Self-assembly MTOR SiRNA-loaded DNA Nanotubes And Its Regulation On Autophagy In Pulmonary Arterial Smooth Muscle Cells

One of the most important research topics in nanomedicine is the development of drug carriers platform that are able to transport various functional agents into cells, due to the huge application potential of gene therapy during several of disease treatment. Many drug delivery systems have been previously reported, such as synthetic polymers, cationic lipids, carbon nanotubes, nanoparticles, cyclodextrin material, and viral capsids. The effectiveness of these systems has been clearly demonstrated. However, a majority of these systems involves unnatural or exogenous molecules, which comprise the performance due to lack of tissue specificity and potential toxicity. DNA is a natural component in the mammal cells. The intracellular DNA is relative stability and biodegradable. Moverver, compared with above drug delivery systems, the advantage of DNA nanoparticles that is structures by short DNA fragments is non-immunogenic and nontoxicity for body. Company with the development of DNA molecular self-assembled nanotechnology, the DNA nanostructures show a graet potential in biological applications. Previous research has shown that some natural, self-assembled DNA nanostructures had been used as carriers for delivery drug and oligonucleotides into cells through changed configuration, invovled binding to signaling molecules, potentially releasing and allowing access to their cargo. Therefore, Selfassembled DNA nanostructures would be become a ideal vehicle for drug and oligonucleotides delivery.Pulmonary hypertension(PAH) is a serious disease to threat the public health and the 5-year survival rate of PAH is only 60%. Pulmonary vascular remodeling(PVR), that forming involved the abnormal proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells(PASMCs) were induced by hypoxia, is one of the important pathophysiological basis of PAH. Autophagy is evolutionarily conserved catabolic mechanism and involved with regulation cellular homeostasis in eukaryotic. Previously study showed autophagy played an important role in pulmonary vascular disease, such as PAH, COPD, cor pulmonale. However, the effect of autophagy in the development of vascular remodeling in PH, especially in PASMCs, is not yet fully understood. Therefore, that was expected to further clarify the molecular mechanism of hypoxic pulmonary vascular remodeling by further study the mutual regulation of autophagy and proliferation on PASMCs under hypoxia, which provide a new targets and strategies for prevention and control pulmonary hypertension.For these reasons, we designed and developed a self-assembled DNA nanotube system by combining four single DNA strands. The functional agent, mTOR siRNA, binds to an overhang on the DNA nanotube by DNA-RNA hybridization at a specific molar ratio. The effectiveness of DNA nanotube was evaluated and the effect of DNA nanotube system on autophagy and proliferation in RPASMCs under normoxia or hypoxia conditions were assayed. These dataes conclusion will further charify the molecular mechanisms of hypoxic pulmonary vascular remodeling. Theywill be to provide new theoretical basis and strategy to prevention PAH.ObjectiveTo design and constructe a self-assembled DNA nanotube loaded m TOR si RNA, and evaluating the effectiveness of the self-assembled DNA nanotube nanoparticles, and analyzing the effect of the DNA nanotube nano-system on the autophagy and proliferation of RPASMCs.Methods1. Self-assemble m TOR si RNA loaded nanotubes nanoparticles and characteristics of the new nano-system(1) The DNA nanotube carrier was assembled by the dimerization of a carefully designed symmetric 3-point-star motif.