Investigate The Role Of MiR-150-5P In Chronic Rhinosinusitis From DC-TH Axis

Chronic rhinosinusitis(CRS) is a common frdquently encountered disease that is characterized by chronic inflammation in the nasal cavity and sinus mucosa in otolaryngology head and neck surgery. The current academia agreed that CRS includes CRS without nasal polyposis(CRSs NP)and with nasal polyposis(CRSw NP). The goal of treatment for CRS is to achieve and maintain clinical control and avoid complications. At present,it still is not to achieve the aim of curing CRS for the pathogenesis of CRS is complex and unclear. Recent researches have shown that CRS may be related to genetic, environment, allergy, Remodeling in CRS, anatomical structure, innate immunity and bacterial biofilm etc, but what kind of factors is the main factor to cause CRS. So it is necessary to study CRS,recent research has shown that dendritic cells(DC), imbalance of Th17/Treg and micro RNAs(mi RNAs) is an important basis of CRS, but its mechanism has not been clarified. Our study would be to investigate the role of mi RNAs in CRS from DC- Th axis. It is still a challenging problem to diagnose and treat CRS for the otolaryngology doctor. Although CRS is related to abnormal anatomy, infection, allergic constitution, immune regulation, environment and genetics, the imbalances of Th17/Treg is an important basis of CRS. However, the mechanism of Th17/Treg imbalances has not yet been elucidated.DCs is a full-time antigen presenting cells in the body for its surface has dendritic protrusions. DCs play an important role in the immune system,different DC can interact and adjust immune response. How do DCs affect the function of the other cells in the immune system? On the one hand, DC plays the role of antimicrobial infection by identification, intake,processing and presenting in natural immunity; on the other hand, DC can activate and interact naive T cells by capturing antigencan. DC can also regulate the induction and differentiation of T helper(Th) cell by secreting IL-17, IL-12, IL-10. DCs have powerful function and can prevent the invasion of the pathogen in microenvironment when they are acted as antigen presenting cells. Mature DC can migrate to the lymph nodes and can stimulate T cell proliferation. CD4 + Th cells is of great significance in cellular immunity by T cell-mediated response. Na?ve T cell can differentiate into Th1 cells and Th2 cells, Th17 cells and Treg cells that have the different function. Mi RNAs is a kind of single noncoding RNA molecules about 19-22 nucleotides long that mediate post-transcriptional gene silencing of target genes and inhibit protein target m RNA translation.Therefore, mi RNAs are a promising class of molecules that may be well positioned to regulate up-airway inflammatory processes, mi RNAs also play a important role in the CRS. We speculate that there are quite a number of mi RNAs to involve in the pathogenesis of CRS according to the role of mi RNA in DC. However, abnormal mi RNA expression profile is how to participate in the regulating of DC-Th axis? Should there be common and specific differential expressed mi RNA in different types of CRS? Whether the balance of DC-Th axis is regulated and achieve the purpose of prevention and treatment by intervening differential expressed mi RNA?It is necessary to explore above scientific problems in order to treat CRS effectively. In this study, CRS patients was designed as study subjects,DC-Th axis was designed as the main line, and the regulation of mi RNA on DC was designed as the entry point. This study aimed to evaluate the upstream events of Th17/Treg imbalance in CRS and determine the mi RNA expression profile in CRS and clarify the role of mi RNA in the pathogenesis of CRS by regulating the DC-Th axis. This study will play an important role in clarifying the pathogenesis of CRS and provide new targets and new ideas for establishing control strategies for CRS.Part one The phenotype and distribution characteristics of DC in peripheral blood with different types of CRS patientsObject: To Detect the phenotypes, quantity and distribution characteristics in DC of peripheral blood with CRS patients,compare the differences among the three kinds of different types of CRS by flow cytometry(FCM) and observe the role of DC in the pathogenesis of CRS.Method: 67 cases CRS patients from 2014.7-2015.8 were collected.These CRS patients was divided into four groups according to EPOS2012 diagnostic criteria:(1)Normal control group: patients only with nasal septum deviation(13 cases);(2)CRSs NP: CRS patients without nasal polyps(18 cases);(3)atopic CRSw NP: CRS patients with allergic constitution and nasal polyps(18 cases);(4)non- atopic CRSw NP: CRS patients with nasal polyps but without allergic constitution vc(18 cases).Peripheral blood mononuclear cells(PBMC)were isolated from these samples using Ficoll-Hypaque density-gradient centrifugation. CD14+monocytes were isolated from PBMCs using human CD14 microbeads.Then CD14+ cells were cultured with granulocyte-macrophage colony stimulating factor(GM-CSF) and interleukin-4(IL-4) to promote differentiation of cells into immature DCs. CD80, CD83, CD1 a and CD86(maturation and activation sign of DC) were detected by FCM and evaluated the phenotype, quantity and distribution characteristics of DCs in peripheral blood in different types of CRS.Results: The distribution of DCs in the peripheral blood with CRS patients: FCM scatter diagram according todouble positive cells: a control 38.98 %; CRSs NP 57.77 %; Atopic CRSw NP 84.87 %; Non-atopic CRSw NP 82.94 %; Compare MFI of mature DCs in different types of CRS.Mature DCs in CRS were higher than in the control group, and mature DCs in polyp group was higher