人FAT1基因启动子的克隆鉴定及其功能浅析
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摘要
目的:克隆人脂肪非典型钙黏蛋白1(FAT atypical cadherin 1,FAT1)基因启动子,找到核心启动子区域并对其转录调控机制进行初步分析。方法:通过PCR方法获得人FAT1基因5′上游1 163 bp(-1 029～+134 bp)的片段,亚克隆至pGL3-basic载体;通过步移缺失构建不同缺失片段的重组质粒。双荧光素酶报告活性分析检测各重组质粒在A549细胞和HEK293T细胞中的活性,找到核心启动子区域。利用生物信息学方法预测核心区域的转录因子结合位点。结果:经测序、酶切鉴定,成功构建了人FAT1启动子荧光素酶活性报告基因重组质粒。与pGL3-basic质粒相比,人FAT1启动子重组质粒的相对荧光素酶活性增加(P <0.05)。通过生物信息学软件预测人FAT1启动子区域(-233～-110 bp)可能含有TFAP2C、KLF5等转录因子结合位点。结论:成功构建人FAT1启动子不同缺失片段的荧光素酶报告基因重组质粒。通过荧光素酶活性比较,推测人FAT1的核心启动子区域位于-233～+134 bp区域,其中可能含有若干转录因子结合位点。
Objective:To clone the promoter sequences of fat atypical cadherin 1(FAT1),and to preliminarily analyze the transcriptional regulatory mechanism of the promoter. Methods:Promoter region was consructed by bioinformatic methods,and the application of 1 163 bp(-1 029～+134 bp)fragment of 5′upstream sequence of FAT1 gene by PCR was conducted,followed by cloning to pGL3-basic vector to establish the luciferase report gene recombinant plasmid. Another four recombinant plasmids of different lengths were obtained through walking deletion and then cloned to pGL3-basic plasmid as before. The resultant plasmids were transfected into A549 cells and HEK293 T cells respectively together with pGL3-basic vector. After this their activities were detected via dual-luciferase reporter assay. Bioinformatic methods was performed to predict the sequences of the potential transcriptional factor binding sites of the core region found in the promoter. Results:The lusiferase reporter gene recombinant plasmids of human FAT1 promoter were successfully conducted. The relative luciferase activities of recombinant promoters,in contrast to pGL3-basic vector,were much higher(P < 0.05). Note the sequences of the binding sites such as TFAP2 C、KLF5 were possibly included in the promoter region(-233～-110 bp)of FAT1 gene,which could bepredicted through bioinformatic means. Conclusion:The construction of the luciferase report recombinant plasmids of FAT1 gene were successfully done,and four of these plasmids had strong luciferase activities in A549 cells and HEK293 T cells,comparing to the activities of pGL3-basic plasmid. Through this the core region was probably found.It is concluded the core promoter region of FAT1 gene was possibly located in the(-233～+134 bp)region,in which may preserve potential important transcriptional binding sites.
Objective:To clone the promoter sequences of fat atypical cadherin 1(FAT1),and to preliminarily analyze the transcriptional regulatory mechanism of the promoter. Methods:Promoter region was consructed by bioinformatic methods,and the application of 1 163 bp(-1 029～+134 bp)fragment of 5′upstream sequence of FAT1 gene by PCR was conducted,followed by cloning to pGL3-basic vector to establish the luciferase report gene recombinant plasmid. Another four recombinant plasmids of different lengths were obtained through walking deletion and then cloned to pGL3-basic plasmid as before. The resultant plasmids were transfected into A549 cells and HEK293 T cells respectively together with pGL3-basic vector. After this their activities were detected via dual-luciferase reporter assay. Bioinformatic methods was performed to predict the sequences of the potential transcriptional factor binding sites of the core region found in the promoter. Results:The lusiferase reporter gene recombinant plasmids of human FAT1 promoter were successfully conducted. The relative luciferase activities of recombinant promoters,in contrast to pGL3-basic vector,were much higher(P < 0.05). Note the sequences of the binding sites such as TFAP2 C、KLF5 were possibly included in the promoter region(-233～-110 bp)of FAT1 gene,which could bepredicted through bioinformatic means. Conclusion:The construction of the luciferase report recombinant plasmids of FAT1 gene were successfully done,and four of these plasmids had strong luciferase activities in A549 cells and HEK293 T cells,comparing to the activities of pGL3-basic plasmid. Through this the core region was probably found.It is concluded the core promoter region of FAT1 gene was possibly located in the(-233～+134 bp)region,in which may preserve potential important transcriptional binding sites.
