RNA干扰介导的端锚聚合酶1表达下调诱导人神经母细胞瘤细胞的凋亡

Down-regulation of tankyrase 1 mediated by RNAi leads to the apoptosis of SH-SY5Y cells

目的探讨RNA干扰(RNAi)介导的端锚聚合酶1(TNKS1)的表达下调对人神经母细胞瘤(NB)细胞SH-SY5Y体外增殖能力和凋亡的影响及机制,为开发治疗NB的新靶点提供一定的实验基础。方法根据TNKS1基因序列,设计合成3个TNKS1基因的特异性短发夹环(shRNA)干扰片段,利用慢病毒作为载体,构建目的基因的RNAi序列。慢病毒感染SH-SY5Y细胞,实时定量PCR(Real-time PCR)法检测TNKS1基因的表达,筛选出最佳干扰序列进行后续实验。然后进行克隆形成实验,检测RNAi-TNKS1后细胞增殖能力的改变。并进一步用Western blotting法检测抗凋亡蛋白Bcl-2、促凋亡蛋白Caspase-3、Wnt/β-连环蛋白(β-catenin)通路的关键蛋白β-catenin及其下游靶蛋白Cyclin D1和c-Myc的表达,最后用透射电镜观察凋亡形态的改变,探讨RNAi-TNKS1对细胞凋亡的影响及其作用机制。结果慢病毒载体构建成功,病毒滴度为5×1011TU/L。Real-time PCR检测结果显示,#3 shRNA为最佳的有效靶点。克隆形成实验的结果显示,RNAi-TNKS1后SH-SY5Y细胞的克隆形成率较对照组明显下降。Western blotting结果显示,RNAi-TNKS1可显著抑制Bcl-2蛋白的表达,促进Caspase-3蛋白的表达。此外,Wnt/β-catenin通路的关键蛋白β-catenin及其下游靶蛋白Cyclin D1和c-Myc的表达也降低。透射电镜结果显示,RNAi-TNKS1后细胞呈现明显的凋亡结构和形态。结论 RNAi介导的TNKS1的表达下调可降低SH-SY5Y细胞的体外增殖能力,诱导其凋亡,可能部分是通过抑制Wnt/β-catenin通路发挥作用。

Objective To investigate the effect and mechanism of tankyrase 1 （TNKS1） down-regulation mediated by RNA interference （RNAi）on the proliferation and apoptosis of human neuroblastoma （NB） SH-SYSY cells, and to provide experimental basis for the development of a new therapeutical target. Methods According to TNKS1 gene sequences, three short hairpin （shRNA） interference segments with gene-specific were designed and synthesized, and lentiviral vectors were used for constructing RNAi sequence. After SH-SYSY cells were transfected with lentiviral vector, Real-time PCR assay was used for detecting the expression of TNKS1 gene, and the best interference sequence was screened out for subsequent experiments. The colony forming assay was used to detect the change of cell proliferation after RNAi- TNKS1. The expression of anti-apoptotic protein Bcl-2, pro-apoptotic protein Caspase-3, the key protein β-catenin of Wnt/ β-catenin pathway and its downstream target proteins Cyclin D1 and c-Myc were detected by the Western blotting method for exploring the mechanism. Apoptotic morphology was observed by transmission electron microscopy. Results The lentiviral vector was constructed successfully, and the virus concentration was 5 ×10^11 TU/L. The results of Real-time PCR test showed that #3 shRNA was the most effective target sequence. The colony forming assay results showed that colony forming rate decreased significantly after RNAi-TNKS1 compared with that of control group. The results of Western blotting showed that the protein expression of Bcl-2 reduced significantly as well as β-catenin, Cyclin D1 and c-Myc after RNAi-TNKS1, while the protein expression of caspase-3 increased. The transmission electron microscopy results showed significant apoptotic structure and morphology after RNAi-TNKS1. Conclusion The down-regulation of TNKS1 mediated by RNAi leads to the proliferation decrease and the apoptosis of SH-SY5Y ceils, and the inhibition of Wnt/β-catenin pathway may play a role in it. [