MiR-124-3p Mediates The Regulation Of IL-1? On Collagen Synthesis In Smooth Muscle Cells And Its Effect On The Atherosclerotic Plaque

BackgroundAccording to the 2017 Global Burden of Disease study,cardiovascular diseases have caused nearly 18 million deaths in this year,an increase of 21.1%from a decade ago.Cardiovascular disease has been the number one killer of humans for several years.Atherosclerosis(AS)is one of the major pathological causes of cardiovascular disease.It is characterized by lipid deposition,fibrosis and calcinosis in the affected arteries and followed by artery calcification,intra-plaque hemorrhage,plaque rupture and local thrombosis.Studies have shown that the stability of atherosclerotic plaque is determined by the size of the lipid core,the infiltration of the inflammatory cell and the degree of inflammatory response,as well as the thickness of the fibrous cap.Unstable plaques are more likely to cause clinical cardiovascular events.Therefore,it is of great clinical significance to explore the factors affecting the stability of atherosclerosis and its specific mechanisms.Vulnerable plaques tend to have thinner fibrous caps,while the formation of fibrous caps and the maintenance of their integrity rely primarily on ECM.Vascualr smooth muscle cells are the main producer of ECM among which collagen is the main component.Among all types of collagen,type I and type III collagen are most abundant in arteries.The metabolism of collagen includes both synthesis and degradation.In recent years,studies on collagen metabolism have focused on the degradation process,and there are few studies about collagen synthesis and its regulation mechanism in smooth muscle cells.The synthesis of collagen requires the assistance of hydroxylases and glycosylases,among which the prolyl-4-hydroxylase(P4H)plays an important role in the formation of a stable triple helix structure of collagen.miRNAs are a class of non-coding RNAs,and recent studies have shown that they are involved in the regulation of a variety of physiological and pathological processes.A variety of miRNAs play important roles in the development of atherosclerosis.miR-124-3p is distributed in various tissues in human body.It was originally discovered in the nervous system and proved to promote the maturation of neurons.It is closely related to various neurological diseases.It has also been confirmed to inhibit the proliferation and migration of different kinds of tumor cells,playing an important role as a tumor suppresser.Recent experiments showed that the serum level of miR-124-3p.was positively correlated with the risk of cardiovascular events in smoking patients with coronary heart disease.However,its specific effects and mechanisms on atherosclerosis remain to be further studied.Objectives1.To clarify the effect of miR-124-3p on atherosclerotic plaque morphology and vulnerability in ApoE-/-mice2.To investigate the effect of miR-124-3p on collagen metabolism in smooth muscle cells and its specific mechanismMaterials and methods1.Lentivirus constructionLentivirus carrying miR-124-3p mimic as well as miR-124-3p inhibitor were constructed with the vector pGLV3/H1/GFP+Puro.2.Animal groupSixty 8-week-old ApoE-/-male mice were fed with normal diet for 2 weeks after obtained from Peking University.They were then fed with high-fat diet for 12 weeks to induce atherosclerosis.They were randomly divided into 3 groups and injected with lentivirus into the tail vein four weeks before the sampling:(1)negative control group(n=20):18 of them were injected with 2×107 TU of control lentivirus(NC group),and 2 mice were randomly selected to receive the same amount of normal saline injection(NS group)for transfection efficiency detection later;(2)miR-124-3p overexpression group(n=20):mice were inj ected with 2x 107 TU lentivirus carrying miR-124-3p mimic;(3)miR-124-3p inhibition group(n=20);mice were injected with 2×107 TU lentivirus carrying miR-124-3p inhibitor.Lentiviral transfection efficiency was measured after 2 weeks and all the mice was sacrificed after 4 weeks.3.Animal tissue collectionThe mice were anesthetized by intraperitoneal injection of sodium pentobarbital.The mice were sacrificed by blood drawing from the apex and the blood was collected to detect total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)levels.The mouse aorta was dissected,and the heart,lung,liver,spleen,and kidney were collected.Those tissues were put into formalin or liquid nitrogen(or-80? freezer)as needed.4%paraformaldehyde was used to fix the aortic roots for 24 hours.The tissues were then embedded with OCT and sliced with a thickness of 