Gene expression profiles in BCL11B-siRNA treated malignant T cells

Samples from three newly diagnosed patients with T-ALL and one patient with T-cell lymphoma/leukemia were obtained after informed consent. The diagnosis of T-ALL was based on cytomorphology, immunohistochemistry, and flowcytometry analyses. The samples were named P1 (55-year-old male with T-ALL), P2 (6-year-old male with T-ALL), P3 (55-year-old female with T-cell lymphoma/leukemia), and P4 (19-year-old male with T-ALL). Peripheral blood was collected with heparin and peripheral mononuclear cells (PBMCs; contained more than 70% leukemic T cells) were separated using the Ficoll-Hypaque gradient centrifugation method. All procedures were conducted in strict accordance with the guidelines of the Medical Ethics committees of the Health Bureau of Guangdong province, China.

Molt-4 cells (Institutes for Biological Sciences Cell Resource Center, Chinese Academy of Sciences, Shanghai, China) and PBMCs collected from the four patients were cultured in complete RPMI 1640 medium with 15% fetal calf serum and were maintained in a sterile incubator at 37°C, 95% humidity, and 5% CO₂.

BCL11B-siRNA935 (Chinese patent application number: 200910193248.3) and the scrambled non-silencing siRNA control (BCL11B-sc) were designed with online software http://​www.​invitrogen.​com and synthesized by Invitrogen (Carlsbad, CA, USA).

Malignant T cells were resuspended at 2.5 × 10⁶ (Molt-4 cells) or 1 × 10⁷ (PBMCs) per 100 μL of the appropriate Nucleofector kit solution (Amaxa Biosystems, Cologne, Germany), and were nucleofected with 3 μg of BCL11B-siRNA or control non-silencing scrambled (sc) RNA using the C-005 (Molt-4 cells) or U-014 (PBMCs) program in the Nucleofection Device II (Amaxa Biosystems). Mock-transfected cells (nucleofected without siRNA) were used as a negative control. After nucleofection, the cells were immediately mixed with 500 μL of pre-warmed culture medium and transferred to culture plates for incubation. Samples were collected for RNA isolation.

Total RNA was isolated using Trizol (Invitrogen), and cDNA was synthesized with a Superscript II RNaseH Reverse Transcriptase kit (Invitrogen).

Total RNA (> 3 μg) was sent for global gene expression profile analysis using an Affymetrix HG U133 Plus 2.0 gene chip (Shanghai Biochip Co., Ltd., Shanghai, China). The Affymetrix microarray analysis was performed using Gene Spring GX10.0 software (Agilent Technologies, Santa Clara, CA, USA).

The primer and probe information for BCL11B and the reference gene β-2-microglobulin (β2-MG), as well as the details of the real-time PCR for BCL11B were described in our previous studies [12, 15]. Expression levels of tumor necrosis factor (ligand) superfamily, member 10 (TNFSF 10; TRAIL), BCL 2-like 1 (BCL2L 1; Bcl-xL), secreted phosphoprotein 1 (SPP 1), cAMP-response element binding protein (CREBBP), and β2-MG were determined by real-time PCR using a SYBR Green I qPCR Master Mix kit [15].

Cells from different groups were prepared according to the protocols, and the BCL2 expression level was measured by flow cytometry (Beckman Coulter, Fullerton, CA, USA). Mouse anti-human BCL2-PE and mouse IgG1-PE (eBioscience, San Diego, CA, USA) were used. Results were analyzed using the Win MDI 2.9 software.

To determine the molecular mechanisms of BCL11B siRNA-mediated cell apoptosis, global gene expression profiling was performed at 24 h post-transfection, when BCL11B mRNA was most effectively suppressed (data not shown). Results were clustered, based on the differential expression level (2-fold up or down), and visualized using a color scale (Figure 1A). Principal component analysis indicated that the changes in the Molt-4 cell gene expression profile could be accounted for primarily by the BCL11B siRNA935 treatment (Figure 1B). A GCOS1.4 software analysis showed that upregulated genes were identified by 142 probe sets, whereas 109 genes were downregulated at least 2-fold, compared with the sc control (Figure 1C). Changes in genes of the same signaling pathways closely related to tumor cell proliferation and apoptosis were analyzed further (Figure 1D).

