Yonsei Med J.  2019 Nov;60(11):1093-1102. 10.3349/ymj.2019.60.11.1093.

Inhibition of miRNA-222-3p Relieves Staphylococcal Enterotoxin B-Induced Liver Inflammatory Injury by Upregulating Suppressors of Cytokine Signaling 1

Affiliations
  • 1Department of Clinical Laboratory, the Third People's Hospital of Dalian, Dalian, China.
  • 2Department of Clinical Laboratory, the Baotou Medical College of Inner Mongolia University of Science and Technology, Inner Mongolia, China.
  • 3Department of Clinical Laboratory, the Hongqi Hospital Affiliated to Mudanjiang Medical University, Mudanjiang, China.
  • 4Department of Clinical Laboratory, the Second Hospital of Dalian Medical University, Dalian, China. zdgkua@163.com

Abstract

PURPOSE
Staphylococcal enterotoxin B (SEB) has been well-documented to induce liver injury. miRNA-222-3p (miR-222-3p) was implicated in SEB-induced lung injury and several liver injuries. This study aimed to explore the role of miR-222-3p in SEB-induced liver injury.
MATERIALS AND METHODS
Expression of miR-222-3p and suppressors of cytokine signaling 1 (SOCS1) was detected using real-time quantitative PCR and western blot. Liver injury was determined by levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and inflammatory cytokines, numbers of infiltrating mononuclear cells using AST/ALT assay kit, enzyme-linked immunosorbent assay (ELISA), and hematoxylin-eosin staining, respectively. Target binding between miR-222-3p and SOCS1 was predicted on targetScan software, and confirmed by luciferase reporter assay.
RESULTS
SEB induced liver injury in D-galactosamine (D-gal)-sensitized mice, as demonstrated by increased serum levels of AST and ALT, elevated release of interferon-gamma (INF-γ), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-2, and promoted infiltrating immune cells into liver. Expression of miR-222-3p was dramatically upregulated, and SOCS1 was downregulated in SEB-induced liver injury both in mice and splenocytes. Moreover, miR-222-3p knockout (KO) mice exhibited alleviated liver injury accompanied with SOCS1 upregulation. Besides, splenocytes under SEB challenge released less INF-γ, TNF-α, IL-6, and IL-2 during miR-222-3p knockdown. Mechanically, SOCS1 was targeted and downregulated by miR-222-3p. Upregulation of SOCS1 attenuated INF-γ, TNF-α, IL-6, and IL-2 release in SEB-induced splenocytes; downregulation of SOCS1 could block the suppressive role of miR-222-3p knockdown in SEB-induced splenocytes.
CONCLUSION
Inhibition of miR-222-3p relieves SEB-induced liver inflammatory injury by upregulating SOCS1, thereby providing the first evidence of miR-222-3p in SEB-induced liver injury.

Keyword

miR-222-3p; SEB; inflammatory cytokine; liver injury

MeSH Terms

Alanine Transaminase
Animals
Aspartate Aminotransferases
Blotting, Western
Cytokines
Down-Regulation
Enterotoxins*
Enzyme-Linked Immunosorbent Assay
Interferon-gamma
Interleukin-2
Interleukin-6
Liver*
Luciferases
Lung Injury
Mice
Polymerase Chain Reaction
Tumor Necrosis Factor-alpha
Up-Regulation
Alanine Transaminase
Aspartate Aminotransferases
Cytokines
Enterotoxins
Interferon-gamma
Interleukin-2
Interleukin-6
Luciferases
Tumor Necrosis Factor-alpha

Figure

  • Fig. 1 The role of miR-222-3p in staphylococcal enterotoxin B (SEB)-mediated liver injury in mice. Wild type (WT) and miR-222 knockout (KO) mice were treated with 20 mg of D-galactosamine (D-gal) for 1 h, followed by 40 µg of SEB injection for 24 h or not. (A) Levels of miR-222 serum were confirmed by real-time quantitative PCR. Data were presented by 2−ΔΔCt value and normalized to WT control. (B and C) Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using special assay kits. (D) Histopathological examination of liver tissues was conducted using hematoxylin-eosin (HE) staining. Magnification: ×100. (E) Liver-infiltrating mononuclear cells were obtained by density gradient centrifugation, and total number of viable cells were counted using a hemocytometer. (F-I) Serum interferon-gamma (INF-γ), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-2 were detected by enzyme-linked immunosorbent assay. *p<0.05. KO control group (without SEB challenge) versus WT control group (without SEB challenge); KO SEB (with standard error of mean challenge) group versus KO control group and WT SEB group, respectively; WT SEB group versus WT control group.

