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. 2020 Sep 29:11:576903.
doi: 10.3389/fimmu.2020.576903. eCollection 2020.

Hsa_circ_0060450 Negatively Regulates Type I Interferon-Induced Inflammation by Serving as miR-199a-5p Sponge in Type 1 Diabetes Mellitus

Lan Yang  1   2 , Xiao Han  1   2 , Caiyan Zhang  1   2 , Chengjun Sun  3 , Saihua Huang  1   2 , Wenfeng Xiao  1   2 , Yajing Gao  1   2 , Qiuyan Liang  1   2 , Feihong Luo  3 , Wei Lu  3 , Jinrong Fu  1 , Yufeng Zhou  1   2
Affiliations

Hsa_circ_0060450 Negatively Regulates Type I Interferon-Induced Inflammation by Serving as miR-199a-5p Sponge in Type 1 Diabetes Mellitus

Lan Yang et al. Front Immunol. .

Abstract

Circular RNAs (circRNAs) constitute a class of covalently circular non-coding RNA molecules formed by 5' and 3' end back-splicing. The rapid development of bioinformatics and large-scale sequencing has led to the identification of functional circRNAs. Despite an overall upward trend, studies focusing on the roles of circRNAs in immune diseases remain relatively scarce. In the present study, we obtained a differential circRNA expression profile based on microarray analysis of peripheral blood mononuclear cells (PBMCs) in children with type 1 diabetes mellitus (T1DM). We characterized one differentially expressed circRNA back-spliced from the MYB Proto-Oncogene Like 2 (MYBL2) gene in patients with T1DM, termed as hsa_circ_0060450. Subsequent assays revealed that hsa_circ_0060450 can serve as the sponge of miR-199a-5p, release its target gene, Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), encoded by the tyrosine-protein phosphatase non-receptor type 11 gene (PTPN11), and further suppress the JAK-STAT signaling pathway triggered by type I interferon (IFN-I) to inhibit macrophage-mediated inflammation, which indicates the important roles of circRNAs in T1DM and represents a promising therapeutic molecule in the treatment of T1DM.

Keywords: SHP2; circular RNA; macrophage; type 1 diabetes mellitus; type I interferon.

