CAMK2A通过激活ERK信号通路调控口腔鳞癌细胞紫杉醇耐药性^*

CAMK2a Regulates Paclitaxel Resistance in Oral Squamous Cell Carcinoma Cells by Activating the ERK Signaling Pathway

摘要

目的 研究钙离子信号通路调控口腔鳞癌（oral squamous cell carcinoma, OSCC）细胞紫杉醇耐药生存的分子机制。方法 通过紫杉醇低剂量持续诱导且剂量递增法诱导构建口腔鳞癌紫杉醇耐药细胞模型；使用Cell Counting Kit-8（CCK-8）和成像流式评估耐药模型的稳定性；通过RNA-seq测序分析差异基因和功能富集；使用实时荧光定量PCR（quantitative real-time PCR, qPCR）分析CAMK2a基因差异表达；通过蛋白质免疫印迹法（Western blot）检测蛋白质表达水平；用KN93及CAMK2a siRNA抑制CAMK2a表达。结果 成功构建紫杉醇耐药口腔鳞癌细胞模型；测序的GO富集结果显示耐药细胞在钙离子信号通路具有差异，其中CAMK2家族多种基因表达增加，CAMK2a分子表达增加12%（P<0.05）；通过使用CAMK2a抑制剂KN93和siRNA基因敲低技术抑制CAMK2a后，耐药细胞增殖能力降低35%（P<0.000 1），同时p-Erk1/2和下游分子表达降低。结论 CAMK2a通过激活MAPK-Erk1/2通路，调控耐药细胞的耐药性和增殖能力，是临床耐药患者治疗的潜在新靶点。

Abstract

Objective To investigate the molecular mechanisms by which calcium signaling pathways regulate the paclitaxel resistance and the survival of oral squamous cell carcinoma (OSCC) cells. Methods A paclitaxel-resistant OSCC cell model was established through continuous low-dose paclitaxel induction with dose escalation. The stability of the paclitaxel-resistant model was assessed using the Cell Counting Kit-8 (CCK-8) and imaging flow cytometry. RNA sequencing (RNA-seq) was performed to analyze differentially expressed genes and functional enrichment. Quantitative real-time PCR (qPCR) was conducted to analyze the differential expression of theCAMK2a gene. Protein expression levels were assessed by Western blot (WB). CAMK2a expression was inhibited using KN93 and CAMK2a small interfering RNA (siRNA). Results A paclitaxel-resistant OSCC cell model was successfully established. Gene Ontology (GO) enrichment analysis of the sequencing data revealed differences in the calcium signaling pathways in resistant cells. The expression of multiple genes of the CAMK2 family was upregulated, and CAMK2a expression increased by 12% (P < 0.05). After inhibiting CAMK2a by treatment with KN93, an inhibitor, and CAMK2a siRNA-mediated gene knockdown, the proliferative capacity of paclitaxel-resistant cells decreased by 35% (P < 0.000 1). Furthermore, the expression levels of phosphorylated Erk1/2 (p-Erk1/2) and its downstream signaling molecules also decreased following CAMK2a inhibition. Conclusion CAMK2a regulates drug resistance and the proliferative capacity of paclitaxel-resistant cells by activating the MAPK-Erk1/2 pathway and represents a potential new therapeutic target for clinical treatment of drug-resistant patients.

关键词

口腔鳞癌 / 钙离子信号通路 / 紫杉醇耐药 / CAMK2a / 靶点

Key words

Oral squamous cell carcinoma / Calcium signaling pathway / Paclitaxel resistance / CAMK2a / Target

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基金资助

*四川省科技计划项目(2023NSFSC0555)

四川大学华西口腔医院基础与应用基础研究项目(RD-02-202105)