Objective: To study the effects and mechanisms of activation of human lung fibroblasts (MRC-5) by exosomal RNA hsa _ circ _ 0006357 (circEZH2) derived from non-small cell lung cancer. Methods: Western blot was used to detect exosome molecular markers, reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and cell invasion assays to detect the effect of non-small cell lung cancer-derived exosomes on MRC-5 activation. A circRNA microarray analysis was performed in serum exosomes from patients with non-small cell lung cancer (collected at Ningbo University People's Hospital, September 2023), and levels of circEZH2 were measured in serum exosomes from non-small cell lung cancer by RT-qPCR analysis. The effects of circEZH2 on MRC-5 activation were explored using wound healing assays, Transwell assays, RT-qPCR, cellular immunofluorescence, and western blot. The regulatory effect of circEZH2 on miR-495-3p/TPD52 axis and NF-κB pathway through dual-luciferase assay, immunofluorescence, and western blot. Results: Exosomes derived from non-small cell lung cancer cells were shown to promote MRC-5 cell invasiveness, the number of cells invaded in co-culture with exosomes derived from normal human bronchial epithelial cells was (42±5), and the number of cells invaded in co-culture with exosomes derived from non-small cell lung cancer cells (SPC-A1, H1299, A549 cells) was (246±7), (89±4), (69±14), expression of markers of fibroblast activation (α-SMA, FAP), and cytokines (IL-6, IL-8, P＜0.05). CircEZH2 expression was significantly upregulated in the serum exosomes of non-small cell lung cancer (P＜0.01). Alternatively, co-culture of exosomes derived from non-small cell lung cancer cells with MRC-5 cells promoted circEZH2 expression (P＜0.05). Functionally, overexpression of circEZH2 promoted MRC-5 cell migration and invasion [the cell migration rates were (30.81±2.54)% and (60.29±8.34)%, respectively, and the cell invasion numbers were (48.00±13.58) and (115.00±9.50), respectively, P＜0.05]. RT-qPCR, western blot, and immunofluorescence assays demonstrated a significant increase in the expression of the pro-inflammatory genes IL-6 and IL-8 in MRC-5 cells as well as the activation markers α-SMA and FAP in fibroblasts (P＜0.05) following the expression of circEZH2. Mechanistically, circEZH2 may function as ceRNA to regulate miR-495-3p, promote the expression of TPD52, and activate the NF-κB pathway to promote the activation of MRC-5. Conclusion: Exosomal circEZH2, derived from non-small cell lung cancer, may promote the activation of fibroblasts by activating the NF-κB pathway through the miR-495-3p/TPD52 axis.
目的： 探讨非小细胞肺癌来源外泌体环状RNA hsa_circ_0006357（circEZH2）对人肺成纤维细胞（MRC-5细胞）的激活影响及其作用机制。 方法： 应用Western blot检测外泌体分子标志物，实时荧光定量聚合酶链反应（RT-qPCR）和细胞侵袭实验检测肺腺癌来源外泌体对MRC-5细胞活化的影响。通过高通量测序对肺腺癌患者血清（2023年9月于宁波大学附属人民医院收集）外泌体进行circRNA分析，并通过RT-qPCR检测肺腺癌患者血清外泌体中circEZH2 mRNA的水平。细胞划痕实验、Transwell侵袭实验、RT-qPCR、细胞免疫荧光实验和Western blot探究circEZH2对MRC-5活化的影响。通过荧光素酶报告基因、免疫荧光实验和Western blot分析circEZH2对miR-495-3p/肿瘤蛋白D52（TPD52）轴和核因子κB（NF-κB）通路的调控作用。 结果： 非小细胞肺癌来源外泌体促进了MRC-5细胞侵袭能力提升［与正常人支气管上皮细胞来源外泌体共培养细胞侵袭数为（42±5）个，与非小细胞肺癌细胞（SPC-A1、H1299、A549细胞）外泌体共培养细胞侵袭数为（246±7）个、（89±4）个、（69±14）个］，成纤维细胞活化标志物［α-平滑肌肌动蛋白（α-SMA）、成纤维细胞活化蛋白（FAP）］和细胞促炎基因［白细胞介素6（IL-6）、IL-8］的表达升高（均P＜0.05）。非小细胞肺癌患者血清外泌体中circEZH2 mRNA的表达水平增加（P＜0.01）。非小细胞肺癌细胞来源外泌体与MRC-5细胞共培养促进了circEZH2表达（P＜0.05）。在功能上，过表达circEZH2促进了MRC-5细胞迁移和侵袭［细胞迁移率分别为（30.81±2.54）%和（60.29±8.34）%，细胞侵袭数分别为（48.00±13.58）个和（115.00±9.50）个，均P＜0.05］。RT-qPCR、western blot和免疫荧光检测结果显示，过表达circEZH2后，MRC-5细胞促炎因子IL-6、IL-8的mRNA和蛋白水平增加（P＜0.05），成纤维细胞活化标志物α-SMA、FAP蛋白表达增加（P＜0.05）。在机制上，circEZH2可能作为ceRNA调节miR-495-3p，促进TPD52表达，同时激活NF-κB通路促进MRC-5活化。 结论： 非小细胞肺癌细胞来源外泌体circEZH2可能通过miR-495-3p/TPD52轴激活NF-κB通路促进成纤维细胞活化。.