| Inflammatory demyelinating diseases(IDDs)of the central nervous system(CNS)are a heterogeneous group of diseases characterized by myelin loss and inflammation-related axonal damage,of which multiple sclerosis(MS)and neuromyelitis optica spectrum disorders(NMOSD)are the most common types.MS and NMOSD have a high prevalence in the young and middle-aged population and involve mainly the spinal cord,optic nerve and brain,with patients presenting with motor and visual impairment,sensory abnormalities and psychiatric abnormalities.The exact etiology and patho-genesis of IDDs are unknown,and a combination of genetic and environ-mental factors contribute to the autoimmune response.Immune-mediated damage in the CNS of demyelinating patients is caused by a complex interaction of multiple immune cells,of which T lymphocytes and B lymphocytes are key mediators,mediating humoral and cellular immune responses,respectively.CD4~+T cells play a crucial role in the pathogenesis of MS.Naive CD4~+T cells can differentiate into regulatory T cells(Treg)with immunosuppressive effects and helper T cells(Th)that promote inflammation,such as Th1 and Th17 cells.Th17 cells secrete a variety of cytokines such as GM-CSF and IL-17 and are one of the major cell types that contribute to the pathogenesis of MS.In contrast,Treg suppresses autoimmune responses by mediating immune tolerance to autoantigens.In MS,the balance between Th17 and Treg cells is disrupted.Although NMOSD is an autoimmune disease with a predominantly humoral immunity,there is much evidence that auto-reactive T cells that specifically recognize aquaporin4 antibody(AQP4)are present in NMOSD and that the Th17/Treg ratio is imbalanced.Th17,Treg and their associated cytokines also play an important role in the pathogenesis of NMOSD.Autophagy is an intracellular degradation pathway that maintains intrace-llular homeostasis by eliminating defective or redundant proteins,complexes and organelles.Numerous studies have shown that autophagy plays a key role in the pathogenesis of experimental autoimmune encephalomyelitis(EAE),an animal model of MS and CNS demyelination.Autophagy maintains CNS homeostasis by degrading damaged organelles and abnormal proteins,and participates in the inflammatory response by regulating the activation of immune cells and the secretion of inflammatory factors.It has been shown that regulation of autophagy can affect Th17/Treg homeostasis,and activation of autophagy with autophagy inducers such as rapamycin can reduce neurological deficits and inflammatory responses in EAE mice by increasing the number of Treg cells.Micro RNAs(miRNAs)are non-coding RNA of 21-25 nucleotides in length those can interact with their target messenger RNA(m RNA)match sites to induce m RNA degradation and repress translation,and regulate gene expression at the post-transcriptional level,and are important molecules involved in the regulation of cellular functions.miRNAs are involved as epigenetic factors in the development of CNS autoimmune diseases.A large number of aberrantly expressed miRNA have been identified in the immune cells and CNS of MS patients,and dysregulated miRNA expression is closely associated with CNS lesions.miR-133 is a miRNA family containing two isoforms,miR-133a and miR-133b,in the human genome.There are two subtypes miR-133a,miR-133a-1 and miR-133a-2,both of which are transcribed and processed to form the same mature sequence.miR-133a is a muscle-specific miRNA that also plays a critical role in the inflammatory response.Recent studies have shown that miR-133a can inhibit autophagy and alter the expression of autophagy-related proteins such as Beclin1,LC3B and P62 in a variety of tumors.And whether miR-133a is involved in the pathogenesis of demyelinating diseases is unclear.Studies exploring the expression,regulation and function of miR-133a in patients with CNS demyelination will provide a basis for the future development of specific miRNAs as diagnostic markers and therapeutic targets.miRNAs have