| Background: Airway mucus hypersecretion is one of the most importantpathophysiological features of chronic airway inflammatory diseases, andmeanwhile a main problem of respiratory diseases which needs to besolved. Currently，as mucus hypersecretion related diseases are verycommon and critical, great attention has been paid to MUC，or mucingradually. At present,21kinds of mucins have been found, at least12kinds of which are expressed in the airway. Raise of MUC5AC secretedby goblet cells is the main change of airway mucin in pathologicalconditions. As increased expression of MUC5AC is closely related to thepathophysiological changes in lot of mucus hypersecretion relateddiseases, such as bronchial asthma, chronic obstructive pulmonarydisease(COPD), bronchiectasis, cystic fibrosis(CF) and so on, understandthe regulatory mechanism of MUC5AC expression is particularlyimportant.AGR2(human anterior gradient-2, HAG-2or Gob-4) is one of theprotein disulfide isomerase family members (PDIs). As a a smallmolecules secretory protein，AGR2is widely expressed in human tissues，however， its function is unclear now. MUC2is the main component of intestinal mucus. Animal study outcome showed that AGR2is closelyrelated to the synthesis of intestinal mucus. AGR2is essential forproduction of intestinal mucus. As MUC5AC is the most importantcomponent of airway mucus，we assume that there is a close relationshipbetween AGR2and airway mucus secretion. To evaluate the effect ofAGR2on the expression of MUC5AC mRNA and MUC5AC protein inhuman airway epithelium, we make CMV-Sport6-AGR2plasmidtransfection in airway epithelium using NCI-H292cells, and then detectthe expression of airway mucin MUC5AC mRNA and protein.Objective:To observe the expression of AGR2mRNA, MUC5ACmRNA and mucin MUC5AC after the CMV-Sport6-AGR2plasmidtransfection on the NCI-H292airway mucus epithelial cells. Toinvestigate the effects of AGR2gene on MUC5AC gene expression in thehuman airway epithelial.Methods: In vitro model, the cultured human airway mucus epithelialcancer cell lines NCI-H292cells were transfected by CMV-Sport6-AGR2plasmid with different concentration for24hours. Thenimmunocytochemical method was used to detect the transfection ofCMV-Sport6-AGR2plasmid with different concentration on NCI-H292cells, that was AGR2protein expression. NCI-H292cells transfected byCMV-Sport6-AGR2plasmid with the best concentration were assigned tothe transfection group, and NCI-H292cells with no treatment as a control group. The expression of AGR2mRNA and MUC5ACmRNA in the twogroup cells were detected by reverse trancription-polymerase chainreaction (RT-PCR). The expression of MUC5AC protein in the two groupcells was examined by immuncocytochemistry SP staining.Results:1.The best concentration of CMV-Sport6-AGR2plasmid transfectingNCI-H292cells is1.33μg/ml.NCI-H292cells transfected byCMV-Sport6-AGR2plasmid with this concentration were assigned to thetransfection group.2.The expresssion of AGR2mRNA and MUC5ACmRNA in cells:2.1RT-PCR results showed that the expression of AGR2mRNA inCMV-Sport6-AGR2plasmid transfection group was positive (0.61±0.05), whereas in control group was negative. The difference wasstatistically significant (P＜0．01)2.2RT-PCR results showed that the expression of MUC5ACmRNAinCMV-Sport6-AGR2plasmid transfection group (0.85±0.09) washigher than that in control group（0.35±0.04）. The difference wasstatistically significant(P＜0．01）.3.The protein of MUC5AC measured by immunocytochemistry in cells:Immunocytochemistry results found that MUC5AC protein-positivesignals were located in the cytoplasm and the cytoplasm of cells stainedyellow or brown. Only a very small proportion cells in control group displayed cytosol staining.,but MUC5AC staining significantly enhancedto be positive in CMV-Sport6-AGR2plasmid transfection group cells.Compared with control group (2.6±0.4), the percentage of positive cellsincreased significantly in CMV-Sport6-AGR2plasmid transfectiongroup(21.2±5). The difference was statistically significant（P＜0．01）.Conclusion: AGR2may induce over-expression of MUC5ACmRNA andMUC5AC protein of airway epithelial cell line NCI-H292cells. AGR2plays an important role in airway mucus hypersecretion. |
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