Construction Of New DNA-RNA Nanocarrier And Its Regulation On The Growth Of Pulmonary Artery Endothelial Cells

Research Background:Pulmonary hypertension(PAH)is a progressive clinical syndrome,which with the main feature of pulmonary artery pressure increased,this is an important pathophysiological basis of chronic obstructive pulmonary disease,pulmonary heart disease and other clinical lung disease.The hypoxic pulmonary hypertension(HPH)is one of the main types of clinical classification of pulmonary hypertension,with main pathological changes of abnormal proliferation of pulmonary vascular smooth muscle cells and endometrial tissue which hypoxic conditions stimulated,Resulting in vascular stenosis,increased pulmonary vascular resistance,pulmonary vascular remodeling,Studies show that the function of abnormal pulmonary artery endothelial cells is the key to the disease occurrence and development,now that the endothelial dysfunction include inhibition of endothelial cell apoptosis,increased proliferation,Then in order to seek HPH therapeutic target,we will study the specific mechanisms of the regulation of endothelial cell proliferation under hypoxic conditions,which can explore new ways of HPH treatment.Autophagy is a process of self-digestion,under normal physiological conditions,autophagy is conducive to maintain its cell homeostasis,When under stress conditions,such as under hypoxia,starvation conditions,the change of the state of autophagy will affect cell homeostasis,which will affect cell proliferation,apoptosis,differentiation,migration and invasion,etc.Autophagy is an important factor in the regulation of endothelial cells nder hypoxic conditions,and ATG101 is important gene which regulation of autophagy.This paper aims to study hypoxia in pulmonary artery endothelial cell autophagy and its mechanism of regulation of endothelial cell proliferation.For a common clinical disease or refractory disease,traditional drug treatment is often symptomatic treatment,there are side effects of drugs,and easy to relapse and other issues,In order to meet the growing demand for treatment increased,new treatment modalities have emerged on the DNA level,Gene therapy which can introduce therapeutic effect gene into normal human target cells to achieve therapeutic purposes,Including small interfering RNA,non-coding RNA,mi RNA and the like.So how to efficiently transfer gene,whichhas become one of the main challenges faced by gene therapy.and the nano-carrier systems for gene has been more and more attention,especially the DNA nanocarriers which are natural biological materials,relative to traditional viral vectors,the advantages of low-immunogenic nanocarrier is low toxicity or nontoxic,biodegradable,stable structure,Simple synthesisetc.,non-viral delivery systems is the most important way to a new carrier.In DNA molecules as a scaffold,self-assembled DNA triangle-shaped nanocarriers,and the use of DNA-RNA hybridization techniques,it will carry the gene of ATG101 si RNA complementary pairing of DNA nano-assembled as a set square,formation of a new DNA-RNA nano-delivery systems.To study its transport within the cell,and to explore the impact of the target gene on the proliferation and autophagy of PAECs under hypoxic conditions and its molecular mechanism of regulation of target gene therapy of pulmonary hypertension,which provide a new theoretical basis for the treatment of pulmonary arterial hypertension treatment.Research purposesSelf-assembly of DNA nano-triangle of carrying ATG101 si RNA,analyzing intracellular transfection efficiency of the DNA-RNA nanocarrier,and to study the impact of nanometer system on autophagy and proliferation of PAECs.Research methods1.Preparation of DNA nano-triangle carrying ATG101 si RNA and to study the properties of this nano-systems.