miR-766-3p/PNCK/mTOR通路在气道上皮细胞机械损伤中的作用研究

doi:10.3877/cma.j.issn.1674-6902.2026.01.003

目的

探讨机械损伤对气道上皮细胞生长影响及miR-766-3p/PNCK/mTOR通路的作用。

方法

选择气道上皮细胞转染has-miR-766-3p mimics、inhibitor及阴性对照组(negative control, NC)，通过机械划痕对气道上皮细胞进行损伤造模，采用细胞计数试剂盒-8(cell counting kit-8, CCK8)检测细胞活性；苏木精-伊红染色法(hematoxylin-eosin staining, HE)染色分析细胞形态；Transwell法检测细胞迁移能力；蛋白印迹法(Western blot, WB)检测蛋白表达水平；逆转录-定量聚合酶链反应(quantitative reverse transcription polymerase chain reaction, RT-qPCR)检测RNA表达水平。

结果

造模后细胞hsa-miR-766-3p RNA表达下降(1 .01比0.31)，侵袭能力下降(180比123)、活性下降(0.97比0.72)，差异有统计学意义(P<0.05)。细胞造模过表达miR-766-3p，细胞侵袭能力(127比94.5)、活性(0.7比0.52)下降，抑制性表达miR-766-3p后细胞侵袭能力(118比217)、活性(0.69比0.87)上升，差异有统计学意义(P<0.05)。抑制性表达miR-766-3p后，妊娠上调非泛素性钙调蛋白激酶(pregnancy-upregulated nonubiquitous calmodulin kinase, PNCK)(0.34比0.84)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)(0.45比1.05)表达水平上升，差异有统计学意义(P<0.05)。

结论

体外细胞研究显示，机械损伤影响miR-766-3p表达，miR-766-3p通过PNCK、mTOR表达影响气道上皮细胞活性及迁移能力。

关键词: 良性气道狭窄, 机械划痕, miR-766-3p, 妊娠上调非泛素性钙调蛋白激酶, 哺乳动物雷帕霉素靶蛋白

Objective

To explore the impact of mechanical injury on the growth of airway epithelial cells by simulating mechanical damage through mechanical scratches, and to investigate the role of the miR-766-3p/PNCK/m-TOR pathway in this process.

Methods

The airway epithelial cells were transfected with has-miR-766-3p mimics, inhibitors, and corresponding negative control(NC), and the airway epithelial cells were mechanically scratched to create a damage model; cell counting kit-8(CCK8) method was used to detect cell viability; hematoxylin-eosin staining(HE) staining analysis of cell morphology; Transwell method for detecting cell migration ability; Western blot(WB) was used to detect protein expression levels; Quantitative reverse transcription polymerase chain reaction(RT qPCR)was used to detect RNA expression levels.

Results

After modeling, the hsa-miR-766-3p RNA expression of cells decreased (1.01 vs. 0.31), the invasion ability decreased (180 vs. 123), and the cell activity decreased (0.97 vs. 0.72). The differences were statistically significant (P<0.05). After cell modeling, overexpression of miR-766-3p decreased cell invasion ability (127 vs. 94.5) and cell activity (0.7 vs. 0.52). After inhibitory expression of miR-766-3p, cell invasion ability (118 vs. 217) and activity (0.69 vs. 0.87) increased. The differences were statistically significant (P<0.05). After inhibitory expression of miR-766-3p, the expression of Pregnancy-upregulated nonubiquitous calmodulin kinase(PNCK)(0.34 vs. 0.84) and mammalian Target of rapamycin(mTOR)(0.45 vs. 1.05) increased, and the difference was statistically significant (P<0.05).

Conclusion

Through in vitro cell studies, it is believed that mechanical injury can affect the expression of miR-766-3p, and miR-766-3p further influences the activity and migration ability of airway epithelial cells by affecting the expression of PNCK and mTOR.

Key words: Benign airway stenosis, Mechanical scratch, miR-766-3p, Pregnancy-upregulated nonubiquitous calmodulin kinase, Mammalian target of rapamycin

图1 细胞造模后生长情况注：①16HBE；②16HBE＋miRNA NC；③16HBE＋miRNA mimics；④16HBE＋miRNA inhibitor NC；⑤16HBE＋miRNA inhibitor；⑥16HBE；②~⑥机械划痕处理

图2 每组miR-766-3p RNA分子表达量注：realtive expression为相对表达；control为对照组；NC为非特异性对照组；mimics为模拟物；inhibitor为抑制剂；*、***、****为差异有统计学意义

图3 细胞迁移检测结果注：A为16HBE；B为16HBE＋miRNA NC＋造模；C为16HBE＋miRNA mimics＋造模；D为16HBE＋miRNA inhibitor NC＋造模；E为16HBE＋miRNA inhibitor＋造模；F为16HBE＋造模

图4 CCK8检测细胞增殖活性注：control为对照组；NC为阴性对照组；mimics为模拟物；inhibitor为抑制剂；****为差异有统计学意义

图5 WB蛋白质印迹法PNCK及mTOR表达注：control为对照组；NC为阴性对照组；mimics为模拟物；inhibitor为抑制剂；ns为无统计学意义；*~***为有统计学意义

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Study on the mechanism of the miR-766-3p/PNCK/mTOR signaling pathway in mechanical injury of airway epithelial cells

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Study on the mechanism of the miR-766-3p/PNCK/mTOR signaling pathway in mechanical injury of airway epithelial cells