Regulation Of Pulmonary Artery Smooth Muscle Cell Growth With Self-assembled DNA Nanomaterials Carrying MiR-135a And The Underlying Mechanism

Background:Dysfunction of human pulmonary artery smooth muscle cells（HPASMCs）is an important factor in pulmonary vascular remodeling.The abnormal differentiation,proliferation,and migration of HPASMCs induced by hypoxia are the key pathological bases of continuous contraction of pulmonary arteriole and increased arterial pressure.Our group has found that the expression of lnc RNA-RP11 increased,the expression of miR-135a decreased,and the expression of Rab26 increased in the lung tissues of patients with chronic obstructive pulmonary disease（COPD）and HPASMCs under hypoxia.Therefore,we propose that under hypoxia,the lnc RNA-RP11 promoter was demethylated,subsequently up-regulated lnc RNA-RP11 expression.The elevated lnc RNA-RP11 adsorb miR-135a through competitive binding,resulting in the up-regulation of Rab26 expression,which led to phenotype transformation and abnormal proliferation of HPASMCs and pulmonary vascular remodeling.Self-assembled DNA nanomaterials with excellent biocompatibility,versatility,and programmability are vastly promising nanomedicine platforms in biomedicine.The canonical assembly of DNA nanostructures depends on Mg²⁺and has poor stability in physical conditions.Therefore,new assembly strategies and functionalization are urgently needed to be applied to biomedical applications.Basic amino acids,such as lysine,arginine,and histidine which carry positive charges in physiological conditions have the potential to mediate DNA self-assembly into defined structures.Therefore,we used basic amino acids instead of canonical Mg²⁺to mediate the assembly of DNA nanostructures.At the same time,we decorated DNA nanomaterials with si RNAs or miRNAs and deliver them to tumor cells to verify nanomaterials’biocompatibilities and functions,then apply them to HPASMCs as a new nano-intervention strategy for the treatment of pulmonary vascular remodeling caused by the abnormal growth of HPASMCs.Research Methods:1.Design and self-assembly of DNA nanomaterials.（1）DNA nanotubes were designed using SEQUIN software.By extending one of the DNA strands,they were able to conjugate with target si RNAs or miRNAs by base pairing.DNA nanorectangle was designed by Cadnano2.0 software.（2）Native polyacrylamide gel electrophoresis（PAGE）,agarose gel electrophoresis,and laser dynamic scattering（DLS）were performed to characterize DNA nanotubes and DNA nanorectangle.（3）Construction of noncanonically self-assembled DNA nanomaterialsDNA nanotubes and DNA nanorectangle were self-assembled by basic amino acids and characterized by PAGE,agarose gel electrophoresis,DLS,and atomic force microscope（AFM）.The concentration,temperature,and p H values required for the synthesis of DNA nanomaterials were studied.2.Biocompatibility of self-assembled DNA nanomaterials and preliminary verification of their applications in cells（1）Stability of amino acid/DNA composite nanomaterials under low ion concentration was characterized by PAGE.（2）The cytotoxicity of amino acid/DNA composite nanomaterials in various cancerous cell lines was detected by MTT assay.（3）The cellular uptake efficiency of amino acid/DNA composite nanomaterials by tumor cells was detected by flow cytometry and confocal laser scanning microscope（CLSM）,and the subcellular location of the nanomaterials was investigated.（4）The cellular uptake of DNA nanotubes by A549 and other tumor cells was detected by flow cytometry and CLSM.（5）The expression and subcellular distribution of lnc RNA PVT1in tumor cells were determined by RT-q PCR.（6）The effect of DNA nanotubes with lnc RNA PVT1 si RNA on tumor cell proliferation was detected by MTT assay.Western blot（WB）was used to detect the expression of related proteins in tumor cells.3.Regulation of pulmonary artery smooth muscle cell growth with self-assembled DNA nanomaterials via miR-135a-Rab26 pathway.