5 ?m using a cryostat.4.Tissue protein extraction and concentration determinationThe mouse aortic arch preserved at-80? was added with protein lysate(RIPA)lOul per mg of tissue,and the tissue was immersed in the corresponding volume of RIPA.After minced with a homogenizer,we centrifudged the fluid,and collected the supernatant.To determine the protein concentration,a BCA kit was used,then protein loading buffer was added to the supernatant and then boiled for denaturation.5.Western blotCalculate the corresponding sample volume according to the measured protein concentration,and finally add 10mg sample into per well.After SDS-PAGE electrophoresis and transmembrane,the membrane was blocked by 5%skim milk-TBST and then incubated overnight at 4? with the diluted primary antibody.Next morning,the membrane was washed with 1×TBST,and then incubated with secondary antibody at room temperature for 1 hour for chemiluminescence.After taken the images,the bands were analyzed for the grayscale value by Photoshop.6.General oil red O stainingThe fixed aorta was longitudinally dissected and stained with oil red O staining solution for 2 hours at room temperature.After washed with warm water,it was observed under a general microscope and the out membrane of the vascular was gently removed,and then the blood vessel was flattened and photographed.7.Tissue section stainingAortic root cryosections were used to observe indicators related to atherosclerotic plaque.The plaque size and morphology were observed by HE stain.The lipid content in the plaque was observed by oil red O staining.The collagen content in the plaque was observed by Sirius red staining.The specific antibodies MOMA-2 and a-SMA of macrophages and smooth muscle cells were used to observe the content of macrophages and smooth muscle cells in the plaque.In addition,immunohistochemical staining of the corresponding proteins in the plaques was performed to calculate the expression of type I and type III collagen,P4HA1,MMP2 and MMP9.8.Fluorescence in situ hybridization(FISH)and immunofluorescence stainingFISH was used to show the distribution of miR-124-3p in plaques,and immunofluorescence staining was performed using antibodies targeting smooth muscle cells(a-SMA),endothelial cells(endomucin)and macrophages(MOMA-2)to show the cell distribution.miR-124-3p was co-localized to observe its distribution in different cell types.9.Smooth muscle cell culture and treatmentHuman aortic smooth muscle cells(HASMCs)were purchased from Sciencell Corporation and cultured in smooth muscle cell medium(SMCM).The miR-124-3p mimic and inhibitor sequences were constructed and transfected into the smooth muscle cells by lipo2000 to regulate the expression of miR-124-3p in HASMCs.10.Reverse transcription quantitative PCR(RT-qPCR)Animal tissue RNA is extracted using Qiagen's miRNA extraction kit,following the instructions.After stimulation,total RNA of the cells was extracted by TRIzol.RNA concentrations were measured using Nanodrop.Reverse transcription of mRNA and miR-124-3p was performed according to the instruction of the reverse transcription kit and then real-time quantitative PCR reaction was performed.The expression of mRNAs of type I collagen,type III collagen and P4HA1 as well as miR-124-3p were measured.11.Construction of luciferase reporter plasmids containing P4HA1 mRNA 3'-UTRLuciferase reporter plasmids containing the 3' untranslated region(UTR)sequence of the wild-type P4HA1 mRNA and the 3' UTR sequence of the mutant P4HA1 mRNA was constructed:P4HA1 3'UTR wild and P4HA1 3'UTR mut.The miR-124-3p mimic and its control as well as the two kinds of plasmid were co-transfected into HASMCs by lipo2000.The expression of the fluorescence intensity was detected to confirm the direct binding of miR-124-3p to the 3' UTR of the P4HA1 mRNA.12.RNA binding protein immunoprecipitation(RIP)The miRNA binds to the mRNA of the target gene by forming RNA-induced silencing complex(RISC)with the argonaute(Ago)protein,of which Ago2 is most commonly seen.The smooth muscle cells transfected with miR-124-3p mimic or control RNA were respectively subjected to the RIP kit.The RNA bound to Ago2 was pulled down by Ago2 antibody,and then the content of P4HA1 mRNA was detected by RT-qPCR.13.Statistical analysisThe experimental data were presented as mean ± SD.Statistical analysis and statistical charting were performed using Graphpad prism 6.0.Paired or unpaired t-test was used to compare between two samples.One-way ANOVA was used for comparison between multiple samples.P<0.05 was considered to be statistically significant.Results1.The general condition of animalsThere was no