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Figures

Figure 1
Figure 1
Hsa_circ_0060450 is upregulated in the PBMCs of T1DM patients. (A) Heatmap of circRNAs with significantly altered expression in the PBMCs of 3 normal controls and 3 T1DM patients. (B) Volcano plot of circRNAs with significantly altered expression in the PBMCs of 3 normal controls and 3 T1DM patients. (C) The expression of hsa_circ_0060450 in PBMCs of 20 children with T1DM and 20 healthy controls, detected by RT-qPCR. (D) Receiver operating curve (ROC) analysis of hsa_circ_0060450 levels in the study population. (E) The genomic loci of the MYBL2 and hsa_circ_0060450. The expression of hsa_circ_0060450 was detected by RT-PCR and validated by Sanger sequencing. (F) RT-qPCR analyses of β-actin, MYBL2, and hsa_circ_0060450 after treatment with RNase R in THP1-derived macrophages. (G) RT-qPCR analyses of MYBL2 and hsa_circ_0060450 after treatment with actinomycin D at the indicated time points in THP1-derived macrophages. (H) The expressions of hsa_circ_0060450 in T cells, B cells and monocytes of human PBMCs, detected by RT-qPCR. (I) The relative distribution of hsa_circ_0060450 in the nucleus and cytoplasm in THP1-derived macrophages. circ_0450 is the abbreviation of hsa_circ_0060450. ActD, actinomycin D. Data represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significance.
Figure 2
Figure 2
Silencing of hsa_circ_0060450 promotes IFN-I-induced inflammation through the JAK-STAT1/3 pathway. (A) RT-qPCR analyses of hsa_circ_0060450 to confirm the silencing efficiencies of two siRNAs. (B) RT-qPCR analyses of IFIT1, IFIH1, CXCL10, and iNOS under IFN-I stimulation after treatment with two hsa_circ_0060450 siRNAs. (C) STAT1 and STAT3 protein phosphorylation assessment at 15 and 30 min of IFN-I stimulation after treatment with two hsa_circ_0060450 siRNAs. NC, negative control. Data represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
Hsa_circ_0060450 serves as a sponge of miR-199a-5p. (A) The predicted target sequence of miR-199a-5p, miR-133a or miR-133b in hsa_circ_0060450 and corresponding mutant target sequence for binding sites-mutant luciferase assay. (B) The construction of hsa_circ_0060450 fragment-containing psicheck2 luciferase vector. (C) A luciferase reporter assay was used to detect the luciferase activity of 293T cells co-transfected with blank psicheck2 vector or psicheck2 recombinant vector containing hsa_circ_0060450 fragment and miR-199a-5p, miR-133a, miR-133b mimics, or NC. (D) A luciferase reporter assay was used to detect the luciferase activity of 293T cells co-transfected with psicheck2 recombinant vector containing mutant hsa_circ_0060450 fragment and miR-199a-5p or NC. (E) STAT1 and STAT3 protein phosphorylation assessment and quantification analyses at 15 and 30 min of IFN-I stimulation after treatment with miR-199a-5p mimics. (F) RT-qPCR analyses of IFIT1, IFIH1, CXCL10, and iNOS under IFN-I stimulation after treatment with miR-199a-5p mimics. NC, negative control. circ_0450-v, psicheck2 recombinant vector containing WT hsa_circ_0060450 fragment. circ_0450-miR199a-mut-v, psicheck2 recombinant vector containing miR-199a-5p binding site-mutant hsa_circ_0060450 fragment. Data represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significance.
Figure 4
Figure 4
miR-199a-5p promotes IFN-I-induced inflammation by targeting SHP2. (A) RT-qPCR analyses of five predicted miR-199a-5p target genes after treatment with miR-199a-5p mimics. (B) SHP2 protein assessment at 0, 15, and 30 min of IFN-I stimulation after treatment with miR-199a-5p mimics. (C) Three miR-199a-5p binding sites within SHP2 3′ UTR predicted using the starBase database. (D) Luciferase activities of 293T cells co-transfected with blank psicheck2 vector or psicheck2 recombinant vector containing SHP2 3′ UTR fragment and miR-199a-5p mimics or NC. NC, negative control. SHP2-v, psicheck2 recombinant vector containing SHP2 3′ UTR fragment. Data represent means ± SEM. *p < 0.05, **p < 0.01. ns, not significance.
Figure 5
Figure 5
Hsa_circ_0060450 upregulates SHP2 by adsorbing miR-199a-5p. (A) SHP2 protein assessment at 0, 15, and 30 min of IFN-I stimulation after treatment with two hsa_circ_0060450 siRNAs. (B) RT-qPCR analyses of SHP2 expression after treatment with two hsa_circ_0060450 siRNAs. (C) RT-qPCR analysis of SHP2 expression to confirm the silencing efficiency of the SHP2 siRNA. (D) STAT1 and STAT3 phosphorylation level assessment at 15 and 30 min of IFN-I stimulation after treatment with the SHP2 siRNA. (E) RT-qPCR analyses of IFIT1, IFIH1, CXCL10, and iNOS under IFN-I stimulation after treatment with the SHP2 siRNA. (F) A schematic model of the function of hsa_circ_0060450 in IFN-I-stimulated macrophages and in T1DM. NC, negative control. Data represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
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References

    1. Pozzilli P, Maddaloni E, Buzzetti R. Combination immunotherapies for type 1 diabetes mellitus. Nat Rev Endocrinol. (2015) 11:289–97. 10.1038/nrendo.2015.8 - DOI - PubMed
    1. Ni Q, Pham NB, Meng WS, Zhu G, Chen X. Advances in immunotherapy of type I diabetes. Adv Drug Deliv Rev. (2018) 139:83–91. 10.1016/j.addr.2018.12.003 - DOI - PubMed
    1. Roep BO, Tree TI. Immune modulation in humans: implications for type 1 diabetes mellitus. Nat Rev Endocrinol. (2014) 10:229–42. 10.1038/nrendo.2014.2 - DOI - PubMed
    1. Marwaha AK, Crome SQ, Panagiotopoulos C, Berg KB, Qin H, Ouyang Q, et al. . Cutting edge: increased IL-17-secreting T cells in children with new-onset type 1 diabetes. J Immunol. (2010) 185:3814–8. 10.4049/jimmunol.1001860 - DOI - PubMed
    1. Ferraro A, Socci C, Stabilini A, Valle A, Monti P, Piemonti L, et al. . Expansion of Th17 cells and functional defects in T regulatory cells are key features of the pancreatic lymph nodes in patients with type 1 diabetes. Diabetes. (2011) 60:2903–13. 10.2337/db11-0090 - DOI - PMC - PubMed

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