（1）The expression of miR-135a and Rab26 in HPASMCs was detected by RT-q PCR and WB.（2）DNA nanotubes were used to deliver miR-135a,and the effect of up-regulation of miR-135a on the proliferation of HPASMCs was detected by CCK-8 method under hypoxia conditions.（3）Knocked down Rab26 by DNA nanotubes with si RNA,and the knockdown efficiency was measured by RT-q PCR,and the cell viability was measured by CCK-8.（4）RT-q PCR was used to detect the effects of hypoxia and nanomaterials onα-SMA,the contractile marker of smooth muscle,HPASMCs phenotype,and AT2R receptors on HPASMCs surfaces.Research Results1.DNA nanotubes and DNA nanorectangle were successfully designed and synthesized.The synthesis yield was at a high level and the particle size was in accordance with the theoretical designs.2.Arginine and lysine could mediate the synthesis of DNA nanotubes and nanorectangle in a certain concentration range,and their particle sizes and morphology conformed to the theoretical designs.Arginine and lysine had a wide p H range of synthesis of DNA nanomaterials and could self-assemble DNA nanostructures isothermally.Histidine failed to synthesize DNA nanostructures.3.Biocompatibility assessment of self-assembled DNA nanomaterials:（1）DNA nanomaterials assembled by arginine had high structural and thermal stabilities.（2）The cytotoxicity of amino acid-mediated self-assembled DNA nanomaterials was at a low level.（3）Compared to Mg²⁺assembled DNA materials’,the uptake efficiency of amino acid assembled DNA nanomaterials was higher.Arginine-assembled DNA nanotubes mainly concentrated on the cell membrane,while Mg²⁺assembled DNA nanotubes located in the cytoplasm.4.（1）Expression of lnc RNA PVT1 was significantly increased in A549 and other lung adenocarcinoma cells.（2）The uptake efficiency of DNA nanotubes was higher in lung adenocarcinoma cells.（3）It was found that lnc RNA PVT1 was expressed in both nucleus and cytoplasm according to nucleocytoplasmic separation experiment.（4）DNA nanotubes carrying lnc RNA PVT1 si RNA could inhibit the proliferation of tumor cells effectively by up-regulating Bax protein.5.Regulation of pulmonary vascular remodeling with self-assembled DNA nanomaterials via miR-135a-Rab26 pathway:（1）The expression of miR-135a andα-SMA in HPASMCs was decreased while Rab26 was increased under hypoxia.（2）Under hypoxia,delivery of miR-135a with DNA nanotubes suppressed HPASMCs proliferation.（3）DNA nanotubes with Rab26 si RNA had high knock-down efficiency and inhibited HPASMCs proliferation significantly.（4）Silencing Rab26 by DNA nanotubes up-regulatedα-SMA and AT2R,down-regulated VIM and MMP2,resulting in the phenotype transformation of HPASMCs to contractile type,and the reduced proliferation.Conclusions:1.DNA nanotubes and DNA nanorectangle were successfully designed and synthesized.DNA nanotubes could carry multiple functional units such as si RNAs and miRNAs.Arginine and lysine could successfully synthesize DNA nanostructures at a certain concentration,p H value,and temperature.2.Compared with Mg²⁺,DNA nanomaterials assembled with amino acids have excellent biocompatibility,low toxicity,and are ready to be absorbed by tumor cells.DNA nanotubes with si RNAs can effectively silence target genes in tumor cells and inhibit the proliferation of tumor cells.3.Rab26 can be knocked down by DNA nanotubes with si RNA Rab26 or indirectly by delivering miR-135a using DNA nanotubes.The following hypothesis was verified:under hypoxia,the methylation level of lnc RNA-Rp11 promoter was down-regulated,the expression of lnc RNA-Rp11 was up-regulated,miR-135a was down-regulated through ce RNA theory,and the expression of Rab26 was indirectly up-regulated,which promoting the abnormal proliferation and phenotype transformation of HPASMCs.Meanwhile,our study shows that DNA nanomaterials carrying nucleic acid drugs are a potential novel strategy for the prevention and treatment of pulmonary artery smooth muscle cell growth targeting Rab26and miR-135a.