转录因子KLF4与miR-106a的调控关系及其对人胃癌细胞迁移的影响

doi:10.3969/j.issn.1004-616x.2018.05.002

癌变·畸变·突变 ›› 2018, Vol. 30 ›› Issue (5): 339-344.doi: 10.3969/j.issn.1004-616x.2018.05.002

转录因子KLF4与miR-106a的调控关系及其对人胃癌细胞迁移的影响

朱萌^1,4, 翟华亮², 赵婉莹³, 张宁², 和水祥⁴

1. 宁夏医科大学总医院消化内科, 宁夏银川 750004;
2. 宁夏医科大学总医院病理科, 宁夏银川 750004;
3. 解放军第三二三医院消化内科, 陕西西安 710061;
4. 西安交通大学医学部第一附属医院消化内科, 陕西西安 710061

收稿日期:2017-11-07 修回日期:2018-09-06 出版日期:2018-09-30 发布日期:2018-09-30
通讯作者: 张宁,E-mail:zhangning_1028@163.com;和水祥,E-mail:hesx123@126.com E-mail:zhangning_1028@163.com;hesx123@126.com
作者简介:朱萌,E-mail:ricc_003@sina.com.cn。
基金资助:
宁夏自然科学基金资助项目（NZ16148，NZ16276）；宁夏高等学校优秀青年教师培育基金项目（NGY2016123）

Regulation of transcription factor KLF4 on miR-106a and their effects on gastric cancer cell migration

ZHU Meng^1,4, ZHAI Hualiang², ZHAO Wanying³, ZHANG Ning², HE Shuixiang⁴

1. Department of Gastroenterology, General Hospital of Ningxia Medical University, Yinchuan 750004;
2. Department of Pathology, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia;
3. Department of Gastroenterology, 323 Military Hospital of China, Xi'an Jiaotong University, Xi'an 710061;
4. Department of Gastroenterology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China

Received:2017-11-07 Revised:2018-09-06 Online:2018-09-30 Published:2018-09-30

摘要/Abstract

摘要： 目的：探讨转录因子Krüppel样因子4（KLF4）与miR-106a表达的调控关系及其对人胃癌细胞迁移的影响。方法：使用基因组数据库UCSC预测miR-106a启动子，插入pGL3-Basic报告载体，构建野生型报告质粒pmiR-106a-WT-luc和点突变报告质粒pmiR-106a-MUT-luc。使用转录因子数据库JASPAR预测并筛选调控miR-106a的转录因子KLF4，克隆至pcDNA3.0载体，构建KLF4过表达质粒KLF4-pcDNA3.0。将KLF4^+/--pcDNA3.0与pmiR-106a-WT/MUT-luc共同转染HEK293T细胞，双荧光素酶系统检测荧光值。收集30对胃癌和癌旁组织标本，采用实时定量PCR（qPCR）检测miR-106a、KLF4 mRNA表达水平。Transwell法检测人胃癌SGC-7901细胞的迁移能力。结果：miR-106a报告质粒及KLF4过表达质粒经双酶切、PCR扩增、测序及Blast比对提示构建正确。KLF4-pcDNA3.0显著抑制pmiR-106a-WT-luc荧光素酶活性（P=0.000），但对pmiR-106a-MUT-luc影响较小（P=0.553）。胃癌组织中KLF4 mRNA平均相对表达量为0.716±0.624，miR-106a为3.367±2.165。KLF4-pcDNA3.0部分阻遏miR-106a对胃癌SGC-7901细胞迁移的促进作用（P=0.038）。结论：转录因子KLF4与miR-106a启动子区存在直接结合位点，KLF4可能在上游转录水平负调控miR-106a成熟体表达，以此发挥部分阻遏miR-106a促胃癌细胞迁移的作用。