significant difference in body weight and serum TC,TG,LDL-C and HDL-C content between the three groups of ApoE-/-mice,indicating that miR-124-3p did not affect the blood lipid and body weight of mice.2.Detection of lentivirus transfection efficiency and expression efficiency in vivoTwo weeks after lentivirus injection,two mice each in the NC group and the NS group underwent cryosection of the aortic root.It was found that green fluorescence appeared in the plaque of the NC group injected with blank lentivirus while the NS group had not.Four weeks after lentivirus injection,the levels of miR-124-3p were detected by RT-qPCR in the aortic arch of mice from NC group,miR-124-3p mimic group and miR-124-3p inhibitor group.Compared with the NC group,the expression level of miR-124-3p was upregulated in the miR-124-3p mimic group and downregulated in the miR-124-3p inhibitor group.3.miR-124-3p increases plaque vulnerabilityCryosections of the aortic roots of the three groups of mice showed no significant statistical difference in atherosclerotic plaque area after oil red O and HE stain.However,Sirius red staining showed that compared with the NC group,the collagen content in the miR-124-3p mimic group decreased,and the smooth muscle cell content in the plaque also decreased,while the collagen content in the plaque of the miR-124-3p inhibitor group increased and the smooth muscle cell content increased,too.In addition,there was no significant difference between the three groups in lipid content and macrophage content in the plaques.4.miR-124-3p inhibits the expression of type I and type III collagen in atherosclerotic plaquesImmunohistochemical staining of aortic root cryosections showed that type I and type III collagen expression was decreased in miR-124-3p mimic group compared with NC group,while in the miR-124-3p inhibitor group the expression of type I and type III collagen increased.We also examined the expression of collagen synthesis-related enzyme P4HA1 and degradation-related enzymes MMP2 and MMP9 in the plaques and found that the expression of P4HA1 decreased in the miR-124-3p overexpression group and increased in the miR-124-3p inhibition group,however there was no significant difference between MMP2 and MMP9 expression.5.miR-124-3p is mainly distributed in smooth muscle cellsCryosections of the aortic roots of the NC group mice were used for co-localization of miR-124-3p and different cell types.In the atherosclerotic plaque,the number of fluorescence co-localization of a-SMA and miR-124-3p was the highest,indicating that the miR-124-3p was mainly distributed in smooth muscle cells.6.miR-124-3p reduces collagen and P4HA1 expression in smooth muscle cellsHuman aortic smooth muscle cells were transfected with miR-124-3p mimic,miR-124-3p inhibitor or scrambled negative control.WB results showed that compared with NC group,the expression of type I and type III collagen as well as P4HA1 in the miR-124-3p mimic group was decreased while the expression of type I and type III collagen and the expression of P4HA1 in the miR-124-3p inhibitor group was increased.The expression of MMP2 and MMP9 in the cells were also detected but no significant difference was found between the three groups.7.P4HA1 is a target protein of miR-124-3pThe possible binding sites of miR-124-3p were present in the 3'UTR region of both human and mouse P4HA1 mRNA as predicted by miRNA target gene software.The double luciferase assay carried out in HASMCs showed that the fluorescence expression in the cells co-transfected with miR-124-3p mimic and wild-type P4HA1 mRNA 3'UTR dual luciferase reporter plasmids was decreased,confirming that miR-124-3p could directly bind to the 3'UTR of P4HA1 mRNAWhat's more,we also carried out RIP test and the results showed that after tranfecting with miR-124-3p mimic,there were more P4HA1 mRNA pulled down by the Ago2 antibody,proving that miR-124-3p could enhance the binding of P4HA1 mRNA and Ago2 protein.Conclusions1.miR-124-3p reduces the stability of atherosclerotic plaque by reducing the content of smooth muscle cells and collagen in plaques;2.miR-124-3p is mainly distributed in the SMCs and affects type ? and type ?collagen synthesis by targeting P4HA1.BackgroundThe inflammatory theory is one of the most important mechanisms for the development of atherosclerosis.The inflammation takes a part in various stages of atherosclerosis.Traditional risk factors such as hyperlipidemia,hypertension,and diabetes can promote atherosclerosis through inflammation.During these processes,a variety of inflammatory cells and inflammatory factors are involved.IL-1? plays an important role