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Observational Study
. 2020 Jul 31;40(7):BSR20201267.
doi: 10.1042/BSR20201267.

MiR-96-5p alleviates inflammatory responses by targeting NAMPT and regulating the NF-κB pathway in neonatal sepsis

Xueli Chen  1 , Ying Chen  1 , Li Dai  1 , Na Wang  2
Affiliations
Observational Study

MiR-96-5p alleviates inflammatory responses by targeting NAMPT and regulating the NF-κB pathway in neonatal sepsis

Xueli Chen et al. Biosci Rep. .

Abstract

Neonatal septicemia is a serious infectious disease in the neonatal period. MicroRNAs (miRNAs) have been reported to participate in the inflammatory responses in neonatal sepsis. The aim of the present study was to explore the effects and molecular mechanism of miR-96-5p on regulating the inflammatory responses in neonatal sepsis. MiR-96-5p was low expressed while nicotinamide phosphoribosyltransferase (NAMPT) was high expressed in the serum of neonatal septicemia patients. The expression of miR-96-5p was decreased in LPS-induced inflammatory responses. Besides, miR-95-5p relieved LPS-induced inflammatory responses in RAW264.7 cells. NAMPT was demonstrated as a potential target of miR-96-5p, and knockdown of NAMPT reduced inflammatory in RAW264.7 cells stimulated with LPS. Moreover, overexpression of NAMPT reversed the effects of miR-96-5p on LPS-induced inflammatory responses. In addition, miR-96-5p inhibited nuclear factor (NF)-κB signaling pathway in RAW264.7 cells stimulated with LPS. MiR-96-5p alleviated inflammatory responses via targeting NAMPT and inhibiting NF-κB pathway in neonatal sepsis.

Keywords: NAMPT; NF-κB pathway; inflammatory; miR-96-5p; neonatal sepsis.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. The expression levels of miR-96-5p and NAMPT in serum of neonatal sepsis patients
(A) miR-96-5p level in serum of neonatal sepsis patients was detected by qRT-PCR. (B) the mRNA level of NAMPT in serum of neonatal sepsis patients was detected by qRT-PCR. (C) the negative correlation miR-96-5p and NAMPT mRNA in serum of neonatal sepsis patients was showed. (D and E) Western blot analysis was performed to measure the protein level of NAMPT in serum of neonatal sepsis patients; *P<0.05.
Figure 2
Figure 2. The expression level of miR-96-5p was suppressed and inflammatory response was induced by LPS
(A and B) The level of miR-96-5p was measured in RAW264.7 cells treated with LPS at different dose and treatment hours. (C and D) The mRNA levels of TNF-α and IL-6 in RAW264.7 cells treated with LPS were detected by qRT-PCR. (E and F) The levels of TNF-α and IL-6 in RAW264.7 cells treated with LPS were detected by ELISA; *P<0.05.
Figure 3
Figure 3. MiR-96-5p inhibited inflammatory response in RAW264.7 cells stimulated with LPS
(A) The expression levels of miR-96-5p in RAW264.7 cells treated with LPS or LPS + miR-96-5p mimic were detected by qRT-PCR. (B and C) The mRNA levels of TNF-α and IL-6 in RAW264.7 cells treated with LPS or LPS + miR-96-5p mimic were detected by qRT-PCR. (D and E) The levels of TNF-α and IL-6 in RAW264.7 cells treated with LPS or LPS + miR-96-5p mimic were detected by ELISA; *P<0.05.
Figure 4
Figure 4. MiR-96-5p regulated NAMPT by directly binding NAMPT
(A) The binding sites of miR-96-5p on NAMPT 3′-UTR were predicted by Targetscan software. (B and C) The luciferase activity was detected in HEK293T and RAW264.7 cells co-transfected with miR-96-5p and NAMPT-WT or NAMPT-MUT. (D and E) The mRNA levels of NAMPT in RAW264.7 cells transfected with miR-96-5p mimic or anti-miR-96-5p were detected by qRT-PCR. (F) The protein levels of NAMPT in RAW264.7 cells transfected with miR-96-5p mimic or anti-miR-96-5p were detected by Western blot analysis; *P<0.05.
Figure 5
Figure 5. Knockdown of NAMPT relieved LPS-induced inflammatory response
(A and B) The level of NAMPT in RAW264.7 cells treated with LPS or LPS + si-NAMPT was detected by qRT-PCR and Western blot. (C and D) The mRNA levels of TNF-α and IL-6 in RAW264.7 cells treated with LPS or LPS + si-NAMPT were detected by qRT-PCR. (E and F) The levels of TNF-α and IL-6 in RAW264.7 cells treated with LPS or LPS + si-NAMPT were detected by ELISA; *P<0.05.
Figure 6
Figure 6. NAMPT overexpression reversed effects of miR-96-5p on LPS-induced inflammatory response
(A and B) The level of NAMPT in RAW264.7 cells transfected with miR-96-5p mimic or miR-96-5p mimic + NAMPT was detected by qRT-PCR and Western blot. (C and D) The mRNA levels of TNF-α and IL-6 in RAW264.7 cells transfected with miR-96-5p or miR-96-5p + NAMPT were detected by qRT-PCR. (E and F) The levels of TNF-αand IL-6 in RAW264.7 cells transfected with miR-96-5p or miR-96-5p + NAMPT were detected by ELISA; *P<0.05.
Figure 7
Figure 7. miR-96-5p inhibited NF-κB pathway in RAW264.7 cells stimulated with LPS
The levels of p65, p-p65, IKKβ and p-IKKα/β in RAW264.7 cells treated with LPS or LPS + miR-96-5p were detected by Western blot. The ratios of p-IKKα/β/IKKβ and p-p65/p65 were analyzed; *P<0.05.
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