  • Fig. 2 Knockdown of miR-222-3p inhibited staphylococcal enterotoxin B (SEB)-induced inflammatory injury in splenocytes. Splenocytes from C57BL/6 mice were transfected with miR-222-3p/NC inhibitor (miR-222-3p/NC-in), and then challenged with 1 µg/mL of SEB for 24 h. (A) Expression levels of miR-222-3p were measured using real-time quantitative PCR. Data was presented by 2−ΔΔCt value and normalized to miR-NC-in. (B) Treated splenocytes were collected, and total cell number was determined by a hemocytometer. (C-F) Interferon-gamma (INF-γ), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-2 in culture supernatant were detected by enzyme-linked immunosorbent assay. *p<0.05. miR-222-3p-in group (without SEB challenge) versus miR-NC-in group; SEB+miR-222-3p-in group versus miR-222-3p-in group and SEB+miR-NC-in group, respectively; SEB+miR-NC-in group versus miR-NC-in group.

  • Fig. 3 Suppressors of cytokine signaling 1 (SOCS1) was negatively regulated by miR-222-3p in splenocytes. (A and B) Real-time quantitative PCR (RT-qPCR) detected expression of SOCS1 in staphylococcal enterotoxin B (SEB)-induced mice and splenocytes. Data was presented by 2−ΔΔCt value and normalized to control. (C) The predicted miR-222-3p binding sites in mouse SOCS1 gene wild type (SOCS1-Wt) according to targetScan software. Corresponding sequence in the mutated version (SOCS1-Mut) was also shown. (D) Levels of miR-222-3p were confirmed in splenocytes when transfected with miR-222-3p mimic (miR-222-3p), inhibitor, or its corresponding control. (E and F) Luciferase activity of SOCS1 wild type (SOCS1-Wt) or SOCS1-Mut in splenocyte cells transfected with miR-222-3p/NC mimic (miR-222-3p/NC) or miR-222-3p/NC-in. (G and H) Expression levels of SOCS1 were confirmed by RT-qPCR and western blot in splenocytes when transfected with miR-222-3p, miR-222-3p-in, or its corresponding control. *p<0.05 compared to controls. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

  • Fig. 4 Overexpression of suppressors of cytokine signaling 1 (SOCS1) suppressed inflammatory cytokines release in staphylococcal enterotoxin B (SEB)-induced splenocytes ex-vivo. Splenocytes from C57BL/6 mice were transfected with pcDNA-SOCS1/Vector (SOCS1/Vector), and then challenged with 1 µg/mL of SEB for 24 h. (A) Expression of SOCS1 was measured by western blot. GAPDH expression was used as internal reference. (B) Treated splenocytes were collected, and total cell number was determined by a hemocytometer. (C–F) Interferon-gamma (INF-γ), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-2 in culture supernatant were detected by enzyme-linked immunosorbent assay. *p<0.05. SOCS1 group (without SEB challenge) versus Vector group; SEB+SOCS1 group versus SOCS1 group and SEB+Vector group, respectively; SEB+ SOCS1 group versus Vector group.

  • Fig. 5 Suppressors of cytokine signaling 1 (SOCS1) downregulation blocked the suppressive effect of miR-222-3p knockdown in staphylococcal enterotoxin B (SEB)-induced splenocytes. Splenocytes were transfected with miR-222-3p/NC, or co-transfected with miR-222-3p-in and si-SOCS1/NC. (A) Western blot showed SOCS1 levels after transfection. GAPDH level was used as internal reference. (B) Transfected splenocytes were collected, and total cell number was determined by a hemocytometer. (C-F) Interferon-gamma (INF-γ), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-2 in culture supernatant were detected by enzyme-linked immunosorbent assay. *p<0.05 compared to miR-NC-in group or miR-222-3p+si-NC group.


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