Huang Bin-Bin, Gao Qing-Mei, Liang Wei, Xiu Bing, Zhang Wen-Jun, Liang Ai-Bin
Department of Hematology, Tongji Hospital of Tongji University, Shanghai, China.
Asian Pac J Cancer Prev. 2012;13(5):2045-9. doi: 10.7314/apjcp.2012.13.5.2045.
To investigate the effect of down-regulation of Sentrin/SUMO-specific protease 1 (SENP1) expression on the apoptosis of human Burkitt lymphoma cells (Daudi cells) and potential mechanisms.
Short hairpin RNA (shRNA) targeting SENP1 was designed and synthesized and then cloned into a lentiviral vector. A lentiviral packaging plasmid was used to transfect Daudi cells (sh-SENP1-Daudi group). Daudi cells without transfection (Daudi group) and Daudi cells transfected with blank plasmid (sh-NC-Daudi group) served as control groups. Flow cytometry was performed to screen GFP positive cells and semiquantitative PCR and Western blot assays were employed to detect the inference efficiency. The morphology of cells was observed under a microscope before and after transfection. Fluorescence quantitative PCR and Western blot assays were conducted to measure the mRNA and protein expression of apoptosis related molecules (caspase-3, 8 and 9). After treatment with COCl2 for 24 h, the mRNA and protein expression of hypoxia inducible factor -1α (HIF-1α) was determined.
Sequencing showed the expression vectors of shRNA targeting SENP1 to be successfully constructed. Following screening of GFP positive cells by FCM, semiqualitative PCR showed the interference efficiency was 79.2±0.026%. At 48 h after transfection, the Daudi cells became shrunken, had irregular edges and presented apoptotic bodies. Western blot assay revealed increase in expression of caspase-3, 8 and 9 with prolongation of transfection (P<0.05). Following hypoxia treatment, mRNA expression of HIF-1α remained unchanged in three groups (P>0.05) but the protein expression of HIF-1α markedly increased (P<0.05). However, in the sh-SENP1-Daudi group, the protein expression of HIF-1α remained unchanged.
SENP1-shRNA can efficiently inhibit SENP1 expression in Daudi cells. SENP1 inhibition may promote cell apoptosis. These findings suggest that SENP1 may serve as an important target in the gene therapy of Burkitts lymphoma.
探讨下调Sentrin/SUMO特异性蛋白酶1（SENP1）表达对人伯基特淋巴瘤细胞（Daudi细胞）凋亡的影响及其潜在机制。
设计并合成靶向SENP1的短发夹RNA（shRNA），然后将其克隆到慢病毒载体中。使用慢病毒包装质粒转染Daudi细胞（sh-SENP1-Daudi组）。未转染的Daudi细胞（Daudi组）和转染空质粒的Daudi细胞（sh-NC-Daudi组）作为对照组。通过流式细胞术筛选绿色荧光蛋白（GFP）阳性细胞，采用半定量聚合酶链反应（PCR）和蛋白质印迹法检测干扰效率。在转染前后于显微镜下观察细胞形态。进行荧光定量PCR和蛋白质印迹法检测凋亡相关分子（半胱天冬酶-3、8和9）的mRNA和蛋白质表达。用氯化钴（COCl2）处理24小时后，测定缺氧诱导因子-1α（HIF-1α）的mRNA和蛋白质表达。
测序显示成功构建了靶向SENP1的shRNA表达载体。通过流式细胞术筛选GFP阳性细胞后，半定量PCR显示干扰效率为79.2±0.026%。转染后48小时，Daudi细胞体积缩小，边缘不规则，并出现凋亡小体。蛋白质印迹法显示，随着转染时间延长，半胱天冬酶-3、8和9的表达增加（P<0.05）。缺氧处理后，三组HIF-1α的mRNA表达无变化（P>0.05），但HIF-1α的蛋白质表达显著增加（P<0.05）。然而，在sh-SENP1-Daudi组中，HIF-1α的蛋白质表达无变化。
SENP1-shRNA可有效抑制Daudi细胞中SENP1的表达。抑制SENP1可能促进细胞凋亡。这些发现提示SENP1可能成为伯基特淋巴瘤基因治疗的重要靶点。
