摘要
目的探讨转录因子kruppel样因子4(Krüppel-like factor 4, KLF4)对微小RNA-106a(miR-106a)表达的调控及其相互作用对人胃癌细胞侵袭能力的影响。方法通过转录因子结合位点数据库JASPAR检索与miR-106a启动子区结合的转录因子KLF4。构建KLF4过表达载体(KLF4-pcDNA)和miR-106a报告基因(pmiR-106a-WT/MUT-luc),双荧光素酶报告基因检测KLF4对miR-106a启动子活性的影响。收集人胃癌和癌旁石蜡包埋-甲醛固定(formalin-fixed paraffin-embedded, FFPE)标本各40例,实时定量PCR和免疫组织化学法检测KLF4表达。人胃癌细胞株BGC-823培养并按miR-106a mimic组、mimic NC组、KLF4+pcDNA组、pcDNA basic组分别进行处理,Transwell法检测各组胃癌细胞侵袭能力变化。结果双荧光素酶报告基因检测显示KLF4-pcDNA具有拮抗miR-106a启动子活性的作用,表现为pmiR-106a-WT-luc荧光素酶活性被抑制(pmiR-106a-WT+pcDNA-basic与pmiR-106a-WT+pcDNA-KLF4比较,P<0.001),但对pmiR-106a-MUT-luc荧光素酶活性影响不明显(pmiR-106a-MUT+pcDNA-basic与pmiR-106a-MUT+pcDNA-KLF4比较,P>0.05)。FFPE样本显示KLF4在胃癌组织中的相对表达量为0.69±0.59,与癌旁组织相比,差异有统计学意义(P<0.001),且与胃癌的细胞分化程度、淋巴转移和浸润深度相关(P<0.05);KLF4阳性表达主要位于胃黏膜上皮细胞核及胞质。Transwell实验表明各处理组胃癌细胞的穿膜数量不全相同(P<0.001),miR-106a mimic组为89.00±14.85,mimic NC组为50.90±17.94,KLF4+pcDNA组为37.70±12.60,pcDNA basic组为66.20±3.19。结论转录因子KLF4与miR-106a启动子区至少存在体外的直接结合,可能通过在上游转录水平负调控miR-106a表达而参与影响胃癌细胞的侵袭转移进程。
Objective To investigate the effect of Krüppel-like factor 4 (KLF4) on the expression of miR-106a and their interaction on the invasive activity of human gastric cancer cells. Methods The JASPAR database was used to screen the transcriptional factor combined with miR-106a promoter.KLF4 over-expression vector KLF4-pcDNA and miR-106a reporter gene pmiR-106a-WT/MUT-luc were both constructed,and the dual luciferase reporter assay was used to detect the effect of KLF4 on the activity of miR-106a promoter.Forty specimens of human gastric cancer and their adjacent formalin-fixed paraffin-embedded (FFPE) specimens were collected,and Real-time PCR and immunohistochemistry were both used to detect the expression of KLF4.Human gastric cancer cell line BGC-823 was divided into four groups:miR-106a mimic,mimic NC,KLF4+pcDNA,and pcDNA basic.Transwell assay was employed to detect the invasive ability of the gastric cancer cells after four different treatments. Results Dual luciferase reporter assay indicated that KLF4-pcDNA could antagonize the activity of miR-106a promoter,which suggested that the activity of pmiR-106a-WT-luc luciferase was inhibited (pmiR-106a-WT+pcDNA-basic compared with pmiR-106a-WT+pcDNA-KLF4,P <0.001),but the pmiR-106a-MUT-luc luciferase was not significantly influenced (pmiR-106a-MUT+pcDNA-basic compared with pmiR-106a-MUT+pcDNA-KLF4,P >0.05).FFPE samples showed that the relative expression of KLF4 in gastric cancer was 0.69±0.59,which significantly differed from that in adjacent paracancerous tissues ( P <0.001).The expression of KLF4 was correlated with differentiation degree,lymph node metastasis,and infiltration depth of human gastric cancer ( P <0.05).The positive expression of KLF4 was mainly located in the nucleus and cytoplasm of gastric mucosal epithelial cells.Transwell essay showed that the number of invasive cells in the four different treatment groups was not entirely the same ( P <0.001):89.00±14.85 for miR-106a mimic,50.90±17.94 for mimic NC,37.70±12.60 for KLF4+pcDNA,and 66.20±3.19
作者
朱萌
张宁
和水祥
张丹
张志勇
ZHU Meng;ZHANG Ning;HE Shui-xiang;ZHANG Dan;ZHANG Zhi-yong(Department of Gastroenterology,The First Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710061;Department of Gastroenterology,General Hospital of Ningxia Medical University,Yinchuan 750004;Department of Pathology,General Hospital of Ningxia Medical University,Yinchuan 750004,China)
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2019年第3期356-361,共6页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.81802416
No.81602611)
中国博士后科学基金(No.2018M633529)
宁夏自然科学基金资助项目(No.NZ16276
No.NZ16148)~~
