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. 2020 Mar 6;19:1123-1133.
doi: 10.1016/j.omtn.2020.01.005. Epub 2020 Jan 18.

Identification of the Regulatory Role of lncRNA SNHG16 in Myasthenia Gravis by Constructing a Competing Endogenous RNA Network

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Free PMC article

Identification of the Regulatory Role of lncRNA SNHG16 in Myasthenia Gravis by Constructing a Competing Endogenous RNA Network

Jianjian Wang et al. Mol Ther Nucleic Acids. .
Free PMC article

Abstract

Myasthenia gravis (MG) is an autoimmune disorder resulting from antibodies against the proteins at the neuromuscular junction. Emerging evidence indicates that long non-coding RNAs (lncRNAs), acting as competing endogenous RNAs (ceRNAs), are involved in various diseases. However, the regulatory mechanisms of ceRNAs underlying MG remain largely unknown. In this study, we constructed a lncRNA-mediated ceRNA network involved in MG using a multi-step computational strategy. Functional annotation analysis suggests that these lncRNAs may play crucial roles in the immunological mechanism underlying MG. Importantly, through manual literature mining, we found that lncRNA SNHG16 (small nucleolar RNA host gene 16), acting as a ceRNA, plays important roles in the immune processes. Further experiments showed that SNHG16 expression was upregulated in peripheral blood mononuclear cells (PBMCs) from MG patients compared to healthy controls. Luciferase reporter assays confirmed that SNHG16 is a target of the microRNA (miRNA) let-7c-5p. Subsequent experiments indicated that SNHG16 regulates the expression of the key MG gene interleukin (IL)-10 by sponging let-7c-5p in a ceRNA manner. Furthermore, functional assays showed that SNHG16 inhibits Jurkat cell apoptosis and promotes cell proliferation by sponging let-7c-5p. Our study will contribute to a deeper understanding of the regulatory mechanism of MG and will potentially provide new therapeutic targets for MG patients.

Keywords: IL-10; SNHG16; ceRNA network; let-7c-5p; myasthenia gravis.

Figures

Figure 1
Figure 1
The lncRNA-Mediated MG-Associated ceRNA Network (LMGCN) (A) Schematic workflow to construct the LMGCN based on the ceRNA hypothesis. (B) LMGCN. Red circles represent miRNAs, blue triangles represent mRNAs, and yellow rhombi represent lncRNAs. Lines represent their regulatory interactions. (C) Pathway enrichment analysis (left) and GO annotation (right) of co-expressed mRNAs with lncRNAs.
Figure 2
Figure 2
The Sub-Network of lncRNA Acting as a ceRNA to Regulate miRNA–Gene Pairs Has Been Verified in MG. (A) The sub-network of let-7c-5p/IL-10 pair. (B) The sub-network of miR-145-5p/CD28 pair. (C) The sub-network of miR-15a-5p/CXCL10 pair. (D)The sub-network of miR-181c-5p/IL-7 pair. The left pipeline are the sub-networks of four experimentally-supported miRNA–gene pairs in MG and their linked lncRNAs. The right pipeline are the summarized functions of lncRNAs.
Figure 3
Figure 3
Upregulation of SNHG16 Is a Target of let-7c-5p in MG (A) SNHG16 expression was examined in 24 MG patients and 29 control subjects by real-time PCR. (B) Transfection efficiency of let-7c-5p mimics was measured by real-time PCR. (C) The relative expression level of SNHG16 in Jurkat cells transfected with miRNA NC or let-7c-5p mimics was measured using real-time PCR. (D) The putative let-7c-5p binding sequence of the wild-type and mutation sequence of SNHG16. (E) The luciferase reporter plasmid containing SNHG16-WT or SNHG16-MUT was co-transfected with let-7c-5p mimics or miRNA NC into HEK293T cells. Luciferase activities were calculated as the ratio of firefly/Renilla activities. The experiment was repeated at least three times, and data are presented as the mean ± SD. **p < 0.01.
Figure 4
Figure 4
SNHG16 Regulates IL-10 Expression by Binding let-7c-5p in a ceRNA Manner (A) Relative mRNA levels of IL-10 were determined by real-time PCR after transfection with negative control or let-7c-5p mimics in Jurkat cells. (B) Relative protein expression levels of IL-10 were determined by western blotting after transfection with negative control or let-7c-5p mimics in Jurkat cells. (C) Relative mRNA levels of IL-10 were determined by real-time PCR analysis after transfection with negative control, siSNHG16, and siSNHG16 + let-7c-5p inhibitor in Jurkat cells. (D) Relative protein expression levels of IL-10 were determined by western blotting after transfection with negative control, siSNHG16, and siSNHG16 + let-7c-5p inhibitor in Jurkat cells. The experiment was repeated at least three times, and data are presented as the mean ± SD. **p < 0.01.
Figure 5
Figure 5
SNHG16 Inhibits Apoptosis and Promotes Cell Proliferation by Sponging let-7c-5p (A) After transfecting negative control, siSNHG16 or siSNHG16+let-7c-5p inhibitor, Jurkat cells were stained with Annexin-V-FITC/PI, and the apoptosis was detected by flow cytometric analysis. (B) The apoptosis rate of Jurkat cells after transfecting negative control, siSNHG16 or siSNHG16+let-7c-5p inhibitor. (C) Cell proliferation was analyzed using CCK-8 assays by transfecting negative control, siSNHG16, or siSNHG16 + let-7c-5p inhibitor into Jurkat cells. (D) Schematic diagram of SNHG16 involved in the immunological pathogenesis of MG. The experiment was repeated at least three times, and data are presented as the mean ± SD. **p < 0.01.

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