(1)Water bath cooling method for preparing DNA nano-triangle which load ATG101 si RNA(2)Using atomic force microscopy to observe ultrastructureand in order to demonstrate the success and efficient synthesis of nano-system by gel electrophoresis,2.Detecting the uptake and transfection efficiency of DNA triangle loading si RNA in lung cancer cells.Cultured lung cancer cells(A549,H292)in vitro,After DNA Nanosystems transfection of A549,H292,observeing lung cancer cell uptake of DNA nanosystems time-and dose-dependent by laser scanning confocal microscopy and flow cytometry,and then using RT-PCR and Western blot to detect expression of target protein.3.The influence of ATG101 si RNA-loaded DNA triangleon on the growth of A549ATG101 si RNA-TNP transfected A549 cells,then electron microscopy,CLSM and Western blot were taken to detect its influence on autophagy of A549,cell activity were testing by MTT assay,At last apoptosis was detected by flow cytometry.4.The influence of ATG101 si RNA-carryed DNA triangleon on the growth of PAECsCultured PAECs in vitro,then transfecting ATG101 si RNA-TNP nanosystems into PAECs,CLSM to observe the fluorescence expression of autophagy,Western blot to detect the expression of autophagy and proliferation marker protein?5.The influence of ATG101 si RNA-carryed DNA triangleon on the growth of PAECs under hypoxic conditions.PAECs were cultured in vitro,which placed in a hypoxic incubator(1% O2,5% CO2,94% N2),after transfection of ATG101 si RNA-TNP nanosystems in PAECs,we were respectively detected the influence on autophagy,proliferation and apoptosis of PAECs.6.ATG101 regulate apoptosis of PAECs under hypoxic by Hedgehog signaling pathway ATG101 si RNA-TNP were add to PAECs,Using Western blot to detect the expression ofHedgehog and Gli after ATG101 silence.Research results1.Successfully self-assemble DNA nano-triangle which use a single-stranded DNA as a skeleton and si RNA single-stranded DNA as the carrier,AFM shows that nanostructures is triangular,and the distribution is uniform;Gel electrophoresis analysis that self-assembled nano-system with high yield and stable structure synthesising by cooling water bath ladder method.2.The system of lung cancer cell line is stable,so the two lung cancer cells as pre-experimental objects,the results show that the two cells(A549,H292)both can take up DNA nano-systems,And with respect to individual si RNA,si RNA which loaded by Nanocarriers more susceptible to cellular uptake,and in a time and concentration-dependent manner.CLSM and flow cytometry results all showed that with the increase of concentration and time,its transfection efficiency enhanced,and that nanosystems uptake into cells occurs by macropinocytosis-and clathrin-mediated endocytosis.Nanocarriers carrying si RNA efficiently into cells and exert their biological effects,RT-PCR and Western blot showed that the decreased expression of the target protein in PCR and protein levels.3.ATG101 si RNA-TNP transfected into A549 cells,which can inhibit the expression of ATG101 with autophagy inhibition,and activate cell apoptosis,leading to inhibition of growth of A549 cells.4.DNA nano-triangle carrying ATG101 si RNA can be efficiently intake PAECs,and silence the target protein ATG101 in a time and concentration-dependent manner,CLSM and Western blot results showed decreased levels of autophagy,while inhibition of cell growth after suppression of the target protein.5.Under hypoxic conditions,PAECs autophagy increased and proliferation activation,but after suppression of the target protein,Western blot and CLSM display that autophagy levels decreased,MTT showed cell growth was inhibited,apoptosis detected by flow cytometry activated.6.Autophagy inhibition after ATG101 silence under hypoxic conditions,which cativated apoptosis by Hedgehog-GLi signaling pathway,thereby inhibiting cell growth.Conclusion1.Successfully synthesis self-assemble DNA nano-triangle which load ATG101 si RNA with high yield and stable structure,This nanosystem can successfully and efficiently transferred to tumor cells and normal cells,and exert biological effects of si RNA,indicating this stable nanosystem has broad application prospects.2.ATG101 si RNA loaded by DNA nano-triangle inhibit autophagy of A549 with induction of apoptosis,an inhibit cell growth.3.DNA nano-triangle carry ATG101 si RNA enter PAECs,can inhibit autophagy under hypoxic conditions,and induce apoptosisby Hedgehog-GLi signaling pathway,which lead to cell growth inhibition,this indicate that autophagy can be adjusted proliferation and apoptosis imbalance of PAECs under hypoxic conditions.