| Objective: Chronic sinusitis(CRS)refers to a chronic inflammatory disease of the sinus mucosa,clinically manifested by nasal congestion,nasal discharge,loss of smell,head and facial swelling and pain.CRS is divided into chronic sinusitis with nasal polyps CRS(CRSw NP)and chronic sinusitis without nasal polyps CRS(CRS without nasal polyps,CRSs NP)according to the presence or absence of nasal polyps.The most common CRS classification by endotype divides CRS into 2 subtypes,ECRSw NP and n ECRSw NP according to the degree of eosinophil infiltration.ECRSw NP shows significant inflammation of type 2 T helper cells(Th),na?ve CD4+ T cells with differentiation into Th2 cells,tissue eosinophilia,accompanied by elevated interleukin(IL)-4,IL-5,IL-13,usually presenting with more severe clinical symptoms and high recurrence rate.Patients with ECRSw NP are often associated with allergic rhinitis,asthma,and aspirin intolerance,and the prognosis is high in the propensity for recurrence.Current treatment modalities for CRSw NP include topical or systemic use of corticosteroid and nasal endoscopic surgery.However,the high recurrence of nasal polyps leads to the need for long-term corticosteroid therapy and multiple endoscopic surgeries,which bring non-negligible risks and seriously affect the quality of life of patients.Therefore,exploring markers for the diagnosis and treatment of CRSw NP is essential to reduce the social and economic burden caused by CRSw NP.Long non-coding RNAs(lnc RNAs)are more than 200 nt transcripts involving in various cellular processes such as transcriptional regulation,post-transcriptional regulation,organelle and structural organization,and genome integrity.Metastasisassociated lung adenocarcinoma transcript-1(MALAT1)is an important member of lnc RNAs,located at 11q13.1,with 8700 nucleotides,which is the first in non-small cell lung cancer(NSCLC)found in lung neoplasm,and involved in the regulation of cell cycle and migration.MALAT1 is widely expressed in human normal tissues and cells,with distinct tissue-specific and temporal expression patterns.MALAT1 plays key roles in transcriptional and cell cycle regulation,epigenetics,inflammation,and tumor metastasis.The current research on MALAT1 mainly focuses on tumors.Reseaches have shown that its expression is dysregulated in head and neck malignant tumors,lung cancer,liver cancer,colon cancer,breast cancer,gastric cancer and other cancers,and its expression is related to tumor size,stage,and prognosis.It is closely related and can be used to evaluate the clinicopathological characteristics and prognosis of tumor patients.Several studies have shown that lnc RNAs are involved in the regulation of Th1/Th2 balance,and MALAT1 is involved in regulating the Th1/Th2 balance in CD4+ T cells of asthma patients by sponging miR-155.MALAT1 was significantly under expressed in different isoforms of CRSw NP,which may be involved in the regulation of CRSw NP.However,its specific mechanism needs further analysis.Overall,the current reseachs show that the lnc RNA MALAT1 is related to the regulation of CRSw NP.Whether it plays a role by regulating Th1/Th2 balance and the specific mechanism need to be further studied.This study explored the effect and mechanism of MALAT1 on the Th1/Th2 balance of nasal polyps in patients with CRSw NP,and provided a theoretical basis for the development of therapeutic drugs for early diagnosis of CRSw NP.Methods:1.Expression of MALAT1 in patients with chronic sinusitis and nasal polyps and its effect on Th1/Th2 balanceThe nasal polyp tissues of 20 CRSw NP patients were collected as the experimental group,including 10 cases of n ECRSw NP and ECRSw NP,and the inferior turbinate tissues or middle turbinate of 9 patients with deviated nasal septum as the control group.The clinical data were collected.The m RNA and protein expressions of Th1 cytokines IFN-γ and IL-2 and Th2 cytokines IL-4,IL-5 and IL-13 in tissues were detected by qRTPCR and Western blot;tissue and peripheral blood were detected by flow cytometry The proportion of CD4+ IFN-γ+ Th1 cells and the proportion of CD4+ IL-4+ Th2 cells in the medium;the expression level of MALAT1 in the tissue was detected by qRT-PCR.CD4+T cells were isolated and cultured in nasal polyp tissue of ECRSw NP patients,and the cells were divided into two groups,which were transfected with negative control and MALAT1 siRNA respectively.The m RNA expressions of Th1 cytokines IFN-γ and IL-2and Th2 cytokines IL-4,IL-5 and IL-13 were detected by qRT-PCR;Western blot was used to detect the Th1 cytokines IFN-γ and IL-2 and The protein expression levels of Th2 cytokines IL-4,IL-5 and IL-13;the proportion of CD4+ IFN-γ+ Th1 cells and the proportion of CD4+ IL-4+ Th2 cells were detected by flow cytometry.2.MALAT1 affects