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Published on: December 23, 2020
LINC01002 functions as a ceRNA to regulate FRMD8 by sponging miR-4324 for the development of COVID-19
Xinyi Kong1, Qinjin Wang1, Xumeng Wang1,2
1Department of Laboratory Medicine, The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, 518033, China.
Background:
Syndrome coronavirus-2 (SARS-CoV-2) has developed various strategies to evade the antiviral impact of type I IFN. Non-structural proteins and auxiliary proteins have been extensively researched on their role in immune escape. Nevertheless, the detailed mechanisms of structural protein-induced immune evasion have not been well elucidated.
Methods:
Human alveolar basal epithelial carcinoma cell line (A549) was stimulated with polyinosinic-polycytidylic acid (PIC) and independently transfected with four structural proteins expression plasmids, including nucleocapsid (N), spike (S), membrane (M) and envelope (E) proteins. By RT-qPCR and ELISA, the structural protein with the most pronounced inhibitory effects on IFN-β induction was screened. RNA-sequencing (RNA-Seq) and two differential analysis strategies were used to obtain differentially expressed genes associated with N protein inhibition of IFN-β induction. Based on DIANA-LncBase and StarBase databases, the interactive competitive endogenous RNA (ceRNA) network for N protein-associated genes was constructed. By combining single-cell sequencing data (GSE158055), lncRNA-miRNA-mRNA axis was further determined. Finally, RT-qPCR was utilized to illustrate the regulatory functions among components of the ceRNA axis.
Results:
SARS-CoV-2 N protein inhibited IFN-β induction in human alveolar epithelial cells most significantly compared with other structural proteins. RNA-Seq data analysis revealed genes related to N protein inhibiting IFNs induction. The obtained 858 differentially expressed genes formed the reliable ceRNA network. The function of LINC01002-miR-4324-FRMD8 axis in the IFN-dominated immune evasion was further demonstrated through integrating single-cell sequencing data. Moreover, we validated that N protein could reverse the effect of PIC on LINC01002, FRMD8 and miR-4324 expression, and subsequently on IFN-β expression level. And LINC01002 could regulate the production of FRMD8 by inhibiting miR-4324.
Conclusion:
SARS-CoV-2 N protein suppressed the induction of IFN-β by regulating LINC01002 which was as a ceRNA, sponging miR-4324 and participating in the regulation of FRMD8 mRNA. Our discovery provides new insights into early intervention therapy and drug development on SARS-CoV-2 infection.
Insights
The SARS-CoV-2 nucleocapsid (N) protein significantly inhibits interferon-beta (IFN-β) induction by regulating the LINC01002-miR-4324-FRMD8 axis. This discovery offers new avenues for developing early intervention therapies against SARS-CoV-2 infection.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) employs strategies to evade host type I Interferon (IFN) responses.
- While non-structural and auxiliary proteins' roles in immune evasion are known, mechanisms of structural protein-induced immune evasion remain unclear.
Purpose of the Study:
- To investigate the role of SARS-CoV-2 structural proteins in evading type I IFN-mediated immunity.
- To elucidate the molecular mechanisms by which SARS-CoV-2 structural proteins interfere with IFN-β induction.
Main Methods:
- Human alveolar epithelial cells (A549) were stimulated and transfected with SARS-CoV-2 structural protein expression plasmids (N, S, M, E).
- RT-qPCR, ELISA, and RNA-sequencing (RNA-Seq) were used to identify the protein with the strongest inhibitory effect on IFN-β and associated genes.
- A competitive endogenous RNA (ceRNA) network and lncRNA-miRNA-mRNA axis were constructed and validated using bioinformatics databases and single-cell sequencing data.
Main Results:
- SARS-CoV-2 nucleocapsid (N) protein demonstrated the most significant inhibition of IFN-β induction.
- RNA-Seq identified 858 differentially expressed genes linked to N protein's inhibition of IFN-β.
- The LINC01002-miR-4324-FRMD8 axis was identified as a key player in N protein-mediated immune evasion, with N protein reversing PIC-induced expression changes.
Conclusions:
- SARS-CoV-2 N protein suppresses IFN-β induction by acting as a ceRNA, sponging miR-4324 to regulate FRMD8 mRNA.
- This study provides novel insights into SARS-CoV-2 immune evasion mechanisms, potentially guiding early intervention strategies and drug development.
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