Hsa_circRNA_100791 Modulates Trim13 Through Sponging miR-487b-5p to Facilitate Inflammation in Allergic Rhinitis

Abstract

Background: Circular RNAs (circRNAs) are a novel class of endogenous non-coding RNA molecules in eukaryotes, involved in many essential biological processes. However, their role in allergic rhinitis (AR) has not been extensively studied.

Methods: The expression levels of hsa_circRNA_100791 were measured using qRT-PCR in peripheral blood mononuclear cells (PBMCs) and nasal mucosa from AR patients. The biological function of hsa_circRNA_100791 in AR was investigated through RNA-seq and a series of in vitro experiments. Western blotting, luciferase reporter assays, and rescue experiments were conducted to elucidate the molecular mechanisms underlying hsa_circRNA_100791. Additionally, a mouse model was used to assess the functional role of hsa_circRNA_100791 in vivo.

Results: Upregulation of hsa_circRNA_100791 was observed in both PBMCs and nasal mucosa of AR patients. In vitro, increased expression of hsa_circRNA_100791 promoted the production of pro-inflammatory mediators (IL-1β, IL-4, IL-5, IL-6, IL-8, IL-13, IL-17, IL-18, IL-33, TNF-α, and NF-κB) and inhibited IL-2 and IFN-γ. Conversely, knockdown of hsa_circRNA_100791 both in vitro and in vivo alleviated AR symptoms, reduced pro-inflammatory mediators, and enhanced IL-2 and IFN-γ levels. Mechanistically, we found hsa_circRNA_100791 contributing to the pathological processes of AR, which upregulate TRIM13 via sponging miR-487b-5p.

Conclusion: Our study demonstrated that hsa_circRNA_100791 mitigates the inhibitory effect of miR-487b-5p on Trim13 by directly binding to miR-487b-5p. This interaction regulates the expression of inflammatory factors and facilitates AR. Thus, hsa_circRNA_100791 could be a promising new therapeutic target for AR.

Keywords: NF-κB; Trim13; allergic rhinitis; diagnosis; hsa_circ_100791; inflammation; miR-487b-5p.

Figure 1

Hsa_circ_100791 expression was increased in AR patients compared to healthy controls. (A and B) hsa_circ_100791 expression was elevated in both PBMCs and nasal mucosa from AR patients compared to healthy controls. (C) Schematic diagram illustrating that hsa_circ_100791 is generated from exons 13, 14, and 15 of the CAPRIN1 gene. (D) Sanger sequencing of hsa_circ_100791 PCR products, with the red arrow indicating the head-to-tail splicing site. (E and F) FISH analysis showing abundant expression of 18S and hsa_circ_100791 in the cytoplasm of human nasal epithelial cells (HNEpCs). Nuclei were stained with DAPI, and 18S and hsa_circ_100791 were labeled with Cy3. (G and H) FISH analysis demonstrating the abundant expression of hsa_circ_100791 in the cytoplasm of nasal mucosa from both healthy controls and AR patients. (I) PCR amplification of hsa_circ_100791 using convergent and divergent primers on cDNA and genomic DNA from HNEpCs. Results indicate successful amplification of hsa_circ_100791 with divergent primers using cDNA, but not with genomic DNA. (J) qRT-PCR analysis of RNA extracted from HNEpCs treated with RNase R, showing that hsa_circ_100791 is more stable than linear CAPRIN1 and GAPDH. Data are presented as mean ± SEM.

Figure 2

Hsa_circ_100791 regulates the expression of inflammatory factors in HNEpCs. (A) qRT-PCR confirmed the transfection efficiency of the hsa_circ_100791 overexpression vector or siRNA in HNEpCs. (B) The heatmap shows the differentially expressed genes between si-circ-100791 groups and NC groups, as analyzed by RNA-seq. (C) The biological process of gene ontology (GO) analysis of DEGs related to hsa_circ_100791 knockdown. (D) KEGG analysis of the DEGs related to hsa_circ_100791 knockdown. (E–N) qRT-PCR analysis shows the mRNA expression level of IL-1β, IL-4, IL-5, IL-6, IL-8, IL-13, IL-17, IL-18, IL-33 and TNF-α was significantly enhanced in hsa_circ_100791 overexpression group and obviously reduced in hsa_circ_100791 knockdown group. (O and P) qRT-PCR analysis shows the mRNA expression level of IL-2, IFN-γ was significantly reduced in hsa_circ_100791 overexpression group and obviously enhanced in hsa_circ_100791 knockdown group. (Q) Western blot analysis further revealed that the protein expression levels of IL-4, IL-5 and IL-13 was enhanced in hsa_circ_100791 overexpression group and reduced in hsa_circ_100791 knockdown group. Data were presented as mean ± SEM. N= 3 for each group.

Figure 3

Continued.

Figure 3

MiR-487b-5p is low expression in AR patients and mediates expression of inflammatory cytokines. (A) The 5 candidate miRNAs have binding sites for hsa_circ_100791. (B and C) qRT-PCR analysis shows the expression level of 5 candidate miRNAs after being transfected with si-hsa_circ_100791 and NC or hsa_circ_100791 overexpression and control vector into HNEpCs. (D and E) qRT-PCR analysis shows that the miR-487b-5p was decreased in nasal mucosa of the AR patients. And statistical analysis indicated that negative correlation between hsa_circ_100791 and miR-487b-5p. (F) The qRT-PCR verified transfection efficiency of transfected the miR-487b-5p mimics or inhibitor into HNEpCs. (G–P) qRT-PCR analysis shows the mRNA expression level of IL-1β, IL-4, IL-5, IL-6, IL-8, IL-13, IL-17, IL-18, IL-33 and TNF-α was significantly reduced in miR-487b-5p mimics group, but obviously enhanced in miR-487b-5p inhibitor group. (Q and R) qRT-PCR analysis shows the mRNA expression level of IL-2, IFN-γ significantly enhanced in miR-487b-5p mimics group, but obviously reduced in miR-487b-5p inhibitor group. (S) Western blot analysis further revealed that the protein expression levels of IL-4, IL-5 and IL-13 was significantly reduced in miR-487b-5p mimics group, but obviously enhanced in miR-487b-5p inhibitor group. Data were presented as mean ± SEM. N= 3 for each group.

