LncRNA JPX contributes to Treg/Th17 imbalance in allergic rhinitis <i>via</i> targeting the miR-378g/CCL5 axis

Aim: T-regulatory (Treg)/T-helper (Th) 17 imbalance contributes to the pathogenesis of allergic rhinitis (AR). Long non-coding RNAs (lncRNAs) participate in the progression of AR. Herein, the effect of lncRNA JP X on Treg/Th17 balance in AR was explored. Methods: CD4+ T cells were isolated from patients with AR and healthy control. The percentage of Treg and Th17 cells were examined by flow cytometry. The levels of JP X, miR-378g, CCL5, T GF-β, and IL-17A were tested using qRT-P CR. The protein expression of Foxp3 and RORγt was measured by western blot. Results: The data showed that an imbalance of Treg/Th17 was associated with AR. Upregulation of JP X was found in AR, and knockdown of which improved the imbalance of Treg/Th17. Furthermore, JP X functioned as a sponge of miR-378g to upregulate CCL5. Inhibition of miR-378g reversed the effects on Treg/Th17 induced by silencing of JP X. Moreover, overexpression of CCL5 reversed miR-378g-induced effects. Conclusion: In conclusion, depletion of JP X promoted Treg/Th17 balance in AR via regulating the miR-378g/CCL5 axis. The findings provided a novel therapeutic insight for AR.</p

Morphological change of SMMC-7721 cells and HUVECs in a 3D co-culture model.

<p>(A) SMMC-7721 cells and HUVECs were seeded and cultured in Matrigel. Morphogenesis was observed by using an inverted phase-contrast microscope. (B) These 3D-cultured cell sheets were fixed and cut into sections which were stained with anti CD147 (green) and KDR/Flk (red) antibodies, and DAPI (blue), respectively, and observed using a laser scanning confocal microscope.</p

The angiogenic phenotype was inhibited by knock-down of CD147 in either SMMC-7721 cells (TC) or endothelial cells (EC) in a 3D co-culture model.

<p>(A) CD147 expression was analyzed by western blot in SMMC-7721 cells and HUVECs after transfected with CD147 siRNA or control siRNA for 48 h. (B) <i>In vitro</i> tube formation analysis in Matrigel. After SMMC-7721 cells and HUVECs were transfected with CD147 siRNA or control siRNA respectively, SMMC-7721 cells were co-cultured with HUVEC cells for 48 h in Matrigel. Images were acquired at 6, 24 and 48 h, using an inverted microscope (Olympus CKX41) fitted with a 10× phase-contrast objective lens. (C) Semi-quantitative assessment of tube formation was performed by determining the number of branches per field. Results are based on four randomly selected fields and are expressed as the mean ± SD of three independent experiments. Statistical significance was determined by the Student’s t-test. *<i>P</i><0.05, **<i>P</i><0.001, compared with the control level.</p

HUVEC proliferation, migration and formation of capillary-like structures were assessed.

<p>HUVECs were cultured with TCMs obtained from SMMC-7221 cells transfected with CD147 siRNA or control siRNA in either normoxia or hypoxia conditions for 24 h. (A) HUVECs proliferation was measured using the BrdU incorporation assay. HUVECs were cultured with these TCMs for 3days. The data were presented as absorbance at 490 nm (mean ± SD of three replicate experiments). *<i>P</i><0.05, compared with the control level. (B) The migration ability of HUVECs was examined by the <i>in vitro</i> invasion assay. Cells were first planted on the upper compartment of the Boyden chambers; the lower compartment was filled with various TCMs. After 12h and 24 h of incubation, the number of cells migrating through the filter was counted and plotted as the mean number of migrating cells in an optic field in three independent experiments. *<i>P</i><0.05, compared with the control level. (C) and (D) The formation of capillary-like structures were induced in different TCMs. HUVECs were seeded on top of the Matrigel and cultured for 4 h with TCMs which were mixed with EBM-2 in a 1∶1 ratio. The formation of capillary-like structures was photographed in (C), and Semi-quantitative assessment of tube formation was performed by counting the number of branches per field. *<i>P</i><0.05, compared with the control level.</p

IGF-I induced CD147 expression in dose-dependent fashion in multiple tumor cells.

<p>(A, B) qRT-PCR (A) and Western blot (B) analysis of CD147 expression level in SMMC-7721, HepG-2, A549, and MCF-7 cells in response to stimulation by IGF-I for 24 h.</p

Knock-down of CD147 down-regulated the expression of VEGF, IGF-I and HIF-1α in normoxia and hypoxia conditions.

<p>All figures are a representation of three trials. (A) RT-PCR was performed to examine the transcription levels of CD147, VEGF, IGF-I, HIF-1α and GAPDH in SMMC-7721 cells transfected with CD147 siRNA or control siRNA. (B) Western blots were performed to examine the expression level of CD147, HIF-1α, and β-actin proteins in SMMC-7721 cells transfected with CD147 siRNA or control siRNA. The expression of β-actin was used as internal control. (C) qRT-PCR was performed to examine the transcription levels of these molecular in SMMC-7721 cells transfected with CD147 siRNA or control siRNA. *<i>P</i><0.05, **<i>P</i><0.001, compared with the control level.</p

IGF-I was a specific up-regulator of CD147 expression to promote angiogenesis.

<p>We removed IGF-I from TCMs by using immunoprecipitation and observed the change of proliferation (A) and migration (B) and formation tube-like structures (C and D) of HUVECs response to TCM, IGF-I and siRNA CD147.</p

Knock-down of CD147 in SMMC-7721 cells decreased the levels of VEGF and IGF-I in TCMs under normoxia and hypoxia conditions.

<p>All figures are a representation of three trials. The concentrations of VEGF (A), IGF-I (B), bFGF (C) and EGF (D) in TCMs were determined by ELISA analysis. *<i>P</i><0.05, ^#<i>P</i>>0.05, compared to the control level.</p

IGF-I induced CD147 expression in HUVECs and SMMC-7721 cells.

<p>CD147 expression level was determined by qRT-PCR and western blot in HUVECs (A and B) or SMMC-7721 (C and D) which were treated with VEGF, bFGF, EGF and IGF-I (100ng/mL) for 24 h. *<i>P</i><0.05, **<i>P</i><0.0001, compared with the control level.</p

Frequencies of alleles and genotypes of FCRL3 polymorphisms in AR patients and controls.

<p>AR, allergic rhinitis; SNP, single-nucleotide polymorphism. Pc: Corrected p value; OR: odds ratios</p><p>Frequencies of alleles and genotypes of FCRL3 polymorphisms in AR patients and controls.</p