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Cited by 26

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MiR-132-3p Modulates MEKK3-Dependent NF-κB and p38/JNK Signaling Pathways to Alleviate Spinal Cord Ischemia-Reperfusion Injury by Hindering M1 Polarization of Macrophages

Fang, Hua, Li, Hua-Feng, Pan, Qin, Jin, Hon-Ling, Yang, Miao, Wang, Ru-Rong, Wang, Quan-Yun, Zhang, Jian-Ping·2021·Frontiers in Cell and Developmental Biology
DOI 10.3389/fcell.2021.570451PMC PMC7905026
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Abstract

Spinal cord ischemia-reperfusion (SCIR) injury is a serious complication of open surgical and endovascular aortic procedures. MicroRNA-132-3p (miR-132-3p) has been reported to be involved in the progression of various diseases, but its role in SCIR injury is unclear. Thus, we aimed in this study to investigate the mechanism of miR-132-3p in SCIR injury and explore its pathway as a therapeutic target for SCIR injury. We first constructed a SCIR injury rat model and documented motor function in the model. Reverse transcription quantitative polymerase chain reaction (RT-qPC)R and Western blot analysis were used to detect the expression of miR-132-3p and mitogen-activated protein kinase kinase kinase 3 (MEKK3) in SCIR injury rats. The interaction between miR-132-3p and MEKK3 was identified by dual-luciferase reporter gene assay. Then, the effects of miR-132-3p and MEKK3 on macrophage M1 polarization were evaluated in vitro and in vivo by altering their expression in macrophages of SCIR injury rats, with treatments altering the nuclear factor-kappaB (NF-κB) and c-Jun N-terminal kinase (JNK)/p38 signaling pathways using SP600125, SB203580, or PDTC. The SCIR injury rats had a high Tarlov score and low miR-132-3p expression along with high MEKK3 expression. miR-132-3p could directly bind to MEKK3, and that macrophage M1 polarization and inflammation could be inhibited by overexpression of miR-132-3p through downregulating MEKK3 and inactivating the NF-κB and p38/JNK signaling pathways. Besides, increased miR-132-3p expression could decrease the injured rat Tarlov score. Overall, our study demonstrated that miR-132-3p can suppress M1 polarization of macrophages and alleviate SCIR injury by blocking the MEKK3-dependent activation of the NF-κB and p38/JNK signaling pathway. Thus, miR-132-3p and its downstream pathways may be useful targets to alleviate the symptoms of SCIR injury.

