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

作　　者：朱萌[1,2] 翟华亮 赵婉莹 张宁 和水祥[2] ZHU Meng;ZHM Hualiang;ZHAO Wanying;ZHANG Ning;HE Shuixiang(Department of Gastroenterology,General Hospital of Ningxia Medical Universiy,Yinchuan 750004;Department of Pathology,General Hospital of Ningxia Medical Universiy,Yinchnan 750004,Ningxia;Department of Gastroenterology,323 Military Hospital of China,Xi＇an Jiaotong University,Xi＇an 710061;Department of Gastroenterology,First Affiliated Hospital of Xi＇an Jiaotong University,Xi＇an 710061,Shaanxi,China)

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

出　　处：《癌变．畸变．突变》2018年第5期339-344,共6页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：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)

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。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.

关 键 词：miR-106a KLF4 启动子 转录因子 报告质粒 

分 类 号：R735.2[医药卫生—肿瘤]