靶向抑制Molt-4 T细胞增殖的Smo-siRNA:筛选和评价

摘要

背景:研究表明,T淋巴细胞白血病的发生发展与Hedgehog通路的异常有关.Smo基因是该信号通路中的关键基因,控制着Hedgehog信号向细胞膜内的传递.目的:筛选一种可以高效抑制Molt-4细胞株增殖和诱导凋亡的小干扰RNA(Smo-siRNA).方法:①根据siRNA设计原理,设计并化学合成Smo-siRNA 1,2,3以及无关干扰序列的阴性对照siRNA;②利用NuclefectorTM核转染仪将以上Smo-siRNA分别转入人T淋巴细胞白血病细胞株(Molt-4细胞),分别在转染24,48,72 h 采用 qRT-PCR 检测 Smo mRNA 相对表达水平,CCK-8法检测细胞生长抑制率, Hoechst33258 染色观察细胞凋亡形态,流式细胞仪(AnnexinV/PI法)检测细胞凋亡率.结果与结论:①利用NuclefectorTM核转染仪成功将Smo-siRNA转入Molt-4细胞,Smo-siRNA 1沉默效果最佳,有效降低Molt-4细胞Smo mRNA表达水平(P < 0.05),并且以48 h作用效果最明显;②转染后24 h, Smo-siRNA可明显抑制Molt-4细胞生长(P < 0.05);③Hoechst染色证实Molt-4细胞符合凋亡的细胞形态学变化;④与对照组比较,Smo-siRNA1组细胞的凋亡率明显增加(P < 0.05);⑤结果表明,小干扰RNA下调Molt-4细胞Smo基因表达可明显抑制Molt-4细胞增殖,并促进凋亡,提示Smo-siRNA具有作为T细胞白血病靶向基因治疗或者协同治疗的潜能.%BACKGROUND: Studies have shown that the occurrence and development of T lymphocytic leukemia is related to the abnormality of Hedgehog pathway. The Smo gene is a key gene in this signaling pathway and controls the transmission of Hedgehog signaling into the cell membrane. OBJECTIVE: To design and screen a highly efficient and specific Smo-siRNA which is able to downregulate the Smo gene expression in Molt-4 cells, thereby inhibiting the Molt-4 cells proliferation and inducing apoptosis. METHODS: (1) Smo-siRNAs numbered 1, 2 or 3, and the scrambled non-siRNA control (SC) were obtained by chemosynthesis. Untreated and sc-treated cells were used as controls. (2) Smo expression levels in Molt-4 cells were analyzed using qRT-PCR at 24, 48, 72 hours after siRNAs delivered by NuclefectorTM.Cell proliferation in vitro was assayed by the cell counting kit-8.The morphology and percentage of apoptotic cells were revealed by Hoechst33258 staining and flow cytometry, respectively. RESULTS AND CONCLUSION: (1) Smo-siRNAs were successfully transferred into Molt-4 cells, and exhibited best silencing results. After transfection with Smo-siRNA1, the mRNA level of Smo was significantly reduced (P < 0.05), and the lowest level was at 48 hours after transfection. (2) Cell proliferation of Molt-4 cells was significantly inhibited by Smo-siRNA at 24 hours after transfection. (3) Hoechst staining results showed morphological changes of Molt-4 were in accordance with those of apoptotic cells. (4) The apoptotic rate was significantly increased in the Smo-siRNA group compared with the control group (P < 0.05). Findings from this study showed that suppression of Smo by RNA interference could effectively inhibit proliferation and induce apoptosis in Molt-4 cells, indicating that Smo-siRNA as gene targeted therapy or synergistic treatment has therapeutic potential in T-cell malignancies.