| BackgroundThe Antrochoanal polyp(ACP)is a special type of nasal polyp(NP)that originates in the maxillary sinus and extends backward through natural or accessory opening of the maxillary sinus to the posterior nostril and nasopharynx.The incidence of ACP accounts for about 4～6%of common NP.Compared with NP,ACP is usually unilateral,it is more common in children and adolescents,the incidence of ACP is slightly higher in male than in female,and the clinical symptoms are relatively slighter.Our previous study found that when compared with the common NP,the epithelial remodeling of ACP was less severe and more related to the non-Th2 inflammatory infiltration.However,there is a lack of in-depth research on the etiology and pathogenesis of ACP,the treatment of ACP is relatively simple,and the postoperative recurrence rate of ACP is high,it brings heavy economic and medical burden to the ACP patients,families and society.The mucociliary clearance(MCC)system which consist of cilia and mucus secreted by goblet cells,is an important intrinsic defense mechanism of the upper respiratory tract to protect the body against pathogens,allergens,toxins and other environmental factors.Impaired MCC caused by ciliary abnormalities may lead to chronic inflammation of nasal mucosa.It has been found that cilia arrangement disorder,cilia length and density change existed in NP,as well as the abnormal expression of ciliogenesis markers including centrosomal protein 110(CP 110)and forkhead box J1(FOXJ1).Other studies found that the aberrant localization of FOXJ1 was correlated with the severity and co-existence of allergic rhinitis(AR)or asthma in patients with NP.Aberrant localization of cilia ultrastructural markers(Dynein axonemal Heavy chain 5,DNAH5;Radial Spoke head protein 1,RSPH1;Radial spoke head protein 4A,RSPH4A;Radial spoke head protein 9,RSPH9)has also been reported in the epithelium of NP and is closely associated with ciliogenesis markers(CP110 and FOXJ1).Moreover,ciliary abnormalities were observed in allergic rhinitis,as well as the down-regulation and aberrant localization of FOXJ1.To date any study has not investigated the morphology and function of cilia in the development of ACPs.This study aims to explore the pathogenesis of ACP from the gene level by analyzing the transcriptome gene expression profile of ACP,and further conducted polymerase chain reaction(qRT-PCR),immunofluorescence staining(IF)and scanning electron microscope(SEM)to confirm the findings of the RNA-seq.Our findings might help elucidate the roles of ciliogenesis and ciliary ultrastructural markers in ACP pathogenesis and would help identify disease targets and novel treatment options(such as biologics)for ACP.Methods1.In this study,lesion tissues of 6 ACP patients(ACP group)and uncinate mucosa of 6 patients with nasal septum deviation or maxillary sinus cyst(control group)were randomly selected for transcriptome sequencing analysis.HISAT2v2.0.5 was used to compare the paired terminal clean reads with the reference genome,and the gene expression level was quantified as log2 value of reads per kilobase million(RPKM)to evaluate inter-group differences;Differentially expressed genes(DEGs)between groups were analyzed by DESeq2 software.ClusterProfiler R software was used for gene ontology[(GO)including biological process,cell composition and molecular function]analysis and KEGG pathway enrichment analysis of DEGs.GSEA official software package was used for gene set enrichment analysis.2.mRNA was extracted from lesion tissue of 18 ACP patients and uncinate process of 20 patients with simple nasal septum deviation or maxillary cyst(control group),the mRNA expression levels of ciliary ultrastructural markers(DNALI1,DNAH5,DNAI1,DNAH9,RSPH1,RSPH4A,RSPH9)and ciliogenesis markers(FOXJ1,CP 110)were detected by real-time fluorescence quantitative polymerase chain reaction.3.13 ACP tissues and 6 control group were collected to perform immunofluorescence staining.A semi-quantitative scoring system was used to evaluate the distribution pattern of cilia,0 point:>70%of epithelial areas with ciliary staining,1 point:≤ 70%of epithelial areas with ciliary staining.The expression pattern of ciliary ultrastructure(DNAI1 and RSPH4A)between the two groups was evaluated by a semi-quantitative scoring system according to the results of immunofluorescence double staining,each area was assigned a score of 0(normal expression)or 1(aberrant expression).0 point:>70%of ciliary distribution,and 1 point:≤ 70%of ciliary distribution.The protein expression level of ciliogenesis marker(FOXJ1)between the two groups was assessed by measuring total fluorescence intensity(TFI).4.The uncinate process mucosa of 3 ACP patients and 3 control group were examined by scanning electron microscopy,to evaluate the distribution and morphology of cilia between the two groups.Results1.There were significant differences in overall gene expression patterns between ACP group and control group,with a total of 3739 DEGs,which including 2377 upregulated DEGs and 1362 down-regulated DEGs of ACP group.2.GO analysis of the DEGs was performed,which find out the TOP20 BP,CC and MF.BP show that about 1/5 DEGs were related to ciliogenesis and ciliary function,including cilium movement and intraciliary transport involved in cilium assembly,etc.In addition,DEGs also involved in inflammatory response and immune response processes such as neutrophil chemotaxis and neutrophil degranulation.In CC,about 1/4 of DEGs were related to the ultrastructure of cilia,including the microtubule structure of cilia and the dynein arm structure of cilia.In MF,more than 1/5 of DEGs are involved in processes such as ciliary microtubule movement and ciliary dynein chain binding.3.KEGG pathway analysis was performed,and the representative pathways included cytokine-cytokine receptor interaction,chemokine signaling pathway,arachidonic acid metabolism,pathways in cancer,IL-17 pathway,malaria and other classic pathways.4.The concordant gene expression patterns of ACP group with control group.were screened by the GSEA.Representative candidate enriched gene sets revealed cilium organization,cilium morphogenesis,cilium movement,axoneme assembly,axonemal dynein complex assembly,cell projection assembly,etc.5.Compared with the control group,mRNA levels of ciliary ultrastructural markers including DNAI1,DNAH9,RSPH1,RSPH4A and RSPH9 were found upregulated in ACP group(all P<0.05).According to our semi-quantitative scoring system,the median staining scores of DNAI1 and RSPH4A in paraffin sections of the control group were 0(0,0.05)and 0.5(0.35,0.6).The median score of ACP group was significantly higher than that of control group[0.6(0.2,0.8),0.8(0.5,1),both P<0.05].6.Compared with the control group,the mRNA level of ciliogenesis marker FOXJ1 in ACP was up-regulated(P<0.05).Immunofluorescence staining showed that FOXJ1 was positive in the nuclei of ciliated and non-ciliated epithelial cells in both groups.The TFI of FOXJ1 was significantly decreased in ACP group[8254276(4934874,15732939)vs.2403926(818453,3721310),P<0.005].7.According to the semi-quantitative scoring system,the cilia distribution pattern between the two groups was scored.The median score of the control group was 0.1(0,0.35),and the median score of the ACP group was significantly higher than control group[0.8(0.6,1),P<0.05].8.Decrease of cilium density,partial loss of cilia and abnormal cilium morphology(shortened length and disordered arrangement)in ACP samples was observed by SEM.ConclusionAberrant expression pattern of cilia-related genes in ACP and the control was shown,the abnormal ciliary ultrastructure may be related to the abnormal expression of ciliogenesis marker,loss of ciliary as well as shortened cilia,imply that the occurrence,development and prognosis of ACP may be closely related to its chronic inflammatory response,defective host-defenses(such as immune intolerance,abnormal cilium structure and function).Our findings may provide important references for the accurate and effective treatment of ACP. |
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