外泌体miR-125a-5p对NIH/3T3细胞增殖和凋亡的影响
Effect of exosomal miR-125a-5p on proliferation and apoptosis of NIH/3T3 cells
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摘要:
背景 矽肺发生涉及多细胞、多因子的变化和相互作用，其中成纤维细胞是肺纤维过程中关键的效应细胞。近年来研究发现，成纤维细胞的增殖除了受转化生长因子-β等细胞因子的调节之外，外泌体作为细胞间信号的载体也在其中发挥重要的作用。外泌体miRNAs介导的细胞间信号可能在SiO2诱导的成纤维细胞转分化和增殖中发挥作用。前期研究发现miR-125a-5p是一条差异表达的miRNAs。
目的 探讨外泌体miR-125a-5p对小鼠胚胎成纤维细胞NIH/3T3增殖和凋亡的影响。
方法 培养RAW264.7细胞，收取上清提取外泌体，并采用透射电镜、纳米颗粒跟踪分析和Western blot对其进行鉴定。设立对照组和SiO2组（100 μg/mL SiO2），观察SiO2粉尘对RAW264.7细胞外泌体及其中miR-125a-5p表达的影响。建立RAW264.7与NIH/3T3的共培养模型，分为对照组、SiO2组（100 μg/mL）和外泌体组（10 μg/mL外泌体作用于NIH/3T3，并且该组无RAW264.7），24、48 h时采用Western blot和CCK-8检测NIH/3T3细胞Ⅰ型胶原（collagenⅠ）和α-平滑肌肌动蛋白（α-SMA）的表达和增殖活性。培养NIH/3T3细胞，分为模拟剂组、抑制剂组和对照组，于转染24、48 h后收集细胞，采用荧光定量PCR、CCK-8和流式细胞术分别检测NIH/3T3细胞中miR-125a-5p的表达、细胞的增殖活性、细胞周期和凋亡的情况。
结果 透射电镜下观察到外泌体具有圆形或茶托样的膜结构。纳米颗粒跟踪分析结果显示外泌体呈聚团或散在分布，粒径大多在30~150 nm。Western blot可检测到外泌体膜标志蛋白TSG101、Alix，未检测到内质网蛋白calnexin。与对照组相比，SiO2粉尘刺激后RAW264.7细胞外泌体含量增多；外泌体膜标志蛋白TSG101和Alix相对表达量升高；miR-125a-5p的相对表达量上升（P < 0.05）。共培养24、48 h检测结果显示，与对照相比，SiO2组和外泌体组collagenⅠ、α-SMA相对表达量均上升；细胞增殖活性升高（P < 0.05）。NIH/3T3细胞转染24 h和48 h后，与对照组比较，模拟剂组miR-125a-5p相对表达量上升至82.969±5.570和64.934±19.972（对照组为1.000）（P < 0.01），细胞增殖活性上升了18.66%和25.92%，G2/M期细胞数目下降至（2.15±0.35）%和（2.99%±0.61）%对照组分别为（6.30±0.87）%和（6.67±0.59）%，凋亡率下降至（23.15±4.21）%和（19.20±4.75）%对照组为（35.15±3.65）%和（37.09±3.52）%（P < 0.05）；抑制剂组miR-125a-5p相对表达量下降至0.008±0.001和0.038±0.003（P < 0.01），细胞增殖活性下降了17.33%和25.13%，G2/M期细胞数目上升至（10.72±2.06）%和（15.67±2.12）%，凋亡率上升至（50.23±4.06）%和（57.38±3.72）%（P < 0.05）。
结论 SiO2粉尘引起RAW264.7细胞外泌体中miR-125a-5p升高，且miR-125a-5p可以促进NIH/3T3细胞增殖并抑制凋亡。
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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