Yu Kuaiyun
Cell Physiol Biochem. 2017;43(3):1235-1246. doi: 10.1159/000481764. Epub 2017 Oct 6.
In respect to the effect of MET1 upon the recurrence of Early gastric cancer (EGC) after endoscopic dissection (ESD) treatment, we aimed to investigate the molecular mechanism, including the potential regulator and signaling pathways of MET1 in this study.
We searched the miRNA database online (www.mirdb.org) with the "seed sequence" located within the 3'-UTR of the target gene, and then validated MET1 to be the direct gene via luciferase reporter assay system. Real-time PCR and western-blot were used to determine the expression of miR-34b mRNA and MET1 mRNA and protein in different treating group.
MET1 was the direct gene of miR-34b by searching the miRNA database online and constructing luciferase reporter. We also investigated the negative regulatory relationship between miR-34b and MET1 via studying the relative luciferase activity at different concentrations of miR-34b mimics. Further, since rs4938723 polymorphism was previously reported to be interfering with the expression of miR-34b, we investigated the expression level of different genotypes including TT (N=20), TC (N=9) and CC (N=3), which supported the hypothesis that the presence of minor allele (C) of rs4938723 polymorphism compromised the expression of miR-34b. Meanwhile, we also conducted real time PCR and Western blot analysis to study the mRNA and protein expression level of MET1 among different genotypes or cells treated with different concentration of miR-34b mimics/inhibitors, indicating the negative regulatory relationship between miR-34b and MET1.We also investigated the relative viability of EGC cells when transfected with miR-34b mimics (50nM and 100nM) and miR-34b inhibitors (100nM) to validate miR-34b to be negatively interfering with the viability of EGC cells.
These data confirmed miR-34b rs4938723 polymorphism was also recognized as a biomarker to predict recurrence after ESD in EGC patients via analysis upon the recurrence-free rate among different genotypes of EGC patients.
关于MET1对早期胃癌（EGC）内镜下黏膜剥离术（ESD）治疗后复发的影响，本研究旨在探究其分子机制，包括MET1的潜在调节因子和信号通路。
我们利用位于靶基因3'-UTR内的“种子序列”在线搜索miRNA数据库（www.mirdb.org），然后通过荧光素酶报告基因检测系统验证MET1为直接作用基因。采用实时定量PCR和蛋白质免疫印迹法检测不同处理组中miR-34b mRNA、MET1 mRNA和蛋白质的表达。
通过在线搜索miRNA数据库并构建荧光素酶报告基因，证实MET1是miR-34b的直接作用基因。我们还通过研究不同浓度miR-34b模拟物下的相对荧光素酶活性，探究了miR-34b与MET1之间的负调控关系。此外，由于先前报道rs4938723多态性会干扰miR-34b的表达，我们研究了不同基因型（TT，n = 20；TC，n = 9；CC，n = 3）的表达水平，支持了rs4938723多态性的次要等位基因（C）的存在会损害miR-34b表达的假设。同时，我们还进行了实时定量PCR和蛋白质免疫印迹分析，以研究不同基因型或用不同浓度miR-34b模拟物/抑制剂处理的细胞中MET1的mRNA和蛋白质表达水平，表明miR-34b与MET1之间存在负调控关系。我们还研究了用miR-34b模拟物（50 nM和100 nM）和miR-34b抑制剂（100 nM）转染时EGC细胞的相对活力，以验证miR-34b对EGC细胞活力有负向干扰作用。
通过分析EGC患者不同基因型的无复发生存率，这些数据证实miR-34b rs4938723多态性也可作为预测EGC患者ESD术后复发的生物标志物。
