摘要
目的:探究miR-218-5p靶向磷酸二酯酶7A(PDE7A)调节人非小细胞肺癌(NSCLC)A549细胞糖酵解过程的机制。方法:常规培养A549细胞,用Lipo3000将miR-218-5pmimic、mimic-NC、PDE7A过表达质粒(PDE7A-oe)和PDE7A对照质粒(PDE7A-NC)转染A549细胞,记为miR-218-5pmimic组、mimic-NC组、PDE7A-oe组和PDE7A-NC组。qPCR法验证转染效率,WB法检测糖酵解关键酶蛋白的表达,葡萄糖测定法和乳酸生成测定法检测各转染组A549细胞中2脱氧葡萄糖和乳酸含量,双萤光素酶报告基因实验验证miR-218-5p与PDE7A靶向结合关系,用TCGA数据库数据分析PDE7AmRNA在肺癌组织中的表达水平。结果:在A549细胞中成功地过表达了miR-218-5p(P<0.01)。过表达miR-218-5p均能显著抑制A549细胞中PDE7A、HK2、PKM2蛋白的表达(均P<0.01)、葡萄糖摄取量和乳酸生成量(均P<0.01)。过表达PDE7A均可显著促进A549细胞中PDE7A、HK2、PKM2蛋白的表达(均P<0.01),以及葡萄糖摄取量和乳酸生成量(均P<0.01)。A549细胞中miR-218-5p可与PDE7AmRNA的3´-UTR直接结合。数据库数据分析结果显示,PDE7AmRNA在肺鳞状细胞癌组织中呈高表达(P<0.01)。结论:miR-218-5p靶向PDE7A调控A549细胞中HK2和PKM2的表达水平,进而抑制糖酵解过程,miR-218-5p/PDE7A可能是NSCLC临床诊断和治疗的潜在靶点。
Objective:To investigate the mechanism of miR-218-5p regulating the glycolytic process in human non-small cell lung cancer A549 cells by targeting phosphodiesterase 7A(PDE7A).Methods:A549 cells were routinely cultured,and miR-218-5p mimic,mimic-NC,PDE7A overexpression plasmid(PDE7A-oe)and PDE7A control plasmid(PDE7A-NC)were transfected into A549 cells using Lipo3000,and recorded as the miR-218-5p mimic group,the mimic-NC group,the PDE7A-oe group and the PDE7A-NC group.The transfection efficiency was verified by qPCR assay;the expressions of glycolysis key enzyme proteins were detected by WB assay;the 2-deoxyglucose and lactate contents in A549 cells of each transfection group were detected by glucose assay and lactate production assay;the target binding relationship between miR-218-5p and PDE7A was verified by dual-luciferase reporter gene assay,and the data from the TCGA database were used to analyze the expression level of PDE7A mRNA in lung cancer tissues.Results:miR-218-5p was successfully overexpressed in A549 cells(P<0.01).Overexpression of miR-218-5p significantly inhibited the expressions of PDE7A,HK2,PKM2 proteins(all P<0.01),glucose uptake and lactate production(both P<0.01)in A549 cells.Overexpression of PDE7A significantly promoted the expressions of PDE7A,HK2,and PKM2 proteins(all P<0.01),as well as glucose uptake and lactate production(both P<0.01)in A549 cells.miR-218-5p in A549 cells could directly bind to the 3´-UTR of PDE7A mRNA.Database data analysis showed that PDE7A mRNA was highly expressed in lung squamous cell carcinoma tissues(P<0.01).Conclusion:miR-218-5p targets PDE7A to regulate the expression levels of HK2 and PKM2 in A549 cells,which in turn inhibits the glycolytic process.miR-218-5p/PDE7A may be a potential target for clinical diagnosis and treatment of NSCLC.
作者
牛海英
赵刚
苏姗娜
白荣荣
穆培娟
张冬
NIU Haiying;ZHAO Gang;SU Shanna;BAI Rongrong;MU Peijuan;ZHANG Dong(Department of Pulmonary and Critical Care Medicine,the First Affiliated Hospital of Baotou Medical College,Inner Mongolia University of Science&Technology,Baotou 014010,Inner Mongolia,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2024年第6期592-597,共6页
Chinese Journal of Cancer Biotherapy
基金
内蒙古自治区卫生健康科技计划项目(No.202202242)
内蒙古医学科学院公立医院科研联合基金科技项目(No.2023GLLH0206)。
