| Part I mi R-223 Expression and its Effect on NF-k B Activity of Human Bronchial Epithelial Cells in Iinflammatory Responses Inducedby LPSObjectiveWith the aid of endotoxin lipopolysaccharide(LPS)stimulation of human airway epithelial cells to establish the inflammatory responses in vitro model.The purpose of this study is to detect mi R-223 and p120(p120)expression change and its influence on the NF-kappa B signaling pathways,and to primarily explore the molecular mechanisms of airway inflammation caused by LPS.MethodsAccording to our previous research and relevant literature,CCK8 was used to detect the effects of different concentrations of LPS(0.1?g/ml,1?g/ml,5?g/ml,10?g/ml,20?g/ml,40?g/ml,100?g/ml and 200?g/ml)on cell viability.According to the results,20?g/ml LPS with different stimulation time was chosen to set up airway inflammation model in vitro.Western Blot was performed to examine the expressions of p120 and NF-?B p65.Real-time PCR was applied to detect the levels of p120 m RNA and mi R-223.After the transfection of mi R-223 mimics,mi R-223 inhibitor respectively,ELISA or Real-time PCR was conducted to detect the expressions of IL-1?,IL-6,IL-8 and COX-2.Western Blot was to detect the expression of proteins within the NF-k B signaling pathway.NF-?B p65 nuclear translocation was examined by separate isolation of cytoplasmic and nuclearproteins.The expression and distribution of p65 was detected by immunofluorescence.Real-time PCR was applied to detect the expression changes of p120 m RNA and mi R-223.Results(1)The results of CCK8 showed that different concentrations of LPS could stimulate the proliferation of bronchial epithelial cells slightly at different time,but there was no significant difference among different groups(p> 0.05).(2)Western Blot detection showed that after the 20?g/ml treatment of LPS,NF-?B signaling was activated with the increased p-IKK,p-I?B and p-p-65 expression,while p120 expression was decreased in a time dependent manner.Real Time PCR showed the expression of mi R-223 was increased upon LPS stimulation while the level of p120 m RNA was decreased.(3)To further confirm the relationship between mi R-223 and NF-?B signaling pathway in human bronchial epithelial cells,16 HBE cells were treated with LPS for 6 hours after the transfection of mi R-223 mimics,mi R-223 inhibitor and their relevant negative control for24 hours.RT-PCR showed that the cells transfected with mi R-223 mimics showed higher levels of COX-2,IL-6 and IL-8 m RNA compared with the LPS stimulated group(p< 0.05).(4)Western Blot experiment showed p-IKK,p-I?B? and p-p65 expression in mi R-223 mimics group was higher than that in the group of only LPS stimulated.Isolation of cytoplasmic and nuclear proteins showed that the p65 nuclear expression was also much higher.Immunohistochemistry verified that in mi R-223 mimics group more p65 protein translocated into the nucleus.ELISA showed the phosphorylation of NF-?B p65 was significantly increased.(5)The p120 expression was detected by Western Blot and RT-PCR,both results showed that mi R-223 mimics group had decreased p120 at both protein and m RNA level(p< 0.05).ConclusionsIn LPS-induced airway inflammatory responses,mi R-223 may influence the inflammatory response by regulating NF-k B signaling pathway.Part II The Possible Mechanisms of mi R-223 on NF-?B Signaling Pathway in LPS-induced Inflammatory Responses of HumanBronchial Epithelial CellsObjectiveTo investigate the effects of mi R-223 and p120 on the activation of NF-?B signaling pathway in LPS-induced airway inflammatory responses in cultured human bronchial epithelial cell,and to further explore the possible molecular mechanism of mi R-223 regulating NF-?B signaling pathway in the airway inflammatory responses.MethodsThe human bronchial epithelial cells were transfected with mi R-223 mimics or its negative control,or co-transfected with mi R-223 mimics and either plasmids p120 1A or plasmids 3A,followed by 6h LPS treatment.ELISA was used to detect the secretion of IL-8.The m RNA levesl of IL-8,COX-2,IL-6,p120 and the expression of mi R-223 were observed by RT-PCR.Western blot was performed to detect the expression of NF-?B pathway proteins.The distribution of p65 was investigated by immunofluorescence.Rho A activity was detected with G-LISA.Results(1)ELISA results showed that the expression of IL-8 was decreased in both groups co-transfected with p120 1A or 3A,compared with groups stimulated by LPS after transfected only with mi R-223 mimics.The m RNA levels of IL-8,COX-2 and IL-6 were also decreased after the transfection of p120 1A and 3A(p< 0.05).(2)The results of Western Blot and RT-PCR verified the increased expression of p120 in the cells after transfection of p120 1A or 3A.After co-transfection of mi R-223 mimics with1 A or 3A,p-p65 expression was significantly decreased,compared with groups stimulatedby LPS after transfected only with mi R-223 mimics.(3)Immunofluorescence experiments showed that in the groups of co-transfected with p120 1A or 3A,the nuclear translocation of p65 was reduced.the co-transfected groups was significantly increased.(4)ELISA showed that the phosphorylation of NF-?B p65(Ser 536)was decreased after the co-transfection of mi R-223 mimics with 1A or 3A,compared with the groups of solo-transfected.(5)G-LISA showed that the co-transfection of mi R-223 mimics with 1A or 3A had an impaired Rho A activity,compared with the groups of solo-transfected.Compared with the co-transfection of p120 3A,p120 DRDD plasmids co-transfection enhanced Rho A activity..ConclusionsIn the process of inflammatory responses,mi R-223 may negatively regulate the expression of p120,which activates Rho A and NF-?B signaling pathway consequently. |
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