摘要
目的研究瞬时受体多囊蛋白2(TRPP2)在口腔鳞状上皮细胞的表达及其对口腔鳞状细胞癌(OSCC)侵袭和迁移的影响,初步探讨TRPP2影响OSCC转移的潜在信号通路。方法利用规律性成簇间隔短回文重复序列核酸酶9(CRISPR-Cas9)慢病毒质粒转染技术,构建TRPP2敲低的OSCC模型。免疫蛋白印迹技术(Western blot)验证TRPP2蛋白敲低效果。CCK-8实验和克隆形成实验检测TRPP2对OSCC增殖的影响。RT-qPCR法检测TRPP2对OSCC转移相关的靶基因。Western blot和qRT-PCR检测与靶基因EpCAM及其与未折叠蛋白反应(UPR)相关转录因子表达。利用侵袭实验和划痕实验检测TRPP2对OSCC侵袭和迁移能力的影响。结果与口腔上皮细胞HOK相比,OSCC中TRPP2显著高表达。敲低TRPP2,OSCC增殖和克隆形成能力显著增强。与对照组相比,TRPP2敲低转录谱中共494个差异基因显著表达,其中234个基因表达上调,260个基因下调。其中与细胞黏附相关的上皮细胞黏附分子(EpCAM)基因上调表达。此外,与UPR相关基因PERK、ATF6、GRP78表达上调,而与HOK细胞相比,OSCC中ATF6与EpCAM表达下调。敲低TRPP2,OSCC中ATF6与EpCAM表达上调,并降低细胞迁移和侵袭能力。ATF6抑制剂ceapin-A7(5.0μmol/L)可恢复TRPP2敲低的OSCC迁移和侵袭能力。结论TRPP2在OSCC中显著高表达。敲低TRPP2,OSCC增殖能力增强,迁移和侵袭能力受抑制。TRPP2通过激活UPR介导EpCAM的表达,进而影响OSCC的侵袭和迁移能力。
Objective To investigate the expression of transient receptor polycystic protein 2(TRPP2)in oral squamous epithelial cell and its effect on the invasion and migration of oral squamous cell carcinoma(OSCC),and to explore the potential signaling pathway of TRPP2 affecting OSCC metastasis.Methods The OSCC model with TRPP2 knockdown was constructed by CRISPR-Cas9 lentivirus plasmid transfection technique.The effect of TRPP2 protein knockdown was verified by Western blot.The effect of TRPP2 on OSCC proliferation was detected by CCK-8 assay and clone formation assay.RT-qPCR was used to detect the target genes associated with TRPP2 metastasis to OSCC.Western blot and RT-qPCR were used to detect the expression of EpCAM and its transcription factors associated with unfolded protein response(UPR).The effects of TRPP2 on the invasion and migration of OSCC were examined by invasion test and scratch test.Results Compared with HOK in oral epithelial cells,the expression of TRPP2 in OSCC was significantly higher.When TRPP2 was knocked down,OSCC proliferation and clonal formation were significantly enhanced.Compared with the control group,a total of 494 differential genes were significantly expressed in TRPP2 knockdown transcription profile,among which 234 genes were up-regulated and 260 genes were down-regulated.The expression of EpCAM gene,which is related to cell adhesion,was up-regulated.In addition,UPR related genes PERK,ATF6,GRP78 were up-regulated,while ATF6 and EpCAM were down-regulated in OSCC compared to HOK cells.The expression of ATF6 and EpCAM in oral squamous cell carcinoma cells was up-regulated by TRPP2 knockdown,and the cell migration and invasion ability decreased.The ATF6 inhibitor ceapin-A7(5μmol/L)restored the OSCC migration and invasion ability of TRPP2 knockdown.Conclusion TRPP2 is highly expressed in OSCC.When TRPP2 is knocked down,OSCC proliferation ability is enhanced,migration and invasion ability are inhibited.TRPP2 mediates the expression of EpCAM through activation of UPR,thus affecting the invasion and migration of oral squamous cell carcinoma.
作者
梁珠珠
陈澍
孙倩玉
沈兵
薛浩伟
Liang Zhuzhu;Chen Shu;Sun Qianyu;Shen Bing;Xue Haowei(Dept of Oral and Maxillofacial Surgery,The First Affiliated Hospital of Anhui Medical University,Hefei230022;State Key Laboratory of Quality Research in Chinese Medicine,Macao University of Science and Technology,Macao SAR999078)
出处
《安徽医科大学学报》
CAS
北大核心
2024年第11期2024-2032,2064,共10页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金联合基金安徽省卫生健康科研项目(编号:AHWJ2023A20133、U22A20272)。
关键词
口腔鳞状细胞癌
瞬时受体多囊蛋白2
未折叠蛋白反应
内质网应激
上皮细胞黏附分子
细胞迁移和侵袭
oral squamous cell carcinoma
transient receptor potential polycystic 2
unfolded protein response
endoplasmic reticulumepithelial stress
cell adhesion molecule
cell migration and invasion
