Calcitonin gene‑related peptide induces IL‑6 expression in RAW264.7 macrophages mediated by mmu_circRNA_007893

Tian Deng, Lan Yang, Zhichao Zheng, Yuanjing Li, Wen Ren, Caijuan Wu, Lvhua Guo, Tian Deng, Lan Yang, Zhichao Zheng, Yuanjing Li, Wen Ren, Caijuan Wu, Lvhua Guo

Abstract

Several circular RNAs (circRNAs) may have role important roles in biological processes, however, there is limited knowledge of circRNAs and their potential functions in RAW264.7 macrophages. The present study aimed to examine the expression of circRNAs and explore their effects on interleukin‑6 (IL‑6) expression induced by calcitonin gene‑related peptide (CGRP) in RAW264.7 macrophages. To identify circRNAs, the circRNA expression was measured in macrophages with or without CGRP stimulation. The interaction between circRNAs and microRNAs (miRs) were then identified using bioinformatic software and networks. In the current study, it was demonstrated that CGRP increased the expression of IL‑6 mRNA in a dose‑ and time‑dependent manner. Furthermore, mmu_circRNA_007893 was significantly increased in the CGRP‑stimulated macrophages. Silencing of mmu_circRNA_007893, IL‑6 mRNA expression was significantly decreased, whereas mmu‑miR‑485‑5p expression was markedly increased. Furthermore, when overexpression of mmu‑miR‑485‑5p, IL‑6 mRNA was markedly decreased. The results demonstrated that CGRP‑induced IL‑6 mRNA expression was mediated by mmu_circRNA_007893, and mmu_circRNA_007893 functioned as an endogenous mmu‑miR‑485‑5p sponge as part of induction of IL‑6 mRNA expression.

Figures

Figure 1.
Figure 1.
CGRP induced changes to the IL-6 mRNA levels in a dose- and time-dependent manner. (A) Cells were treated for up to 2 h with CGRP at concentration ranging from 0.01 to 100 nM. The optimum concentration was 1 nM and the IL-6 mRNA expression in following treatment with 0.1 nM (P=0.019) and 1 nM (P=0.011) were significantly higher than that in the 0 nM group. (B) Cells were treated with CGRP at concentration of 1 nM for 1–4 h. The optimum treatment duration was 2 h and the IL-6 mRNA expression at 2 h (P=0.011) and 3 h (P=0.044) was significantly higher than that in the 0 h group. The IL-6 mRNA was detected by reverse transcription-quantitative polymerase chain reaction. The values are presented as the mean ± standard error. *P

Figure 2.

Upregulated circRNAs identified by in…

Figure 2.

Upregulated circRNAs identified by in the circRNA microarray. (A) Top four upregulated circRNAs…

Figure 2.
Upregulated circRNAs identified by in the circRNA microarray. (A) Top four upregulated circRNAs according to fold change. (B) Top four upregulated circRNA according to maximum miRNA binding sites. (C) Eight circRNAs and 24 predicted target miRNAs. In this network, the circle represents the circRNA and the diamond represents miRNAs. mmu, Mus musculus; circRNA, circular RNA; miRNA, microRNA.

Figure 3.

Validation of mmu_circRNA_007893 expression. (A)…

Figure 3.

Validation of mmu_circRNA_007893 expression. (A) Fold change in expression of mmu_circRNA_007893 in RAW264.7…

Figure 3.
Validation of mmu_circRNA_007893 expression. (A) Fold change in expression of mmu_circRNA_007893 in RAW264.7 macrophages is determined by reverse transcription-quantitative polymerase chain reaction. The result was in accordance with the results of the microarray. Fold change in subject group normalized to the result of the microarray which showed no significant difference (P=0.107; n=4 for each group). (B) Fluorescence in situ hybridization images of mmu_circRNA_007893 from the circRNA microarray, demonstrating the localization of circRNAs and nuclei of RAW264.7 macrophages (scale bar, 7.5 µm). circRNA, circular RNA; mmu, Mus musculus.

Figure 4.

Effects of mmu_circRNA_007893 inhibition on…

Figure 4.

Effects of mmu_circRNA_007893 inhibition on IL-6 mRNA expression in RAW264.7 macrophages. (A) Cells…

Figure 4.
Effects of mmu_circRNA_007893 inhibition on IL-6 mRNA expression in RAW264.7 macrophages. (A) Cells were treated with three siRNAs targeting mmu_circRNA_007893. circRNA expressions in siRNA1 groups (P=0.001), siRNA2 groups (P=0.003) and siRNA3 groups (PMus musculus; circRNA, circular RNA; siRNA, small interfering RNA; NC, negative control; CGRP, calcitonin gene-related peptide; IL-6, interleukin-6.

