Expression Of TIPE2 In Adenoids Of OSA Children And The Mechanism Of LncRNA HOXA-AS2/MiR-17-5p/TIPE2 Axis In Improving Pulmonary Inflammation In OSA Rats

Part one:Expression of TIPE2 in adenoids of OS A childrenObstructive Sleep Apnea(OSA)is one of the most common and frequent diseases in children and adolescents,it is primarily due to the intermittent appearance of children during sleep,part or all of the upper airway obstruction,collapse caused.The common clinical symptoms of OSA are snoring,suffocating,mouth-opening breathing,apnea,postnasal drip and so on,which can change the normal ventilation and sleep structure of children,this leads to growth restriction,behavioral and cognitive impairment.It was found that airway stricture caused by adenoidal hypertrophy is a very important cause of OSA in children.The prevalence of adenoid hypertrophy in pediatrics is as high as 34%,according to a new meta-analysis.However,the exact pathogenesis of OSA in children has not been fully understood.Therefore,more attention should be paid to the adenoidal hypertrophy of OSA in children,and further study on its pathogenesis will provide a new theoretical method for clinical diagnosis and treatment.Recent studies have shown that the immune response plays an important role in OSA in children.Tipe2 is an Tumor necrosis factor immune protein that inhibits the activation of inflammatory pathways.TIPE2 is abundantly expressed in human immune cells and organs,especially in lymphoid tissue or thymus,beside has been reported to play a key role in sustaining the firmness of the immune system.Tipe2 has a negative administrative effect on the inflammatory reaction induced by various factors,intervening in the over-activated inflammatory response through a negative regulatory function,participating in the maintenance of immune balance,and playing an important role in these processes.Nuclear Factor-kappa B(NF-KAPPA B)is a considerable transcription factor in human cells.NF-κB consists of five subunits:P50(NF-kappa b 1),RELB,P52(NF-KAPPA B 2),Rel(Crel)and p65(RelA,NF-KAPPA B 3).It can regulate the course of apoptosis,inflammation,migration and carcinogenesis,and participate in the process of expression of many genes in human body,and play a very important role in it,it also plays a pivotal role in the regulation of autoimmune disease.When the human body is in the inflammation infection,the body mainly through the antigen and the Pattern recognition receptor binding,then induces the activation of the nuclear factor kappa B signaling pathway,thus further increases the expression of the body’s inflammatory factors.Up to now,there is no literature about the expression of Tipe2 in adenoid tissues of children with OSA,and whether there is any regulation or intervention mechanism of Tipe2 in children with OSA.In this study,we collected adenoids from children with OSA and non-OSA,and examined the TIPE2 expressing and its related inflamed molecule,pivotal proteins of NFκb signaling pathway,to investigate the expression of Tipe2 in adenoid tissues of OSA children and the mechanism of Tipe2 in regulating the immune response in OSA children,the activity and apoptosis of adenoid monocytes overexpressing TIPE2 were detected at the cellular level,to further study the pathogenesis of OSA in children.We hope to provide a new theoretical basis and feasible method for the prevention,clinical diagnosis,treatment and scientific research of OSA in children.Objective:The purpose of this research is to investigate the expression of Tipe2 in adenoid,in OSA children,and to explore the role of Tipe2 in the upper airway inflammation of OSA children.Methods:63 cases of adenoidectomy in our hospital were selected.Of these,48 were children with OSA(21 females,27 males,male:female=1.29:1)and 15 without OSA(6 females,9 males,male:female=3:2 in the Con group).Apnea-hypopnea index(AHI)was more than 5 times per hour,and the parents were asked to fill in the clinical rating scale Specimens of adenoids were collected during operation.The expression of Tipe2 in adenoid tissues was detected by immunohistochemistry,and the transcription levels of TIPE2,TNF-α,IL-6 RNA in adenoid tissues were detected by RT-PCR.The protein expression of TNF-α and IL-6 were detected by ELISA,and the protein expression of TIPE2 was detected by Western blot,primary cell culture in vitro,plasmid transfection,construction,and Western blot were used to verify the overexpression of TIPE2 in monocytes.The expression of TNF-α and IL-6 were detected in monocytes overexpressed with TIPE2,and the