(2) The successful synthesis of DNA nanotube and si RNA-loaded nanotube was demonstrated by native polyacrylamide gel electrophoresis(PAGE). Using atomic force microscopy(AFM) to observe the morphology and distribution of DNA nanotubes nanoparticles.2. Intracellular uptake analyse by Confocal laser scanning microscopy(CLSM) and transfection efficiency study by flow cytometry.Rat pulmonary arterial smooth muscle cells(RPASMCs) were cultured in vitro. The various concentration of mTOR si RNACy3-loaded nanotubes were then added to examine the dose-dependent cellular internalization, while it was kept at 50 nM to investigate the time-dependent intracellular uptake and endocytosis process. Confocal laser scanning microscope(CLSM) images were taken on a Leica DMRA2 fluorescence microscope. The transfection efficiency of si RNACy3-loaded nanotubes at dose- and time-dependent was evaluated by fluorescence-activated cell sorting(FACS) technique. The data were analyzed using Flowjo software.3.The effect of m TOR siRNA-loaded nanotubes on the autophagy of RPASMCsRPASMCs were cultured in vitro. Various formulations with free mTOR si RNA, nanotubes alone or m TOR si RNA-loaded nanotubes were added, and then exposed to normoxia. Using Cyto-ID®Green autophagy dye kits to observe cell autophagy induced by siRNA-loaded nanotubes via CLSM images, and using transmission electron microscope(TEM) to observe the ultrastructure of autophagosome in RPASMCs. Using RT-PCR to detect mTOR m RNA levels after incubated with si RNA-loaded nanotubes at various of concentration and predetermined time in RPASMCs, and western blot to test the protein expression of m TOR(T-m TOR,p-m TOR), LC3 B, PCNA in RPASMCs. Using MTT to test the growth inhibition rate incubated with siRNA-loaded nanotubes at dose- and time dependent manner in RPAMCs.4. The influence of the m TOR siRNA-loaded nanotubes on the autophagy of hypoxia-treated RPASMCs.RPASMCs were cultured in vitro. Various formulations with free mTOR si RNA, nanotubes alone or m TOR si RNA-loaded nanotubes were added, and then exposed to hypoxia(1% O2). CLSM to observe RPASMCs autophagy by the Cyto-ID®Green autophagy dye kits. Using [3H]-thymidine incorporation assay to detect the impact of different formulations on proliferation of RPASMCs, MTT analyse to test cell viability, Western blot to detect the protein expression of p-m TOR, LC3 B, PCNA in RPASMCs by treated 48 h in hypoxic condition with different formulations, and the influence of mTOR siRNA and p-mTOR on the autophagy and proliferation of RPASMCs.Results1. Successfully self-assemble DNA nanotube and m TOR si RNA loaded DNA nanotubes(si RNA-NTs). It was clearly observed that both DNA nanotube and si RNA anchored nanotube have a single, sharp band with expected mobility on the native polyacrylamide gel. AFM imaging further confirmed the individual nanoparticles were uniformly dispersed on the mica surface and had a uniform size distribution. The result of MTT verified the excellent cell compatibility of self-assembled DNA nanoparticles.2. Cellular uptake of siRNACy3-loaded nanotubes at dose- and time dependent manner in RPAMCs.(1) CLSM to observe uptake of si RNACy3-loaded nanotubes at doseand time dependent manner. The intensity of the fluorescence was significantly enhanced with increasing dose and incubation times. When the dose of siRNACy3-loaded nanotubes at 50 nM and incubation time for 12 or 24 h, cellular uptake of red fluorescence significantly improved comparable with si RNA alone group(P<0.05).(2) Flow cytometry analysis of the transfection efficiency and effectiveness of DNA nanotubes(si RNA-NTs) at dose- and time dependent manner in RPAMCs. The transfection efficiency gradually increased with increasing dose and incubation times. At the same time, the mean fluorescence intensity of per cell of siRNA-NTs group was significantly enhanced comparable with free siRNA group(P<0.05). These results positively demonstrated that self-assemble si RNA-loaded nanotubes can effectively promote mTOR si RNA, which was Cy3 red fluorescence labeling, cellular uptake and transfection by RPASMCs.(3) CLSM imaging showed that the siRNACy3-NTs nanoparticles from the cell membrane into the cytoplasm with the extension of incubation time, and locate in the endosome.3. Effects of self-assembled siRNA-loaded nanostructures on PASMC autophagy and proliferation.