Objective      To study the effect of self-assemb

led 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 mTOR siRNA with Cy3-labeling 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 mTOR 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 MTT and

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H-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 (P0.05). Green fluorescence intensity that labels autophagosome was found significantly higher in siRNA-NPs compared to other 3 groups (P0.05). 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 (P0.05) in siRNA-NPs vs. other three groups. In MTT and

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H-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 (P0.01).       Conclusion      Our data demonstrated that siRNA-NPs

self-assembled

nucleic acid nanoparticle-loaded mTOR siRNA

could be effectively uptaken in PASMCs and target mTOR to reduce its expression

thereby inducing autophagy and inhibiting proliferation.