The Expression Of MicroRNAs Related To Notch Signaling Pathway In Immune Cells Of Mice With Collagen-induced Arthritis

Objective:Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by symmetry of the joints of the hands and feet,multi-articularity,invasive joint inflammation,often accompanied by abnormal proliferation of synovial tissue,and eventually lead to joint and cartilage destruction.Its pathogenesis is complex and affected by environmental,infection,genetic and other factors.In recent years,a large number of studies had shown that microRNAs(miRNAs)are differentially expressed in RA patients and collagen-induced arthritis(CIA)mouse models.Our previous study found that Notch signaling pathway was involved in the pathogenesis of RA by regulating the functional differentiation of helper T cells.The purpose of this study was to detect the expression of Notch signaling pathway-related miRNAs in peripheral blood,spleen and lymph node T cells of CIA mice and to screen differentially expressed miRNAs to analyze the correlation between the differential expression of miRNAs and the functional differentiation of helper T cells regulated by Notch signaling pathway,and lay a foundation for the development of RA treatment strategy based on RNA interference.Methods:(1)DBA/1J mice were immunized with bovine type II collagen and an adjuvant to construct a collagen-induced arthritis(CIA)mouse model.(2)Peripheral blood of CIA mice was taken on the 40th day of modeling,and quantitative Real-time fluorescent PCR was used to detect the expression levels of miR-1a-3p,miR-20a-5p,miR-23b-3p,miR-30b-5p,miR-30c-5p,miR-107-3p,miR-141-3p,miR-144-3p,miR-146a-5p,miR-150-5p,miR-199b-5p,miR-200b-3p,miR-200c-3p and miR-449a-5p in peripheral blood mononuclear cells of CIA mice and normal control mice.(3)Flow cytometry(FCM)was used to sort CD4~+T and CD8~+T cells in peripheral blood,spleen and lymph nodes of CIA mice and normal control mice.The qRT-PCR was used to detect the expression levels of miR-23b-3p,miR-30b-5p,miR-30c-5p and miR-200b-3p in CD4~+T and CD8~+T cells.(4)GraphPad Prism 5.0 software was used for mapping and statistical analysis,and the data between the two groups were analyzed by t-test.Results:(1)Compared with normal controls,the expression levels of miR-200b-3p,miR-30c-5p,miR-30b-5p and miR-23b-3p in CIA mice PBMC were significantly decreased(P<0.05).(2)Compared with normal controls,the expression level of miR-23b-3p in peripheral blood CD4~+T cells of CIA mice was significantly decreased(P<0.05),although miR-30c-5p showed a decreasing trend but no statistical significance.There was no significant difference between miR-30b-5p and miR-200b-3p(P>0.05).There was no significant difference in the expression level of miR-23b-3p,miR-30b-5p,miR-30c-5p,miR-200b-3p in peripheral blood CD8~+T cells of CIA mice(P>0.05).(3)There was no significant difference in the expression levels of miR-23b-3p,miR-30b-5p,miR-30c-5p,miR-200b-3p in lymph node CD4~+T cells and CD8~+T cells of CIA mice compared with normal controls(P>0.05).(4)Compared with normal controls,the expression level of miR-23b-3p in spleen CD4~+T cells of CIA mice was significantly decreased(P<0.05).There was no significant difference in expression levels of miR-30b-5p,miR-30c-5p and miR-200b-3p between the two groups(P>0.05).The expression levels of miR-23b-3p,miR-30b-5p,miR-30c-5p and miR-200b-3p in spleen CD8~+T cells of CIA mice were not obvious difference(P>0.05).Conclusions:(1)The expression levels of miR-200b-3p,miR-30c-5p,miR-30b-5p and miR-23b-3p in the PBMC of CIA mice were significantly lower than those in normal control mice(P<0.05).These four miRNAs may play a role in the pathogenesis of CIA.(2)Compared with normal control mice,miR-23b-3p was significantly decreased in the PBMC of CIA mice(P<0.05),and the expression level of miR-23b-3p in spleen CD4~+T cells of CIA mice was significantly decreased(P<0.05),suggesting that miR-23b-3p in peripheral blood CD4~+T cells may be derived from the spleen.(3)miR-23b-3p is significantly decreased in CIA mouse PBMC and spleen CD4~+T(P<0.05).It is suggested that miR-23b-3p may immunoregulate CIA mouse T cells through the Notch signaling pathway.