Nicotine-induced CHRNA5 activation modulates CES1 expression, impacting head and neck squamous cell carcinoma recurrence and metastasis via MEK/ERK pathway.

IF 8.1 1区 生物学 Q1 CELL BIOLOGY
Chen Feng, Wei Mao, Chenyang Yuan, Pin Dong, Yuying Liu
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引用次数: 0

Abstract

The mucosal epithelium of the head and neck region (including the oral cavity, nasal cavity, pharynx, nasopharynx, and larynx) is the primary site exposed to tobacco smoke, and its presence of nicotinic acetylcholine receptors (nAChRs) has been observed in the mucosal epithelial cells of this area. It remains unclear whether HNSC cells can migrate and invade through nAChR signaling. A model of HNSC cells exposed to nicotine is established. Cell proliferation following nicotine exposure is assessed using the CCK-8 assay, while migration and invasion are evaluated through wound healing and Transwell assays. The effects of CHRNA5 knockdown and overexpression are also investigated. Immunofluorescence staining is used to analyze CHRNA5 expression and localization, and clonogenic assays are performed to measure colony proliferation after CHRNA5 knockdown and overexpression. The interaction between CHRNA5 and CES1 is examined using molecular docking, co-immunoprecipitation, and immunofluorescence. Differentially expressed genes are subjected to pathway enrichment analysis, and MEK/ERK protein expression and phosphorylation are validated via western blot. Tumor formation assays are performed in nude mice using sh-CHRNA5 Cal27 cells, followed by western blot and immunohistochemical staining. Additionally, laryngeal and hypopharyngeal cancer tissues are analyzed through immunohistochemistry. Nicotine significantly enhanced the proliferation, migration, and invasion capabilities of head and neck tumor cells, including Cal27, Fadu, HN6, and Tu686 cells, through the expression of CHRNA5. Knockdown of CHRNA5 can reduce cell migration, invasion, and proliferation, whereas nicotine exposure can reverse this trend. Additionally, the mRNA and protein expression of CES1 decreases with the knockdown of CHRNA5, indicating a regulatory relationship between the two. Transcriptomics revealed that the knockdown of CHRNA5 is associated with the MEK/ERK signaling pathway. Further cellular- and tissue-level evidence confirmed that the levels of p-MEK/MEK, p-ERK/ERK, and CES1 decreased following knockdown of CHRNA5, a trend that nicotine can reverse. Nicotine promotes the proliferation, migration, and invasion of HNSC by upregulating CHRNA5 expression. Knockdown of CHRNA5 reduces these effects, which can be reversed by nicotine. Nicotine exposure activates CHRNA5, regulating CES1 expression via the MEK/ERK pathway, contributing to the recurrence and metastasis of head and neck squamous carcinoma.

尼古丁诱导的 CHRNA5 激活可调节 CES1 的表达,通过 MEK/ERK 通路影响头颈部鳞状细胞癌的复发和转移。
头颈部(包括口腔、鼻腔、咽、鼻咽和喉)的粘膜上皮是暴露于烟草烟雾的主要部位,在该部位的粘膜上皮细胞中已观察到烟碱乙酰胆碱受体(nAChR)的存在。目前仍不清楚 HNSC 细胞是否能通过 nAChR 信号迁移和入侵。我们建立了一个暴露于尼古丁的 HNSC 细胞模型。细胞暴露于尼古丁后的增殖通过 CCK-8 试验进行评估,迁移和侵袭则通过伤口愈合和 Transwell 试验进行评估。此外还研究了 CHRNA5 基因敲除和过表达的影响。免疫荧光染色用于分析 CHRNA5 的表达和定位,克隆生成试验用于测量 CHRNA5 敲除和过表达后的集落增殖。采用分子对接、共免疫沉淀和免疫荧光等方法检测 CHRNA5 和 CES1 之间的相互作用。对差异表达的基因进行通路富集分析,并通过 Western 印迹验证 MEK/ERK 蛋白的表达和磷酸化。使用 sh-CHRNA5 Cal27 细胞在裸鼠体内进行肿瘤形成试验,然后进行免疫印迹和免疫组化染色。此外,还通过免疫组化对喉癌和下咽癌组织进行了分析。尼古丁通过表达 CHRNA5 能明显增强头颈部肿瘤细胞(包括 Cal27、Fadu、HN6 和 Tu686 细胞)的增殖、迁移和侵袭能力。敲除 CHRNA5 可减少细胞的迁移、侵袭和增殖,而尼古丁暴露则可逆转这一趋势。此外,CES1的mRNA和蛋白表达随着CHRNA5的敲除而减少,这表明两者之间存在调控关系。转录组学发现,CHRNA5的敲除与MEK/ERK信号通路有关。进一步的细胞和组织水平证据证实,在敲除 CHRNA5 后,p-MEK/MEK、p-ERK/ERK 和 CES1 的水平下降,而尼古丁可以逆转这一趋势。尼古丁通过上调 CHRNA5 的表达促进 HNSC 的增殖、迁移和侵袭。敲除 CHRNA5 可减少这些影响,而尼古丁可逆转这些影响。尼古丁暴露会激活CHRNA5,通过MEK/ERK途径调节CES1的表达,从而导致头颈部鳞癌的复发和转移。
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来源期刊
Cell Death & Disease
Cell Death & Disease CELL BIOLOGY-
CiteScore
15.10
自引率
2.20%
发文量
935
审稿时长
2 months
期刊介绍: Brought to readers by the editorial team of Cell Death & Differentiation, Cell Death & Disease is an online peer-reviewed journal specializing in translational cell death research. It covers a wide range of topics in experimental and internal medicine, including cancer, immunity, neuroscience, and now cancer metabolism. Cell Death & Disease seeks to encompass the breadth of translational implications of cell death, and topics of particular concentration will include, but are not limited to, the following: Experimental medicine Cancer Immunity Internal medicine Neuroscience Cancer metabolism
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