| IntrductionIntervertebral disc degeneration(IDD)is the pathological basis of various spinal degenerative diseases,which can induce disc herniation,spinal canal stenosis,spinal stability decline et al.To date,the detailed pathogenesis of IDD has not been elucidated.It is reported that comprehensive factors induce IDD,such as heredity,gene polymorphism,and dysfunction of nucleus pulposus cells,activation of inflammatory factors and degradation of extracellular matrix.It should be noted that the intervertebral disc tissue is based on the nucleus pulposus cells,and endogenous or exogenous factors affect the signal pathway and physiological function of the nucleus pulposus cells.Then the degenerated nucleus pulposus cells are involved in the inflammatory reaction,matrix degradation and apoptosis of the intervertebral disc.Therefore,the study of the molecular mechanism of nucleus pulposus cell degeneration will help to clarify the pathological mechanism of IDD.More and more studies have confirmed that nucleus pulposus cells of intervertebral disc can be regulated by microRNAs(miRNAs).MiRNAs play an important role in many physiological and pathological processes of the body,which are involved in the transcriptional process of target mRNA.At present,the mechanism of miRNAs,such as miR-155,miR-193a-3p and miR-223,has been widely reported in intervertebral disc degeneration.Ji ML et al found that miR-486-5p also showed obvious multiple changes after Solexa sequencing of IDD tissues.However,the specific mechanism has not been studied.In addition,our preliminary target prediction revealed that the 3’-UTR of FOXO1 is a potential target of miR-486-5p.FOX01,as an important transcription factor,is mainly located in the nucleus through its post-translational modification.These pathways,including phosphorylation,acetylation and ubiquitin,play an important role in many physiological processes,such as regulating cell cycle and glucose and lipid metabolism,inducing cell apoptosis,and participating in the process of resisting oxidative stress.Recent studies have also reported that the expression of FOXO1 is up-regulated in IDD,but the specific mechanism is not clear.In this study,we first determined the expression of miR-486-5p in IDD samples by qRT-PCR.The role of miR-486-5p in the inflammatory response of nucleus pulposus cells induced by lipopolysaccharide(LPS)in vitro and the degeneration of nucleus pulposus induced by annulus fibrosus(AF)puncture in vivo were systematically verified.By studying the mechanism of miR-486-5p in IDD,we can deepen our understanding of the pathophysiological mechanism of IDD and help us understand the relationship between intervertebral disc degeneration and miRNA pathway.It provides further insight that miR-486-5p can be used as a new diagnostic marker or therapeutic target for IDD patientsin the future.Part oneExpression and significance of miR-486-5p and FOXO1 in Human Nucleus pulposus and LPS-induced Nucleus pulposus cellsPurpose1.To detect the differential expression of miR-486-5p,FOXO1 mRNA and its protein in healthy and IDD nucleus pulposus tissues,and explore the relationship between the expression of miR-486-5p,FOXO1 mRNA and its protein and the pathological grade of IDD.2.To investigate the isolation and culture of human intervertebral disc nucleus pulposus cells in vitro,and investigate the effects of LPS on apoptosis,inflammatory reaction,matrix synthesis and expression of stromal enzymes in human intervertebral disc nucleus pulposus cells,and construct LPS-induced degeneration model of nucleus pulposus cells.3.To investigate the expression of miR-486-5p,FOXO1 mRNA and its protein in human intervertebral disc nucleus pulposus cells by LPS.Methods1.30 samples of nucleus pulposus from IDD patients and 5 samples from spontaneous scoliosis death patients were collected,respectively.The expression of miR-486-5p,FOXO1 mRNA in the two groups was measured by real-time quantitative PCR.The protein expression of two groups of FOXOl was identified by Western blot.2.The collected nucleus pulposus was isolated in aseptic condition,then trimmed into fragments.The nucleus pulposus cells were digested by trypsin and typeⅡ collagenase,and filtered,centrifuged.Then discarded the supernatant,and cultured the cells.The culture medium was changed twice per week,and the adherent and growth of the cells were observed by inverted microscope during culture.3.Nucleus pulposus cells were stimulated with different concentrations of LPS(0,10,100,200,500 ng/ml)at different time(0,12,24,48 h).The optimal concentration of LPS and