Study on the mechanism of let-7e-5p regulating Fas/FasL to affect the pathogenesis of allergic rhinitis in mice

doi:10.11958/20221110

Abstract

Abstract:

Objective To investigate the effect and mechanism of let-7e-5p on allergic rhinitis in mice. Methods Eighteen BALB/c mice were randomly divided into the control group, the allergic rhinitis (AR) group, the let-7e-5p agonist negative control (AR+agomir-NC) group, the let-7e-5p agonist (AR+agomir) group, the let-7e-5p inhibitor negative control (AR+antagomir-NC) group and the let-7e-5p inhibitor (AR+antagomir) group. Except the control group, the other groups were sensitized with ovalbumin (OVA) to establish AR model. The AR+agomir group and the AR+antagomir group were treated with let-7e-5p agomir or let-7e-5p antagomir at the same time for intervention. After the last sensitization, the allergic rhinitis of mice were scored. HE staining was used to observe nasal mucosa. The number of eosinophils was counted. Real-time PCR was used to detect mRNA expression levels of let-7e-5p, Fas and FasL, and Western blot assay was used to detect protein expression levels of Fas and FasL. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of immunoglobulin E (IgE), specific immunoglobulin E (sIgE), interferon gamma (IFN-γ), interleukin (IL)-12, IL-4 and IL-13. The targeting relationship between let-7e-5p and Fas and FasL were analyzed by dual luciferase assay. Results Compared with the control group, the allergic rhinitis score was increased in the AR group, nasal mucosa hyperplasia was obvious and the number of eosinophils was increased. The expression levels of let-7e-5p and IFN-γ and IL-12 were decreased, while the expression of Fas and FasL and levels of IgE, sIgE, IL-4 and IL-13 were increased (all P<0.05). Compared with the AR+agomir-NC group, the allergic rhinitis score was decreased in the AR+agomir group, the nasal mucosa structure was clear and the number of eosinophils was reduced. The expression of let-7e-5p and levels of IFN-γ and IL-12 were increased, while the expression of Fas and FasL and levels of IgE, sIgE, IL-4 and IL-13 were decreased (all P<0.05). Compared with the AR+antagomir-NC group, the allergic rhinitis score was increased in the AR+antagomir group, the nasal mucosa was significantly thickened, and the number of eosinophils was increased. The expression of let-7e-5p was decreased, and the expression of Fas, FasL and levels of IgE, sIgE, IL-4 and IL-13 were increased (all P<0.05). There were no significant differences in IL-12 and IFN-γ between the AR+antagomir-NC group and the AR+antagomir group (P>0.05). Compared with the NC+3'UTR-WT group, luciferase activity was decreased in the let-7e-5p agomir+3'UTR-WT group (P<0.05). Conclusion Let-7e-5p may affect the balance of T helper type 1 (Th1)/ T helper type 2 (Th2) cells by targeting Fas/FasL, and thus participate in the progression of AR in mice.

Key words: rhinitis, allergic, perennial, disease models, animal, let-7e-5p, Fas, FasL, T helper type 1, T helper type 2

CLC Number:

