槟榔提取物对口腔上皮细胞生物功能影响的研究

Effect of areca nut extract on the biological function of oral epithelial cells

目的探讨槟榔提取物(ANE)对人口腔上皮细胞的生物学功能影响。方法本研究首先分析了梯度浓度ANE下对人口腔角质细胞(HOK)增殖的影响;采用第二代RNA测序分析对照组与ANE干预组HOK细胞的差异表达基因。随后,流式细胞术用于检测HOK细胞周期和凋亡;β-半乳糖苷酶(β-Gal)染色试剂盒用于检测细胞衰老;免疫荧光实验检测HOK细胞中Ki67和γH2AX蛋白的表达;实时聚合酶链反应(RT-PCR)用于检测衰老相关表型基因白细胞介素(IL)-6、基质金属蛋白酶-2(MMP2)和TGFB1表达;免疫印迹用于检测自噬相关蛋白(LC3-Ⅰ/Ⅱ和p62)和上皮间充质转化(EMT)相关蛋白(SMAD2、p-SMAD2、E-Cadherin和Vimentin)的表达;酶联免疫吸附实验用于检测HOK细胞外分泌蛋白TGFB1表达。结果测序结果显示ANE可引起HOK细胞细胞周期、p53、自噬、FoxO、细胞衰老等信号通路相关基因异常表达。流式细胞术显示ANE可诱导HOK细胞凋亡和G2/M期阻滞。与对照组相比,ANE处理后的HOK细胞衰老细胞数量增加,衰老相关表型基因IL-6、MMP2和TGFB1表达上调。此外,相较于对照组,ANE组中HOK细胞的γH2AX、LC3-Ⅱ、TGFB1、p-SMAD2和Vimentin蛋白表达增加,Ki67蛋白、LC3-Ⅰ和E-Cadherin蛋白表达下降。结论ANE可诱导HOK细胞凋亡、G2/M期阻滞、衰老、DNA损伤、自噬和EMT,形成促癌的细胞微环境。

Objective This study aimed to investigate the effects of areca nut extract(ANE)on the biological function of human oral epithelial cells.Methods First,we analyzed the effects of ANE with gradient concentrations on the proliferation of human oral keratinocytes(HOKs).Next-generation RNA sequencing was used to examine the differential genes between control HOKs and those treated with ANE.Flow cytometry was employed to evaluate the apoptosis and cycle arrest of HOKs.β-galactosidase staining kit was used to detect cell senescence.Immunofluorescence was applied to detect the expression of Ki67 andγH2AX protein in HOKs.Real-time PCR(RT-PCR)was utilized to detect the expression of senescence-related phenotypic genes interleukin(IL)-6,matrix metalloproteinase-2(MMP2),and TGFB1.Western blot was adopted to detect the expression of autophagy-related proteins(LC3-Ⅰ/Ⅱand p62)and epithelialmesenchymal transition(EMT)-related proteins(SMAD2,p-SMAD2,E-cadherin,and Vimentin).Enzyme-linked immunosorbent assay was employed to detect the expression of exocrine TGFB1 protein in HOKs.Results Sequencing results showed that ANE caused abnormality in cell cycle,p53,autophagy,FoxO,cellular senescence,and other signal pathways.Flow cytometry showed that ANE induced HOK apoptosis and G2/M phase arrest.The number of senescent cells and the expression of senescent phenotypic secretion genes(IL-6,MMP2,and TGFB1)increased in the ANE group.Compared with those in the control group,the expression levels ofγH2AX,LC3-Ⅱ,TGFB1,p-SMAD2,and Vimentin protein increased and those of Ki67,LC3-Ⅰ,and E-cadherin protein decreased in the ANE group.Conclusion ANE could induce HOK apoptosis,G2/M phase arrest,senescence,DNA double-strand damage,autophagy,and EMT,possibly forming a cell microenvironment that promotes tumor occurrence.