in atherosclerosis.It could activate secondary inflammatory factors downstream such as IL-6 and TNF-a.Experiments have confirmed that in IL-1?knockout or IL-1R(IL-1 receptor)knockout ApoE-/-mouse,atherosclerotic plaque is significantly smaller than the control group.IL-1? antibodies have also been applied to clinical treatment.The famous CANTOS experiment showed that IL-1? antibody-Canakinumab can reduce the risk of cardiovascular disease.However,the specific mechanism of IL-1? on atherosclerosis remains to be further studied.In previous experiments,we found that miR-124-3p affects collagen synthesis by acting on collagen synthesis rate-limiting enzyme P4HA1 in smooth muscle cells.Inflammation is also closely related to collagen synthesis.Previous studies have confirmed that IL-1? can activate matrix metalloproteinases(MMPs),thereby accelerating collagen degradation and reducing the stability of atherosclerotic plaque.However,an experiment using Canakinumab in ApoE-/-mice with advanced arteriosclerotic plaques showed that the application of IL-1? antibody reduced collagen and smooth muscle content in late plaques.Therefore,the specific regulation mechanism of IL-1? on collagen metabolism remains to be further studied.miRNAs participate in a variety of pathophysiological processes and play important regulatory roles.In recent years,it has been found that IL-1? can affect the expression and activity of MMP and TIMP enzymes through various miRNAs in different cells.However,whether IL-1? can cause miR-124-3p changes and its effects on collagen synthesis has not been reported.Objectives1.To determine the expression of miR-124-3p in smooth muscle cells stimulated by IL-1??TNF-? or IL-6;2.To explore whether miR-124-3p is involved in the regulation of IL-1? on collagen metabolism in smooth muscle cells;3.To explore the specific mechanism of IL-1? regulating miR-124-3p expression.Methods1.Culture of human aortic smooth muscle cells(HASMCs)in vitroHASMCs were purchased from Sciencell Corporation of the United States,and they were cultured in smooth muscle cell medium(SMCM)which was also purchased from the same company.Cell resuscitation and passage were performed according to the protocol,and the cells were cultured in a cell incubator at 37 ?,5%CO2.6-10 generation cells were used for the experiment.2.HASMCs stimulation by inflammatory factors to observe miR-124-3p expression6-10 generation HASMCs were stimulated with TNF-? 100ng/ml for 0,1,3,6 hours;IL-6 100ng/ml for 0,1,3,6 hours;or IL-1? 10ng/ml for 0,1,3,6,12,and 24 hours.The effects of different inflammatory factors on miR-124-3p expression were observed at different time points.RNA was collected and the expression level of miR-124-3p was detected by RT-qPCR.After knowing the appropriate time for IL-1?stimulation to induce miR-124-3p expression,we selected 4 kinds of concentrations(0,2.5,5,10 ng/ml)of IL-1? to stimulate HASMCs.The total RNA of SMCs were also collected and went through RT-qPCR for the determination of miR-124-3p expression.The appropriate stimulation time and concentration were chosen for subsequent study.3.Cell transfection6-10 passage HASMCs were transfected with miR-124-3p mimic,miR-124-3p inhibitor or respective control using lipo2000,and stimulation was performed according to the experimental group,and cells were collected after 24-48 hours.6-10 passage HASMCs were transfected with c-Rel siRNA or c-Rel overexpression plasmid by lipo2000,and the corresponding stimulation was given according to the group,and the cells were collected after transfection for 24-48 hours.4.Reverse transcription quantitative PCR(RT-qPCR)After 24 hours of cell stimulation,the total RNA was extracted by TRIzol method.The reverse transcription kit was used to reverse the cDNA,and then qPCR was performed using SYBR Green fluorescence quantitative kit to detect the RNA content of miR-124-3p and c-Rel mRNA.5.Western Blot(WB)After the end of the cell stimulation,the total protein was extracted by RIPA.After SDS-PAGE,it was transferred to a PVDF membrane.After blocking,the primary antibody was incubated at 4 ? overnight,and the next day,the secondary antibody was incubated at room temperature for 1 h.Then the membrane was washed for chemiluminescence.The expression of collagen I,collagen III,P4HA1,c-Rel and other proteins in the cells was detected.6.ELISAAfter the cells were stimulated with IL-1?,the cell culture medium was collected,and the procollagen I ELISA detection kit was used to detect the content of type I procollagen in the cell culture medium.7.Dual luciferase assayThe firefly luciferase gene reporter plasmid(Promoter)containing