人源LDHA基因启动子质粒的构建及在肺癌细胞中Nrf2对其转录表达调控和功能分析
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摘要
为探索核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)调节肿瘤代谢进而影响肿瘤细胞迁移的分子机制,着重研究了Nrf2对乳酸脱氢酶A(lactate dehydrogenase A,LDHA)表达的调控。首先成功构建了人源LDHA基因启动子萤火虫荧光素酶报告基因质粒LDHA-luc。将质粒LDHA-luc与Nrf2表达载体共同转染A549细胞,转染后经双荧光素酶报告基因系统检测,显示共转Nrf2质粒时LDHA-luc活性明显要比对照组高;同时,转染Nrf2质粒时LDHA的蛋白质表达水平明显高于对照组,表明在A549细胞中Nrf2促进LDHA的转录和表达。而LDHA的上调会促进酸性肿瘤微环境的形成,促进细胞迁移。因此,成功构建的人源LDHA基因启动子萤火虫荧光素酶报告基因质粒LDHA-luc及探索出的Nrf2对LDHA基因表达的调控,为进一步研究Nrf2通过LDHA影响细胞代谢进而促进细胞迁移奠定了基础。
In order to investigate the effect of nuclear factor erythroid 2-related factor 2(Nrf2) on tumor metabolism and, consequently, tumor migration, we focused on regulation of lactate dehydrogenase A(LDHA) ex-pression by Nrf2. Herein, the plasmid LDHA-luc containing the firefly luciferase reporter gene driven by human LDHA gene promoter was successfully constructed, and transfected into A549 cells together with Nrf2 expresion vector. After transfection, the dual luciferase reporter gene assay system showed that the LD-HA promoter activity in cells transfected with Nrf2 plasmid was significantly higher than that of the control.Meanwhile, the expression level of LDHA in cells transfected with Nrf2 plasmid was also higher, indicating that Nrf2 promoted transcription and expression of LDHA in A549 cells. Up-regulation of LDHA promotes the formation of acidic tumor microenvironment and cell migration. Therefore, successful construction of the plasmid LDHA-luc and demonstration of LDHA gene expression regulation by Nrf2 lay a foundation for further research on how Nrf2 influences tumor metabolism to promote cell migration through LDHA.
In order to investigate the effect of nuclear factor erythroid 2-related factor 2(Nrf2) on tumor metabolism and, consequently, tumor migration, we focused on regulation of lactate dehydrogenase A(LDHA) ex-pression by Nrf2. Herein, the plasmid LDHA-luc containing the firefly luciferase reporter gene driven by human LDHA gene promoter was successfully constructed, and transfected into A549 cells together with Nrf2 expresion vector. After transfection, the dual luciferase reporter gene assay system showed that the LD-HA promoter activity in cells transfected with Nrf2 plasmid was significantly higher than that of the control.Meanwhile, the expression level of LDHA in cells transfected with Nrf2 plasmid was also higher, indicating that Nrf2 promoted transcription and expression of LDHA in A549 cells. Up-regulation of LDHA promotes the formation of acidic tumor microenvironment and cell migration. Therefore, successful construction of the plasmid LDHA-luc and demonstration of LDHA gene expression regulation by Nrf2 lay a foundation for further research on how Nrf2 influences tumor metabolism to promote cell migration through LDHA.
引文
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[9]Gonzalez-Donquiles C,Alonso-Molero J,Fernandez-Villa T.The NRF2 transcription factor plays a dual role in colorectal cancer:a systematic review[J].PLo S One,2017,12(5):e0177549.
[10]Claudia G,Alexander A,Susanne S,et al.Cytoprotection“gone astray”:Nrf2 and its role in cancer[J].Onco Targets and Therapy,2014,7:1497-1518.
[11]Jin C,Ye Q H,Yuan F L,et al.Involvement of acid-sensing ion channel 1αin hepatic carcinoma cell migration and invasion[J].Tumor Biology,2015,36(6):4309-4317.
[2]Olayioye M A,Neve R M,Lane H A,et al.The Erb B signaling network:receptor heterodimerization in development and cancer[J].The EMBO Journal,2000,19(13):3159-3167.
[3]Lv X B,Liu L J,Cheng C,et al.SUN2 exerts tumor suppressor functions by suppressing the Warburg effect in lung cancer[J].Scientific Reports,2015,5:17940.
[4]Untereiner A A,Oláh G,Módis K,et al.H2S-induced S-sulfhydration of lactate dehydrogenase a(LDHA)stimulates cellular bioenergetics in HCT116 colon cancer cells[J].Biochemical Pharmacology,2017,136:86-98.
[5]周鑫,潘泓,杨日荣,等.非小细胞肺癌患者肿瘤组织乳酸脱氢酶A和Sox-2基因的表达及其临床意义[J].广西医学(Zhou Xin,Pang Hong,Yang Ri-rong,et al.Expression of lactate dehydrogenase-A and SRY-related HMG box-2 in tumor tissues of patients with non-small cell lung cancer and its clinical significance[J].Guangxi Medical Journal),2016,38(11):1529-1531.
[6]Li L,Kang L,Zhao W,et al.mi R-30a-5p suppresses breast tumor growth and metastasis through inhibition of LDHA-mediated Warburg effect[J].Cancer Letters,2017,400:89-98.
[7]倪静,秦丽娟,吴拥军,等.肺癌组织中Nrf2、Keap1和NQO1蛋白的表达[J].郑州大学学报(Ni Jing,Qin Li-juan,Wu Yongjun,et al.Expression of Nrf2,Keap1 and NQO1 proteins in lung cancer tissues[J].Journal of Zhengzhou University),2012,47(4):456-459.
[8]金科华,吴昕瑞,王苗苗,等.人LDHA原核表达、纯化及体外抑制剂筛选模型的建立[J].厦门大学学报(Jin Ke-hua,Wu Xin-rui,Wang Miao-miao,et al.Prokaryotic expression and purification of human LDHA and establishment of an inhibitor screening model in vitro[J].Journal of Xiamen University),2015,54(1):36-40.
[9]Gonzalez-Donquiles C,Alonso-Molero J,Fernandez-Villa T.The NRF2 transcription factor plays a dual role in colorectal cancer:a systematic review[J].PLo S One,2017,12(5):e0177549.
[10]Claudia G,Alexander A,Susanne S,et al.Cytoprotection“gone astray”:Nrf2 and its role in cancer[J].Onco Targets and Therapy,2014,7:1497-1518.
[11]Jin C,Ye Q H,Yuan F L,et al.Involvement of acid-sensing ion channel 1αin hepatic carcinoma cell migration and invasion[J].Tumor Biology,2015,36(6):4309-4317.
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