关键词: miR-106a, KLF4, 启动子, 转录因子, 报告质粒

Abstract: OBJECTIVE:To construct miR-106a promoter reporting plasmids and to explore their regulatory effects on the migration of human gastric cancer cells with transcription factor Krüppel-like factor 4. METHODS:Using UCSC genomic database to predict the promoter region of miR-106a,and to insert it into pGL3-basic reporter vector for construction of the wild-type reporter plasmid,pmiR-106a-WT-luc,and the point mutation reporter plasmid,pmiR-106a-MUT-luc. Using transcription factor database JASPAR to predict and to screen the transcription factor KLF4 that regulates the promoter region of miR-106a,and to clone it into pcDNA3.0 vector for construction of the over-expression plasmid KLF4-pcDNA3.0. KLF4^+/- -pcDNA3.0 and pmiR-106a-WT/MUT-luc were co-transferred into HEK293T cells and their fluorescence activities were detected by dual luciferase assay. 30 pairs of gastric cancer and adjacent non-tumor tissues were collected and qPCR was used to detect the expression level of miR-106a and KLF4. Transwell assay was used to detect the migratory capacity of human gastric cancer SGC-7901 cells. RESULTS:Both miR-106a reporter and KLF over-expression plasmids were constructed correctly through enzyme digestion,PCR amplification,sequencing and Blast comparison. KLF4-pcDNA3.0 significantly inhibited the luciferase activity of pmiR-106a-WT-luc (P=0.000);whereas, the effect on pmiR-106a-MUT-luc was little (P=0.553). The relative expression of KLF4 was 0.716±0.624 corresponding to 3.367±2.165 for miR-106a in gastric cancer tissues. KLF4-pcDNA3.0 partially blocked the stimulative effect of miR-106a on the migration of gastric cancer SGC-7901 cells (P=0.038). CONCLUSION:Our data show that there was a direct binding site between transcription factor KLF4 and the promoter region of miR-106a. In addition,KLF4 negatively regulated the expression of mature miR-106a at upstream transcription level. Thereby,it played a role in inhibiting the migration of gastric cancer cells induced by miR-106a.

Key words: miR-106a, KLF4, promoter, transcriptional factor, reporter plasmid

中图分类号:

R735.2

朱萌, 翟华亮, 赵婉莹, 张宁, 和水祥. 转录因子KLF4与miR-106a的调控关系及其对人胃癌细胞迁移的影响[J]. 癌变·畸变·突变, 2018, 30(5): 339-344.

ZHU Meng, ZHAI Hualiang, ZHAO Wanying, ZHANG Ning, HE Shuixiang. Regulation of transcription factor KLF4 on miR-106a and their effects on gastric cancer cell migration[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(5): 339-344.

参考文献

[1] HAMMOND S M. An overview of microRNAs[J]. Adv Drug Deliv Rev,2015,87(1):3-14.
[2] ZHENG Y,LIU L,SHUKLA G C. A comprehensive review of web-based non-coding RNA resources for cancer research[J]. Cancer Lett,2017,407(1):1-8.
[3] ZHU M,ZHANG N,HE S,et al. MicroRNA-106a functions as an oncogene in human gastric cancer and contributes to proliferation and metastasis in vitro and in vivo[J]. Clin Exp Metastasis,2016,33(5):509-519.
[4] LUJAMBIO A, LOWE S W. The microcosmos of cancer[J]. Nature,2012,482(7385):347-355.
[5] CHO W C. OncomiRs:the discovery and progress of microRNAs in cancers[J]. Mol Cancer,2007,6(1):60.
[6] 黄可婷,吴昊,吴敏丹,等. 小鼠pre-miRNA-122启动子荧光素酶报告质粒的构建及表达调控[J]. 生物技术,2017, 27(3):211-217.
[7] LEE Y, KIM M, HAN J, et al. MicroRNA genes are transcribed by RNA polymerase Ⅱ[J]. EMBO J, 2004, 23(20):4051-4060.
[8] JOCELYN E K, ELLIOTT S G, STEPHEN T K. Lewin's essential genes[M]. Canada:Jones & Bartlett Publishers, 2013:554-557.
[9] KIM C K,HE P,BIALKOWSKA A B,et al. SP and KLF Transcription Factors in Digestive Physiology and Diseases[J]. Gastroenterology,2017,152(8):1845-1875.
[10] SHIELDS J M,CHRISTY R J,YANG V W. Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest[J]. J Biol Chem,1996, 271(33):20009-20017.
[11] LI J,ZHENG H,YU F,et al. Deficiency of the Kruppel-like factor KLF4 correlates with increased cell proliferation and enhanced skin tumorigenesis[J]. Carcinogenesis, 2012, 33(6):1239-1246.
[12] MURGAI M, JU W, EASON M, et al. KLF4-dependent perivascular cell plasticity mediates pre-metastatic niche formation and metastasis[J]. Nat Med,2017,23(10):1176-1190.
[13] MAO L, ZHANG Y, DENG X, et al. Transcription factor KLF4 regulates microRNA-544 that targets YWHAZ in cervical cancer[J]. Am J Cancer Res,2015,5(6):1939-1953.