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Figures

FIGURE 1 Spinal cord tissues of SCIR injury rats exhibit low expression of miR-132-3p. (A) Tarlov score of SCIR injury rats and sham-operated rats. (B) Pathological features of spinal cord neurons in spinal cord tissues of SCIR injury rats and sham-operated rats observed by HE staining. (C) Expression of CD68 (a macrophage marker) in spinal cord tissues of SCIR injury rats and sham-operated rats detected by immunohistochemistry. (D) Expression of miR-132-3p, CD206, and CD86 in spinal cord tissues of SCIR injury rats and sham-operated rats measured by RT-qPCR. *p < 0.05 vs. sham-operated rats. Measurement data were expressed as mean ± standard deviation. n = 8 for rats following each treatment. Comparisons of data in panel (A) were conducted by repeated measures ANOVA and those in panel (D) were conducted by one-way ANOVA.
FIGURE 1 Spinal cord tissues of SCIR injury rats exhibit low expression of miR-132-3p. (A) Tarlov score of SCIR injury rats and sham-operated rats. (B) Pathological features of spinal cord neurons in spinal cord tissues of SCIR injury rats and sham-operated rats observed by HE staining. (C) Expression of CD68 (a macrophage marker) in spinal cord tissues of SCIR injury rats and sham-operated rats detected by immunohistochemistry. (D) Expression of miR-132-3p, CD206, and CD86 in spinal cord tissues of SCIR injury rats and sham-operated rats measured by RT-qPCR. *p < 0.05 vs. sham-operated rats. Measurement data were expressed as mean ± standard deviation. n = 8 for rats following each treatment. Comparisons of data in panel (A) were conducted by repeated measures ANOVA and those in panel (D) were conducted by one-way ANOVA.CC BY · PubMed Central
FIGURE 2 miR-132-3p inhibits M1 polarization of macrophages from SCIR injury rats. The macrophages from SCIR injury rats were transfected with miR-132-3p mimic, miR-132-3p inhibitor, or their corresponding NC plasmids. (A) Expression of miR-132-3p in macrophages detected by RT-qPCR. (B) Expression of CD86 (a specific markers of M1 phenotype) and CD206 (a specific marker of M2 phenotype) in macrophages detected by flow cytometry. (C) Expression of iNOS, TNF-α, IL-10, and Arg-1 in macrophages measured by RT-qPCR. (D) Contents of TNF-α and IL-10 in macrophages determined by ELISA. (E) Expression of iNOS and Arg-1 in macrophages measured by immunofluorescence staining. *p < 0.05 vs. macrophages transfected with NC mimic. #p < 0.05 vs. macrophages transfected with NC inhibitor. Measurement data were expressed as mean ± standard deviation. Differences between two groups were compared by unpaired t-test and those among multiple groups were compared by one-way ANOVA. The experiment was repeated three times independently.
FIGURE 2 miR-132-3p inhibits M1 polarization of macrophages from SCIR injury rats. The macrophages from SCIR injury rats were transfected with miR-132-3p mimic, miR-132-3p inhibitor, or their corresponding NC plasmids. (A) Expression of miR-132-3p in macrophages detected by RT-qPCR. (B) Expression of CD86 (a specific markers of M1 phenotype) and CD206 (a specific marker of M2 phenotype) in macrophages detected by flow cytometry. (C) Expression of iNOS, TNF-α, IL-10, and Arg-1 in macrophages measured by RT-qPCR. (D) Contents of TNF-α and IL-10 in macrophages determined by ELISA. (E) Expression of iNOS and Arg-1 in macrophages measured by immunofluorescence staining. *p < 0.05 vs. macrophages transfected with NC mimic. #p < 0.05 vs. macrophages transfected with NC inhibitor. Measurement data were expressed as mean ± standard deviation. Differences between two groups were compared by unpaired t-test and those among multiple groups were compared by one-way ANOVA. The experiment was repeated three times independently.CC BY · PubMed Central
FIGURE 3 Spinal cord tissues of SCIR injury rats present with high expression of MEKK3 which is a target gene of miR-132-3p. (A) mRNA expression of MEKK3 in spinal cord tissues of sham-operated rats and SCIR injury rats measured by RT-qPCR. (B) Protein expression of MEKK3 in spinal cord tissues of sham-operated rats and SCIR injury rats measured by Western blot analysis; n = 8. (C) Expression of MEKK3 and CD68 in spinal cord tissues of sham-operated rats and SCIR injury rats detected by immunofluorescence staining. (D) Binding sites of miR-132-3p to the 3′UTR of MEKK3 mRNA predicated by a bioinformatics database. (E) Luciferase activity of cells co-transfected with miR-132-3p mimic and MEKK3-WT or MEKK3-MUT detected by dual-luciferase reporter gene assay. (F) mRNA expression of MEKK3 in cells treated with miR-132-3p mimic, miR-132-3p inhibitor, or their NC plasmids detected by RT-qPCR. (G) Protein expression of MEKK3 in cells treated with miR-132-3p mimic, miR-132-3p inhibitor, or their NC plasmids measured by Western blot analysis. ∗p < 0.05 vs. macrophages transfected with NC mimic. #p < 0.05 vs. macrophages transfected with NC inhibitor. n = 8 for rats following each treatment. Measurement data were expressed as mean ± standard deviation. Differences between two groups were compared by unpaired t-test and those among multiple groups were compared by one-way ANOVA. The experiment was repeated three times independently.
