The Effect And Mechanism Of KIF3A Gene On Airway Inflammation In Asthma

PART Ⅰ THE EFFECT OF THE EXPRESSION OF KIF3A GENE ON AIRWAY INFLAMMATION IN ASTHMATIC MICEObjective: To detect the impact of the expression of KIF3 A gene on airway inflammation in asthmatic mice.Methods: 1)Female C57BL/6 mice were randomly divided into asthma group(n=10)and control group(n=10).Asthma group were sensitized and challenged with house dust mite(HDM)to establish asthma model,while those in control group were replaced by phosphate buffer solution(PBS).2)Invasive lung function test was performed within 24 hours after the last challenge to assess airway hyperresponsiveness(AHR).Bronchoalveolar lavage fluid(BALF)was collected for counting the total number of cells and eosinophils.Lung tissue was stained with hematoxylin and eosin(HE)staining to observe inflammatory cell infiltration to verify the successful establishment of mouse asthma model;Western blot and immunohistochemistry(IHC)to detect the expression of KIF3 A in the two groups.3)Adeno-associated virus(AAV)carrying KIF3 A gene was used to construct KIF3 A overexpression group(AAV-KIF3A),and negative control virus(AAV-control)was used to infect mice to construct AAV-CON group,Western blot and RT-PCR to detect KIF3 A expression level.4)After the establishment of KIF3 A overexpression,the asthma model was established by the same method,that is,AAV-KIF3A+HDM group and AAV-CON+HDM group,ICG-001 5mg/(kg· per)was given to establish HDM+ICG-001 group for 15 days after the completion of sensitization.The inflammatory infiltration of lung tissue was observed by HE staining.The protein expression of KIF3 A and β-catenin was detected by Western blot,and the m RNA by RT-PCR.Results: 1)Successfully constructed the mouse asthma model,and the AHR 、 the total number of cells and eosinophils in the BALF and HE staining in Asthma group were significantly higher than those in Control group.The expression of KIF3 A was significantly lower in the asthma mice than the control mice.2)The overexpression KIF3 A model of AAV transfected mice was successfully constructed,Western blot and RT-PCR showed that the expression level of KIF3 A in AAV-KIF3 A group was significantly higher than AAV-CON group.3)HE staining showed that the inflammatory infiltration in AAV-KIF3A+HDM group and HDM+ICG-001 group was less than control group;Western blot showed that the level of KIF3 A in AAV-CON+HDM group was significantly lower than AAV-KIF3A+HDM group,and there was no significant difference between HDM+ICG-001 group and Asthma group,while the level of β-catenin in Asthma group was significantly higher than Control,AAV-CON+HDM group was significantly higher than AAV-KIF3A+HDM group,and the expression of β-catenin in ICG-001+HDM group was the lowest.RT-PCR showed that the expression levels of KIF3 A and β-catenin were corresponding to the results of Western blot.Conclusion: KIF3 A expression waslower in asthmatic mice,and overexpression of KIF3 A could reduce the inflammatory infiltration of lung tissue,which may be related to β-catenin-dependent Wnt signal pathway,however,the specific mechanism needs further study.PART Ⅱ STUDY ON THE MECHANISM BETWEEN KIF3A GENE AND AIRWAY INFLAMMATION IN ASTHMAAbjective: To investigate the possible mechanism of kinesin family member 3A(KIF3A)taking part in the regulation of asthma airway inflammation,so as to provide a new target and direction for asthma treatment.Methods: 1)After human bronchial epithelial 16 HBE cells were intervened using phosphate buffer solution(PBS)and house dust mite(HDM),Western blotting and RT-PCR were adopted to detect the KIF3 A expression.2)Moreover,the lentiviral vectors of KIF3 A overexpression(LV-KIF3A-45894-J2,LV-KIF3A)and knock-down(LV-KIF3A-RNAi-81183-1,RNAi)were constructed and then transfected into 16 HBE cells,and the blank lentiviral vectors and those containing negative control sequence served as controls,respectively.3)The binding of KIF3 A to β-arrestin in the overexpressed cells was detected by immunoprecipitation,4)while the expression level of β-catenin in each group of cells was determined by Western blotting and RT-PCR.5)The effect of differential expression of KIF3 A on the expression of chemokines CCL-17 and CCL-26 was also determined.Results: 1)In vitro study indicated that HDM intervention decreased the expression of KIF3 A at protein and m RNA levels in 16 HBE cells,though there was no statistical difference(P>0.05).2)The RT-PCR and Western blotting results showed that the expression of KIF3 A were increased in the LV-KIF3 A transfected cells(P<0.05)and decreased in the RNAi treated cells(P<0.05).3)Immunoprecipitation confirmed the binding of KIF3 A to β-arrestin.4)No significant difference was found in the total amount of β-catenin protein between the LV-KIF3 A transfected cells and the RNAi treated cells.Compared with the corresponding cells,the m RNA level of β-catenin was significantly lower in the RNAi treated cells(P<0.0001),but no such difference was seen in the LV-KIF3 A transfected cells.5)The RT-PCR results suggested that the levels of chemokines CCL-17 and CCL-26 were increased in the RNAi treated cells(P<0.001),while decreased in the LV-KIF3 A transfecyed cells(P<0.05)as compred with their control cells.Conclusion: KIF3 A binds to β-arrestin in the Wnt/β-catenin pathway,and is involved in asthmatic airway epithelial inflammation by regulating the expression of chemokines CCL-17 and CCL-26.