Department of Gastroenterology, First Affiliated Hospital of Xi'an Jiaotong University, 277 West Yanta Road, Xi'an, 710061, Shaanxi, People's Republic of China.
Department of Pathology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People's Republic of China.
Dig Dis Sci. 2018 Oct;63(10):2604-2616. doi: 10.1007/s10620-018-5143-z. Epub 2018 Jun 8.
MicroRNAs are classes of endogenous noncoding RNAs that play a substantial role in tumor processes through regulating the targets at posttranscriptional level. However, little is known about the upstream transcription regulatory mechanism although it is a prerequisite for investigation of its aberrant expression and function.
This report evaluates miR-106a's direct transcriptional factor from upstream level to in depth elucidate their communication in gastric cancer development.
Gastric cancer tissues were collected to analyze the miR-106a expression using real-time PCR methods. The combination of Kruppel (or Krüppel)-like factor 4 (KLF4) to miR-106a promoter was testified through bioinformatics followed by construction of luciferase reporter plasmid and chromatin immunoprecipitation assay. Functional experiments and mouse models for evaluating cell growth and metastasis were conducted to observe the biological effect of KLF4 on miR-106a. The interplay between KLF4 and miR-106a was tested with Wnt activator and confirmed in clinical specimens.
The up-regulated miR-106a linked to gastric cancer metastasis and epithelial-mesenchymal transition. UCSC and JASPAR predicted the promoter sequence of miR-106a and its binding site with transcriptional factor KLF4. Construction of reporter gene further verified their direct combination at upstream level. Moreover, the inhibitory effect of KLF4 on the phenotype of gastric cancer cells could be restored by miR-106a. CHIR-induced experiment and clinical specimens confirmed the negative regulation of KLF4 on miR-106a.
Our findings provide novel direct insights into molecular mechanisms for interaction of KLF4 and miR-106a at upstream level and new ways for clinical application of KLF4-miR-106a axis in advanced gastric cancer metastasis.
MicroRNAs 是一类内源性非编码 RNA，通过在转录后水平调节靶基因发挥在肿瘤过程中的重要作用。然而，尽管它是研究其异常表达和功能的前提，但对于其上游转录调控机制知之甚少。
本报告从上游水平评估 miR-106a 的直接转录因子，深入阐明其在胃癌发生发展中的相互作用。
采用实时 PCR 方法分析胃癌组织中 miR-106a 的表达。通过生物信息学验证 Krüppel 样因子 4（KLF4）与 miR-106a 启动子的结合，构建荧光素酶报告质粒和染色质免疫沉淀实验。通过细胞生长和转移实验及小鼠模型评估 KLF4 对 miR-106a 的生物学效应。通过 Wnt 激活剂检测 KLF4 和 miR-106a 之间的相互作用，并在临床标本中进行验证。
上调的 miR-106a 与胃癌转移和上皮-间充质转化有关。UCSC 和 JASPAR 预测了 miR-106a 的启动子序列及其与转录因子 KLF4 的结合位点。报告基因的构建进一步验证了它们在转录水平的直接结合。此外，miR-106a 可恢复 KLF4 对胃癌细胞表型的抑制作用。CHIR 诱导实验和临床标本证实了 KLF4 对 miR-106a 的负调控作用。
本研究结果为 KLF4 和 miR-106a 在转录水平相互作用的分子机制提供了新的直接见解，并为 KLF4-miR-106a 轴在晚期胃癌转移中的临床应用提供了新的途径。