not protein-coding functions and need to function through transcriptional regulation of target genes.Previous studies have reported the targeting of miR-133a with silent information regulation 1(SIRT1)and suppressors of cytokine signaling 2(SOCS2),while SIRT1 and SOCS2 are both associated with inflammatory signaling pathways and autophagy levels.It was demonstrated that miR-133a promotes inflammatory responses in sepsis by inhibiting SIRT1 expression,and silencing SIRT1 reverses the anti-inflammatory effects of miR-133a inhibitors,and that dual luciferase assays confirmed the targeting relationship between miR-133a and SIRT1.The targeting relationship between miR-133a and SIRT1 was also confirmed in osteoarthritis models and hyperlipidemia models.miR-133a interaction with SOCS2 was validated in myocardial injury.and studies showed that miR-133a in myocardial reperfusion injury inhibited autophagy by downregulating SOCS2.Based on the above literature,we speculate that miR-133a may be involved in regulating autophagy and inflammation levels through negative regulation of SOCS2 and/or SIRT1 in demyelinating diseases.In this study,we first explored the expression of miR-133a and related genes in CNS demyelinating patients and EAE mice.miR-133a was confirmed to be upregulated in PBMC of NMOSD and MS patients and in spleen and lymph-nodes of EAE mice,and miR-133a was suppressed or overexpressed in vivo experiments and vitro CD4~+T cell culture in EAE mice.Exploring whether miR-133a and its target gene play a role in the progression of EAE by regulating autophagy and inflammation will provide an experimental basis for the future development of specific miRNA as diagnostic markers and therapeutic targets.Part One Expression and biological function of miR-133a in PBMC from patients with IDDs of the central nervous systemObjective:This study aimed to explore the expression of miR-133a and its potential target genes in peripheral blood mononuclear cells(PBMC)from NMOSD and MS patients and its clinical relevance,and to investigate the potential mechanism of miR-133a action.Methods:1.Study Subjects:NMOSD and MS patients from the Department of Neurology,Second Hospital of Hebei Medical University and age-and sex-matched healthy controls were enrolled,with peripheral blood drawn and clinical information recorded.2.Extraction of PBMC and total RNA:Peripheral blood was collected from all participants after an overnight fast,and mononuclear cells were isolated using Ficoll solution.Samples were then dissolved in TRIzol and rapid frozen in liquid nitrogen for future use.Total RNA was extracted from the samples by the chloroform,isopropyl and ethanol methods.3.RT-q PCR was used to detect the expression of miR-133a-1,miR-133a-2,miR-30d-5p,miR-204-5p and miR-29b-3p in PBMC from NMOSD and MS patients and controls,as well as the levels of SOCS2 and SIRT1m RNA,potential target genes of miR-133a.4.Analysis of the correlation between miR-133a-1,miR-133a-2,SOCS2m RNA,SIRT1 m RNA and clinical characteristics(EDSS scores and inflammation levels).5.Analysis of transcript levels of SOCS2,SIRT1 and autophagy-related genes in ce RNA microarrays.Results:1.miR-133a-1 and miR-133a-2 levels were significantly higher in PBMC from patients with NMOSD in the acute phase compared with controls(miR-133a-1,P=0.005;miR-133a-2,P=0.001).miR-133a-1 and miR-133a-2 expression levels were positively correlated(r=0.553,P=0.005).The expression of miR-133a-1 in MS was higher than that in controls,but not statistically significant(P=0.126),and miR-133a-2 was significantly higher than that in controls(P=0.03).miR-133a-1 expression had a positive trend with miR-133a-2(r=0.691,P=0.196).2.No significant association was observed between miR-133a-1,miR-133a-2 levels and EDSS scores in acute-phase NMOSD patients.miR-133a-1 showed a positive trend with NLR(r=0.319,P=0.129).miR-133a-2was positively correlated with serum IL-2(r=0.493,P=0.038)and had a positive trend with TNF-α(r=0.437,P=0.070).3.SOCS2 m RNA level in