CircN4bp1 Facilitates Sepsis-Induced Acute Respiratory Distress Syndrome Through Mediating Macrophage Polarization via the miR-138-5p/EZH2 Axis

Background: We recently reported the differential circRNAs expression patterns of the pulmonary macrophages in sepsis induced ARDS mice model by microarray analysis; However, their function and hidden molecular mechanism in regulation of macrophage activation and inflammation remains poorly understood. Methods: Serum were obtained from ARDS patients post sepsis and control subjects. Mice were subjected to Cecal ligation and puncture (CLP) surgery and intravenously injected with si-circN4bp1 plasmid transfected macrophages. Raw264.7 cells and MH-S cells were transfected with circN4bp1 overexpression or silence vectors and then polarized as M1 or M2 macrophages in vitro.  Findings: CircN4bp1 was overexpressed in PBMC and monocytes and its expression levels were correlated with a poor prognosis in ARDS patients induced by sepsis. Knockdown of circN4bp1 inhibited the lung injury and improved the long-time survival through blunting the M1 macrophage activation in ARDS mice. Moreover, bioinformatics analysis predicated a circN4bp1/miR-138-5p ceRNA network, which was confirmed by Luciferase reporter assay and RNA binding protein immunoprecipitation (RIP). CircN4bp1 affected macrophage differentiation by binding to miR-138-5p, thus regulating the expression of EZH2 in vivo and ex vivo . Lastly, the m6A level of circN4bp1 was found to be elevated in ARDS mice; inhibition of m6A methyltransferases METTL3 blocked this response in vitro. Interpretation: CircN4bp1 can function as a miR-138-5p sponge for the modulation of macrophage polarization through regulation the expression of EZH2 and may serve as a potential target and/or prognostic marker for ARDS patients following sepsis. Funding: The National Natural Science Foundation of China (81970072 and 81670690), the leading medical talent project of Shanghai Pudong heath bureau (PWRI2019‐05 and PWR12020-07), the Shanghai Scientific Committee of China (20Y11901200, 21ZR1452400, 20ZR1445800 and 13PJ1406900). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The manuscript contains animal experiments that have been approved by the Animal Use Committee of East Hospital/Tongji University, China. The manuscript contains human subjects that have been approved by the Research Ethics Board of East Hospital, Tongji University. All recruited patients or their authorized family members were given a written consent document.