MiR-155 acts as an inhibitory factor in atherosclerosis-associated arterial pathogenesis by down-regulating NoxA1 related signaling pathway in ApoE−/− mouse

Abstract

Background: To investigate the protective efficacy of miR-155 on down regulating NADPH oxidase isoform subunit A1 (NoxA1) gene expression, resulting in inhibition of VSMC migration and over proliferation and thus ameliorating the progression of arterial atherosclerosis in AS mouse model. Therefore, to further explore the regulatory effect of miR-155 on neointima formation in AS and locate potential antiatherosclerosis target. Methods: The mouse vascular aorta smooth muscle cell (MOVAS) was cultured and transfected with recombinant Pad2YFG adenovirus fluorescent vector with miR-155 fragment into 4 groups. Western blotting and RT-PCR were performed to identify the expression of NoxA1 under different circumstances. Fluorescence microscope was applied to observe the transfection rate of miR-155 into adenovirus. Twelveweek fatty food induced atherosclerotic ApoE −/− mouse model was established as host to accept miR-155 transfected adenovirus transplantation to observe its effect on VSMC in AS progression. Carotid and thoracic artery were extracted at 1 month after dosing. Distribution of miR-155 was quantified via expression levels of protein and RNA to detect NoxA1, Nox1, p47phox and NADPH expression. Immunohistochemistry, fluorescence imaging and other methods were performed in arteries section to compare the thickness of neointima and assess the severity of AS in each group.Results: Luciferase reporter gene assay showed significant expression of miR-155 in mimic group indicating that miR-155 had target binding effect with NoxA1 gene. Western blotting and RT-PCR results both showed significantly decreased NoxA1 expression in miR-155 mimic group while increased with its inhibitor. The miR-155 distribution was observed varied at 1 month after in control, miR-155 mimic and inhibitor groups. The NoxA1, NADPH, Nox1 and pp47phox protein expression in VSMC was decreased in mimic group vs control and inhibitor groups (P<0.05); no significant difference of NADPH expression was observed in all groups. The NoxA1, Nox1 and p47phox gene expression in VSMC were both found reduced compared with those of control group at week 4 (P<0.05). Immunohistochemistry staining of artery frozen sections figured out that the thickness of neointima of carotid artery in miR-155 mimic group was significantly lower vs control and inhibitor groups (P<0.01) at week 4.Conclusions: miR-155 played an important role in NoxA1-related signaling pathway. miR-155 transfection into VSMC may have anti-inflammatory regulatory effect on NoxA1 expression in vivo and resulting in amelioration of atherosclerotic lesion in AS mouse model. In summary, miR-155 specifically plays in a negative feedback loop and demonstrates a protective role during atherosclerosis-associated VSMC proliferation and neointima formation through the miR-155-NoxA1-p47phox complex signaling pathway.

Extracellular vesicles in atherosclerosis and vascular calcification: the versatile non-coding RNAs from endothelial cells and vascular smooth muscle cells

“…Zheng et al ( 150) found miR-155 encapsulated in KLF5induced exosomes from VSMCs to ECs, which destroyed the integrity of endothelial barriers, resulting in AS. This result is consistent with the research mentioned above that elevated miR-155 enhanced AS progress (54,(151)(152)(153)(154). González et al (144) suggested that miR-155-5p might be implicated in plaque instability by targeting AKT, contributing to AS.…”

“…MiR-155 activated the NLRP3 by regulating NF-κB in the AS progression of ApoE (−/−) mice ( 155 ). However, evidence showed that miR-155 exerted inhibition on AS in ApoE (−/−) mice ( 156 ). Park et al ( 157 ) suggested that miR-155 was a novel negative regulator in the soluble guanylyl cyclase (sGC)/cGMP pathway, providing a novel therapeutic target for AS.…”

Identification and Interaction Analysis of Molecular Markers in Myocardial Infarction by Integrated Bioinformatics Analysis

“…As shown in Fig. 7, CFTR, CDK1, RPS13, RPS15A, RPS27, NOTCH1, MRPL12, NOS2, CCDC85B and ATN1 achieved an AUC value of >0.88, demonstrating NOXA1 [76] are involved in progression of atherosclerosis. A previous study reported that S100A9 [77], ADORA3 [78], IL1R2 [79], FPR1 [80], CCL20 [ [96], AQP4 [97], FCER1G [98], CCL18 [99], HP (haptoglobin) [100], CDK1 [101], SLC7A11 [102], CFTR (CF transmembrane conductance regulator) [103], F8 [104], STC1 [44], IL18RAP [90], TIMP3 [105], PDE4D [106], CYP4A11 [107], SCN10A [108], APOB (apolipoprotein B) [109], ACE (angiotensin I converting enzyme) [110], PENK (proenkephalin) [111], HSPB6 [112], TLR9 [113], EGR1 [114], CACNG8 [115], FOXD3 [116], DBH (dopamine beta-hydroxylase) [117], FOXP3 [118] [189], MAOA (monoamine oxidase A) [190], BMPR1B (bone morphogenetic protein receptor type 1B) [191], FGF7 [192], CALCRL (calcitonin receptor like receptor) [193], MARK3 [194], ADH1B [195], AMH (anti-Mullerian hormone) [196], RET (ret proto-oncogene) [197], IGF2 [198], SLC6A9 [199], NPPA (natriuretic peptide A) [200], SCT (secretin) [201], DCX (doublecortin) …”

“…Immune system [46], innate immune system [47], neutrophil degranulation [48], toll-like receptor cascades [49], metabolism of amino acids and derivatives [50], neuronal system [51], extracellular matrix organization [52], degradation of the extracellular matrix [53], diseases of glycosylation [54], response to stimulus [55], cell communication [56], cell periphery [57], membrane [58], anatomical structure development [59] and plasma membrane [60] were responsible for progression of MI. PLA2G2A [61], CCL23 [62], CD53 [63], TREML4 [64], TREM2 [65], CD180 [66], HPSE (heparanase) [67], CELA2A [68], TNFRSF4 [69], AMBP (alpha-1-microglobulin/bikunin precursor) [70], SOX18 [71], PANX2 [72], RSPO2 [73], COMP (cartilage oligomeric matrix protein) [74], ASGR1 [75] and NOXA1 [76] are involved in progression of atherosclerosis. A previous study reported that S100A9 [77], ADORA3 [78], IL1R2 [79], FPR1 [80], CCL20 [81], CD163 [82], S100A8 [83], TLR2 [84], HAS2 [85], PTX3 [86], TIMP4 [87], AREG (amphiregulin) [88], LBP (lipopolysaccharide binding protein) [89], IL18R1 [90], ALOX5AP [91], RETN (resistin) [92], F13A1 [93], FPR2 [94], SAA1 [95], FLT3 [96], AQP4 [97], FCER1G [98], CCL18 [99], HP (haptoglobin) [100], CDK1 [101], SLC7A11 [102], CFTR (CF transmembrane conductance regulator) [103], F8 [104], STC1[44], IL18RAP [90], TIMP3 [105], PDE4D [106], CYP4A11 [107], SCN10A [108], APOB (apolipoprotein B) [109], ACE (angiotensin I converting enzyme) [110], PENK (proenkephalin) [111], HSPB6 [112], TLR9 […”