activity of monocytes transfected with TIPE2 was detected by MTT assay The Flow cytometry analyzed apoptosis in adenoid monocytes overexpressing Tipe2.Results:1.Clinical data:there was observably difference in A/N ratio between OSA Group and Con group,there was no prominently difference in age,sex and BMI between the two groups(P>0.05).2.The average clinical score of OSA group was 69.98±22.02,and that of Con group was 22.43±3.59(P<0.001).3.Immunohistochemistry showed that TIPE2 was abundantly expressed in the adenoid tissue cells of Con group,and it was mainly expressed in the protoplasm of the adenoid tissue cells.The expression of Tipe2 in OSA group was lighter than that in control group(p<0.001).4.The results of Elisa showed that the expression level of TNF-α and IL-6 in OS A group were dramatically higher than those in Con group(p<0.01).In OSA Group,the expression level of TNF-α was positively correlated with the clinical score(p=0.001),the expression level of TNF-α was positively correlated with the AHI value(p=0.0212),the expression level of IL6 was positively correlated with clinical scoring(p<0.001),there was a positive correlation between AHI and IL-6 expression(p=0.0035).5.The results of Western blot showed that the TIPE2 express in OSA group was dramatically lower than that in Con group((p<0.001).The consequences of statistic analysis showed that the expression of TIPE2 in adenoid tissue of OSA group was negatively correlated with clinical score(r=-0.5639,(p<0.001).It was negatively correlated with AHI(r=-0.4172,(p=0.0032).6.The results of Real-time PCR showed that the RNA transcription level of TIPE2 in the adenoid tissues of OSA children was significantly down-regulated(p<0.001).Compared with the CON,while the RNA transcription level of TNF-α IL-6 was significantly up-regulated(p<0.001).7.In this study,we extracted the primary monocytes from hypertrophic adenoid tissue of children with OSA and transfected them with plasmid pRX5-TIPE2 to increase the protein expression level of TIPE2.The transfection effect was confirmed by Western blot,it was confirmed that the overexpression of TIPE2 in monocyte was successfully constructed.8.The results of Elisa showed that the expression level of TNF-α and IL-6 were dramatically lower in adenoid monocytes(PRK5-TIPE2 group)than those in PRK5-NC group(p<0.01)after overexpression of Tipe2(p<0.01).It was confirmed that TIPE2 regulated the expression of TNF-α and IL-6 in adenoid monocytes.ELISA test results showed that the expression levels of TNF-α and IL-6 in overexpressed TIPE2 cells(pRK5-TIPE2 group)were significantly lower than those in group pRK5-NC(p<0.01).9.The results of Western blot showed that compared with pRK5-NC Group,P-P65/P65 was decreased in TIPE2-overexpressed monocytes(pRK5-TIPE2 group),while the expression of IκB-α was significantly up-regulated(p<0.01).It was confirmed that Tipe2 was involved in the activation of NF-κb signal pathway.10.The results of MTT assay showed that the monocyte activity of pRK5-TIPE2 group was significantly lower than that of control group(pRK5-NC group)(p<0.001).It was confirmed that TIPE2 overexpression reduced the activity of adenoid monocytes.11.The results of Flow cytometry showed that the apoptosis rates of adenoid monocytes after TIPE2 overexpress were remarkably higher than that in control group(p<0.001).That were ensured which Tipe2 overexpress had promoted the apoptosis of adenoid monocytes.Conclusion:1.The expression of Tipe2 was down-regulated in adenoid tissues of OS A children,which confirmed that Tipe2 was related to adenoid hyperplasia and upper airway inflammation in OSA children,the function and mechanism of Tipe2 were introduced into the field of airway inflammation of OSA.2.The lower expression of Tipe2 increased the release of inflammatory cytokines,promoted the inflammation and proliferation of adenoids,and aggravated the upper airway inflammation in OSA children.The expression of Tipe2 was negatively correlated with the severity of symptoms in OSA children.The down-regulation of Tipe2 may be the cause of adenoid hyperplasia and upper airway inflammation in OSA children.3.In vitro studies had shown that overexpression of TIPE2 had inhibited the excitation of NF-κB signaling pathway,and reduced the expression of TNF-α and IL-6 in adenoid monocytes of children with OSA,and TIPE2 negatively regulated the NF-κB signaling pathway.Part two:The mechanism of LncRNA HOXA-AS2/MiR-17-5p/TIPE2 axis in improving pulmonary inflammation in OSA ratsBackground:OSA is a kind of sickness caused by fractional