目的构建带FLAG标签的醛缩酶A(ALDOA)基因的真核表达载体,对其表达产物进行功能检测。方法利用PCR技术从人乳腺文库中扩增ALDOA基因,将其克隆至真核载体pcDNA3.0-FLAG中。经过酶切与测序验证,转染人胚肾293T细胞系,利用蛋白质免疫印迹...目的构建带FLAG标签的醛缩酶A(ALDOA)基因的真核表达载体,对其表达产物进行功能检测。方法利用PCR技术从人乳腺文库中扩增ALDOA基因,将其克隆至真核载体pcDNA3.0-FLAG中。经过酶切与测序验证,转染人胚肾293T细胞系,利用蛋白质免疫印迹实验完成其表达鉴定。转染人乳腺癌细胞MCF7和ZR75-1,使用葡萄糖摄取试剂盒检测细胞糖摄取情况,乳酸试剂盒检测细胞外乳酸产量。通过细胞生长曲线及克隆形成实验探究pcDNA3.0-FLAG-ALDOA对乳腺癌细胞生长的影响,通过划痕实验探究ALDOA对乳腺癌细胞迁移能力的影响。结果通过PCR技术从人乳腺文库中扩增得到约1100 bp cDNA片段,克隆到pcDNA3.0-FLAG载体上,测序结果与目的序列完全一致。转染人胚肾293T细胞后,pcDNA3.0-FLAG-ALDOA基因表达成功。糖摄取及乳酸含量鉴定结果表明,pcDNA3.0-FLAG-ALDOA对乳腺癌MCF7和ZR75-1细胞糖摄取及乳酸的生成有促进作用;生长曲线及克隆形成实验结果表明,pcDNA3.0-FLAG-ALDOA可促进乳腺癌MCF7和ZR75-1细胞生长;迁移实验结果表明,pcDNA3.0-FLAG-ALDOA可促进乳腺癌MCF7和ZR75-1细胞迁移。结论FLAG-ALDOA真核表达载体构建成功,表达产物ALDOA具有生物学活性,可促进乳腺癌细胞的葡萄糖摄取、乳酸生成、细胞生长及迁移,为进一步研究ALDOA在肿瘤发展中的意义奠定了理论基础。展开更多
Background:Our previous study demonstrated that aldolase A(ALDOA)is overexpressed in clinical human lung squamous cell carcinoma and that ALDOA promotes epithelial-mesenchymal transition and tumorigenesis.The pre-sent...Background:Our previous study demonstrated that aldolase A(ALDOA)is overexpressed in clinical human lung squamous cell carcinoma and that ALDOA promotes epithelial-mesenchymal transition and tumorigenesis.The pre-sent study aimed to explore the function of ALDOA in the modulation of non-small cell lung cancer(NSCLC)prolifera-tion and cell cycle progression and the potential mechanism.Methods:ALDOA was knocked down by short hairpin RNA in H520 and H1299 cells.ALDOA was overexpressed with vectors carrying the full-length ALDOA sequence in H1299 and H157 cells.The proliferation capacities were assessed with immunohistochemical staining,Cell Counting Kit-8 and colony formation assays.The cell cycle distribution was examined by flow cytometry,and molecular alterations were determined by western blotting.Cell synchronization was induced with nocodazole.The stability of cyclin D1 mRNA was tested.The pyruvate kinase M2 and ALDOA protein distributions were examined.Aerobic glycolysis was evaluated with Cell Titer-Glo assay,glucose colorimetric assay and lactate colorimetric assay.Results:ALDOA knockdown inhibited the proliferation and G1/S transition in H520 cells.Conversely,ALDOA over-expression promoted the proliferation and G1/S transition in H157 cells.The cell cycle synchronization assay showed that ALDOA expression increased in the G1 phase and G1/S transition.Furthermore,ALDOA knockdown reduced cyclin D1 expression by regulating epidermal growth factor receptor/mitogen-activated protein kinase(EGFR/MAPK)pathway.Similar results were found in H1299 and H157 cells.The inhibition of mitogen-activated protein kinase kinase 1/2 prompted the nuclear distribution of ALDOA.Additionally,ALDOA knockdown reduced nuclear distribution of PKM2,the extracellular lactate and intracellular adenosine triphosphate concentrations and elevated the extracellular glucose concentration.Conclusions:ALDOA contributed to activation of the EGFR/MAPK pathway,thus promoting cyclin D1 expression and enhancing proliferation and G1/S transition in NSCLC.Additionally,ALDOA facilitated NSCLC aerobic glycolysis.展开更多
