Inhibition of DLEU1 Following siRNA Transfection in Burkett’s Lymphoma (BL): Implication for Tumor Repressor Role of DLEU1 in C-Myc-Activated BL Lymphomagenesis

Background: The progress of childhood BL and DLBCL has improved dramatically in the past three decades; however, patients with a 13q-deletion have a significantly poorer outcome (Cairo/Patte et al Blood, 2007 and Patte/Cairo et al Blood, 2007; Poirel/Cairo et al Leukemia 2008). DLEU1, a potential tumor suppressor gene, is located within the 13q-deletion. DLEU1 was reported to be a key gene in the Burkitt classifier genes (Dave/Staudt et al NEJM, 2006) and c-myc binds to the promoter region of DLEU1. DLEU1-network proteins include, among others, E3 ubiquitin-protein ligase (UBR1), Tubulin beta-2C (TUBB2C) and RASSF1A. We previously demonstrated that UBR1, TUBB2C, and RASSF1A, were differentially expressed in BL vs DLBCL patients and cell lines by global gene profiles and real time RT-PCR studies (Day/Cairo et al AACR 2008; Day/Cairo et al ICML 2008). We further demonstrated decreased expression of UBR1 (33.2±4.5% reduction compared to control (p<0.02)) and TUBB2C (30.0±3.5% reduction compared to control (p<0.001)) by DLEU1 gene siRNA knock down, while expression of RASSF1A was not changed (Day/Cairo, et al SIOP 2008). Taken together, these data suggest the hypothesis that DLEU1 interacting with UBR1 may interfere with microtubule function, and therefore act as a tumor repressor in c-myc-activated BL lymphomagenesis, by arrest of the cell cycle at G2/M and subsequent inducion of apoptosis.

Objective: In this study, we investigated the role of DLEU1 in regulation of apoptosis in BL by inhibition of DLEU1 gene expression by a DLEU1 siRNA and evaluated it effects on the apoptotic rate in a BL cell line.

Methods: The Ramos BL cell line was transiently transfected with a 25-nucleotide modified DLEU1 siRNA (5′-AUACUUGGCAUGAAUGAACUUAUGU-3′ and 3′-UAUGAACCGUACUUACUUGAAUACA-5′). Stealth RNAi whose GC content is similar to that of this DLEU1 siRNA was used as negative control. The transient transfection of DLEU1 siRNA (10 – 20 nM) was achieved using Lipofectamine RNAiMAX. The transfection efficiency of siRNA was evaluated using Alexa Fluor Red Fluorescent Oligo. DLEU1 contents were measured by qRT-PCR with ddCt relative quantitative determination. GAPDH was used as endogenous control. Statistical analysis was conducted by one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test. To determine the early and late stages of apoptosis, we transfected Ramos BL cells with DLEU1 siRNA, and then incubated cells with Annexin V-FITC and Propidium Iodide for 15 minutes, respectively (BD Pharmingen), followed by FACS using BD LSRII with FACSDiva.

Results: The DLEU1 siRNA decreased the expression of DLEU1 RNA (52±13%; p<0.0006). The transfection efficiency of siRNA was 85 – 90%. Comparing to untreated cells, DLEU1 siRNA treatment significantly reduced early apoptosis (16.90±0.37%; p<0.001) and late stage apoptosis (14.70±0.27%; p<0.0001).

Conclusion: These results suggest that when DLEU1 gene expression is decreased in BL cells, there is a significant reduction in both early and late apoptosis. The results strongly support a relationship between DLEU1 gene and regulation of BL apoptotic mechanisms. In concert with previous investigations, this data suggests that DLEU1 may function as a tumor growth repressor via UBR1 and TUBB2C-regulated mechanism in the cellular apoptotic process. Since c-myc binds promoter region of DLEU1 and these two genes are a part of the c-myc signaling network, this further underscores the importance of DLEU1 and its network proteins may play in c-myc-activated BL lymphomagenesis.