香烟烟雾提取物诱导16HBE细胞MUC5AC上调的机制

Mechanism of CSE⁃induced up⁃regulation of MUC5AC in 16HBE cells by transcriptomics

目的本实验旨在利用转录组测序技术探索参与香烟烟雾提取物(CSE)诱导16HBE细胞MUC5AC上调的信号通路及其分子机制,为寻找新的慢性阻塞性肺疾病(COPD)黏液高分泌药物作用靶点提供理论依据。方法利用转录组测序技术检测CSE引起16HBE细胞差异表达的基因(DEGs),通过生物信息学分析挖掘出可能参与CSE诱导16HBE细胞MUC5AC上调的信号通路及相关基因。利用RT⁃qPCR技术验证所筛选的信号通路中相关基因转录水平;利用信号通路激动剂及siRNA干扰技术分别验证该信号通路及相关基因是否参与CSE诱导的16HBE细胞MUC5AC上调过程。结果转录组测序共检测到2567个DEGs,生物信息学分析结果显示16HBE细胞中MAPK信号通路、WNT信号通路、PI3K⁃Akt等信号通路在受到CSE刺激后表达异常。RT⁃qPCR结果显示CSE刺激16HBE后WNT4、WNT5B、WNT6、WNT10A基因的转录水平较对照组分别下调了0.885、0.893、0.705、0.618倍(P<0.05),WNT9A较对照组上调3.727倍(P<0.05),与转录组测序结果一致。激活经典WNT信号通路后,16HBE细胞MUC5AC转录水平较对照组上调3.197倍(P=0.0313)。而在小分子干扰实验中,WNT5B siRNA组+CSE组MUC5AC mRNA较阴性对照+CSE组上调2.092倍(P=0.0162)。结论非经典WNT信号通路中的WNT5B在CSE刺激16HBE细胞后下调,并参与CSE诱导的MUC5AC的表达上调。

Objective To explore the signaling pathway and molecular mechanism involved in up⁃regulation of MUC5AC in 16HBE cells induced by CSE using the method of transcriptome sequencing,and to provide theoreti⁃cal basis for finding new targets of COPD.Methods Differently expressed genes(DEGs)were detected by tran⁃scriptome sequencing in 16HBE cells induced by CSE.Excavating out which potential signal pathway involved in up⁃regulation of MUC5AC in 16HBE cells induced by CSE was excavated through bioinformatics analysis.The transcript levels of relevant genes in the screened signaling pathways were verified by RT⁃qPCR,and whether the signaling pathway and related genes were involved in up⁃regulation of MUC5AC in 16HBE cells induced by CSE was verified using the method of signaling pathway agonists and siRNA interference.Results A total of 2567 DEGs were detected by transcriptome sequencing,and the bioinformatics analysis showed that MAPK signaling,WNT signaling,and PI3K⁃Akt in 16HBE cells were abnormally expressed after CSE stimulation.RT⁃qPCR showed that the tran⁃script levels of WNT4,WNT5B,WNT6 and WNT10A genes were down⁃regulated when compared with control group(0.885 times,0.893 times,0.705 times,0.618 times,P<0.05),and WNT9A were up⁃regulated(3.727 times,P<0.05),which were consistent with transcriptome sequencing.After the activation of the classical WNT signaling pathway,the MUC5AC transcript level of 16HBE cells was up⁃regulated by 3.197 times when compared with the control group(P=0.0313).However,in the small molecule interference experiment,the transcript level of MUC5AC in WNT5B siRNA+CSE group was 2.092 times higher than that in negative control+CSE group(P=0.0162).Conclusion WNT5B in the non⁃classical WNT signaling pathway is down⁃regulated and participates in CSE induced up⁃regulation of MUC5AC in 16HBE cells.