| Chronic Obstructive Pulmonary Disease (COPD) is a preventable and treatable disease with some significant extrapulmonary effects that may contribute to the severity in individual patients. Its pulmonary component is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lung to noxious particles or gases. Not only it affects seriously the quality of life but also exerts a heavy financial burden on the family and society. It is confirmed that there exists abnormality in the structure and function of the respiratory cilia, which leads to impairment of the mucociliary clearance (MCC), clog of secretions, obstruction of small airways, and recurrent infections.All of these are the key factors of recurrence and exacerbation of COPD.It has been proved that COPD affects structure and function of the respiratory cilia, although the pathogenesis especially the molecular mechanism is not yet clear, research on the mechanism is helpful for prevention and treatment of COPD.MIP-T3{Tumor necrosis factor receptor associated factor 3 (TRAF3)-interacting protein 1,TRAF3IP1 or MIP-T3}is a 83-KDa ciliary protein, which is speculated that may play an important role in the process of ciliary proteins transporting toward the ciliary tip with the help of intraflagellar transport (IFT) complex B. MIP-T3 and TRAF3 are co-localized to microtubule structure when co-expressed. MIP-T3 also constitutively associates with IL-13Rα1 and suppresses IL-13/IL-4-induced signal transducer and activator of transcription 6(STAT6) phosphorylation. It has been proved that MIP-T3 is closely associated with the biogenesis and function of cilia in many model organisms such as Chlamydomonas, Caenorhabditis elegans, Zebrafish, Drosophila and Mouse.In the mutant, defective and knock-down of MIP-T3, some changes were found in structure of cilia such as short, few and scattered cilia, defective microtubes and disordered arrangement. MIP-T3 mRNA was expressed in many human tissues such as bronchus, lung, testis, ovarian, spinal cord, thyroid, prostate, and so on, especially rich in testis whose cilia is abundant. Protein databases revealed that MIP-T3 is highly evolution-conserved from worm to human.In view of the crucial role in biogenesis of cilia, we speculate that MIP-T3 is likely to perform an important role in the formation of cilia. At present, most research is focused on model organisms and cell lines, however, the relationship between MIP-T3 and diseases especially respiratory diseases has not been explored.Foxj 1 and Rfx3 are markers of ciliary biogenesis, which play regulatory roles as nuclear factors. We measured the level of MIP-T3 mRNA, along with the expression of Foxj 1 and Rfx3 as control. The percentage of abnormal cilia and clinical data were also studied in order to explore the role of MIP-T3 played in the formation of cilia and impact on the clinical parameters, to achieve a deeper understanding of cilia and COPD,to pay attention to the impact of ciliary disfunction on diseases, and to persue some new treatments of COPD.ObjectiveTo compare the differences of expression of MIP-T3 mRNA and ciliary ultrastructure in bronchus from above lung segment among group NS(Nonsmokers with normal lung function), group S(Smokers with normal lung function) and group COPD.To explore the role of MIP-T3 played in the abnormal cilia in COPD patients.Materials and Methods56 patients who accepted pulmonary lobectomy in Lung Cancer Research Institute of Guangdong General Hospital,were divided into 3 groups:group NS, group COPD, and group S according to smoking history and pulmonary function. All patients in group COPD(smoking index≥10 pack-years,n=18) had been diagnosed as COPD according guidelines, including 17 males and 1 female, with a mean age of 62.2years±9.4years. Lung carcinoma consisted of adenocarcinoma(n=14), squamous cell carcinoma(n=3) and lung tuberculosis(n=1). All patients in group S(smoking index≥10 pack-years, n=17) had normal lung function, including 16 males and 1 female, with a mean age of 57.4 years±10.4years. Lung carcinoma consisted of adenocarcinoma(n=15), squamous cell carcinoma(n=1) and lung tuberculosis(n=1). All patients in group NS(n=21)without a smoking history had normal lung function, including 18 males and 3 females, with a mean age of 58.6years±11.2years.Lung carcinoma consisted of adenocarcinoma(n=18), squamous cell carcinoma(n=l) and lung tuberculosis(n=2). There was no significant difference in sex, age, and composition of diseases. Bronchus and lung tissue were obtained from lung segment more than 5 cm away from lung neoplasm and immersed into liquid nitrogen immediately, another piece of tissue was placed into electron microscopy stationary liquid for pre-fixation. After preparation of samples was done, the ultrastructure of cilia was observed with transmission electron microscope. The main targets of observation were axonemal ultrastructure