(1) CLSM imaging showed that the intracytoplasmic green fluorescence was observed in almost the same cellular site as the red fluorescence, which indicated that the autophagosomes were colocalized with the siRNACy3-loaded nanotubes.(2) mTOR si RNA induces autophagy of RPASMCs. CLSM imaging revealed that siRNA-NTs group showed very strong green fluorescence compared with the control group, the free si RNA group and the positive control group(P<0.05). TEM images display that si RNA-NTs group showed a lot of typical autophagic vacuoles in the cytoplasm of RPASMCs compared with the control group, the free siRNA group and the nanotubes-alone group(P<0.05). Westernblot and semi-quantitative analysis showed that the levels of LC3 B II/I protein expression in the group with siRNA-loaded nanotubes significantly increased compared with the free siRNA group and the nanotubes-alone group.(3) mTOR siRNA-loaded nanotubes enhance LC3 B expression in RPASMCs by suppressing mTOR signaling. RT-PCR results showed the levels of m TOR m RNA decreased in a time- and concentration-dependent manner. When the dose of si RNA-loaded nanotubes at 50 nM and after incubation for 48 h, the m TOR m RNA level was significantly reduced comparable with others group(P<0.05). Westernblot showed self-assembled mTOR si RNA-loaded nanotubes inhibited m TOR protein levels in a time-dependent manner. When the dose of siRNA-loaded nanotubes at 50 n M and after incubation for 48, 72 h, The mTOR protein expression level was significantly reduced comparable with 24h(P<0.05). Meanwhile, westernblot showed LC3 B protein levels was remarkably upregulated as the mTOR protein level was decreased. after treatment with mTOR siRNA-loaded nanotubes for predetermine time. Especially at 48 h, the LC3 B level was significant increased and the PCNA expression was prominent inhibited. These results demonstrated that the m TOR si RNA-loaded nanotubes effectively suppressed the expression of mTOR, allowing a stronger induction of PASMC autophagy, thus leading to cell growth inhibition.(4) MTT assay showed cell growth inhibition by m TOR siRNA-loaded nanotubes in a time- and dose-dependent manner.4. mTOR coordinates hypoxia-induced cell autophagy and proliferation in RPASMCs.(1) mTOR siRNA-loaded nanotubes promote hypoxia-induced autophagy in RPASMCs. CLSM imaging revealed that si RNA-NTs group exhibited higher green fluorescence expression than the hypoxia-alone or free si RNA treatment groups(P<0.05). Westernblot showed the LC3BII/I protein levels in the hypoxia-alone was increased compared with the normoxic group(P<0.05), and the LC3BII/I protein levels in siRNA-NTs group significant increased compared with the others group(P<0.05).(2) mTOR si RNA-loaded nanotubes inhibit the cell viability and proliferation of RPASMCs under hypoxia. MTT result suggested the cell viability of RPASMCs in the si RNA-NTs group obvious reduction compared with the others group under hypoxia. The data of [3H]Td R revealed that hypoxia-induced PASMC proliferation in the siRNA-NTs group significant reduction compared with the hypoxia-alone or free si RNA treatment groups(P<0.05).(3) m TOR regulates hypoxia-induced the protein expression of LC3 B and PCNA in RPASMCs. Westernblot showed the si RNA-loaded nanotubes suppressed the protein expression of p-mTOR and PCNA, which meantime increased LC3 B protein levels under hypoxia. These results further demonstrated that siRNA-loaded nanotubes suppress cell proliferation by enhancing hypoxia-induced PASMC autophagy, resulting in autophagic cell death.Conclusions1. Self-assemble mTOR si RNA loaded DNA nanotubes is successfully constructed, the character of this is much more stable structure and higher biocompatibility. Cellular uptake of siRNACy3-loaded nanotbues by RPASMCs via endocytosis. Cellular uptake and transfection of mTOR siRNA-loaded nanotubes at dose- and time dependent manner in RPAMCs.2. Self-assemble m TOR siRNA loaded DNA nanotubes induced autophagy and inhibited proliferation in RPASMCs via m TOR inhibition.