Figure 4

MiR-487b-5p induces allergic responses of HNEpCs in vitro by targeting TRIM13. (A) The sequences of 3ʹUTR for TRIM13, which are complementary to miR-487b-5p seed sequence. WT represent wild-type and Mut represent mutant sequences. (B and C) qRT-PCR analysis shows that the expression of TRIM13 was increased in nasal mucosa of the AR patients (n=23) compared with nasal mucosal tissues of healthy controls (n=21). And statistical analysis indicated that negative correlation between TRIM13 and miR-487b-5p in nasal mucosal tissues. (D and E) The luciferase reporter assays results demonstrated that the luciferase activity of TRIM13-wild-type version significantly increased when miR-487b-5p knockdown in HNEpCs, while decreased when miR-487b-5p mimics in HNEpCs. However, the luciferase activity of Trim13- mutant version was unchanged. (F) qRT-PCR analysis result showed that the expression mRNA level of TRIM13 was significantly reduced in miR-487b-5p mimics group, but obviously enhanced in miR-487b-5p inhibitor group. (G) Western blot analysis result showed that the expression protein level of Trim13, p65 and p-p65 was significantly reduced in miR-487b-5p mimics group, but obviously enhanced in miR-487b-5p inhibitor group. Data were presented as mean ± SEM. Data are pooled from 3 independent experiments.

Figure 5

Hsa_circ_100791 acts as a sponge for miR-487b-5p to regulate TRIM13 in HNEpCs. (A) The databases predicted the sequences of hsa_circ_100791, which are complementary to miR-487b-5p seed sequence. (B and C) The luciferase reporter assays results showed that the luciferase activity of wild-type version significantly reduced when miR-487b-5p mimics in HNEpCs, while enhanced when miR-487b-5p knockdown in HNEpCs. But, the luciferase activity of mutant version was obviously different. (D and E) FISH assay revealed that hsa_circ_100791 (red) localized with miR-487b-5p (green) in the cytoplasm. Nuclei were stained with DAPI. Hsa_circ_100791 and miR-487b-5p were labeled with Cy3 and FAM, respectively. (F) qRT-PCR analysis result showed that the expression mRNA level of TRIM13 was significantly enhanced in hsa_circ_100791 overexpression group, but obviously reduced in hsa_circ_100791 knockdown group. (G and H) Western blot analysis result showed that the expression protein level of Trim13, p65 and p-p65 was significantly increased in hsa_circ_100791 overexpression group, whereas decreased in hsa_circ_100791 knockdown group. Data were presented as mean ± SEM. N=3 for each group.

Figure 6

Hsa_circ_100791 exerts biological functions in AR by regulating the expression of miR-487b-5p. (A) qRT-PCR analysis of the expression protein level of TRIM13 in HNEpCs co-transfected with vector+ mimics NC or oe- circ-100791 + mimics NC or oe- circ-100791 + miR-487b-5p mimics. (B) Western blot analysis of the expression protein level of Trim13 in HNEpCs co-transfected with vector+ mimics NC or oe- circ-100791 + mimics NC or oe- circ-100791 + miR-487b-5p mimics. (C–G) qRT-PCR analysis of the expression protein level of IL-4, IL-5, IL-33, TNF-α and IFN-γ in HNEpCs co-transfected with vector+ mimics NC or oe- circ-100791 + mimics NC or oe- circ-100791 + miR-487b-5p mimics. Data were presented as mean ± SEM. N=3 for each group.

Figure 7

Knockdown hsa_circ_100791 increase the expression of miR-487b-5p and reduced the nasal symptoms of AR in OVA-induced AR mice. (A) Schematic diagram of mouse model. (B–D) qRT-PCR analysis of the expression of hsa_circ_100791, miR-487b-5p and TRIM13 in the OVA group or the OVA+ si-circ100791 or the OVA+ circ-NC group. (E and F) The frequencies of nose rubbing and sneezing were record in the OVA group or the OVA+ si-circ100791 or the OVA+ circ-NC group. (G–I) qRT-PCR analysis of the expression of IL-4, IL-5 and IL-6 in the OVA group or the OVA+ si-circ100791 or the OVA+ circ-NC group. (J and K) HE analysis of the number of eosinophils in the OVA group or the OVA+ si-circ100791 or the OVA+ circ-NC group. (L and M) IHC analysis of the expression protein level of Trim13 in the OVA group or the OVA+ si-circ100791 or the OVA+ circ-NC group. (N) IF analysis showed that the Trim13 colocalized in the cytoplasm and the expression protein level of Trim13 in the OVA group or the OVA+ si-circ100791 or the OVA+ circ-NC group. Data were presented as mean ± SEM. N=6 for each group.

Figure 8

Schematic show that the mechanism of hsa_circ_100791 promote the progression of allergic rhinitis. Hsa_circ_100791 could increase the expression of TRIM13 by sponging miR-487b-5p, and consequently activate the NF-κB signaling pathway, and upregulates the expression of pro-inflammatory factors (IL-4, IL-5 and IL-13, IL-1β, IL-6, IL-8, IL-17, IL-18, IL-33 and TNF-α), inhibits the expression of Th1-related inflammatory factors (IL-2 and IFN-γ), and ultimately promotes the the inflammatory response of AR.