Bronchial Epithelial MiR-146 Induces Airway Mucus Production By Regulating IL-13 In COPD

Background & objective:Mi RNAs are potent modulators of cellular responses and have been widely documented in inflammatory and neurological responses,but their role in airway mucus regulation in chronic obstructive pulmonary disease(COPD)is not completely clear yet.In this study,we investigated the role of mi R-146 in COPD by observing the regulatory effect of mi R-146 on mucus secretion in chronic airway inflammatory epithelial cells.Methods:(1)A total of 20 COPD patients and 20 healthy subjects were included in this study,and serum samples were collected from the two groups of subjects for detection.The cell line used in this study was 16 HBE,and the cells were cultured under strictly sterile conditions.IL-13 was used to induce the occurrence of cellular inflammatory response.The concentration of IL-13 was 0,1,2,5,20 and 40 mg/L,and dimethyl sulfoxide(DMSO)was used to induce the cells in the control group.CCK-8 was used to detect the effect of IL-13 on the proliferation of cells in different groups.The expression levels of mi R-146 in cells and levels were analyzed by reverse transcription quantitative real-time PCR(RT-q PCR);the expression levels of intracellular inflammatory cytokines IL-6,IL-1β and TNF-α were detected by Human Cytokine Multiplex ELISA Kit.The above studies confirmed the feasibility of using IL-13 to construct a COPD model in 16 HBE cells.On this basis,we used 16 HBE cells to regulate the expression of mi R-146 and knocked down crimidium 146 by Cytokine 9;Western Blot was used to detect the expression levels of cellular inflammatory factors;Assay annexin V binding buffer was used to detect apoptosis;(ii)flow cytometry was used to detect mucus secretory proteins induced by IL-13 in16 HBE cells Correlative cellular expression;expression of mucus production-associated proteins was analyzed using dot blot;in this study,male C57BL/6 mice aged 5 – 8 weeks were used to construct a mi R-146 knockdown model(mi R-146-/-)and a mouse model of chronic obstructive pulmonary disease was constructed using smoke exposure;cellular and horizontal levels of mi R-146 expression were analyzed by RT-q PCR.Lung tissue lesions were detected by immunohistochemistry,and mean linear intercept(MLI),mean alveolar septal thickness(MAST),and destruction index(DI)were collected to assess emphysema changes in mice.Results:(1)Compared with healthy subjects,the expression of mi R-146 in serum samples of COPD patients was significantly higher than that in the control group,and the difference was significant(P < 0.05);2.The concentration of IL-13 was set at 0 mg/L,1 mg/L,2 mg/L,5 mg/L,20 mg/L,and 40 mg/L using reference to previous studies.Cells were stimulated with different concentrations of IL-13 immediately after attachment.CCK-8 was used to detect cell proliferation,and the results showed that the growth and proliferation of 16 HBE cells treated with different concentrations of IL-13 were affected by IL-13 compared with cells treated with CDMO in the control group,and the inhibition rate increased with the increase of IL-13 concentration.Combined with the inhibition rate data,the half inhibitory concentration of IL-13 was calculated.The results showed that the half inhibitory concentration of IL-13 in16 HBE cells in this study was 19.17 ± 7.11 mg/L;3,after IL-13 stimulation,the m RNA and concentration of IL-1β,IL-6 and TNF-α in 16 HBE and supernatant were significantly increased,respectively(P < 0.01),and after IL-13 concentration of 5mg/L,the increase of IL-13 concentration did not gain the release of inflammatory factors.Therefore,we used the concentration of IL-13 of 5 mg/L for subsequent validation studies;4.The relative expression level of mi R-146 in 16 HBE was set as 1,the relative expression level of mi R-146-inhibitor-1 in NC group was 1.09,the relative expression level of mi R-146-inhibitor-2 was 0.09,and the expression level of mi RNA in knockdown group was significantly different from that in control group;5.analysis of apoptosis-related proteins by Western Blotting.The results showed that IL-13 significantly increased the protein levels of Bax and cleaved caspase 3/9,but decreased the protein expression of Bcl-2,which was significantly reversed after mi R-146 knockdown.6.The results of cell proliferation detected by CCK-8 showed that the proliferation level of cells was significantly increased when the expression of mi R-146 was inhibited;(ii)To investigate the effect of MIR-146 gene editing in primary 16 HBE cells on IL-13-induced airway mucin MUC5 AC.Using intracellular flow cytometry,MUC5AC-expressing cells were significantly increased after IL-13 induction,but significantly decreased after mi R-146 knockdown,and MUC5AC-expressing cells were significantly less abundant in the knockdown group than in the Mock group,and gene editing decreased the frequency and mean fluorescence intensity of MUC5AC-expressing cells;mi R-146 regulated IL-13-induced MUC5 AC production in epithelial cells,and mi R-146 knockdown helped 16 HBE cells reduce mucus production.Comparing with cells in the high mi R-146 gene expression group,knockdown of mi R-146 resulted in a significantly lower frequency of tetraspanin 8(TSPAN8 +)secreting cells(defined as acetylatedα-tubulin – NGFR – CEACAM6 + TSPAN8 +)in stimulated 16 HBE cells.After modeling,the body weight of the mice was detected,and the body weight gain of the mice in the model group and the model + mi R-146 knockdown group was slower,especially in the model group at the early stage of body weight intervention,the body weight even showed a downward trend.However,after 2 months of modeling,the body weight gain of mice in each group tended to be stable,and the body weight of mice in the model group was less than that in the model + mi R-146 knockdown and control groups;immunohistochemical results showed that the alveolar space of mice in the model group was enlarged,the alveolar septa were thinned,and the alveolar wall was significantly destroyed,and the trend in the COPD group was more severe than that in the COPD + mi R-146 inhibition group;the changes in the MAST and the MLI as well as the changes in the DI of mice in different groups were measured.The results showed that the lung function of mice in the model group was severely impaired;the expression level of mi R-146 in the lung tissue was significantly reduced after targeted antagomir administration using mi R-146,and the MUC5 AC gene expression in the whole lung homogenate showed a decreasing trend in mice receiving mi R-146 antagomir.Mice challenged with cigarette smoke and receiving mi R-146 antagomir showed a significant decrease in the number of mucus-expressing epithelial cells in the large and small airways.In addition,compared with the cigarette smoke group,mi R-146 antagomir treatment reduced the secretion of mucus in the airway lumen,indicating that inhibition of mi R-146 expression has a therapeutic effect in COPD mucus hypersecretion.Conclusions:We developed a CRISPR/Cas9-based targeted mi R-146 knockout protocol to target mi R-146 in primary human bronchial epithelial cells differentiated at the air-liquid interface and induce goblet cell hyperplasia by IL-13 stimulation.mi R-146 knockdown resulted in decreased goblet cell frequency,intracellular MUC5 AC,and total secreted mucus.Intranasal application of sequence-specific inhibitors in mice reduced mucus protein production.Furthermore,mi R-146 antagomir treatment was followed by mucus reduction in airway lumen endocrine compared to scramble antagomir treatment,indicating that inhibition of mi R-146 expression has a therapeutic effect in mucus hypersecretion in COPD.