the acute NMOSD group was lower that in control group(P=0.003).SOCS2 m RNA level was significantly positively cor-related with lymphocyte ratios(r=0.365,P=0.029)and negatively corre-lated with neutrophil ratios(r=-0.409,P=0.013)and NLR(r=-0.374,P=0.025).4.The level of SIRT1 m RNA in the acute NMOSD group was lower than that in control group(P<0.0001).SIRT1 m RNA level was significantly positively correlated with lymphocyte ratio(r=0.5137,P=0.0006),negatively correlated with neutrophil ratio(r=-0.4785,P=0.0016)and NLR(r=-0.4988,P=0.0006).5.Ce RNA chip analysis showed SOCS2 expression was significantly downregulated in NMOSD compared to the control group(P=0.031)and SIRT1 expression was significantly downregulated(P<0.001).Autophagy-related gene Beclin1 was significantly upregulated(P=0.005),LC3B was downregulated(P=0.006),and P62 was significantly upregulated(P=0.04)in NMOSD.Conclusions:In this study,we investigated the expression of miR-133a in PBMC from NMOSD and MS patients and its correlation with clinical indicators.miR-133a-1 and miR-133a-2 were significantly upregulated in the acute NMOSD group and MS group,which correlated with inflammatory indexes.SOCS2 and SIRT1 m RNA levels were decreased and autophagy was abnormal in PBMC from the acute NMOSD patients.miR-133a might affect autophagy and participate in the pathogenesis of NMOSD and MS by targeting SOCS2 and/or SIRT1.Part Two Inhibition of miR-133a plays a protective role in EAE by regulating autophagyObjective:EAE will be intervened with recombinant lentiviral plasmids to overexpress or inhibit miR-133a-3p.This study aimed to explore the expression of miR-133a-3p in EAE and to investigate the potential mechanism of miR-133a-3p.In addition,we explored the protective effect of anti-miR-133a-3p by intervention with 3-methyladenine(3-MA)blocking autophagy.Methods:1.Experiment 1 Increased miR-133a expression in experimental autoim-mune encephalomyelitis1)Experimental animals and EAE model establishment:Female C57BL/6 mice(8-10 weeks of age,18-20g)were used.MOG35-55 peptide was diluted to 10mg/ml with physiological saline,and an equal volume of com-plete Freund’s adjuvant(CFA)and a certain amount of Mycobacterium tuber-culosis H37Ra(its final concentration was 4mg/ml)were added.0.1ml of the fully emulsified mixture was injected subcutaneously into the mice at any four points on either side of the back spine.Subsequently,pertussis toxin(500 ng)was given through intraperitoneal injection,and again the same amount was injected after 48 h.2)EAE mice were evaluate daily using Jager clinical score after immunization(Jager method).The tissues were removed at the peak of the disease,rapid frozen in liquid nitrogen and stored in-80℃refrigerator.3)RNA was extracted and the expression levels of miR-133a-3p,as well as SOCS2,SIRT1,Beclin1,LC3B and P62 m RNA were detected by RT-q PCR.2.Experiment 2 miR-133a involved in the pathogenesis of experimental autoimmune encephalomyelitis by influencing autophagy1)Experimental animals and EAE model establishment(same as Experiment 1)2)Animal grouping and interventions:Lentiviral vectors were used to infect EAE mice:mmu-miR-133a-3p mimics group(LV-miR-133a),mmu-miR-133a-3p inhibitor group(LV-anti-miR-133a),and empty(untransformed)lentiviral vector(LV-Con)being the endogenous reference.The mice were infected with lentiviral vector carrying above plasmids one week before EAE model establishment.Evaluate the EAE mice using Jager clinical score daily.3)The tissues were removed at the peak of the disease.The lumbar enlargement of spinal cord was embedded with paraffin.Following that,HE and LFB staining was performed to measure demyelination and infiltration of inflam-matory cells separately.4)RNA was extracted and RT-q PCR was used to detect the expression of miR-133a-3p,SOCS2,SIRT1,FOXP3,IFN-γ,RORC and IL-17 m RNA in EAE spleen tissue.5)Protein were extracted and the expression of SOCS2,SIRT1,Beclin1,LC3II/I and P62 proteins in spleen tissues were detected by Western