than in without polyps group, allergic constitution group was significantly higher than without allergic constitution. P<0.05 were considered significant when theywere compared with control group.Conclusion: DCs is increased in peripheral blood of CRS and may play an important role in the pathogenesis of CRS. And it is shown more DC distribution in atopic group, prompt allergic factors could lead to increase DCs. Therefore, antianaphylaxis treatment is necessary for CRS patients with allergic constitution apart from surgery.Part two To study mi RNA expression profile of DC in peripheral blood with CRS patientsObject: Total RNA was extracted using Trizol and detected by micro RNA microarrays after DCs were isolated from peripheral blood with CRS patients. Mi RNA expression in CRS were verified by q PCR. It can be To determine The mi RNA expression profile in peripheral blood with CRS patients and screen out differential expressed mi RNAs. It is evaluated that there be common and specific differential expressed mi RNAs in different types of CRS. This study aimed to explore out the typical differential expressed mi RNAs that could play a important role in the development of CRS.Method: 3 CRS patients groups and control group samples in peripheral blood were collected separately.3 CRS patients groups of CRS patients and control group in peripheral blood samples. PBMCs were isolated from these samples using Ficoll-Hypaque density-gradient centrifugation. CD14+ monocytes were isolated from PBMCs using human CD14 microbeads. Then CD14+ cells were cultured with GM-CSF, IL-4and lipopolysaccharide(LPS) to promote differentiation of cells into immature DCs. Total RNA was extracted using Trizol and detected by micro RNA microarrays after DCs were isolated from peripheral blood with CRS patients. Mi RNA expression in CRS were verified by q PCR. It can be To determine The mi RNA expression profile in peripheral blood with CRS patients and screen out differential expressed mi RNAs.Results: Mi RNA microarrays showed that DCs from different types of patients have different sets of differential expressed mi RNAs when comparing with Controls; they also share 31 commonly changed mi RNAs among all three groups of CRS patients. Of these 31 mi RNAs, 5 mi RNAs were up-regulated and 25 mi RNAs were down-regulated in all three types of CRS, while Mi R-1290 was down-regulated in CRSs NP but up-regulated in both atopic CRSw NP and non-atopic CRSw NP.Conclusion: By comparing mi RNA gene expression patterns in 3types of CRS patients, we have been able to identify candidate mi RNAs that might mediate the core pathogenesis of CRS through regulating dendritic cells. These mi RNAs could serve as potential therapeutic targets for CRS.Part three To investigate the role of mi R-150-5P in CRS from DC-Th axisObject: Flow cytometry was used to evaluate the proliferation of T cells. Interleukin-17(Il-17) cytokines in the supernatants of DCs-na?ve T cells cocultures were assayed by Enzyme-linked immunosorbent assay(ELISA). This study aim to investigate the up-regulation role of mi R-150-5P in CRS on DC-Th axis and evaluate the upstream events of Th17/Treg imbalance in Chronic Rhinosinusitis(CRS) and their immune regulatory factors.Method: DCs were isolated from peripheral blood in different types of CRS and induced as mature DCs. DCs were transfected with mi R-150-5P mimic/mi R-150-5P inhibitor. Na?ve CD4+T cells can be isolated from PBMCs and stain with anti-Human CD3 and anti-Human CD28, and analyzed by flow cytometry. T cell proliferation was assessed by Tcells labeled CFSE. Il-17 in the supernatants of DCs-na?ve T cells cocultures were assayed by ELISA.Results: Compared with not transfection, of T cell proliferation and secretion of Il-17 in transfection mi R- 150-5 p mimic group was increased,and transfection mi R-150-5P inhibitor group was decreased, co-cultured group with DCs was higher than co-cultured group without DCs. T cell proliferation in CRS group was higher than the control group. P<0.05 were considered significant when theywere compared with control group.Conclusion: Our results suggested that the expression of mi R-150-5p was up-regulated in DC of patiens with CRS. Mi R-150-5P mimic could promote T cell proliferation and secretion of IL-17, mi R-150-5P inhibitor could inhibit T cell proliferation and secretion of IL-17. It shows mi R-150-5P lead to CRS from DC-Th axis.Part four To predict and verify possible target genes of mi R-150-5P in DC with CRS patientsObject: To investigate possible target genes and target protein of differential expressed mi R-150-5P that could prevent and treat CRS by interfering mi R-150-5P.Method: The mi R-150-5P targets gene were predicted, several target prediction databases were searched, including Target Scan, Pic Tar, Mi Randa,and mi RBase Targets. Western blot(WB) analysis was performed to determine the protein expression of early growth response 2(EGR2).Luciferase reporter assay was performed to verify EGR2 target genes.EGR2 3’ untranslated region(3’-UTR) containing the the predicted mi R? 150? 5p target sequence and binding site. The seed sequence mutated version(EGR2? 3’UTR? mut) were synthesized. Mi R-150-5p mimic, or control mimic were co-transfected into HEK 293 T cells. The luciferase activity was measured at 48 h after transfection using the dual-luciferase reporter assay system, it was verifed whether the target gene was mi R-150-5P target gene.Results: EGR2 was also identified as a direct target of mi R-150-5P by target prediction databases, and this target was validated via with a dual-luciferase reporter activity assay and western blot analysis.Conclusion: Mi R-150-5P and its identified target EGR2 play important roles in the development of CRS.