the optimal treatment time were determined by CCK-8 at different time(0,12,24,48 h).4.The nucleus pulposus cells were treated with 100 ng/ml LPS for 24 h according to the results of concentration and time dependent experiment.Flow cytometry was used to detect apoptosis,qRT-PCR and Western blot were used to detect the expression of miR-486-5p,FOXO1 mRNA,FOXO1 protein and apoptotic protein,and the expression of related inflammatory factors,matrix degrading enzymes,collagen Ⅱ and aggrecan mRNA and protein were analyzed.Results1.In the degenerated nucleus pulposus samples,the expression of miR-486-5p was down-regulated,and the expression of FOXO1 was up-regulatedThe results of qRT-PCR and Western blot showed that the expression of miR-486-5p in IDD was down-regulated compared with that in 5 healthy Nucleus pulposus tissues.The expression of FOXO1 mRNA and protein were up-regulated significantly(P<0.05).Compared with the nucleus pulposus with mild degeneration,the expression of miR-486-5p was down-regulated in the nucleus pulposus with severe degeneration,while the FOXO1 expression of mRNA and protein was up-regulated(P<0.05).In addition,the expression of miR-486-5p was negatively correlated with Pfirrmann grade(P=0.019),while the expression level of FOXOl mRNA and protein was positively correlated with Pfirrmann grade(P=0.021,P=0.024).2.LPS inhibited the expression of miR-486-5p in nucleus pulposus cells and induced the FOXO1 expressionThe CCK-8 results showed that the proliferation ability of nucleus pulposus cells decreased gradually with the increase of LPS concentration in a time-and dose-dependent manner.When the half inhibitory concentration of LPS was 100 ng/ml,for 24 h,the difference of proliferation ability of the cells was the most significant.The results of qRT-PCR and Western blot showed that compared with the control,the proliferation of nucleus pulposus cells was higher than that of nucleus pulposus.The expression of miR-486-5p was down-regulated in LPS-induced nucleus pulposus cells,while the expression of FOXOl mRNA and protein was up-regulated significantly(P<0.05).3.LPS induced the expression of inflammatory factors and matrix degrading enzymes in nucleus pulposus cellsThe results of qRT-PCR and western blot showed that compared with the control group,LPS significantly upregulated the inflammatory cytokines(IL-1β,IL-6 and TNF-a)mRNA and protein expression in the nucleus pulposus cells.Meanwhile,the expression of Matrix degrading enzyme(MMP-3,MMP-13,ADAMTS-4 and ADAMTS-5)mRNA and protein was also up-regulated by LPS(P<0.01).However,the mRNA and protein expressions of extracellular matrix components,collagen Ⅱand aggrecan were down-regulated by LPS stimulation(P<0.01).4.LPS induced apoptosis of nucleus pulposus cellsThe results of western blot showed that compared with the control group,LPS-stimulated apoptosis-related proteins Caspase-3 and Bax were up-regulated,but anti-apoptotic protein Bcl-2 was significantly down-regulated in LPS-stimulated nucleus pulposus cells.Flow cytometry showed that LPS significantly increased the apoptosis rate of nucleus pulposus cells,which was about twice as high as that of the control group(P<0 01).Conclusion1.With the aggravation of degeneration of nucleus pulposus,the expression of miR-486-5p was down-regulated,on the contrary,the expression of FOXO1 was up-regulated.2.LPS induced the degradation of nucleus pulposus cells,including inhibition of proliferation,apoptosis,decrease of extracellular matrix synthesis,and increase of inflammatory factors and matrix degrading enzymes.Part twoRegulation of miR-486-5p on nucleus pulposus cells in vitroPurpose1.By up-regulating or down-regulating the expression of miR-486-5p in nucleus pulposus cells,the effects of miR-486-5p on apoptosis,expression of inflammatory factors and matrix-degrading enzymes,synthesis of extracellular matrix in LPS-induced nucleus pulposus cells were determined.2.To explore the relationship between miR-486-5p and FOXO1,and to clarify the synergistic or antagonistic effects of miR-486-5p and FOXO1 in LPS-induced nucleus pulposus cells.Methods1.LipofetaminTM 2000 method was used to transfect agomir-486-5p or antagomir-486-5p into nucleus pulposus cells,and the expression of miR-486-5p was detected by qRT-PCR.2.The effect of miR-486-5p expression on proliferation of nucleus pulposus cells was detected by CCK-8 assay,and apoptosis was detected by AnnexinV/PI method.3.FOXO1 mRNA and protein were detected by qRT-PCR and Western blot,and the mRNA and protein expression of related inflammatory factors,matrix degrading