blot.3.Experiment 3 Autophagy inhibitor 3-MA partially offset the protective effect of miR-133a inhibitors on EAE1)Experimental animals and EAE model establishment(same as Experi-ment 1)2)EAE was intervened with lentiviral vectors and 3-MA in the following groups:EAE group,mmu-miR-133a-3p inhibitor group(LV-anti-miR-133a)and mmu-miR-133a-3p inhibitor combined with 3-MA group(LV-anti-miR-133a+3-MA).The mice in the LV-anti-miR-133a and LV-anti-miR-133a+3-MA groups were infected with lentiviral vector carrying above plas-mids one week before EAE model establishment.The mice in the LV-anti-miR-133a+3-MA group were given intraperitoneal injection of 3-MA(10mg/kg)every day,and the mice in the other two groups were given intra-peritoneal injection of an equal volume of saline after immunization.The neurological function of EAE mice were evaluated using Jager clinical score.3)Ficoll was used to isolated the lymphocytes from spleen tissue of the mice at the peak of disease.The expression of Beclin1,LC3II/I,P62 and FOXP3 proteins were detected by Western blot.Results:1.Experiment 1 Increased miR-133a expression in experimental autoim-mune encephalomyelitis1)The EAE mice began to develop the symptom on the 12th day after MOG immunization,and the mean of neurological function deficit score was2.438±1.084 on the 19th day,with a morbidity rate of 100%.2)The expression of miR-133a-3p was significantly upregulated in the spleen and lymphonodus of EAE mice compared to the controls(Spleen:P=0.006;Lymphonodus:P=0.002).miR-133a-3p expression in the brain and spinal cord of the EAE group was not significantly different from that of control group(Brain:P=0.818;Spinal cord:P=0.135).3)The expression of SOCS2 m RNA in EAE mice was lower than that in controls,but not statistically significant(P=0.183),and SIRT1 m RNA was significantly downregulated(P=0.038).Beclin1 m RNA was significantly higher in EAE group than that in control group(P=0.042),and LC3B m RNA was not significantly different from that in control group(P=0.691),and P62m RNA was significantly higher than that in control group(P=0.029).2.Experiment 2 miR-133a involved in the pathogenesis of experimental autoimmune encephalomyelitis by influencing autophagy1)The expression of miR-133a-3p was significantly higher in the LV-miR-133a group than that in LV-Con group(P=0.010),and miR-133a-3p was significantly lower in the LV-anti-miR-133a group(P=0.038).2)Overexpression of miR-133a-3p advanced the onset time of EAE mice,increased neurological function score,delayed the remission of neurological symptoms,and aggravated the severity of inflammation and demyelination damage.In contrast,inhibition of miR-133a-3p had a protective effect on EAE,reduced the score of clinical signs and suppressed the inflammation and demyelination damage.3)Compared with the LV-Con group,the expression of SOCS2 m RNA and protein was statistically decreased in LV-miR-133a group(P=0.010;P=0.011),and the expression of SOCS2 m RNA and protein was significantly increased in LV-anti-miR-133a group(P=0.002;P=0.031).The targeting relationship between miR-133a-3p and SOCS2 was verified by dual luciferase assay.SIRT1 m RNA and protein levels were not significantly different between the three groups.4)The expression of Beclin1 protein was significantly lower in LV-miR-133a group than that in LV-Con group(P=0.005),LC3II/I protein was lower than that LV-Con group but not statistically significant(P=0.053),and P62protein was higher(P<0.001).Inhibition of miR-133a-3p increased the expression of Beclin1 protein(P=0.005)and decreased the expression of P62protein(P<0.001).5)Compared with the LV-Con group,the expression of FOXP3 m RNA was significantly decreased in LV-miR-133a group(P=0.026),and FOXP3m RNA was significantly increased in LV-anti-miR-133a group(P=0.027).3.Experiment 3 Autophagy inhibitor 3-MA partially offset the protective effect of miR-133a inhibitors on EAE1)Inhibition of miR-133a-3p significantly alleviated clinical severity of EAE mice,but the protective