hnRNPUL1-AS

Description

In previous studies, we verified that nonylphenols (NPs) facilitate the occurrence of allergic rhinitis (AR). By analyzing data from the Gene Expression Omnibus database and subsequent verification with clinical samples, we confirmed that the A3SS alternative splicing of TRAF2 and NCK interacting kinase (TNIK) plays a significant role in AR. Meanwhile, we discovered that NP could suppress the expression of hnRNPUL1 and increase the alternative splicing ratio of TNIK in the nasal mucosa of mice. Further studies revealed that downregulation of the splicing factor hnRNPUL1 promoted A3SS alternative splicing in TNIK, leading to the deposition of β-catenin in nasal mucosa tissue and enhanced binding of β-catenin with TCF1 and TCF4. This binding, in turn, increased the expression of GATA3 and decreased that of Th1 cell transcription factor T-bet and Treg cell transcription factor Foxp3, driving the immune response towards Th2 polarization. A mere increase in β-catenin expression sufficiently promoted the proliferation of all helper T cell subsets; however, it did not induce any changes in Th cell differentiation. Under NP intervention, the expression of hnRNPUL1 was more reduced, Th2 polarization in local tissues was more pronounced, and symptoms in mice were more severe than under non-NP intervention. These findings confirm that NP intake can interfere with the transcription process and exacerbate the development of AR.

Steps to reproduce

1. Clinical Samples Nasal mucosal samples (exfoliated cells from 77 AR patients and 16 healthy individuals; tissues from 9 AR and 5 non-AR patients) were collected during endoscopic exams or surgery. AR diagnosis was based on clinical history, nasal endoscopy, allergen tests, and IgE levels. Samples were stored at –80°C. No corticosteroids were used 4 weeks pre-collection. Ethics approval: No. 2024PS695K. 2. Murine AR Model Thirty 8-week-old female C57BL/6 mice (18 ± 2 g) were housed under standard conditions. AR was induced in OVA and NP+OVA groups via intraperitoneal OVA/aluminum hydroxide sensitization (days 1–15) followed by intranasal OVA challenge (days 16–26). NP+OVA mice received 5 µg/kg/day NP in corn oil. AR symptoms (sneezing/rubbing) were assessed 15 min post-final challenge. Ethics approval: No. 2024PS521K. 3. Histopathology Mouse nasal tissues were fixed in 4% paraformaldehyde, decalcified, paraffin-embedded, and coronally sectioned (4 µm). HE-stained sections were analyzed at 400× magnification. 4. Cell Culture and Transfection HNEpCs (Gineo, China) were cultured in RPMI-1640 with 10% FBS. β-catenin overexpression plasmids (pcDNA3.1-β-catenin) and hnRNPUL1-targeting siRNAs (si-hnRNPUL1-1: 5′-GCUUCGAGAUGAAGAUCAATT-3′; si-hnRNPUL1-2: 5′-GAGACAAGUUUGCAGAGAATT-3′) were transfected using jetPRIME. Cells were harvested 48 h post-transfection. 5. RNA Detection Total RNA (RNAiso Plus) was reverse-transcribed (Roche LightCycler 480 II). qPCR primers (Sangon Biotech) normalized to GAPDH. Mouse cDNA was PCR-amplified (SanTaq Mix), electrophoresed, and sequenced. 6. Immunoblotting Proteins (RIPA lysates) were separated via SDS-PAGE, transferred to PVDF membranes, and probed with antibodies (hnRNPUL1, β-catenin, TCF1/7, TCF4/7L2, T-bet, GATA-3, Foxp3, GAPDH). HRP-conjugated secondary antibodies and chemiluminescence (SuperSignal) were used for detection. ImageJ quantified band intensities. 7. Immunoprecipitation Cell lysates incubated with β-catenin antibody (Cell Signaling) and protein-A/G beads. Complexes were washed, eluted, and analyzed via immunoblotting. 8. Statistical Analysis GSE75011 RNA-seq data were processed via FastQC and EdgeR for DEGs. Alternative splicing (ABLas pipeline) and functional enrichment (KOBAS 2.0, Reactome) were analyzed. PCA and heatmaps used R packages. Data (mean ± SEM) were compared via t-test, ANOVA, or non-parametric tests (Prism 10.0). Significance: p < 0.05, p < 0.01, p < 0.001.

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