文摘目的构建带FLAG标签的醛缩酶A(ALDOA)基因的真核表达载体,对其表达产物进行功能检测。方法利用PCR技术从人乳腺文库中扩增ALDOA基因,将其克隆至真核载体pcDNA3.0-FLAG中。经过酶切与测序验证,转染人胚肾293T细胞系,利用蛋白质免疫印迹实验完成其表达鉴定。转染人乳腺癌细胞MCF7和ZR75-1,使用葡萄糖摄取试剂盒检测细胞糖摄取情况,乳酸试剂盒检测细胞外乳酸产量。通过细胞生长曲线及克隆形成实验探究pcDNA3.0-FLAG-ALDOA对乳腺癌细胞生长的影响,通过划痕实验探究ALDOA对乳腺癌细胞迁移能力的影响。结果通过PCR技术从人乳腺文库中扩增得到约1100 bp cDNA片段,克隆到pcDNA3.0-FLAG载体上,测序结果与目的序列完全一致。转染人胚肾293T细胞后,pcDNA3.0-FLAG-ALDOA基因表达成功。糖摄取及乳酸含量鉴定结果表明,pcDNA3.0-FLAG-ALDOA对乳腺癌MCF7和ZR75-1细胞糖摄取及乳酸的生成有促进作用;生长曲线及克隆形成实验结果表明,pcDNA3.0-FLAG-ALDOA可促进乳腺癌MCF7和ZR75-1细胞生长;迁移实验结果表明,pcDNA3.0-FLAG-ALDOA可促进乳腺癌MCF7和ZR75-1细胞迁移。结论FLAG-ALDOA真核表达载体构建成功,表达产物ALDOA具有生物学活性,可促进乳腺癌细胞的葡萄糖摄取、乳酸生成、细胞生长及迁移,为进一步研究ALDOA在肿瘤发展中的意义奠定了理论基础。
基金supported by the National Natural Science Foundation of China(Nos.81470367,81773122 and 81702290).
文摘Background:Our previous study demonstrated that aldolase A(ALDOA)is overexpressed in clinical human lung squamous cell carcinoma and that ALDOA promotes epithelial-mesenchymal transition and tumorigenesis.The pre-sent study aimed to explore the function of ALDOA in the modulation of non-small cell lung cancer(NSCLC)prolifera-tion and cell cycle progression and the potential mechanism.Methods:ALDOA was knocked down by short hairpin RNA in H520 and H1299 cells.ALDOA was overexpressed with vectors carrying the full-length ALDOA sequence in H1299 and H157 cells.The proliferation capacities were assessed with immunohistochemical staining,Cell Counting Kit-8 and colony formation assays.The cell cycle distribution was examined by flow cytometry,and molecular alterations were determined by western blotting.Cell synchronization was induced with nocodazole.The stability of cyclin D1 mRNA was tested.The pyruvate kinase M2 and ALDOA protein distributions were examined.Aerobic glycolysis was evaluated with Cell Titer-Glo assay,glucose colorimetric assay and lactate colorimetric assay.Results:ALDOA knockdown inhibited the proliferation and G1/S transition in H520 cells.Conversely,ALDOA over-expression promoted the proliferation and G1/S transition in H157 cells.The cell cycle synchronization assay showed that ALDOA expression increased in the G1 phase and G1/S transition.Furthermore,ALDOA knockdown reduced cyclin D1 expression by regulating epidermal growth factor receptor/mitogen-activated protein kinase(EGFR/MAPK)pathway.Similar results were found in H1299 and H157 cells.The inhibition of mitogen-activated protein kinase kinase 1/2 prompted the nuclear distribution of ALDOA.Additionally,ALDOA knockdown reduced nuclear distribution of PKM2,the extracellular lactate and intracellular adenosine triphosphate concentrations and elevated the extracellular glucose concentration.Conclusions:ALDOA contributed to activation of the EGFR/MAPK pathway,thus promoting cyclin D1 expression and enhancing proliferation and G1/S transition in NSCLC.Additionally,ALDOA facilitated NSCLC aerobic glycolysis.
