核酸纳米材料携带的mTOR小干扰RNA对肺动脉平滑肌细胞自噬和增殖的影响

Effect of nucleic acid nanoparticle-loaded mTOR siRNA on autophagy and proliferation in pulmonary arterial smooth muscle cells

目的研究自组装核酸纳米材料携带的mTOR小干扰RNA(self-assembled nucleic acid nanoparticles loaded mTOR small interfering RNA,siRNA-NPs)对大鼠肺动脉平滑肌细胞(rat pulmonary arterial smooth muscle cells,RPASMCs)自噬和增殖的影响。方法设计并合成DNA序列m T-2A、m T-1A3以及5'端标记Cy3的mTOR siRNA,共设计mTOR siRNA序列3条,选其中最佳序列,将上述核酸材料按照操作步骤自组装合成三角板型的核酸纳米材料。实验分4组:核酸纳米材料携带的mTOR siRNA组(siRNA-NPs组)、单纯纳米材料组(NPs组)、单独的siRNA组和空白对照组。各组材料分别转染RPASMCs 24 h,激光共聚焦显微镜观察RPASMCs对核酸纳米材料携带的mTOR siRNA的摄取情况;应用RT-PCR检测各组细胞中mTOR mRNA的表达水平,应用Western blot检测p-mTOR、LC3B蛋白的表达水平;激光共聚焦、透射电镜检测细胞自噬水平;MTT及3H-TdR检测各种材料处理后对细胞的生长的影响。结果激光共聚焦显微镜下可见siRNA-NPs组细胞质内均匀分布大量呈颗粒状的红色荧光物质,其荧光强度均显著高于单独的siRNA组(P<0.05);而且siRNA-NPs组处理的mTOR mRNA和p-mTOR蛋白表达显著低于单独的siRNA组、NPs组及空白对照组(P<0.05);而LC3B蛋白表达显著高于其他各组(P<0.05),激光共聚焦检测显示siRNA-NPs组标记自噬小体的绿色荧光明显强于单独的siRNA组、NPs组及空白对照组(P<0.05),电镜结果显示siRNA-NPs能明显诱导细胞自噬小体形成;MTT及3H-TdR检测siRNA-NPs和单独siRNA处理RPASMCs后明显抑制其生长,且siRNA-NPs组细胞生长抑制率又显著高于单独的siRNA组(P<0.01)。结论 siRNA-NPs能被RPASMCs有效摄取并明显抑制靶基因的表达,进而诱导细胞自噬并且抑制其细胞增殖。

Objective To study the effect of self-assembled nucleic acid nanoparticle-loaded mammalian target of rapamycin （mTOR） small interfering RNA （siRNA） on the autophagy and proliferation in rat pulmonary arterial smooth muscle cells （RPASMCs）. Methods DNA oligonucleotides, mT-2A, mT-1A3 and roTOR siRNA with Cy3-1abeling in the 5＇ terminal, were designed and synthesized. Three mTOR siRNA sequences were designed and the best one was used. According to the instructions, these oligonucleotides were assembled to triangular nucleic acid nanoparticles. Four experimental groups were setup： DNA-loaded mTOR siRNA （siRNA-NPs）, DNA nanoparticles （NPs）, mTOR siRNA alone and control.siRNA-NPs, NPs and roTOR siRNA were transfected into rat RPASMCs for 24 h, respectively. Confocal microscopy was used to observe the uptake of mTOR siRNA in PASMCs. The expression of mTOR mRNA was detected by RT-PCR, and the protein levels of p-mTOR and LC3B were detected by Western blotting. The autophagy levels of PASMCs were observed by confocal microscopy and transmission electron microscopy （TEM）. The growth rate of PASMCs was detected by MTY and 3H-TdR assays. Results Confocal microscopy showed a large number of brightly red-fluorescent granules distributed in the cytoplasm of PASMCs in siRNA-NPs group. The red fluorescence intensity was significantly higher than that of mTOR siRNA group （ P 〈 0.05 ）. Green fluorescence intensity that labels autophagosome was found significantly higher in siRNA- NPs compared to other 3 groups （ P 〈 0.05 ）. This was confirmed by TEM imaging that siRNA-NPs induced autophagosome formation in PASMCs. The expression of mTOR mRNA and p-mTOR protein was significantly increase and LC3B protein significantly decreased （P 〈 0.05 ） in siRNA-NPs vs. other three groups. In MTY and 3H-TdR assays, PASMCs transfected with siRNA-NPs and mTOR siRNA had reduced growth rate. The inhibition of cell growth was stronger in siRNA-NPs group than that of mTOR siRNA group （ P 〈 0.01 ）. Conclusion Our data demonstrated that siRNA-NPs, self-assembled nucleic acid nanoparticle-loaded roTOR siRNA, could be effectively uptaken in PASMCs and target mTOR to reduce its expression, thereby inducing autophagy and inhibiting proliferation.