Th1/Th2 balance in eosinophilic chronic rhinosinusitis with nasal polyps by targeting and binding to miR-155-5p to regulate GATA3 expressionThe potential binding sites of MALAT1 and GATA3 m RNA and miR-155-5p were predicted from the online bioinformatics database,and the MALAT1 and miR-155-5p and GATA3 m RNA were verified by dual-luciferase reporter gene assay,AGO2-RIP and MS2-RIP and miR-155-5p targeting relationship.The m RNA expression levels of miR-155-5p and GATA3 in the nasal polyp tissue of n ECRSw NP and ECRSw NP patients and the inferior turbinate mucosa tissue of the control group were detected by qRT-PCR;the protein expression level of GATA3 in the tissue was detected by Western blot;Spearman correlation analysis was performed The correlation between MALAT1 and miR-155-5p,MALAT1 and GATA3,and miR-155-5p and GATA3 in nasal polyps of CRSw NP patients was detected.CD4+ T cells were isolated and cultured in nasal polyp tissue of ECRSw NP patients,and the cells were divided into six groups,respectively transfected with negative control,MALAT1 siRNA,MALAT1 siRNA+inhibitor negative control,MALAT1 siRNA+miR-155-5p inhibitor,MALAT1 siRNA+miR-155-5p inhibitor+negative control and MALAT1 siRNA+miR-155-5p inhibitor+GATA3 siRNA.The m RNA and protein expressions of Th1 cytokines IFN-γ and IL-2 and Th2 cytokines IL-4,IL-5 and IL-13 were detected by qRT-PCR and Western blot;CD4+ IFN-γ was detected by flow cytometry + Th1 cell ratio and CD4+ IL-4+ Th2 cell ratio.Results:1.Expression of MALAT1 in patients with chronic sinusitis and nasal polyps and its effect on Th1/Th2 balanceIn the ECRSw NP group,the peripheral blood eosinophil count and peripheral blood eosinophil percentage were significantly higher than those in the n ECRSw NP group and the control group;the Lund-Mackay CT score and Lund-Kennedy score in the ECRSw NP group and the n ECRSw NP group were significantly higher than the control group,but two groups have no significant difference;all three groups had no significant differences in age,sex,height,weight,BMI,whether smoked,accompanied with asthma or allergic rhinitis,Fn NO or Fe NO.The m RNA and protein expressions of Th1 cellrelated factors IFN-γ and IL-2 and the proportion of CD4+ IFN-γ+ Th1 cells in nasal polyp tissues of patients with n ECRSw NP and ECRSw NP were up-regulated,and the expression of MALAT1 was down-regulated.The m RNA and protein expressions of γand IL-2,the proportion of CD4+ IFN-γ+ Th1 cells were down-regulated,the expression of MALAT1 was up-regulated,and the m RNA and protein expressions of Th2 cellrelated factors IL-4,IL-5 and IL-13 in nasal polyps of ECRSw NP patients The proportion of CD4+ IL-4+ Th2 cells was up-regulated;silencing MALAT1 increased the m RNA and protein expressions of IFN-γ and IL-2 in nasal polyps of ECRSw NP patients,as well as the proportion of CD4+ IFN-γ+ Th1 cells,and decreased the IL-4,IL-5 and IL-13 m RNA and protein expression and the proportion of CD4+ IL-4+ Th2 cells.2.MALAT1 affects Th1/Th2 balance in eosinophilic chronic rhinosinusitis with nasal polyps by targeting and binding to miR-155-5p to regulate GATA3 expression MALAT1 and GATA3 m RNAs have potential binding sites for miR-155-5p,and MALAT1 and GATA3 m RNAs bind to miR-155-5p.The expression of miR-155-5p was up-regulated in the nasal polyp tissue of n ECRSw NP and ECRSw NP patients,and the expression of miR-155-5p was down-regulated in the nasal polyp tissue of ECRSw NP patients compared with that of n ECRSw NP patients;GATA3 m RNA and protein expressions were up-regulated in the nasal polyp tissue of ECRSw NP patients.The expressions of MALAT1 and miR-155-5p,and the expressions of GATA3 m RNA and miR-155-5p were negatively correlated in nasal polyps of CRSw NP patients,and the expressions of GATA3 m RNA and MALAT1 were positively correlated.Transfection of miR-155-5p inhibitor decreased the m RNA and protein expression of IFN-γ and IL-2 in CD4+ T cells and the proportion of CD4+ IFN-γ+ Th1 cells in nasal polyps of ECRSw NP patients with MALAT1 silenced,and increased the proportion of IL-4,IL-4,IL-4 and IL-4 of CD4+ T cells.IL-5 and IL-13 m RNA and protein expression and the proportion of CD4+ IL-4+ Th2 cells.Silencing GATA3 increases the m RNA and protein expression of IFN-γ and IL-2 in CD4+ T cells and the proportion of CD4+ IFN-γ+ Th1 cells in nasal polyps of ECRSw NP patients who silence MALAT1 and administer miR-155-5p inhibitor,and reduce IL-1 in CD4+ T cells-4,IL-5 and IL-13 m RNA and protein expressions and the proportion of CD4+ IL-4+ Th2 cells.Conclusion: MALAT1 promotes Th2 cell excursion in nasal polyps in patients with eosinophilic chronic rhinosinusitis with nasal polyps by up-regulating GATA3 expression by sponging miR-155-5p. |
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