SNHG1 Deficiency Promoting The Senescence Of Vascular Smooth Muscle Cells Mediating The Development Of Abdominal Aortic Aneurysm

Objective: Abdominal aortic aneurysm(AAA)refers to high-risk vascular disease with a diameter larger than 3cm or 50% larger than normal.Its incidence increases with age and exceeds 5% in people over 60 years of age.Most patients usually have no obvious symptoms and are found during physical examination or screening,so early detection and treatment are extremely difficult.However,once abdominal aortic aneurysm ruptures,the mortality rate is as high as 90%,posing a serious threat to the health of patients.Currently,there is no drug therapy that can effectively control the occurrence and development of abdominal aortic aneurysm.The most commonly used treatment means are abdominal aortic aneurysm resection,artificial vascular replacement and endovascular repair.However,studies have shown that early surgical intervention has not significantly improved the quality of life of patients.Therefore,it is of great clinical significance to further study the pathogenesis of abdominal aortic aneurysm and explore effective drug therapeutic targets.Abdominal aortic aneurysm is a chronic degenerative disease characterized by inflammatory infiltration,impaired vascular smooth muscle function and degradation of extracellular matrix(ECM).Current studies have shown that the senescence of vascular smooth muscle cells(VSMC)leads to changes in vascular wall function and the formation of AAA.VSMC,as the main cellular component of the middle layer of aortic wall,maintains vascular structure and function by secreting extracellular matrix,while aging VSMC produces the state of senescence associated secretory phenotype(SASP).The production of more inflammatory cytokines exacerbates the progression of AAA.Chronic increased inflammation has been detected in the cardiovascular system with age,but the molecular mechanisms that link vascular aging and inflammation to AAA formation remain unelucidated.Previous studies have determined that SIRT1-p53-p21 axis maladjustment in VSMC accelerates NF-κB induced vascular inflammation and predisposes abdominal aorta to damage,which is a direct link between vascular cell senescence and vascular inflammation and AAA formation.However,how aging of VSMC produces and accelerates AAA formation needs further study.Long non-coding RNAs(Linc RNAs)are RNA molecules between200 and 100,000 nucleotides in length that do not encode proteins,can be located in the nucleus or cytoplasm,and are involved in a wide range of biological processes.In addition,Linc RNAs have been reported to be important players in the occurrence and development of diseases,such as the regulation of gene transcription and posttranscriptional m RNA modification,thereby influencing age-related physiological and pathological conditions,including cardiovascular disease and cellular senescence.However,whether and how Linc RNAs affect AAA remains unclear.Small nucleolar RNAs(sno RNAs)are conserved non-coding RNAs found in all eukaryotes.Their most common function is to direct site-specific post-transcriptional modifications in ribosomal RNAs(r RNAs).Studies have shown that alterations in r DNA transcription modification in eukaryotic cells trigger nucleolar stress(also known as ribosomal stress).Nucleolar stress induces the activation of p53 pathway,thus promoting cell senescence.In human AAA tissue,increased nucleolar stress in vascular smooth muscle cells is associated with local DNA damage response.In vitro,nucleolar stress induces a DNA damage response that mediates p53 phosphorylation and VSMC senescence.In addition,a few additional sno RNAs such as SNORD3 and SNORD118 are known to play a role in r RNA processing and maturation.Although sno RNA’s typical characteristics,functions,and targets have been well described,a growing body of literature has revealed novel and unexpected biological roles in sno RNA,including important regulatory roles in stress response pathways,regulatory stability of precursor m RNA,and regulation of m RNA 3’-terminal processing.Advances in sno RNA research methods have led to more and more sno RNAs and their host genes being found to be associated with disease.Because SNORNA has been poorly studied in AAA research,it is unclear how much role sno RNA plays in the formation of AAA.Therefore,this study aims to provide experimental evidence for the pathogenesis of AAA by exploring the senescence state of aortic smooth muscle cells in patients with abdominal aortic aneurysm,the role of VSMC senescence regulation in the occurrence and development of mouse elastic protease AAA model,and the role of SNHG1 in vitro and in vivo on AAA development and VSMC senescence.Therefore,it provides a new strategy and target for the clinical treatment of AAA.Methods:(1)Aortic tissue samples from patients with abdominal aortic aneurysm and normal control group were obtained,and the expression levels of senescence associated secretory phenotype inflammatory factors IL6 and TNF-α were detected by PCR.Meanwhile,the expression levels of P53,P16 and SIRT1 proteins were detected by immunohistochemistry and immunofluorescence methods,and the aging situation of VSMC in the vascular wall was observed.The aging mechanism of abdominal aortic aneurysm smooth muscle was mined by bioinformatics method.