[1]	陈敏娜, 李春兰, 刘静. 转录因子STAT5A启动子荧光素酶报告基因质粒的构建及鉴定[J]. 癌变·畸变·突变, 2022, 34(1): 53-56,71.
[2]	孙娇娇, 车碧众, 罗秋林, 翟兵中, 信丽丽. 1,25-二羟基维生素D₃对PM_2.5所致HBE细胞氧化损伤的保护作用[J]. 癌变·畸变·突变, 2020, 32(2): 92-97.
[3]	李慧英, 李洋. RNAi沉默p53基因对HeLa细胞核转录因子3表达及其对细胞增殖和侵袭能力的影响[J]. 癌变·畸变·突变, 2017, 29(1): 46-50.
[4]	Bernd Kaina. 甲基化抗肿瘤药物治疗中的DNA修复:治疗效果与健康结局[J]. 癌变·畸变·突变, 2016, 28(5): 333-341,347.
[5]	何洁, 万经海, 李学记, 钱海鹏, 孟肖利, 郝佳洁, 徐昕, 王明荣. 胶质瘤中端粒酶逆转录酶启动子区突变分析及其预后意义[J]. 癌变·畸变·突变, 2015, 27(5): 361-365.
[6]	刘颖, 傅晗, 师腾瑞, 刘江正, 王欣, 海春旭. 高脂饲料喂饲C57小鼠诱发胰岛素抵抗过程中肝脏氧化应激水平及Nrf2、NRF-1和mtTFA 蛋白表达的变化[J]. 癌变·畸变·突变, 2015, 27(3): 191-196.
[7]	刘海英，黄青，董静文，李莉*. 胚胎癌F9细胞中Oct4对miR-302启动子的调控作用[J]. 癌变·畸变·突变, 2014, 26(4): 258-265.
[8]	陈锡和,邓小玲,尹丽琴,傅玉才,许铭炎. 口腔鳞癌细胞中ITGB6基因主要转录调控区域的定位分析[J]. 癌变·畸变·突变, 2013, 25(3): 190-193.
[9]	吐尔逊帕夏?吾布力卡斯木,阿比达?阿布都卡德尔,盛磊,阿布力孜?阿布杜拉. 维吾尔族妇女宫颈病变与CALCA基因启动子甲基化的关系[J]. 癌变·畸变·突变, 2013, 25(2): 134-138.
[10]	罗邦雨; 熊俊. 转录因子Snail与肿瘤关系的研究进展[J]. , 2012, 24(5): 393-396.
[11]	金彩霞1;2;李文林1;李元杰2;徐方2;胡以平1. 胰岛素启动子在肝干细胞分化研究中的应用[J]. , 2010, 22(3): 171-174.
[12]	吴钊(综述);王子卫(审校). KLF4在正常生理活动与肿瘤中的作用[J]. , 2010, 22(3): 246-246.
[13]	吴　钊;陈小鱼;张　能;王子卫. KLF4蛋白在胃癌组织中的表达及临床意义[J]. , 2009, 21(5): 388-391.
[14]	高书颖1;3;李恩民1;杜则澎1;陆晓峰2;许丽艳2;. 食管癌细胞ezrin基因启动子TPA反应性的研究[J]. , 2009, 21(5): 358-361.
[15]	谢仰民1;谢剑君2;#;周飞1;2;#;侯健2;曹君君1;许丽艳3;李恩民2;. 激活转录因子3的过表达对食管癌细胞生长的抑制作用[J]. , 2009, 21(4): 258-262.

转录因子KLF4与miR-106a的调控关系及其对人胃癌细胞迁移的影响

Regulation of transcription factor KLF4 on miR-106a and their effects on gastric cancer cell migration

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价