摘要
目的探讨磷酸蛋白磷酸酶催化亚基(PPP5C)对人肺腺癌H1299细胞迁移侵袭能力及肿瘤干性的影响及机制。方法构建PPP5C-pcDNA3.1过表达载体,转染PPP5C-pcDNA3.1和pcDNA3.1至H1299细胞,遗传霉素(G418)筛选H1299稳转细胞系。qRT-PCR和Western blot法鉴定PPP5C mRNA和蛋白的表达水平,细胞生长曲线绘制和克隆形成实验检测细胞增殖活性;划痕和Transwell实验检测细胞迁移侵袭能力;诱导干细胞成球实验检测细胞干性。结果成功构建PPP5C-pcDNA3.1真核表达载体,转染H1299细胞后PPP5C的表达水平显著升高。H1299细胞过表达PPP5C后,生长曲线和克隆形成实验结果显示细胞增殖能力无变化;划痕和Transwell实验显示细胞迁移和侵袭能力显著增强,基质金属蛋白酶9(MMP9)的表达升高;干细胞成球结果显示细胞干性显著增强,性别决定区Y-box 2(SOX2)的表达升高。结论人肺腺癌细胞H1299中过表达PPP5C不影响细胞增殖,但可通过调控MMP9和SOX2来增强其迁移侵袭能力及肿瘤干性。
Objective To investigate the effect and mechanism of phosphoprotein phosphatase 5 catalytic(PPP5C)on the migration,invasion and tumor stemness of human lung adenocarcinoma H1299 cells.Methods The PPP5C-pcDNA3.1 overexpression vector was constructed.PPP5C-pcDNA3.1 and pcDNA3.1 were transfected into H1299 cells,and H1299 stable cell lines were screened with G418.The mRNA and protein expression levels of PPP5C were identified by qRT-PCR and Western blot.The proliferation activity of H1299 cells was detected by drawing cell growth curve and cell clonal formation assay.The wound-healing assay and transwell assay were used to test the migration and invasion abilities of H1299 cells,respectively.The stemness of H1299 cells was evaluated by sphere formation assay.Results The PPP5C-pcDNA3.1 eukaryotic expression vector was successfully constructed and the expression levels of PPP5C significantly increased after transfection into H1299 cells.After overexpression of PPP5C in H1299 cells,the cell growth curve and clonal formation assay displayed that the proliferation ability was not affected,the migration and invasion of cells were significantly enhanced through wound-healing assay and transwell assay,accompanied by an increase in the expression of MMP9,stem cell spheroid assay showed a significant increase in stemness of cells,accompanied by increased expression of SOX2.Conclusion The proliferation ability of cells is not affected,the migration and invasion and the stemness of cells are enhanced by regulating MMP9 and SOX2 respectively,after overexpression of PPP5C in human lung adenocarcinoma H1299 cells.
作者
曾莉莉
陈微微
马向瑞
董洪亮
杜静
Zeng Lili;Chen Weiwei;Ma Xiangrui;Dong Hongliang;Du Jing(Medical Research Center,Binzhou Medical University Hospital,Binzhou 256600;Dept of Oral and Maxillofacial Surgery,Binzhou Medical University Hospital,Binzhou 256600)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第1期94-100,共7页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金青年项目(编号:31900441)
山东省自然科学基金面上项目(编号:ZR2019MC026)
山东省中医药发展计划(编号:2019-0514)
泰山学者青年专家、齐鲁卫生与健康杰出青年人才和外专双百项目资金(编号:WSG2018019)。