abnormality(AUA) including vesiculation of ciliary membrane, defects in central microtubes, defects in peripheral microtubes, compound cilia and giant cilia.The percentage of abnormal cilia was also counted. Bronchus and lung tissue were ground with liquid nitrogen, then RNA was extracted by Trizol and quantified. After all treat as above, we investigate the mRNA expession of MIP-T3,Foxj1 and Rfx3 quantitative-PCR according to the instructions from TAKARA. The expression of each sample was computed(ΔCT=CT Target gene-CTGAPDH),then the expression of target gene was 2-ΔCTResults1. Comparison of percentage of abnormal cilia1.1 Axonemal ultrastructure abnormality The percentage of axonemal ultrastructure abnormality among group NS, COPD and S was (4.24±1.07)%vs (12.74±3.17)%vs(4.61±1.90)%(P<0.001). The percentage in group COPD was significantly higher than the other two(P<0.001), but there was no statistical significance between group NS and group S(P>0.05).1.2 Vesiculation of ciliary membrane The percentage of vesiculation of ciliary membrane among group NS,COPD and S was (0.15±0.13)%vs(1.38±0.82)%vs (0.19±0.12)%, the percentage in group COPD was significantly higher than the other two (P<0.01), but there was no statistical significance between group NS and group S(P>0.05).1.3 Defects in central microtubes There was no significant difference in percentage of defects in central microtubes among group COPD, S and NS [(0.19±0.26)%vs(0.03±0.06)%vs(0.01±0.03)%, P> 0.05].1.4 Giant cilia There was no significant difference in percentage of giant cilia among group COPD, S and NS [(1.22±1.40)%vs(0.26±0.37)%vs(0.16±0.26)%, P> 0.05].1.5 Defects in peripheral microtubes The percentage of defects in peripheral microtubes average was significantly higher in group COPD(5.20±1.52)% compared with its in group NS (1.86±0.89)% and group S(3.08±1.09)%, P<0.01. While it was higher in group S than in group NS(P<0.01).1.6 Compound cilia The percentage of defects in peripheral microtubes average was significantly higher in group COPD(4.75±2.12)% compared with its in group NS (2.05±0.69)% and group S(1.04±1.01)%(P<0.05 and P<0.01,respectively). While it was higher in group NS than in group S (P<0.05).1.7 Correlation analysis In group COPD, there was a negative correlation between axonemal ultrastructure abnormality and FEV1(r=-0.766, P<0.05), FEV1Pred(r=-0.777, P<0.05) and FEV1/FVC(r=-0.684, P<0.05).2. Comparison of MIP-T3,Foxj1,Rfx3 mRNA among all groups in bronchial tissue2.1 MIP-T3 There was statistical significance of MIP-T3 mRNA in expression among group NS[0.021(0.013-0.034)], group COPD[0.043(0.033-0.053)] and group S[0.034(0.019-0.052)],P=0.001.The expression of MIP-T3 was significantly lower in group COPD compared with its in group NS and group S (P< 0.05 and P<0.01,respectively).2.2 Foxj1 There was no statistical significance of Foxj1 in mRNA expression among group NS[0.014(0.006-0.028)], group COPD[0.027(0.014-0.063)] and group S[0.018(0.012-0.032)], P>0.05.2.3 Rfx3 There was no statistical significance of Foxj1 in mRNA expression among group NS[0.025(0.007-0.044)], group COPD[0.021(0.013-0.048)] and group S[0.019(0.008-0.058)], P>0.05.2.4 Correlation analysis between Foxj 1,Rfx3 and MIP-T3 Correlation of all data, There was a positive correlation between MIP-T3 and Foxj1(r=0.520, P<0.01), but there was no significant correlation between the levels of MIP-T3 and Rfx3.3. Comparison of MIP-T3,Foxj1,Rfx3 mRNA among all groups in lung tissue3.1 There was no statistical significance of MIP-T3,Foxj1 and Rfx3 in mRNA expression of lung tissue among group NS, group COPD and group S (P>0.05).3.2 There was no significant correlation between the levels of Foxj1,Rfx3 and MIP-T3 in lung tissue.4. Correlation analysis between expression of MIP-T3 and the percentage of abnormal cilia and clinical parameters in COPD4.1 Correlation analysis between levels of MIP-T3 and the percentage of abnormal cilia There was no significant correlation between axonemal ultrastructure abnormality and the levels of MIP-T3. The expression of MIP-T3 in bronchus showed a negative correlation with percentage of defective peripheral microtubes (r=-0.750, P<0.05).4.2 Correlation analysis between expression of MIP-T3 and clinical parameters The expression of MIP-T3 in group COPD showed no correlation with smoking index,FVC,FEV1,FEV,%Pred and FEV,/FVC.Conclusion1. Generally, there existed abnormal cilia in COPD patients and there was an increase in vesiculation of ciliary membrane, defects in peripheral microtubes and compound cilia. The severer COPD was,the more abnormal cilia was.2. There was abnormal ciliary ultrastructure in smokers who has not developed to be COPD, which mainly had defects in peripheral microtubes.3. The abnormal ciliary ultrastructure in COPD had something to do with down-rugulation in MIP-T3,but had no direct link to Foxj1 and Rfx3. |
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