Among apoptosis-related genes, changes in expression levels occurred mainly in three pro-apoptotic genes; TNFSF 10, BCL-2 interacting killer (BIK), and BCL-2/E1B 19 kDa interacting protein 3 (BNIP 3), which were upregulated 2-4 fold, and one anti-apoptotic gene (BCL2L 1) was downregulated by 3-4 fold. The expression levels of SPP 1 and CREBBP genes involved in the transforming growth factor (TGF-β) pathway were down by 16 fold. The changes in the expression levels of the TNFSF10, BCL2L 1, SPP 1, and CREBBP genes were further detected by real-time PCR (Figure 1E). The BH3-only domain proteins BIK and BNIP3, which were located upstream of BCL-2 (Figure 2), may enhance their binding to BCL-2, thereby inhibiting the anti-apoptotic function. Thus, we analyzed the BCL-2 protein expression level by flow cytometry in Molt-4 cells at 72 h after BCL11B-siRNA treatment (Figure 1F). A similar altered expression pattern of these genes, as well as expression of the BCL-2 protein, was confirmed. However, CREBBP did not show downregulation in BCL11B-siRNA treated Molt-4 cells.

The global gene expression profile results suggest that the molecular mechanisms of BCL11B siRNA-mediated cell death may involve BCL-2 family genes in the intrinsic mitochondrial pathway as well as the TNFSF 10 gene in the death receptor signaling pathway (Figure 2) [20]. Upregulation of the TNFSF 10 gene activated the death receptor signaling pathway, whereas upregulation of the two mitochondrial BCL-2 family genes (the BH3-only domain proteins BIK and BNIP 3) enhanced their binding to BCL-2, with a reduction in the anti-apoptotic gene BCL2L 1, thereby inhibiting the anti-apoptotic function and promoting Bax and Bak activation. This in turn activates the downstream caspases 3, 6, and 7, leading to increased apoptosis. Reduced expression of SPP 1 correlated with increased apoptosis in Molt-4 cells, suggesting that the SPP 1 gene may be a BCL11B gene target.

CREBBP overexpression has been detected in Jurkat cells [21]. However, previous studies have not reported a change in CREBBP expression in T cell lines after BCL11B-siRNA treatment. In the present study, downregulation of CREBBP was identified in the microarray analysis, but not confirmed by real-time PCR analysis. The reason may be due to a systemic error on the microarray analysis. Interestingly, unlike the result from Molt-4 cells, the alteration of the CREBBP expression level in primary T-All cells after BCL11B-siRNA treatment was in accordance with the results from the microarray analysis (Figure 3). Thus, the role of CREBBP during BCL11B downregulation in malignant T cells requires further investigation.

After obtaining interesting data from Molt-4 cells, we analyzed the effect of the BCL11B-siRNA in primary T-ALL cells. We examined the expression levels of TNFSF 10, BCL2L 1, SPP 1, and CREBBP in primary leukemic T cells after BCL11B siRNA935 treatment. BCL11B expression level decreased in primary leukemic T cells treated with BCL11B siRNA935 (282.77 ± 247.57 copies/10⁵ β2-MG) as compared with the sc control group (519.48 ± 303.41 copies/10⁵ β2-MG). The TNFSF 10, BCL2L 1, SPP 1, and CREBBP expression levels in BCL11B-siRNA935- treated primary leukemic T cells were 2.7 ± 2.17%, 9.53 ± 15.34%, 3.5 ± 2.95%, and 4.25 ± 5.82%, respectively, whereas the expression levels in primary leukemic T cells in the sc control group were 1.77 ± 1.93%, 6.96 ± 9.88%, 10.23 ± 13.09%, and 4.98 ± 7.2%, respectively. The T-ALL specimen number was too small to perform statistical analysis. The changes in the mRNA levels of TNFSF 10, SPP 1, and CREBBP in the T cells from the four patients agreed in general with those from the microarray analysis results (Figure 3A, C, D). However, the changes in the BCL2L 1 expression levels in the different samples varied (Figure 3B). The reduced BCL2L 1 expression rates in leukemic T cells from patients 1 and 3 were 31.84% and 13.73%, respectively, compared with the sc controls, whereas BCL2L 1 expression in leukemic T cells from patients 2 and 4 was upregulated. Although BCL11B gene overexpression occurred in all samples, it may have been due to the heterogeneity of T-cell malignancies during apoptosis induced by BCL11B downregulation [22], so it remains to be determined whether apoptosis induced by BCL11B downregulation in some cases with T-ALL involves the BCL-2 family genes in the intrinsic mitochondrial pathway.

A previous analysis revealed that overexpression of the BCL11B, BCL2L 1, and CREBBP genes in primary T-ALL samples blocks apoptosis in malignant T cells [15]. This study suggests that inhibition of BCL11B may trigger apoptosis in leukemic T cells by downregulating the downstream genes SPP 1, CREBBP, and TNFSF 10.