Gene expression profiles in BCL11B-siRNA treated malignant T cells
- Xin Huang†1, 2,
- Qi Shen†1,
- Si Chen1,
- Shaohua Chen1,
- Lijian Yang1,
- Jianyu Weng2,
- Xin Du2,
- Piotr Grabarczyk3,
- Grzegorz K Przybylski3, 4,
- Christian A Schmidt3 and
- Yangqiu Li1, 5Email author
© Huang et al; licensee BioMed Central Ltd. 2011
Received: 21 March 2011
Accepted: 15 May 2011
Published: 15 May 2011
Downregulation of the B-cell chronic lymphocytic leukemia (CLL)/lymphoma11B (BCL11B) gene by small interfering RNA (siRNA) leads to growth inhibition and apoptosis of the human T-cell acute lymphoblastic leukemia (T-ALL) cell line Molt-4. To further characterize the molecular mechanism, a global gene expression profile of BCL11B-siRNA -treated Molt-4 cells was established. The expression profiles of several genes were further validated in the BCL11B-siRNA -treated Molt-4 cells and primary T-ALL cells.
142 genes were found to be upregulated and 109 genes downregulated in the BCL11B-siRNA -treated Molt-4 cells by microarray analysis. Among apoptosis-related genes, three pro-apoptotic genes, TNFSF 10, BIK, BNIP 3, were upregulated and one anti-apoptotic gene, BCL2L 1 was downregulated. Moreover, the expression of SPP 1 and CREBBP genes involved in the transforming growth factor (TGF-β) pathway was down 16-fold. Expression levels of TNFSF 10, BCL2L 1, SPP 1, and CREBBP were also examined by real-time PCR. A similar expression pattern of TNFSF 10, BCL2L 1, and SPP 1 was identified. However, CREBBP was not downregulated in the BLC11B-siRNA -treated Molt-4 cells.
BCL11B-siRNA treatment altered expression profiles of TNFSF 10, BCL2L 1, and SPP 1 in both Molt-4 T cell line and primary T-ALL cells.
Although treatment outcome in patients with T-cell acute lymphoblastic leukemia (T-ALL) has improved in recent years, relapsed T-ALL remains a challenge . Monoclonal antibodies, gene inhibitors, and upregulation of microRNAs [2, 3] are promising tools for cancer targeted therapy. However, few targeted therapies are available for T-cell malignancies. For example, transforming Mer signals may contribute to T-cell leukemogenesis, and regulation of Mer expression could be a novel therapeutic target for pediatric ALL therapy . The recent identification of activating Notch1 mutations in the majority of patients with T-ALL has brought interests on targeting the Notch signaling pathway for this disease .
The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B) gene was first identified on human chromosome 14q32.2  and encodes a Krüppel-like C2H2 zinc finger protein initially identified as a transcriptional repressor . BCL11B plays an important role in T-cell differentiation and proliferation [8–11]. Altered expression, mutation, disruption, or rearrangement of BCL11B has been associated with T-cell malignancies [12–14]. In humans, BCL11B overexpression is found primarily in lymphoproliferative disorders, such as T-ALL and adult T-cell leukemia/lymphoma [12, 15–17]. BCL11B mediates transcriptional activation by interacting with the p300 co-activator at the upstream site 1 (US1) of the interleukin (IL)-2 promoter, leading to transcriptional activation of IL-2 expression in activated T cells . Although the interaction partners and binding sequence have been revealed, only a few BCL11B direct target genes have been identified to date. Our previous study in the human T-ALL cell lines Molt-4, Jurkat, and hut78 has shown increased apoptosis upon BCL11B suppression by RNA interference .
In the present study, we further analyzed the global gene expression profiles in Molt-4 and primary T -ALL cells after BCL11B-935-siRNA treatment.
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.
RNA isolation, expression profiling, reverse transcription, and real-time PCR
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 .
Flow cytometry assay
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.
Results and discussion
Global gene expression profile in BCL11B-siRNA935 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) . 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.
Expression of TNFSF 10, BCL2L 1, SPP 1, and CREBBP genes in BCL11B-siRNA935-treated primary leukemic T cells
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 , 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 . 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.
Our findings provide evidence that BCL11 B siRNA-mediated cell apoptosis may be related to the mitochondrial pathway BCL-2 family genes and the TNFSF 10 gene of the death receptor signaling pathway. Moreover, the SPP 1 and CREBBP genes in the TGF-β pathway may also be involved in BCL11B siRNA-mediated cell apoptosis.
The authors thank Dr. Xuesong Yang for critical reading of this manuscript, and thank Dr. Xuchao Zhang from the Cancer Center of Guangdong Provincial Hospital for helpful analysis of the gene-chip data. This work was supported by Grants from National Natural Science Foundation of China (no. 30771980) and the Guangdong Science & Technology Project (no. 2007B030703008; and 2009B050700029).
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