Effects of Down-Regulating BCL11B Expression On the Proliferation, Apoptosis and Global Gene Expression Profiling of Molt-4 Cells.

Abstract 4505

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B) gene plays a crucial role in T-cell development, differentiation, and proliferation. BCL11B disturbed expression, mutation, disruption, or rearrangement has been associated with T-cell malignancies. Here, we described the effects of BCL11B target-specific small interfering RNA (BCL11B-siRNA) on the proliferation, apoptosis, and global gene expression profiling of Molt-4 cells. The BCL11B-siRNA beginning with the 924bp of mRNA sequence, and the scrambled non-silencing siRNA control (sc) were obtained by chemosynthesis. Non-treated, mock-transfected, and sc-treated cells were used as controls. BCL11B expression in mRNA levels were analyzed at 24 h, 48 h and 72 h after siRNA delivered by nucleofection using the real-time quantitative PCR with Taqman technique. At the same time points, cell proliferations were assayed by the cell count kit-8 method. The morphology and the percentage of apoptosis were revealed by Hoechst33258 stain and flow cytometry at 72 h, respectively. An obvious reduction in mRNA level was observed in BCL11B-siRNA treated cells at 24 h, compared to the controls (P< 0.05). The proliferation rates of BCL11B–siRNA treated cells were significantly decreased from 24 h to 72 h (P< 0.05). BCL11B–siRNA treated cells showed a large increase in PI-positive cells, reaching to 84.6%. However, the percentage of each cell phases did not reveal significantly difference. Thus, the apoptosis could be induced in all phases of the cell cycle. The Hoechst33258 stain was used to verify the apoptosis. To elucidate the molecular mechanisms accounting for BCL11B-siRNA-mediated cell death, global gene expression profiling were performed on the BCL11B–siRNA treated cells and non-treated control by the Affymetrix HG U133 Plus 2.0 GeneChip at 24 h after nucleofection. The BCL11B showed 10.1 fold reductions in mRNA levels. Upregulated genes was found in 1982 probe sets, while 1884 genes down-regulated, at least two folds upon Bcl11b suppression. Involvement of the T-cell activation or differentiation as TRAT1, CD3G, CD247, LCK, PTGER4, or CD1D, all revealed at least 7 folds down-regulated, which may contribute to the inhibition of proliferation. Six genes of the TNF receptor family were up-regulated at least 2 folds as well as the BCL-2 gene down-regulated 3.5 folds. The apoptosis was most likely affected by the simultaneous activation of TNF receptor family genes and suppression of the Bcl-2 gene. Those related to the autophagy and the cell cycle did not find significant change. In conclusion, we demonstrated that the survival of Molt-4 cells was critically dependent on BCL11B gene. Suppression of BCL11B by RNA interference selectively inhibited the proliferation and induced the apoptosis effectively in transformed Molt-4 cells. Therefore, down-regulation of Bcl11b might be considered as a new target therapeutic strategy in T-cell malignancies.