FIGURE 3 Spinal cord tissues of SCIR injury rats present with high expression of MEKK3 which is a target gene of miR-132-3p. (A) mRNA expression of MEKK3 in spinal cord tissues of sham-operated rats and SCIR injury rats measured by RT-qPCR. (B) Protein expression of MEKK3 in spinal cord tissues of sham-operated rats and SCIR injury rats measured by Western blot analysis; n = 8. (C) Expression of MEKK3 and CD68 in spinal cord tissues of sham-operated rats and SCIR injury rats detected by immunofluorescence staining. (D) Binding sites of miR-132-3p to the 3′UTR of MEKK3 mRNA predicated by a bioinformatics database. (E) Luciferase activity of cells co-transfected with miR-132-3p mimic and MEKK3-WT or MEKK3-MUT detected by dual-luciferase reporter gene assay. (F) mRNA expression of MEKK3 in cells treated with miR-132-3p mimic, miR-132-3p inhibitor, or their NC plasmids detected by RT-qPCR. (G) Protein expression of MEKK3 in cells treated with miR-132-3p mimic, miR-132-3p inhibitor, or their NC plasmids measured by Western blot analysis. ∗p < 0.05 vs. macrophages transfected with NC mimic. #p < 0.05 vs. macrophages transfected with NC inhibitor. n = 8 for rats following each treatment. Measurement data were expressed as mean ± standard deviation. Differences between two groups were compared by unpaired t-test and those among multiple groups were compared by one-way ANOVA. The experiment was repeated three times independently.CC BY · PubMed Central
FIGURE 4 miR-132-3p inhibits the M1 polarization of macrophages from SCIR injury rats by down-regulating MEKK3. The macrophages were transfected with both si-NC and NC inhibitor, both si-MEKK3 and NC inhibitor, or both miR-132-3p inhibitor and si-MEKK3. (A) Expression of CD86 (a specific marker of M1 phenotype) and CD206 (a specific marker of M2 phenotype) in macrophages detected by flow cytometry. (B) Expression of inflammatory factors iNOS, TNF-α, IL-10, and Arg-1 in macrophages detected by RT-qPCR. (C) The level of TNF-α and IL-10 in macrophages measured by ELISA. (D) Expression of iNOS and Arg-1 in macrophages detected by immunofluorescence staining. *p < 0.05 vs. macrophages transfected with both si-NC and NC inhibitor. Measurement data were expressed as mean ± standard deviation. Comparisons of data among multiple groups were conducted by one-way ANOVA. The experiment was repeated three times independently.
FIGURE 4 miR-132-3p inhibits the M1 polarization of macrophages from SCIR injury rats by down-regulating MEKK3. The macrophages were transfected with both si-NC and NC inhibitor, both si-MEKK3 and NC inhibitor, or both miR-132-3p inhibitor and si-MEKK3. (A) Expression of CD86 (a specific marker of M1 phenotype) and CD206 (a specific marker of M2 phenotype) in macrophages detected by flow cytometry. (B) Expression of inflammatory factors iNOS, TNF-α, IL-10, and Arg-1 in macrophages detected by RT-qPCR. (C) The level of TNF-α and IL-10 in macrophages measured by ELISA. (D) Expression of iNOS and Arg-1 in macrophages detected by immunofluorescence staining. *p < 0.05 vs. macrophages transfected with both si-NC and NC inhibitor. Measurement data were expressed as mean ± standard deviation. Comparisons of data among multiple groups were conducted by one-way ANOVA. The experiment was repeated three times independently.CC BY · PubMed Central
FIGURE 5 miR-132-3p upregulation hinders M1 polarization of macrophage from SCIR injury rats by inactivating the NF-κB and p38/JNK signaling pathways via MEKK3 downregulation. (A) Protein expression of JNK, p38, and IKK-β as well as the extent of JNK, p38, and IKK-β phosphorylation measured by Western blot analysis in macrophages transfected with miR-132-3p mimic or miR-132-3p inhibitor. (B) NF-κB luciferase activity detected by dual-luciferase reporter gene assay in macrophages transfected with miR-132-3p mimic or miR-132-3p inhibitor. (C) Protein expression of JNK, p38, and IKK-β as well as the extent of JNK, p38, and IKK-β phosphorylation measured by western blot analysis in macrophages transfected with si-MEKK3 or in combination with miR-132-3p inhibitor. (D) NF-κB luciferase activity detected by dual-luciferase reporter gene assay in macrophages transfected with si-MEKK3 or in combination with miR-132-3p inhibitor. *p < 0.05 vs. macrophages transfected with NC mimic. #p < 0.05 vs. macrophages transfected with NC inhibitor. &p < 0.05 vs. macrophages transfected with both si-NC and NC inhibitor. (E) Expression of inflammatory factors iNOS, TNF-α, IL-10, and Arg-1 in macrophages after treatment with SP600125, SB203580, or PDTC detected by RT-qPCR. (F) The level of TNF-α and IL-10 in macrophages after treatment with SP600125, SB203580, or PDTC measured by ELISA. *p < 0.05 vs. macrophages treated with DMSO. (G) Expression of iNOS and Arg-1 in macrophages after treatment with SP600125, SB203580, or PDTC detected by immunofluorescence staining. *p < 0.05 vs. macrophages treated with DMSO. Measurement data were expressed as mean ± standard deviation. Differences between two groups were compared by unpaired t-test. Comparisons of data among multiple groups were conducted by one-way ANOVA. The experiment was repeated three times independently.
