| Objective:The aim of the study is to design and screen the highly efficient and specific BCL11B-siRNAs which are able to downregulate the BCL11B gene expression in Molt-4 cells, thereby inhibit the Molt-4 cells proliferation and induce apoptosis, and to analyze the related molecular mechanism of BCL11B-siRNAs.Method:(1) BCL11B-siRNAs numbered 1,2, or 3, and the scrambled non-silencing siRNA control (sc) were obtained by chemosynthesis. Untreated, mock-transfected, and sc-treated cells were used as controls. BCL11B expression in mRNA levels from Molt-4 cells were analyzed after siRNAs delivered by nucleofection using the real-time quantitative PCR with Taqman technique. The BCL11B protein levels were analyzed by Western blotting. Cell proliferation in vitro was assayed by the cell count kit-8 method after treatment. The morphology and the percentage of apoptosis were revealed by Hoechst33258 stain and flow cytometry (FCM). Molt-4 cells transfected with BCL 11 B-siRNA3 were observed by the high-resolution live cell imaging system. (2) Global gene expression profiling was analyzed on the BCL11B-siRNA3 treated cells and controls by the Affymetrix HG U133 plus 2.0 Gene Chips, and verified part of the differentially expressed genes by real-time quantitative PCR. BCL-2 protein levels in Molt-4 cells from BCL11B-siRNA3 group and controls were measured by FCM. (3) CD34+cells were sorted from 3 cases of umbilical cord blood by MACs techinque, and the purity was identified by FCM. BCL11B expressions in mRNA level of umbilical cord blood CD34+cells were analyzed by the real-time quantitative PCR technique. BFU-E, CFU-GM, and CFU-Meg were performed from BCL11B-siRNA3 treated CD34+cells by methyl cellulose semi-solid culturing method, to estimate the role of differentiation and proliferation in CD34+cells after BCL11B-siRNAtransfection.Result:(1) BCL11B-siRNA3 (935) group took best silencing results, BCL11B-siRNA1 (434) group followed. An obvious reduction about 3 to 8 folds in BCL11B mRNA level was observed in BCL11B-siRNA3 treated cells at 24 h, and 2 to 6 folds in BCL11B-siRNA1 group at 48 h. Compared with the SC control, BCL11B protein expression inhibition rate reached 54.08% in BCL11B-siRNA3 group at 48 h, and 41.73% in BCL11B-siRNAl group at 72 h. The proliferation rates of BCL11B-siRNA3 and BCL11B-siRNA1 treated Molt-4 cells were significantly decreased at 72 h (P< 0.05). BCL11B-siRNA3 treated Molt-4 cells showed a significantly increase in AnnexinV/PI-positive cells, reaching to 58.71±2.93%(P< 0.05), similar results in the morphological changes of apoptosis were found by Hoechst staining test and high-resolution imaging system. The BCL11B-siRNA3 and BCL11B-siRNA1 sequences have been applied for patents. (2) Global gene expression profiling analysis showed that up-regulated genes were found in 142 probe sets, while 109 genes down-regulated in BCL11B-siRNA3 group, compared with the SC control. The up-or down-regulation genes mainly involved in the pathway were apoptosis pathway (TNFSF10, BIK, BNIP3, BCL2L1 genes), and TGF beta pathway (SPP1, CREBBP genes). The changes of expression level of TNFSF10, BCL2L1, and SPP1 genes were confirmed by real-time PCR, while the lower expression of BCL-2 protein was confirmed by FCM. (3) The purity of CD34+cell sorted from umbilical cord blood was 97.17±1.45%. BCL11B gene mRNA expression levels in CD34+cells were significantly lower than that in peripheral blood mononuclear cells and Molt-4 cells (P< 0.05). The formation of BFU-E, CFU-GM, and CFU-Meg from CD34+cells showed no significant difference between BCL11B-siRNA3 treatment and MOCK control group (P> 0.05).Conclusion:Suppression of BCL11B by RNA interference could inhibit the proliferation and induce the apoptosis effectively in Molt-4 cells, which might related to BCL-2 family genes in mitochondrial pathways and TNFSF10 gene in death receptor signaling pathway. No significant effect of BCL11B-siRNA on the proliferation and differentiation of CD34+cells in vitro suggest that BCL11B-siRNA might be safe and considered as a new target therapeutic strategy in T-cell malignancies.Key word:Small interfering RNA; BCL11B gene; Molt-4 cells; Nucleofection; Apoptosis; Gene chip; Umbilical cord blood CD34+cells; Proliferation and differentiation in vitro... |
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