the full length of the miR-124-3p promoter was constructed and transfected with the Renilla luciferase plasmid(pRL-TK)into HASMCs for 48 hours and stimulated with IL-1?for 6 hours before collection.The relative intensity of firefly luciferase was detected using a dual luciferase assay kit.In addition,in the Promoter and pRL-TK co-transfected HASMCs,c-Rel siRNA and its control or pEGFP-c-Rel and its control were also tranfected into the cells at the same time.After 48 hours the fluorescence intensity was detected using dual luciferase assay kit.8.Statistical analysisThe experimental data were presented as mean± SD.Statistical analysis and statistical plotting were performed using Graphpad prism 6.0.Unpaired t-test was used to compare between two samples.One-way ANOVA was used for comparison between multiple samples.P<0.05 was considered to be statistically significantResults1.IL-1? up-regulates the expression of miR-124-3p in HASMCsHASMCs were stimulated by different time gradients of TNF-a,IL-6 and IL-1?,and RT-qPCR was performed after RNA extraction.The results showed that the effects of TNF-a and IL-6 on miR-124-3p expression were not obvious.After stimulation with IL-1?,the expression of miR-124-3p in smooth muscle cells was significantly increased after 6h compared with Oh.HASMCs were stimulated with different concentrations of IL-1? for 6 hours.Compared with 0 ng/ml,IL-1?significantly upregulated the expression of miR-124-3p at 10 ng/ml.2.IL-1? reduces procollagen and collagen expression in smooth muscle cellsAfter stimulated by IL-1? for 24h,the cell culture medium was used to detect the type I procollagen content by ELISA.The results showed that compared with the NC group the type I procollagen content in IL-1?-stimulated group was significantly lower.Western blot was used to detect the content of collagen I and collagen III after IL-1? stimulation.The results showed that the type I and type III collagen in the IL-1?-stimulated group were significantly decreased.3.miR-124-3p is involved in IL-1?-induced collagen reduction in HASMCsHASMCs were transfected with miR-124-3p mimic or inhibitor for 24h,and then stimulated with IL-1? lOng/ml.The protein extracted for WB showed that miR-124-3p mimic further aggravated the downregulation effect of IL-1? for P4HA1,while miR-124-3p inhibitor can alleviate the downregulation of IL-1? on P4HA1.The tranfection of miR-124-3p inhibitor could also alleviate the downregulation of IL-1?on type I and type III collagen.4.IL-1? promotes the expression of miR-124-3p by upregulating c-RelWestern blot was used to detect the expression of c-Rel protein in HASMCs stimulated by IL-1? at different time points.The results showed that the expression of c-Rel increased significantly after 1 h of stimulation,and the expression of c-Rel peaked after 3 h,then decreased gradually.c-Rel siRNA was transfected into HASMCs with lipo2000,and the expression of miR-124-3p was detected by RT-qPCR.The results showed that cells transfected with c-Rel siRNA,compared to cells transfected with control siRNA,the expression of miR-124-3p were decreased.The expression of P4HA1,type I and type III collagen were also increased in c-Rel siRNA group compared with the control group.The c-Rel overexpression plasmid was transfected into HASMCs with lipo2000 and the expression of miR-124-3p was increased.The expression of P4HA1 and type I and type III collagen were also decreased in the c-Rel overexpression plasmid group.5.miR-124-3p gene is the target gene of c-RelFull-length of the miR-124-3p promoter was cloned into the luciferase reporter vector pGL3-basic to generate pGL3-miR-124-3p-promoter vector(referred to as"Promoter" in the text below).Promoter and pRL-TK vectors were transfected into HASMCs together.Then the cells were stimulated by IL-1?.Dual luciferase assay showed that IL-1? significantly increased the miR-124-3p promoter activity in HASMCs compared with that in the control groupBesides transfection of Promoter and pRL-TK plasmids,HASMCs were tranfected with c-Rel siRNA or control siRNA at the same time.Dual luciferase assay showed that c-Rel siRNA could decrease miR-124-3p promoter activity.pEGFP-c-Rel plasmids or control pEGFP plasmids were transfected into HASMCs along with Promoter and pRL-TK vectors.Results showed that c-Rel overexpression could upregulate miR-124-3p promoter activity.Conclusion1.IL-1? upregulates the expression of miR-124-3p in smooth muscle cells;2.miR-124-3p is involved in the regulation of IL-1? on collagen expression in smooth muscle cells;3.IL-1? enhances the transcriptional activity of miR-124-3p by the transcription factor c-Rel.