内源性芳香烃配体ITE对宫颈癌细胞生物学行为的影响及机制

Effect and mechanism of endogenous aromatic ligand ITE on the biological behavior of cervical cancer cells

目的 探讨内源性芳香烃配体2-(1’H-吲哚-3’-羰基)噻唑-4-羧酸甲酯(ITE)对宫颈癌细胞生物学行为的影响及其作用机制。方法 免疫组化检测芳香烃受体(AhR)在宫颈癌及正常宫颈组织的表达及定位;采用ITE处理宫颈癌细胞,MTS实验检测增殖能力,Transwell实验检测迁移能力,qRT-PCR及Western blot检测AhR及下游基因CYP1A1、CYP1B1 mRNA和蛋白的表达变化,通过RNA-seq技术和生物信息学筛选参与AhR影响宫颈癌细胞增殖、迁移相关基因及相关信号通路;采用siRNA敲减PKN1后,检测宫颈癌细胞增殖及迁移能力,KEGG分析富集的与增殖及迁移相关的通路,Western blot检测ITE处理后PI3K-AKT通路蛋白表达。结果 AhR在宫颈癌组织中表达高于正常宫颈组织,ITE激活AhR及下游基因CYP1A1和CYP1B1且呈剂量和时间依赖性抑制宫颈癌细胞SiHa和CaSki的增殖和迁移能力(P <0.05)。RNA-seq技术和生物信息学筛选显示,经ITE处理后,与宫颈癌增殖、迁移功能密切相关的PKN1表达下调(P <0.05),且Western blot结果显示,SiHa和CaSki宫颈癌细胞系中PKN1蛋白表达在ITE处理后显著降低(P <0.05)。通过敲低细胞PKN1表达后,SiHa和CaSki细胞的增殖及迁移能力显著下降(P <0.05)。KEGG信号通路富集分析结果显示,差异表达基因相关的信号通路主要集中在PI3K-AKT信号通路。PI3KAKT通路抑制剂LY294002可阻断ITE诱导的P-AKT水平的增加(P <0.05),并逆转ITE对宫颈癌细胞增殖及迁移的抑制作用(P <0.05)。结论 内源性芳香烃配ITE可以抑制宫颈癌细胞增殖和侵袭能力,可能与下调PKN1及激活PI3K-AKT通路有关。

Objective To study the effect of endogenous aromatic ligand ITE(2⁃(1′H⁃indole⁃3′⁃carbonyl)⁃thiazole⁃4⁃carboxylic acid methylester)on the biological behavior and its mechanism of cervical cancer cells.Methods The expression and localization of AhR in cervical cancer and normal cervical tissues were detected by immunohistochemistry.MTS assay detected cell proliferation and Transwell assay detected cell migration.The qRT⁃PCR and Western Blot assays were used to detect relative expression in the mRNA and protein of AhR and its downstream genes CYP1A1,CYP1B1.RNA⁃seq and bioinformatics were used to screen genes related to AhR that affect cervical cancer cell proliferation and migration,as well as related signaling pathways.The ability of cervical cancer cells to proliferate and migrate was observed after siRNA knockdown of PKN1.KEGG was used to analyze the enriched pathways related to proliferation and migration,and Western blot was used to detect protein expres⁃sion in the PI3K⁃AKT pathway after ITE treatment of cervical cancer cells.Results The expression of AhR was higher in cervical cancer tissues than in normal cervical tissues.ITE activated AhR and downstream genes CYP1A1 and CYP1B1 and inhibited the proliferation and migration of cervical cancer cells SiHa and CaSki in a dose⁃and time⁃dependent manner(P<0.05).RNA⁃seq technique and bioinformatics screening showed that the expression of PKN1,which is closely related to the proliferation and migration functions of cervical cancer,was down⁃regulated after ITE treatment(P<0.05),and Western blot results showed that PKN1 protein expression was significantly reduced in SiHa and CaSki cervical cancer cell lines after ITE treatmentr(P<0.05).Proliferation and migration of SiHa and CaSki cells were significantly reduced by knocking down cellular PKN1 expression(P<0.05).KEGG signal pathway enrichment analysis revealed that the differential expression gene signal pathway was primarily concentrated in the PI3K⁃AKT signal pathway.PI3K⁃AKT pathway inhibitor LY294002 blocked ITE⁃induced in⁃crease in P⁃AKT levels(P<0.05),and can reverse the inhibitory effect of ITE on the proliferation and migration of cervical cancer cells(P<0.05).Conclusion Endogenous aromatic hydrocarbons containing ITE can inhibit the proliferation and invasion of cervical cancer cells,which may be related to PKN1 downregulation and PI3K⁃AKT activation.