| Research background and purpose:Population aging is a prominent problem in today’s society.Since 2019,China’s population aging growth rate ranks first in the world;The prevention and treatment of cardiovascular disease,diabetes,chronic kidney disease and other chronic diseases has become an important demand of the country.In 2018,cardiovascular diseases accounted for the first place in all-cause deaths.The incidence rate of cardiovascular diseases increased year by year,and the situation became more and more serious.Cell senescence and even vascular senescence are the risk factors of cardiovascular disease.Vascular senescence is manifested by the senescence of endothelial cells and vascular smooth muscle cells.Atherosclerosis is the basis of cardiovascular and cerebrovascular diseases,and aging cells can be seen in advanced atherosclerosis,which is related to the aging of vascular smooth muscle cells.Vascular smooth muscle cells(VSMC)are the most important part of vascular wall.The phenotypic and secretory transformation of VSMC are related to vascular aging and participate in the process of vascular remodeling and atherosclerosis.Delaying cell senescence,especially VSMC senescence,is one of the most effective means to reduce cardiovascular disease.However,the process of vascular aging is complex,and its effective research and intervention measures are limited.At present,the study of vascular aging,how to improve vascular aging and reduce the incidence of aging related cardiovascular diseases have become a hot research topic.Mi RNA is a small non coding RNA with a length of 19-25 nucleotides.Mi RNA regulates cell growth,differentiation,development and apoptosis.Recent studies have shown that miRNAs are associated with cardiovascular disease,and the expression of miR-21 is increased in right heart failure and myocardial fibrosis;Mi R-21-3p can promote the process of atherosclerosis;In patients with acute myocardial infarction,the levels of circulating miRNAs,including miR-1,miR-133,mir-29 b and mir-192,increased;In myocardial injury associated with PD-1 inhibitor,the expression of mir-34a-5p in cardiomyocytes was up-regulated.It can be seen that miRNAs is related to cardiovascular disease and participates in the regulation of cardiovascular aging.Many studies have found that lnc RNA is associated with cell aging and cardiovascular disease.Lnc RNA regulates the biological effects of miRNA through the sponge effect of miRNA and the competitive inhibition RNA mechanism.For example,up regulation of lnc RNA anril expression can inhibit miR-181 a expression and improve vascular smooth muscle cell aging.It shows that lnc RNA /micro RNA is a key regulator of cell aging.Lnc RNA GAS5 is related to cell proliferation.It was found that lnc RNA GAS5 can improve the process of atherosclerosis in Apo E-/-rats,and the expression of lnc RNA GAS5 decreased in patients with heart failure;In the myocardial ischemiareperfusion model,the expression of lnc RNA GAS5 increased and the expression of mir-188-5p decreased.When silencing the expression of lnc RNA GAS5 or promoting the overexpression of miR-188-5p,myocardial ischemia could be improved;Another study found that the expression of miR-665 was increased in the hearts of patients with acute heart failure,and our previous results showed that the expression of miR-665 was increased in bleomycin induced aging VSMC;Thus,lnc RNA GAS5 and miR-665 can regulate the aging of vascular smooth muscle cells,which is related to cardiovascular disease.However,whether lnc RNA GAS5 is associated with miR-665,and its role and mechanism in VSMC aging are not completely clear.Therefore,this paper intends to explore the mechanism and correlation between lnc RNA GAS5 and miR-665 regulating cell aging.In this paper,by increasing the passage times of VSMC as an aging model,through SA-β-Gal staining,NAD+/NADH and γ-H2 AX staining/protein was used to evaluate aging,and miRNA biochip was used to determine the differentially expressed miRNA,so as to further explore the regulatory relationship between lnc RNA / miRNA / downstream genes,so as to provide a new therapeutic target for vascular aging.Measure:1.VSMC cells were cultured in vitro.VSMCs passage to 15 generations was the aging model,and the corresponding 3-5 generations of HA VSMCs were the young group;2.Aging related-β-Galactosidase(SA)-β-Gal)staining to judge the aging of cells in each group;3.NAD+/NADH ratio was used to evaluate the damage of mitochondrial function in each group;4.