Figure 5.

mmu_circRNA_007893 regulates IL-6 mRNA expression…

Figure 5.

mmu_circRNA_007893 regulates IL-6 mRNA expression by targeting mmu-miR-485-5p. (A) mmu-miR-485-5p matched five regions…

Figure 5.
mmu_circRNA_007893 regulates IL-6 mRNA expression by targeting mmu-miR-485-5p. (A) mmu-miR-485-5p matched five regions in the complete sequence of mmu_circRNA_007893. (B) mmu-miR-485-5p expression in CGRP groups significantly decreased relative to that in the negative control groups (P=0.037). miRNA expression in CGRP + siRNA groups was significantly higher than that in CGRP groups (P=0.001). (C) IL-6 mRNA expression in CGRP groups was significantly higher than that in negative control groups (P=0.014). Expression level in CGRP + mimics groups decreased following overexpression of mmu-miR-485-5p using mimics, which was markedly lower than that in the CGRP groups (P=0.001). IL-6 mRNA expression in CGRP + siRNA group was significantly higher than that in the negative control group (P=0.014). The IL-6 mRNA and mmu-miR-485-5p was detected by reverse transcription-quantitative polymerase chain reaction. The values are presented as the mean ± standard error. *PMus musculus; circRNA, circular RNA; NC, negative control; CGRP, calcitonin gene-related peptide; siRNA, small interfering RNA; IL-6, interleukin-6.
Figure 2.
Figure 2.
Upregulated circRNAs identified by in the circRNA microarray. (A) Top four upregulated circRNAs according to fold change. (B) Top four upregulated circRNA according to maximum miRNA binding sites. (C) Eight circRNAs and 24 predicted target miRNAs. In this network, the circle represents the circRNA and the diamond represents miRNAs. mmu, Mus musculus; circRNA, circular RNA; miRNA, microRNA.
Figure 3.
Figure 3.
Validation of mmu_circRNA_007893 expression. (A) Fold change in expression of mmu_circRNA_007893 in RAW264.7 macrophages is determined by reverse transcription-quantitative polymerase chain reaction. The result was in accordance with the results of the microarray. Fold change in subject group normalized to the result of the microarray which showed no significant difference (P=0.107; n=4 for each group). (B) Fluorescence in situ hybridization images of mmu_circRNA_007893 from the circRNA microarray, demonstrating the localization of circRNAs and nuclei of RAW264.7 macrophages (scale bar, 7.5 µm). circRNA, circular RNA; mmu, Mus musculus.
Figure 4.
Figure 4.
Effects of mmu_circRNA_007893 inhibition on IL-6 mRNA expression in RAW264.7 macrophages. (A) Cells were treated with three siRNAs targeting mmu_circRNA_007893. circRNA expressions in siRNA1 groups (P=0.001), siRNA2 groups (P=0.003) and siRNA3 groups (PMus musculus; circRNA, circular RNA; siRNA, small interfering RNA; NC, negative control; CGRP, calcitonin gene-related peptide; IL-6, interleukin-6.
Figure 5.
Figure 5.
mmu_circRNA_007893 regulates IL-6 mRNA expression by targeting mmu-miR-485-5p. (A) mmu-miR-485-5p matched five regions in the complete sequence of mmu_circRNA_007893. (B) mmu-miR-485-5p expression in CGRP groups significantly decreased relative to that in the negative control groups (P=0.037). miRNA expression in CGRP + siRNA groups was significantly higher than that in CGRP groups (P=0.001). (C) IL-6 mRNA expression in CGRP groups was significantly higher than that in negative control groups (P=0.014). Expression level in CGRP + mimics groups decreased following overexpression of mmu-miR-485-5p using mimics, which was markedly lower than that in the CGRP groups (P=0.001). IL-6 mRNA expression in CGRP + siRNA group was significantly higher than that in the negative control group (P=0.014). The IL-6 mRNA and mmu-miR-485-5p was detected by reverse transcription-quantitative polymerase chain reaction. The values are presented as the mean ± standard error. *PMus musculus; circRNA, circular RNA; NC, negative control; CGRP, calcitonin gene-related peptide; siRNA, small interfering RNA; IL-6, interleukin-6.

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