enzymes,collagen Ⅱ and aggrecan were analyzed at the same time.4.Bioinformatics,HEK-293T cells,virus transfection in vitro and double luciferase reporter gene test were used to detect the target of miR-486-5p and FOXO1 mRNA.5.To upregulate the expression of FOXO1 in nucleus pulposus cells,co-transfect agomir-486-5p or antagomir-486-5p,and detect the inflammatory factors,apoptosis,matrix synthesis and the expression of stroma enzyme in nucleus pulposus cells in vitro.Results1.MiR-486-5p enhanced the activity of nucleus pulposus cells and inhibited LPS-induced inflammatory cytokineThe results of qRT-PCR showed that agomir-486-5p could increase the expression of miR-486-5p in nucleus pulposus cells exogenous(about 3.1 times).Antagomir-486-5p significantly down-regulated the expression of miR-486-5p(about 60%).Compared with the control group,the overexpression of miR-486-5p significantly inhibited the expression of IL-1β,IL-6 and TNF-α mRNA.and protein in the nucleus pulposus cells induced by LPS(P<0.001).However,antagomir-486-5p significantly enhanced the mRNA and protein expression of IL-1β,IL-6 and TNF-a induced by LPS in nucleus pulposus cells(P<0 05).2.MiR-486-5p promoted matrix formation of nucleus pulposus cells inhibited by LPS,and inhibited the expression of LPS-induced matrix degrading enzymesThe nucleus pulposus cells transfected with agomir-486-5p,atagomir-486-5p or their respective control sequences.The results of qRT-PCR and Western blot showed that the levels of collagen Ⅱ and aggrecan in agomir-486-5p group were significantly higher than those in agomir-486-5p control group,while the collagen Ⅱ and aggrecan levels in antagomir-486-5p group were significantly lower than those in antagomir-486-5p control group(P<0.001).In addition,the overexpression of miR-486-5p inhibited the up-regulated expression of LPS-induced matrix-degrading enzymes in nucleus pulposus cells(P<0.001),and the inhibition of miR-486-5p significantly promoted the expression of matrix-degrading enzymes in nucleus pulposus cells induced by LPS.The expression level of matrix degrading enzyme was significantly higher than that of LPS alone(P<0.001).3.MiR-486-5p inhibited LPS-induced apoptosisThe results showed that agomir-486-5p significantly decreased the expression of Bax,Caspase-3 mRNA and protein,while agomir-486-5p significantly increased the expression of Bcl-2(P<0.001).The results showed that agomir-486-5p significantly decreased the expression of Bax and Caspase-3 mRNA and protein in LPS-induced apoptosis cells treated with miR-486-5p(P<0.05).On the contrary,antagomir-486-5p significantly increased the expression of Bax,Caspase-3 mRNA and protein,and decreased the expression of Bcl-2.The results of flow cytometry showed that the apoptosis rate of nucleus pulposus cells transfected with agomir-486-5p was significantly lower than that of agomir-486-5p control group(P<0.01).However,compared with the antagomir-486-5p control group,antagomir-486-5p significantly increased the apoptosis rate of the nucleus pulposus cells(P<0.01).4.FOXO1 is a direct target of miR-486-5p.Bioinformatics results show that 3’-UTR of FOXO1 has complementary sequence structure with miR-486-5p.After transfection of wild-type(WT)or luciferase reporter vector with mutated(MUT)FOXO1 3’-UTR sequence into HEK-293T cells,the fluorescence intensity of HEK-293T cells transfected with wild-type vector was significantly increased(P<0.05).However,the fluorescence intensity of HEK-293T cells transfected with mutant vector had no significant change(P>0 05).In the HEK-293T cells with high expression of miR-486-5p,the mRNA and protein levels of FOXO1 were significantly lower than those of the blank carrier group after transfection of the full-length FOXO1 expression sequence(P<0.05).5.MiR-486-5p plays a role in nucleus pulposus cells by targeting FOXO1After co-transfection of miR-486-5p-overexpressing nucleus pulposus cells with pcDNA-FOXO1 or blank vector via targeting FOXO1,the overexpression of FOXO1 effectively interferes with the enhancement of proliferation of Nucleus pulposus cells by miR-486-5p.At the same time,the overexpression of FOXO1 antagonized the protective effect induced by miR-486-5p overexpression,but induced the expression of inflammatory factors,mRNA and protein of matrix degrading enzymes,and inhibited the expression of aggrecan and collagen II(P<0.001).In addition,the expression of Bax and Caspase-3 in the nucleus pulposus cells overexpressed by FOXO1 and miR-486-5p was about 2 times higher than that of the nucleus pulposus cells overexpressed by miR-486-5p