引文
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[9] Hou R,Sibinga NES. Atrophin proteins interact with the fat1 cadherin and regulate migration and orientation in vascular smooth muscle cells[J]. J Biol Chem,2009,284(11):6955-6965
[10] Qi C,Zhu YT,Hu L,et al. Identification of Fat4 as a candidate tumor suppressor gene in breast cancers[J]. Int J Cancer,2009,124(4):793-798
[11]Gee HY,Saisawat P,Ashraf S,et al. ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling[J]. J Clin Invest,2013,123(8):3243-3253
[12] Scott RP,Hawley SP,Ruston J,et al. Podocyte-specific loss of Cdc42 leads to congenital nephropathy[J]. J Am Soc Nephrol,2012,23(7):1149-1154
[13] Choi SY,Chaconheszele MF,Huang L,et al. Cdc42 deficiency causes ciliary abnormalities and cystic kidneys[J]. J Am Soc Nephrol,2013,24(9):1435-1450
[14] Ma D,Chang LY,Zhao S,et al. KLF5 promotes cervical cancer proliferation,migration and invasion in a manner partly dependent on TNFRSF11a expression[J]. Sci Rep,2017,7(1):15683
[15] Park JM,Wu T,Cyr AR,et al. The role of Tcfap2c in tumorigenesis and cancer growth in an activated Neu model of mammary carcinogenesis[J]. Oncogene,2015,34(50):6105-6114
[2] Zhang X,Liu J,Xiao L,et al. History and progression of Fat cadherins in health and disease[J]. Onco Targets Ther,2016,9:7337-7343
[3] Moeller MJ,Soofi A,Braun GS,et al. Protocadherin FAT1binds Ena/VASP proteins and is necessary for actin dynamics and cell polarization[J]. EMBO J,2004,23(19):3769-3779
[4] Gee HY,Sadowski CE,Aggarwal PK,et al. FAT1 mutations cause a glomerulotubular nephropathy[J]. Nat Commun,2016,7:10822
[5] Kim KT,Bo-Sung Kim,Kim JH. Association between FAT1 mutation and overall survival in patients with human papillomavirus-negative head and neck squamous cell carcinoma[J]. Head Neck,2016,38(S1):E2021-E2029
[6] Hu X,Zhai Y,Shi R,et al. FAT1 inhibits cell migration and invasion by affecting cellular mechanical properties in esophageal squamous cell carcinoma[J]. Oncol Rep,2018,39(5):2136
[7] Neumann M,Seehawer M,Schlee C,et al. FAT1 expression and mutations in adult acute lymphoblastic leukemia[J]. Blood Cancer J,2014,4(6):e224
[8] Valletta D,Czech B,Spruss T,et al. Regulation and function of the atypical cadherin FAT1 in hepatocellular carcinoma[J]. Carcinogenesis,2014,35(6):1407-1415
[9] Hou R,Sibinga NES. Atrophin proteins interact with the fat1 cadherin and regulate migration and orientation in vascular smooth muscle cells[J]. J Biol Chem,2009,284(11):6955-6965
[10] Qi C,Zhu YT,Hu L,et al. Identification of Fat4 as a candidate tumor suppressor gene in breast cancers[J]. Int J Cancer,2009,124(4):793-798
[11]Gee HY,Saisawat P,Ashraf S,et al. ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling[J]. J Clin Invest,2013,123(8):3243-3253
[12] Scott RP,Hawley SP,Ruston J,et al. Podocyte-specific loss of Cdc42 leads to congenital nephropathy[J]. J Am Soc Nephrol,2012,23(7):1149-1154
[13] Choi SY,Chaconheszele MF,Huang L,et al. Cdc42 deficiency causes ciliary abnormalities and cystic kidneys[J]. J Am Soc Nephrol,2013,24(9):1435-1450
[14] Ma D,Chang LY,Zhao S,et al. KLF5 promotes cervical cancer proliferation,migration and invasion in a manner partly dependent on TNFRSF11a expression[J]. Sci Rep,2017,7(1):15683
[15] Park JM,Wu T,Cyr AR,et al. The role of Tcfap2c in tumorigenesis and cancer growth in an activated Neu model of mammary carcinogenesis[J]. Oncogene,2015,34(50):6105-6114