or total upper airway obstruction or collapse during sleeping,and its typical pathological feature is CIH.This kind of pathological change can cause the human body cell to produce the massive inflammation molecule and the inflammation chemokine,induces more cells to produce the inflammation reaction,forms the vicious circle,causes the excessive immune response,this affects multiple systems throughout the body.CIH can produce pulmonary hypertension,leading to pulmonary endothelial dysfunction,lung vascular reconstructing,pneumonia,and eventually lung injury.The transcript length of long non-coding RNA(lncRNA)is representatively more than 200 nucleotides,on account of there is no open reading structure in it,therefore,it is an RNA that does not have a protein-coding function.This class of RNA is a small fraction of the many noncoding RNAs in the human genome.LncRNAs are widely distributed in the cytoplasm and nucleus of cells.They interact with other RNAs and DNA and affect the expression of proteins.The expression of lncRNA is specific and is an important regulator of Epigenetics and transcription.A lot of studies have proved that lncRNA expression participates in and regulates the process of cell growth,metabolism,differentiation and apoptosis in different stages of cells,it played a vital part in adjusting cell homeostasis,organismal metabolism and the appearance and evolution of diseases.The lncRNA Hoxa cluster antisense RNA2(Homeobox gene A-AS2,HOXA-as2),a recently discovered lncRNA,is located in the chromosome 7 long arm and contains 1048 nucleotide lengths.It has been reported that it is related to many inflammatory and tumor diseases.It can accommodate the activity of relating signal pathways by modulating protein modification and inhibiting mRNA expression by sponge adsorption,play a role in alleviating inflammatory damage,cancer-promoting and other functions.However,the expression of lncRNA HOXA-AS2 in OSA,its biological role,clinical significance and specific mechanism of action are not clear.MicroRNAs(mRNAs)are small non-coding RNAs,and it have no-protein-coding functions,but play a role in cellular differentiation,hyperplasia and apoptosis.Some studies have proved that Mir-17-5p expression can improve cellular proliferation,restore mitochondrial function,and ameliorate the effect of hypoxia on hyperplasia and apoptosis of satellite cells.Mir-17-5p inhibits brain hypoxia/reoxygenation injury by regulating PI3K/AKT/mTOR signaling pathway.Up-regulated Mir-17-5p may help ameliorate hypoxia-induced astrocyte damage.Our previous study demonstrated that TIPE2 protein has a negative regulatory function in upper airway phlegmonosis in children with OSA;our preliminary study confirmed that HOXA-AS2 is expressed in OSA rats.The results predicted by ENCORI database show that HOXA-AS2 can bind miR-17-5p and miR-17-5p can target TIPE2 protein.Therefore,we hypothesized that lncRNA HOXA-AS2 could interact with miR-17-5p as antisense RNA(Cerna),thus up-regulate the expression of TIPE2 protein and play a role in lung protection.In this study,we investigated the expression of lncRNA HOXA-AS2 in a rat model induced by chronic intermittent hypoxia,and analyzed its significance,to explore the specific molecular mechanism of lncRNA HOXA-AS2 in OSA regulating down-stream miR-17-5p/TIPE2 gene to improve chronic intermittent hypoxia-induced pulmonary inflammation,this study affords new biomarkers for the clinical diagnosis and therapy of OSA,and affords a new theoretical basis for the further exploitation of clinical targeted therapy.Purpose:1.The rat model of OSA was established according to CIH,and the expression of LncRNA HOXA-AS2 and its downstream genes(miR-17-5p,TIPE2)were detected at the animal level,to investigate the mechanism of LncRNA HOXA-AS2/Mir-17-5 p/Tipe2 axis in relieving lung injury in OSA Rats.2.To predict the targeting relationship between HOXA-AS2 and miR-17-5p,miR-17-5p and TIPE2 by bioinformatics method,and to verify the expression and targeting relationship of lncRNA HOXA-AS2,MiR-17-5p and TIPE2 at the cellular level,the molecular mechanism of lncRNA HOXA-AS2 targeting MIR-17-5p regulating TIPE2 was investigated.Material Method:1.Animal level:the OSA rat model induced by CIH and the OSA rat model with overexpression of HOXA-AS2 were set up,and the pathomorphological changes of the lung tissue in Osa rats were detected by HE.Blood gas analysis was applied to detecting the levels of PaO2 and PaCO2(mmHg)in blood,and Tunel was used to measure the apoptosis of pulmonary tissue cells in rats with