alone(P<0.001).The expression of Bcl-2 decreased by 51.1%(P<0.001).Annexin/PI showed that the overexpression of FOXO1 enhanced the apoptosis induced by LPS and partially affected the inhibitory effect of miR-486-5p on apoptosis(P<0.001).Conclusion1.MiR-486-5p can inhibit LPS-induced inflammation,matrix degradation and apoptosis of nucleus pulposus cells.2.FOXO1 3’-UTR is a direct target of miR-486-5p.3.MiR-486-5p inhibited the expression of FOXO1 and was involved in protecting the normal biological function of nucleus pulposus cells.Part threeRegulation of miR-486-5p in nucleus pulposus cells in vivoPurposeBased on experiment 1 and 2,it has been confirmed that miR-486-5p gene can directly act on the mRNA 3’-UTR target gene of FOXO1,and was verified by in-vitro studies.It should be noted that the form of cultured cells in vitro is usually simple,which can better control the culture conditions,and the cells are rarely affected by external factors.Therefore,the in vitro study is not comparable to the living disc tissue growth environment.In this study,the intervertebral disc degeneration model of SD rats was established by annulus fibrosus puncture,and the effects of miR-486-5p on the inflammatory response,apoptosis and matrix synthesis of the nucleus pulposus cells were investigated.Methods1.Of 30 SD rats,aged 4 weeks,weighing(100± 20)g,10 cases of SD rates were randomly divided into control group and operation group with 5 rats in each group.In the operation group,L3,4,L4,5,L;,6 vertebrae were exposed through the ventral posterolateral approach.The 21G puncture needle was selected from the anterior-lateral parallel cartilage endplate of the annulus fibrosus and the sagittal plane of the spinal column in the direction of about 45°.The whole-layer acupuncture was performed.The needle depth was 2.3 mm.The sham operation group was only sutured by incision and suture of the ventral posterolateral approach2.In order to up-regulate or down-regulate the expression of miR-486-5p in intervertebral discs,the intervertebral discs were transfected with agomir-486-5p,antagomir-486-5p or their control sequences by injection in 4 groups with 5 rats in each group on the basis of the operation group3.The rats were killed at 8 weeks after operation,the intervertebral discs containing the upper and lower vertebrae were removed completely,and the nucleus pulposus tissue was extracted.Real-time quantitative PCR technique was used to determine the expression level of miR-486-5p and the mRNA expression of each factor.Western blot was used to determine the protein expression level of each factor after transfectionResults1.Inhibitory effect of MiR-486-5p on inflammatory response and matrix degradation in rat IDD modelThe results of gene and protein analysis showed that the expression of miR-486-5p decreased,but the expression of FOXO1 increased in puncture group(P<0.001).Compared with puncture group,the expression of miR-486-5p in agomir-486-5p-transfected group was significantly higher than that in antagomir-486-5p-transfected group,while the expression of miR-486-5p in antagomir-486-5p-transfected group was significantly down-regulated(P<0.001).In inflammatory aspect,compared with puncture group,the expression of inflammatory cytokines was down-regulated in agomir-486-5p group,but up-regulated in antagomir-486-5p group.In matrix synthesis,agomir-486-5p restored the down-regulated expression of aggrecan and collagen Ⅱ in the puncture group,and inhibited the expression of matrix degrading enzymes in the puncture group;However,the antagomir-486-5p group showed the opposite result.2.MiR-486-5p inhibited the apoptosis in rat IDD modelThe western blot results showed that annulus fibrosus puncture induced apoptosis in the nucleus pulposus tissue,and the cell apoptosis in the nucleus pulposus was induced by fibrillary annulus puncture.The protein expression of Caspase-3 and bax was up-regulated and the protein expression of bcl-2 was down-regulated.The overexpression of MiR-486-5p partially inhibited the protein expression of Caspase-3 and bax induced by annulus fibrosus puncture,and promoted the protein expression of bcl-2.In contrast,the low expression of miR-486-5p partially enhanced the expression of Caspase-3 and bax induced by annulus fibrosus puncture,and inhibited the expression of bcl-2.ConclusionMiR-486-5p may protect and maintain the normal biological function of the intervertebral disc tissue in vivo,while the down-regulation of miR-486-5p aggravates the degeneration of the rat intervertebral disc. |
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