R765.21

Figures/Tables 11

References 21

[1]	ZHANG Y, LAN F, ZHANG L. Advances and highlights in allergic rhinitis[J]. Allergy, 2021, 76(11):3383-3389. doi:10.1111/all.15044.
[2]	SIDDIQUI Z A, WALKER A, PIRWANI M M, et al. Allergic rhinitis: Diagnosis and management[J]. Br J Hosp Med (Lond), 2022, 83(2):1-9. doi:10.12968/hmed.2021.0570.
[3]	JAIN A, IRIZARRY-CARO R A, MCDANIEL M M, et al. T cells instruct myeloid cells to produce inflammasome-independent IL-1β and cause autoimmunity[J]. Nat Immunol, 2020, 21(1):65-74. doi:10.1038/s41590-019-0559-y.
[4]	KINIKLI G, ATES A, TURGAY M, et al. Serum-soluble Fas antigen level in patients with allergic rhinitis: Its relation to specific immunotherapy[J]. Allergy Asthma Proc, 2006, 27(2):145-147.
[5]	PIRAYESH A, SHAHSAVAN S, ZARGARI SAMANI O Z, et al. Differential expression of Fas in moderate/severe and mild persistent allergic rhinitis and its correlation with pathological parameters[J]. Am J Rhinol Allergy, 2019, 33(3):286-293. doi:10.1177/1945892418824246.
[6]	LI W Q, ZHANG W T, LIU J, et al. Down-regulation of miR-let-7e attenuates LPS-induced acute lung injury in mice via inhibiting pulmonary inflammation by targeting SCOS1/NF-κB pathway[J]. Biosci Rep, 2021, 41(1): BSR20201089. doi:10.1042/BSR20201089.
[7]	SUOJALEHTO H, LINDSTROM I, MAJURI M L, et al. Altered microRNA expression of nasal mucosa in long-term asthma and allergic rhinitis[J]. Int Arch Allergy Immunol, 2014, 163(3):168-178. doi:10.1159/000358486.
[8]	LI L H, ZHANG S R, JIANG X S, et al. MicroRNA-let-7e regulates the progression and development of allergic rhinitis by targeting suppressor of cytokine signaling 4 and activating Janus kinase 1/signal transducer and activator of transcription 3 pathway[J]. Exp Ther Med, 2018, 15(4):3523-3529. doi:10.3892/etm.2018.5827.
[9]	姜玉秋, 唐桥斐, 闫智永, 等. LncRNA PVT1通过竞争miR-149-5p上调CHI3L1并影响变应性鼻炎进展的研究[J]. 天津医药, 2022, 50(3):259-264.
	JIANG Y Q, TANG Q F, YAN Z Y, et al. LncRNA PVT1 upregulates CHI3L1 and affects the progression of allergic rhinitis by competition with miR-149-5p[J]. Tianjin Med J, 2022, 50(3):259-264. doi:10.11958/20211109.
[10]	RONG J P, XU L, HU Y Y, et al. Inhibition of let-7b-5p contributes to an anti-tumorigenic macrophage phenotype through the SOCS1/STAT pathway in prostate cancer[J]. Cancer Cell Int, 2020, 20:470. doi:10.1186/s12935-020-01563-7.
[11]	SUOJALEHTO H, TOSKALA E, KILPELÄINEN M, et al. MicroRNA profiles in nasal mucosa of patients with allergic and nonallergic rhinitis and asthma[J]. Int Forum Allergy Rhinol, 2013, 3(8):612-620. doi:10.1002/alr.21179.
[12]	DONG J H, XU O, WANG J X, et al. Luteolin ameliorates inflammation and Th1/Th2 imbalance via regulating the TLR4/NF-κB pathway in allergic rhinitis rats[J]. Immunopharmacol Immunotoxicol, 2021, 43(3):319-327. doi:10.1080/08923973.2021.1905659.
[13]	LI J J, RAGHAV P, HU C Y. Ajwain oil attenuates allergic response of ovalbumin-induced allergic rhinitis via alteration of inflammatory,oxidative stress,and Th1/Th2 responses[J]. J Food Biochem, 2021, 45(12):e13963. doi:10.1111/jfbc.13963.
[14]	GUAN H B, FAN D P, MRELASHVILI D, et al. MicroRNA let-7e is associated with the pathogenesis of experimental autoimmune encephalomyelitis[J]. Eur J Immunol, 2013, 43(1):104-114. doi:10.1002/eji.201242702.
[15]	KAGAWA T, WATANABE M, INOUE N, et al. Increases of microRNA let-7e in peripheral blood mononuclear cells in Hashimoto's disease[J]. Endocr J, 2016, 63(4):375-380. doi:10.1507/endocrj.EJ15-0577.
[16]	RASQUINHA M T, SUR M, LASRADO N, et al. IL-10 as a Th2 cytokine:Differences between mice and humans[J]. J Immunol, 2021, 207(9):2205-2215. doi:10.4049/jimmunol.2100565.
[17]	KODA Y, TERATANI T, CHU P S, et al. CD8(+) tissue-resident memory T cells promote liver fibrosis resolution by inducing apoptosis of hepatic stellate cells[J]. Nat Commun, 2021, 12(1):4474. doi:10.1038/s41467-021-24734-0.
[18]	JIMBO H, NAGAI H, FUJIWARA S, et al. Fas-FasL interaction in cytotoxic T cell-mediated vitiligo:The role of lesional expression of tumor necrosis factor-alpha and interferon-gamma in Fas-mediated melanocyte apoptosis[J]. Exp Dermatol, 2020, 29(1):61-70. doi:10.1111/exd.14053.
[19]	UPADHYAY R, BOIARSKY J A, PANTSULAIA G, et al. A critical role for fas-mediated off-target tumor killing in T-cell immunotherapy[J]. Cancer Discov, 2021, 11(3):599-613. doi:10.1158/2159-8290.CD-20-0756.
[20]	ZHOU L L, XUE C J, CHEN Z Y, et al. c-Fos is a mechanosensor that regulates inflammatory responses and lung barrier dysfunction during ventilator-induced acute lung injury[J]. BMC Pulm Med, 2022, 22(1):9. doi:10.1186/s12890-021-01801-2.
[21]	侯穗波, 戴勇, 刘明, 等. 变应性鼻炎患者血清可溶性Fas与Fas配体的变化[J]. 中华微生物学和免疫学杂志, 2001, 21(S):111-113.
	HOU S B, DAI Y, LIU M, et al. Changes of serum soluble Fas and Fas ligand in patients with allergic rhinitis[J]. Chinese Journal of Microbiology and Immunology, 2001, 21(S):111-113.