FIGURE 5 miR-132-3p upregulation hinders M1 polarization of macrophage from SCIR injury rats by inactivating the NF-κB and p38/JNK signaling pathways via MEKK3 downregulation. (A) Protein expression of JNK, p38, and IKK-β as well as the extent of JNK, p38, and IKK-β phosphorylation measured by Western blot analysis in macrophages transfected with miR-132-3p mimic or miR-132-3p inhibitor. (B) NF-κB luciferase activity detected by dual-luciferase reporter gene assay in macrophages transfected with miR-132-3p mimic or miR-132-3p inhibitor. (C) Protein expression of JNK, p38, and IKK-β as well as the extent of JNK, p38, and IKK-β phosphorylation measured by western blot analysis in macrophages transfected with si-MEKK3 or in combination with miR-132-3p inhibitor. (D) NF-κB luciferase activity detected by dual-luciferase reporter gene assay in macrophages transfected with si-MEKK3 or in combination with miR-132-3p inhibitor. *p < 0.05 vs. macrophages transfected with NC mimic. #p < 0.05 vs. macrophages transfected with NC inhibitor. &p < 0.05 vs. macrophages transfected with both si-NC and NC inhibitor. (E) Expression of inflammatory factors iNOS, TNF-α, IL-10, and Arg-1 in macrophages after treatment with SP600125, SB203580, or PDTC detected by RT-qPCR. (F) The level of TNF-α and IL-10 in macrophages after treatment with SP600125, SB203580, or PDTC measured by ELISA. *p < 0.05 vs. macrophages treated with DMSO. (G) Expression of iNOS and Arg-1 in macrophages after treatment with SP600125, SB203580, or PDTC detected by immunofluorescence staining. *p < 0.05 vs. macrophages treated with DMSO. Measurement data were expressed as mean ± standard deviation. Differences between two groups were compared by unpaired t-test. Comparisons of data among multiple groups were conducted by one-way ANOVA. The experiment was repeated three times independently.CC BY · PubMed Central
FIGURE 6 miR-132-3p ameliorates SCIR injury in vivo. SCIR injury rats were treated with miR-132-3p agomir or NC agomir. (A) Motor function of rats assessed by Tarlov score. (B) Histological characteristics of spinal cord tissues of rats observed using HE staining. (C) The protein level of MEKK3, JNK, p38, and IKK-β as well as the phosphorylated JNK, p38, and IKK-β levels measured by Western blot analysis in rat spinal cord tissues. (D) Expression of iNOS and Arg-1 in spinal cord tissues of rats detected by immunofluorescence staining. *p < 0.05 vs. rats treated with NC agomir. Measurement data were expressed as mean ± standard deviation. n = 9 for rats following each treatment. Differences between two groups were compared by unpaired t-test. The data in panel (A) were analyzed by repeated measures ANOVA.
FIGURE 6 miR-132-3p ameliorates SCIR injury in vivo. SCIR injury rats were treated with miR-132-3p agomir or NC agomir. (A) Motor function of rats assessed by Tarlov score. (B) Histological characteristics of spinal cord tissues of rats observed using HE staining. (C) The protein level of MEKK3, JNK, p38, and IKK-β as well as the phosphorylated JNK, p38, and IKK-β levels measured by Western blot analysis in rat spinal cord tissues. (D) Expression of iNOS and Arg-1 in spinal cord tissues of rats detected by immunofluorescence staining. *p < 0.05 vs. rats treated with NC agomir. Measurement data were expressed as mean ± standard deviation. n = 9 for rats following each treatment. Differences between two groups were compared by unpaired t-test. The data in panel (A) were analyzed by repeated measures ANOVA.CC BY · PubMed Central
FIGURE 7 Schematic diagram of the mechanism of miR-132-3p in SCIR injury rats. miR-132-3p targets MEKK3 and negatively regulates the MEKK3 expression to inhibit activation of the NF-κB and p38/JNK signaling pathways, thus inhibiting the M1 polarization of macrophages and further alleviating the motor dysfunction caused by SCIR injury.
FIGURE 7 Schematic diagram of the mechanism of miR-132-3p in SCIR injury rats. miR-132-3p targets MEKK3 and negatively regulates the MEKK3 expression to inhibit activation of the NF-κB and p38/JNK signaling pathways, thus inhibiting the M1 polarization of macrophages and further alleviating the motor dysfunction caused by SCIR injury.CC BY · PubMed Central

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Metadata

Authors
Fang, H., Li, H.-F., Pan, Q., Jin, H.-L., Yang, M., Wang, R.-R., Wang, Q.-Y., & Zhang, J.-P.
Venue
Frontiers in Cell and Developmental Biology
Publisher
Frontiers Media SA
Published
2021-02-11
Volume
9
ISSN
2296-634X
DOI
10.3389/fcell.2021.570451
PMC
PMC7905026
Cited by
26
References
42
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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