(2)A mouse AAA model was constructed using elastase,and SIRT1 inhibitor and activator were given to explore the effect of regulation of VSMC aging on experimental abdominal aortic aneurysm.Firstly,the changes of body weight and the maximum diameter of abdominal aorta were recorded during modeling,and then the breakage and destruction of elastic fibers of vascular wall between different groups were observed by HE staining and EVG staining.Then,the expression changes of senescence-related proteins P53,P16,SIRT1,and senescence associated secretory phenotype inflammatory factors IL6 and TNF-α were detected by immunofluorescence and immunohistochemical staining,respectively,to observe the aging state of VSMC during the formation of abdominal aortic aneurysm.Finally,the expressions of SNHG1,SNORD22,IL6 and TNF-α were investigated by PCR.(3)The effect of SNHG1/SNORD22 on hydrogen peroxidation-induced aging of VSMC was explored in cell experiments.After the knockdown of SNHG1 and overexpression of SNORD22,the changes of cell proliferation capacity were detected by senescence-related β-galactosidase(SA-β-gal)staining,CCK8 assay,and the expressions of P53,P16 and SIRT1 proteins were detected by Western blotting.The relative expression levels and locations of P53,P16 and SIRT1 were detected by immunofluorescence;the changes of cell cycle and apoptosis were detected by flow cytometry;the secretion status of TNF-α and IL6 were detected by enzyme linked immunosorbent assay(ELISA)and PCR.In situ hybridization and PCR were used to detect the effect of transfected SNHG1-si RNA and SNORD22 overexpressed plasmid,and RTL-P was used to detect the 2 ’-O-ME levels at different sites of r RNA.(4)SNHG1expression was knocked down in mice using adeno-associated virus.The mechanism of SNHG1 affecting the development of AAA was verified by constructing AAA model with elastase.First,the adeno-associated virus infection status of abdominal aorta was observed in frozen sections,PCR was used to detect the expression levels of SNHG1.And then the changes of abdominal aorta diameter and elastic fiber breakage of mice in each group were detected.The expressions of senescence-related proteins P53,P16 and SIRT1 in each group were detected by immunohistochemistry and immunofluorescence.At the same time,immunohistochemistry and PCR were used to detect the expression of senescence associated secretory phenotype inflammatory factors IL6 and TNF-α,to explore the effect of SNHG1 on the development of AAA and its regulatory mechanism.Results:(1)Compared with the control group,there were significant differences in the expression of P53,P16 and SIRT1 of aortic VSMC in patients with abdominal aortic aneurysm.The expressions of IL6 and TNF-α were higher than those of normal people.Transcriptome sequencing data mining results indicate that there may be an imbalance in the expression of small nucleolar RNA(Sno RNA)and its host gene SNHG1 in abdominal aortic aneurysm.(2)Compared with the sham operation group,the AAA model group showed increased abdominal aortic diameter,elastic fiber rupture,inflammatory infiltration,increased expression of senescence-related proteins,and decreased expression of SNHG1.Compared with the elastase AAA model group,the abdominal aortic diameter dilatation was reduced,the elastic fiber breakage was improved,and the inflammatory infiltration and senescence phenotype severity were decreased in the group that was given SIRT1 activator to delay the aging process of VSMC.(3)Cell experiments showed that hydrogen peroxide could induce senescence of VSMC,which was represented by inhibition of cell cycle,increase of β-galactosidase positive cells,increase of senescence associated secretory phenotype inflammatory cytokines IL6 and TNF-α,increase of senescence-related proteins P53 and P16,and decrease of SIRT1 expression.The knockdown of SNHG1 intensified the cell senescence induced by hydrogen peroxide,and the expression of SNORD22 restored partially alleviated the more severe senescence phenotype caused by SNHG1 knockdown.(4)After adeno-associated virus knockdown SNHG1 expression in mice,compared with the infected control group,AAA formation was more serious,abdominal aortic diameter dilation was more severe,elastic fiber breakage was more serious,inflammatory factor IL6 and TNF-α infiltration were increased,senescence-related proteins P53 and P16 expression were increased,and SIRT1 expression was decreased.The role of SNHG1 in the pathogenesis of abdominal aortic aneurysm was demonstrated.Conclusions: This study demonstrated that there was senescence of VSMC in the abdominal aorta of AAA patients with elevated levels of inflammatory infiltration.The sequencing data showed that the expression of small nucleolar RNA and its host genes was unbalanced.Delaying the aging of VSMC by activating senescence regulatory protein SIRT1 can improve the diameter change and vascular inflammation in AAA mice model induced by elastin.In H2O2-induced senescence VSMC,SNHG1 produces a series of SNORNA through variable shear to regulate the modification of r RNA and ultimately regulate the expression of SIRT1,playing a role in the process of cell senescence.Knockdown of SNHG1 expression in mice accelerates the development of abdominal aortic aneurysms.