分级评分（分）	喷嚏（个/30 min）	清涕	搔鼻（次）
0	0	无	无
1	1~3	流涕至鼻前孔	轻微抓鼻几次
2	4~10	流涕超过鼻前孔	双爪反复挠鼻
3	>11	涕流满面	抓挠鼻面不止、四处摩擦

分级评分（分）	喷嚏（个/30 min）	清涕	搔鼻（次）
0	0	无	无
1	1~3	流涕至鼻前孔	轻微抓鼻几次
2	4~10	流涕超过鼻前孔	双爪反复挠鼻
3	>11	涕流满面	抓挠鼻面不止、四处摩擦

基因名称	引物序列（5'→3'）	产物大小（bp）
let-7e-5p	上游：TGAGGTAGGAGGTTGTATAGTT	54
	下游：GCAGGGTCCGAGGTATTC	54
U6	上游：CGCAAGGATGACACGCAAAT	74
	下游：GCAGGGTCCGAGGTATTC	74
Fas	上游：AACCAGACTTCTACTGCGATTC	144
	下游：TCAACAACCATAGGCGATTT	144
FasL	上游：GCCATCACAACCACTCCC	107
	下游：CCAGAGCCACCAGAACCA	107
β-actin	上游：CTGTGCCCATCTACGAGGGCTAT	155
	下游：TTTGATGTCACGCACGATTTCC	155

基因名称	引物序列（5'→3'）	产物大小（bp）
let-7e-5p	上游：TGAGGTAGGAGGTTGTATAGTT	54
	下游：GCAGGGTCCGAGGTATTC	54
U6	上游：CGCAAGGATGACACGCAAAT	74
	下游：GCAGGGTCCGAGGTATTC	74
Fas	上游：AACCAGACTTCTACTGCGATTC	144
	下游：TCAACAACCATAGGCGATTT	144
FasL	上游：GCCATCACAACCACTCCC	107
	下游：CCAGAGCCACCAGAACCA	107
β-actin	上游：CTGTGCCCATCTACGAGGGCTAT	155
	下游：TTTGATGTCACGCACGATTTCC	155

组别	评分（分）	嗜酸性粒细胞（个/视野）
Control组	1.33±0.58	21.50±2.65
AR组	7.67±0.58^a	147.75±6.75^a
AR+agomir-NC组	7.33±0.58^a	136.25±6.13^a
AR+agomir组	4.00±1.00^b	75.00±10.42^b
AR+antagomir-NC组	7.33±0.58^a	149.75±1.71^a
AR+antagomir组	9.00±0.00^c	226.25±8.18^c
F	63.310^**	441.500^**