Abstract:
Background Silicosis occurrence involves multicellular and multifactorial changes and interactions, in which fibroblasts are the key effector cells in the progression of pulmonary fibrosis. Recent studies have found that, in addition to cytokines such as transforming growth factor-beta, the proliferation of fibroblasts is also regulated by exosomes as carriers of intercellular signals. The intercellular signals mediated by exosomal microRNAs may play a role in the transdifferentiation and proliferation of fibroblasts induced by SiO2. A previous study has proved miR-125a-5p one of the differentially expressed miRNAs.
Objective This study is designed to explore the effect of exosomal miR-125a-5p on proliferation and apoptosis of mouse embryonic fibroblasts NIH/3T3 cells.
Methods Exosomes were extracted from the supernatant of RAW264.7 cell culture and identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot. RAW264.7 cells were divided into a control group and a SiO2 group (100 μg/mL SiO2) to observe the effect of SiO2 dust on exosomes and exosomal miR-125a-5p of RAW264.7 cells. Co-cultured RAW264.7 and NIH/3T3 cells were divided into a control group, a SiO2 group (100 μg/mL SiO2), and an exosome group (10 μg/mL exosomes were administered directly to NIH/3T3 cells, without RAW264.7 cells) to detect the expressions of collagenⅠ and α-smooth muscle actin (α-SMA) by Western blot and cell proliferation by CCK-8 after 24 h and 48 h. NIH/3T3 cells were divided into mimic, inhibitor, and control groups, and after 24 h and 48 h of transfection, the expression of miR-125a-5p, cell proliferation activity, cell cycle, and apoptosis of NIH/3T3 cells were detected by fluorescence quantitative PCR, CCK8, and flow cytometry, respectively.
Results The exosomes exhibited round or saucer-like membrane structure under transmission electron microscopy. They were clustered or scattered with a particle size mainly at 30-150 nm by nanoparticle tracking analysis. The membrane marker proteins TSG101 and Alix were detected and the endoplasmic reticulum protein calnexin was not detected by Western blot. Compared with the control group, the concentration of exosomes, the relative expression levels of membrane marker proteins TSG101 and Alix, and the relative expression level of miR-125a-5p increased after the designed SiO2 dust exposure (P < 0.05). After 24 h and 48 h of co-culture, compared with the control group, the relative expression levels of collagen Ⅰ and α-SMA and the cell proliferation activity increased in the SiO2 group and the exosome group (P < 0.05). After 24 h and 48 h of transfecting into NIH/3T3 cells, compared with the control group, the relative expression levels of miR-125a-5p increased to 82.969±5.570 and 64.934±19.972 (control:1.000) (P < 0.01), the cell proliferation activities increased by 18.66% and 25.92%, the numbers of cells in G2/M decreased to (2.15±0.35)% and (2.99±0.61)%control:(6.30±0.87)% and (6.67±0.59)%, and the apoptosis rates decreased to (23.15±4.21)% and (19.20±4.75)% in the mimic groupcontrol:(35.15±3.65)% and (37.09±3.52)%, respectively (P < 0.05); the relative expression levels of miR-125a-5p declined to 0.008±0.001 and 0.038±0.003 (P < 0.01), the proliferation activities decreased by 17.33% and 25.13%, the numbers of cells in G2/M increased to (10.72±2.06)% and (15.67±2.12)%, and the apoptosis rates increased to (50.23±4.06)% and (57.38±3.72)% in the inhibitor group, respectively (P < 0.05).
Conclusion SiO2 dust induces the increase of exosomal miR-125a-5p in RAW264.7 cells, and miR-125a-5p promotes the proliferation and inhibits the apoptosis of NIH/3T3 cells.
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