γ-H2 AX staining / protein to evaluate DNA damage in each group;5.The total RNA of VSMCs in each group was extracted,the differentially expressed miRNAs were determined by miRNAs biochip,and the quantitative verification was carried out by QRT PCR.The differentially expressed miRNAs were selected for further mechanism study;6.Bioinformatics analysis was used to determine the lnc RNA and downstream candidate target genes targeted to the target miRNA for further verification by double luciferase reporting experiment;7.QRT PCR: expression levels of 20 differentially expressed miRNAs: miR-4742-3p,miR-433-5p,miR-376b-5p,miR-668-3p,miR-6868-3p,miR-665,miR-431-3p,miR-1262,miR-4876-5p,miR-3173-5p,miR-211-3p,miR-276-3p,miR-16-1-3p,miR-548 k,miR-222-5p,miR-1908-5p,miR-296-1-5p,miR-3158-3p,miR-1256-3p;Evaluate the transfection efficacy of miRNA micmic / inhibitor;Evaluate the transfection efficiency of pc DNA;The expression levels of lnc RNA and downstream genes in each group were measured;8.WB : γ-H2 AX,GAPDH,downstream genes;It was used to evaluate the regulatory effect of miRNA on downstream genes;9.The rescue experiment verified the effect of lnc RNA / miRNA / downstream target gene signal pathway on VSMCs aging by overexpression or knockdown of lnc RNA / miRNA / downstream target gene,respectively;Results:1.Mi RNA-665 is the most differentially expressed miRNA in VSMC of young and aging group.Inhibiting mirna-665 can delay VSMC aging.VSMC SA in aging group compared with young group-β-Gal positive cells increased(P<0.05),which was consistent with the characteristics of aging;VSMC SA in aging group compared with young group-β-Gal positive cells increased(P < 0.05),which was consistent with the characteristics of aging;Gene chip detection of the aging group and the aging group was used to analyze the differential expression of miRNAs.The up-regulated expression and the top 10 differential expression of miRNAs in the next week were selected respectively.QRT PCR confirmed that the expression of miR-433-5p,miR-665,miR-433-5p,miR-487b-5p in aging ha VSMCs was up-regulated,and the expression of miR-3173-5p,miR-16-1-3p and miR-1908-5p were down regulated,while the expression of miR-665 was the most significant;The aging group transfected with miR-665 inhibitor was significantly lower than that transfected with miRNC which SA-β-Gal positive cells decreased and NAD+/NADH ratio decreased,γ-H2 AX red staining decreased.2.Lnc RNA GAS5 regulates the negative molecule "sponge" of miR-665Firstly,bioinformatics analysis showed that there was a seed sequence of lnc RNA GAS5 targeting miR-665.Compared with VSMC in young group,the expression level of lnc RNA GAS5 in aging group decreased(P < 0.05);Secondly,double luciferase reporter experiment verified that only in lnc RNA GAS5 wild-type reporter plasmid,miR-665 overexpression or interference could reduce or increase luciferase activity,while there was no change in luciferase activity in lnc RNA GAS5 mutant reporter plasmid;Finally,compared with pc DNA NC,the expression level of miR-665 was down regulated by transfecting pc DNA-GAS5 overexpressing lnc RNA GAS5 in aging VSMC(P < 0.05).3.SDC1 is the downstream target gene of miRNA-665Firstly,bioinformatics analysis showed that SDC1 had a seed sequence targeting miR-665.Compared with VSMC in young group,the expression level of SDC1 in aging group decreased(P < 0.05);Secondly,the double luciferase reporter experiment verified that miR-665 overexpression or interference could reduce or increase luciferase activity only in SDC1 wild-type reporter plasmid,while there was no change in luciferase activity in SDC1 mutant reporter plasmid;Finally,compared with mir-nc,the expression of miR-665 was inhibited by transfection of miR-665 inhibitor in aging VSMC,and the expression level of SDC1 was up-regulated(P < 0.05).4.MiR-665 interacts with lnc RNA GAS5 and SDC1 to regulate VSMC aging.Compared with the young group VSMCs,the aging group VSMCs which transfected with NC,pc DNA-GAS5+mi-NC,pc DNA-GAS5+miR-665 mimic,pc DNA-GAS5+si-NC and pc DNA-GAS5+si SDC1 shows of increasing numbers of SA-β-gal positive cells,increasing of NAD+/NADH ratio,γ-H2 AX red staining and protein level(P<0.05);Compared with NC,among the aging group,the decreasing of SA-β-gal staining positive cells,NAD+/NADH,and the level of γ-H2 AX red staining/protein decreased were abserved in pc DNA-GAS5+mi-NC ground and pc DNAGAS5+si-NC(P<0.05).This effect was reversed in pc DNA-GAS5+miR-665 mimic ground and pc DNA-GAS5+si SDC1 ground(P<0.05).ConclusionThe expression of miR-665 is increased in aging VSMCs,and it plays a regulatory role in delaying VSMC aging by inhibiting the expression of SDC1;lnc RNA GAS5 acts as a competitive endogenous RNA(ce RNA)to regulate the role of miR-665 in VSMC aging. |
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