香烟诱导支气管上皮细胞外泌体释放miR-34a靶向CASP2促进肺成纤维细胞增殖

Cigarette induced release of exo⁃miR⁃34a from 16HBE vesicles targeting CASP2 promoted the proliferation of COPD MRC⁃5 cell

目的:研究香烟模拟物(cigarette smoke extract,CES)通过调控16 HBE支气管上皮细胞中外泌体miR-34a的表达,从而影响MRC-5肺成纤维细胞的增殖。方法:从市售香烟中制备CES,后将6HBE细胞经过CES处理,收集上清中的外泌体miR-34a用于MRC-5细胞培养;使用RT-PCR检测外泌体miR-34a的表达水平;MRC-5细胞增殖能力通过细胞计数试剂盒CCK-8测定;通过Western blot检测CASP2的表达,利用双荧光素酶验证miR-34a与CASP2基因的靶向结合。结果:透射电镜下,16 HBE上清液中的外泌体呈球状,粒径在100 nm左右;经过CES处理后,外泌体miR-34a的表达显著增加。进一步研究表明,通过CES诱导的外泌体miR-34a能够促进MRC-5细胞的增殖;miR-34a与CASP2存在靶向结合关系;miR-34a mimic显著抑制了CASP2的表达。结论:在CES诱导的16HBE细胞中,外泌体miR-34a通过与CASP2基因的靶向结合,对成纤维细胞增殖起着关键作用。

Objective:To explore how cigarette smoke extract(CES)regulates the expression of exosomal miR⁃34a in 16 HBE bronchial epithelial cells,thus affecting the proliferation of MRC⁃5 lung fibroblasts.Methods:CES was prepared from com⁃mercially available cigarettes,and 16HBE cells were treated with CES.The exosomal miR⁃34a collected from the supernatant was used for MRC⁃5 cell culture.The expression level of exosomal miR⁃34a was detected by RT⁃PCR.The proliferation ability of MRC⁃5 cells was determined by CCK⁃8 cell counting kit.The expression of CASP2 was detected by Western blot,and the target binding of miR⁃34a and CASP2 gene was verified by dual luciferase.Results:Under the transmission electron microscope,the exo⁃somes in the supernatant of 16 HBE were spherical,with a particle size of about 100 nm;after CES treatment,the expression of exosomal miR⁃34a significantly increased.Further research showed that the exosomal miR⁃34a induced by CES can promote the proliferation of MRC⁃5 cells;miR⁃34a and CASP2 have a target binding relationship;miR⁃34a mimic significantly inhibited the ex⁃pression of CASP2.Conclusion:In CES⁃induced 16HBE cells,exosomal miR⁃34a plays a key role in fibroblast proliferation through target binding with the CASP2 gene.