this abnormal cell proliferation. However, the low expression level of SPP1 in untreated Molt-4 cells differed from the high expression levels found in mostly solid tumors [26]. Additionally, our findings indicated no significant difference in SPP1 gene expression in the T-ALL group. Comprehensive analysis revealed that T-ALL occurred in the presence of BCL11B, BCL2L1, and CREBBP gene over-expression, which was closely related to blocking apoptosis of malignant T cell, whereas the TNFSF10 gene was also highly expressed in some patients, which may partly correct the imbalance.
Correlation analysis of BCL11B in the T-ALL group revealed that the BCL11B expression level was negatively correlated with that of BCL2L1 (Bcl-xL), although over-expression of both genes was found in T-ALL samples. This suggested that BCL2L1 was affected by the BCL11B gene in transcriptional regulation, and both participated in the same protein-protein interactions, acting as apoptosis regulators along with a competitive target protein downstream. In BCL11Bknockdown T-cell lines, when exposed to growth stimuli, T cells exhibit apoptosis in S phase with concomitant decreases in the cell-cycle inhibitor p27 and the anti-apoptotic protein Bcl-xL, due to transcriptional repression [13]. However, BCL11B and BCL2L1 protein levels in the T-ALL group still remain to be validated. Correlation analysis of BCL11B in the T-ALL group revealed that the BCL11B expression level was positively correlated with the relative SPP1 expression level. The expression of SPP1 was significantly downregulated with BCL11B silencing by RNA interference, suggesting that the SPP1 gene may be a target of the BCL11B gene in transcriptional regulation (unpublished data). SPP1 gene silencing in vitro significantly increased mitochondrial cytochrome c release, and the inhibitory action of the Wnt target gene osteopontin (SPP1) on mitochondrial cytochrome c release determines renal ischemic resistance [27]. Thus, the SPP1 gene may play a consistent role in anti-apoptotic effects with the BCL11B gene, by decreasing mitochondrial cytochrome c release. The hypothetical regulatory network of apoptosis in BCL11B and related genes is shown in Figure 4. However, the role of the SPP1 gene in T-cell malignancies is unclear, because low expression of SPP1 was detected in T-ALL.

Conclusions
The expression pattern of the BCL11B gene and four of its related genes (TNFSF10, BCL2L1, SPP1, and CREBBP) was characterized in T-ALL. Over-expression of BCL11B may play a role in anti-apoptosis in T-ALL cells through up-regulation of its downstream genes BCL2L1 and CREBBP.