OSA.The relative expression levels of HOXA-AS2,Mir17-5p and Tipe2 were measured by RT-QPCR or WB.The number of Th1 cell-associated inflammatory and anti-inflammatory cell in the spleen cell was gauged by Flow cytometry.ELISA was applied to detecting the expression of Th1 interrelated factors in serum and inflammatory factors in pulmonary tissue.2.The bioinformatics method predicted the targeting relationship between HOXA-AS2 and miR-17-5p,MiR-17-5p and TIPE2.3.Cell level:HOXA-AS2 plasmid,miR-17-5p plasmid and TIPE2 plasmid were transfected into 293T cell line.The biosorption of Mir-17-5p and miR-17-5p to Tipe2 by HOXA-AS2 sponge was verified by dual luciferase reporter system.In NHBE cell lines,after overexpression of HOXA-AS2 and knockdown of HOXA-AS2,the changes of HOXA-AS2 and miR-17-5p expression were spotted by RT-QPCR.After transfecting Mir-17-5p-mimic and miR-17-5p inhibitor,the expression of miR-17-5p and Tipe2 were detected by RT-QPCR and WB.After transfected with HOXA-AS2 and HOXA-AS2+miR-17-5p-mimic,the expression of Tipe2 was detected by RT-QPCR and WB.Results:Animal testing1.The expression of HOXA-AS2 was low in the lung tissue of OSA rats.HE proved that the pathological changes of inflammatory injury in the lung tissue of OSA rats.Blood gas analysis showed that the oxygen content in the blood of OSA rats was decreased.however,carbon dioxide was retained.Rt-QPCR showed that the expression of HOxA-AS2 was decreased in lung tissue of OSA rats compared with control group.2.Over-expression of HOXA-AS2 alleviates lung injury in OSA rats:RT-QPCR results recommended that over-expression of HOXA-AS2 lowers the down-regulation affection of CIH by HOXA-AS2 expression compared with control group;The results of HE and Tunel suggested that HOXA-AS2 overexpression could moderate the inflammatory damage in OSA rats,and that HOXA-AS2 overexpression regulated CIH-induced apoptosis in OSA rats Blood gas analysis showed that overexpression of HOXA-AS2 improved hypoxia and decreased carbon dioxide retention in the lungs of OSA rats.3.HOXA-AS2 regulates Thl-induced inflammatory response in lung tissue of OSA rats.Flow cytometry results showed that overexpression of HOXA-AS2 decreased the proportion of CD4+IFN-γ+T cells in TH1 cells of spleen tissue of OSA rats,while increased the proportion of anti-inflammatory CD4+TGF-β1+T cells.ELISA results suggested that overexpression of HOXA-AS2 could increase the expression of TGF-β1 and reduce the expression of IFN-γ and IL-2 in serum of OSA rats.ELISA results suggested that overexpression of HOXA-AS2 could reduce the release of inflammatory factors TNF-α,IL-6 and IL-1β in lung tissue of OSA rats.4.HOXA-AS2 regulates the expression of miR-17-5p and Tipe2.The results of RT-QPCR indicated that the expression of miR-17-5p was up-regulated in the lung tissue of OSA rats.Overexpression of HOXA-AS2 could down-regulate the expression of miR-17-5p Overexpression of HOXA-AS2 could up-regulate the expression of Tipe2 in the lung tissue of OSA rats.WB showed that the expression of Tipe2 was down-regulated in the lung tissue of OSA rats,however,overexpress HOXA-AS2 in OSA rats could increase the expression of TIPE2.Cell experiment1.HOXA-AS2 combined with miR-17-5p,and HOXA-AS2 negatively regulated the miR17-5p expression.ENCORI predicted that HOXA-AS2 combined with miR-17-5p.The existence of the same binding target between HOXAAS2 and miR-17-5p was discovered using double luciferase reporter.RT-QPCR resulted indicated that overexpression of HOXA-AS2 could downregulate miR-17-5p and down-regulate HOXA-AS2 could up-regulate miR-17-5p.2.MiR-17-5p targeted TIPE2,MiR-17-5p negatively regulates the TIPE2 expression:ENCORI predicted MIR-17-5P targets TIPE2;double luciferase reporter system results confirmed that miR-17-5p and TIPE2 have the same combine site;RT-QPCR and WB results showed that overexpression of miR-17-5p down-regulated the expression of TIPE2 and inhibited miR-17-5p up-regulated the expression of TIPE2.3.HOXA-AS2 accommodates the expression of Tipe2 via miR-17-5p:RT-QPCR and WB results confirmed that HOXA-AS2 could target miR-17-5p to regulate the expression of Tipe2 after coinfection with HOXA-AS2+miR-17-5p-mimic.Conclusion:1.Low expression of HOXA-AS2 in CIH-induced OSA rat lung negatively regulated inflammatory lung injury in OSA rats.2.The downstream genes of HOXA-AS2 were miR-17-5p and TIPE2,which had the same binding targets.3.HOXA-AS2 targeted miR-17-5p,attenuated miR-17-5p’s negative regulation of TIPE2,meliorating the pulmonary inflammatory injury in OSA Rats.