effect of anti-miR-133a-3p was partially offset by the autophagy inhibitor 3-MA.The neurological function score was higher in the anti-miR-133a+3-MA group than that in the anti-miR-133a group(P=0.013).2)LC3II/I protein was increased in the anti-miR-133a-3p group com-pared to EAE group(P=0.026),and P62 protein was decreased(P=0.022).Compared with the anti-miR-133a group,LC3II/I protein was decreased in the anti-miR-133a+3-MA group(P=0.003),and the expression of P62 protein was increased but not statistically significant(P=0.539).3)The expression of FOXP3 protein was significantly higher in anti-miR-133a-3p group than that in EAE group(P=0.043).FOXP3 protein was lower in anti-miR-133a+3-MA group than that in anti-miR-133a-3p group but not statistically significant(P=0.120).Conclusions:In this study,we found that the expression of miR-133a-3p was significantly upregulated in the spleen and lymphonodus of EAE mice.miR-133a might be involved in the pathogenesis of EAE by targeting SOCS2,and inhibition of miR-133a-3p possessed a protective effect on EAE.Autophagy might be involved in the protective effect of miR-133a-3p inhibitors on EAE,and the autophagy inhibitor 3-MA could partially offset the protective effect of miR-133a-3p inhibitors.Part Three Mechanism of miR-133a in CD4+cells of EAEObjective:After the establishment of EAE model,isolate CD4~+cells from spleen tissue of the EAE mice at the peak of disease,and explore the mechanism of miR-133a-3p in CD4~+cells.Methods:1.Experimental animals and EAE model establishment(same as Part Two,Experiment 1)2.Cell transfection:The splenocytes were isolated at the peak of disease,and the mouse CD4~+T cell Isolation Kit was utilized to isolate naive CD4~+T cells from EAE mouse spleens.CD4~+T cells were stimulated with MOG35-55peptide,anti-CD3 and anti-CD28 antibodies.Thereafter,CD4~+T cells were respectively transfected with miR-133a-3p mimics,mimics control(mimics NC),miR-133a-3p inhibitors and inhibitor control(inhibitor NC)for 72h.3.RT-q PCR was used to detect the expression of miR-133a-3p,SOCS2and FOXP3 m RNA.5.Western blot was used to detect the expression of SOCS2,Beclin1,LC3II/I and P62 protein.6.The production levels of IFN-γ,IL-17,IL-10,IL-6 were measured by ELISA kits.Results:1.The expression of miR-133a-3p was significantly increased in the miR-133a mimics group compared to the mimics NC group(P=0.029),and miR-133a-3p was significantly decreased in the miR-133a inhibitor group compared to the inhibitor NC group(P=0.030).2.Overexpression of miR-133a-3p in CD4~+cell suppressed SOCS2m RNA(P=0.087)and protein expression(P=0.036).Inhibition of miR-133a-3p upregulated SOCS2 m RNA(P=0.007)and SOCS2 protein expres-sion(P=0.009).3.Overexpression of miR-133a-3p in CD4~+cell suppressed Beclin1protein(P=0.236)and LC3II/I protein expression(P=0.002),but increased P62 protein expression(P=0.007).Compared with the inhibitor NC group,Beclin1 protein was increased in the miR-133a inhibitor group but not statistically significant(P=0.400),LC3 II/I protein was increased(P=0.013),and P62 protein was decreased(P=0.025).4.Overexpression of miR-133a-3p decreased FOXP3 m RNA(P=0.039)and protein(P=0.012)expression.FOXP3 m RNA(P=0.003)and protein(P=0.003)were significantly increased in the miR-133a inhibitor group compared to inhibitor NC group.5.IL-6 was significantly higher in the miR-133a-3p mimics group compared to the mimics NC group(P=0.042),while IL-17A,IFN-γand IL-10 were not significantly different from that in mimics NC group.IL-10 was significantly higher in the miR-133a-3p inhibitor group compared to the inhibitor NC group(P=0.014),while IL-6,IL-17A,IFN-γand IL-10 were not significantly different from that in inhibitor NC group.Conclusions:Consistent with the findings in vivo,we found that miR-133a-3p negatively regulated the expression of SOCS2 and autophagy-related proteins in CD4~+T cells from EAE mice.Inhibition of miR-133a-3p could restore autophagic flow,and increase the expression of FOXP3. |
PDF Full Text Request