Objective: To investigate the mechanism of miRNA-340 for regulating the proliferation of gastric cancer (GC) cells and predict its interacting circular RNAs (circRNAs), its downstream target genes and the involved signaling pathways.
Methods: The differentially expressed miRNAs in GC cell lines were analyzed and screened using miRNA microarrays. The expression level of miRNA-340 in 21 pairs of GC tissues and adjacent normal tissues was detected using real-time PCR. MTT and EdU assays were performed to examine the effect of miRNA-340 on the proliferation ability of HFE145 and BGC-823 cells. We also tested the effect of miRNA-340 inhibition on subcutaneous tumorigenesis of GC cells in a nude mouse model. The downstream target genes of miRNA-340 and the probable signal pathways were predicted online using Targetscan and DAVID database, respectively. The interacting circRNAs of miRNA-340 were analyzed using starBase platform.
Results: Among the differentially expressed miRNAs, miRNA-340 was significantly down-regulated in GC cell lines. Real-time PCR results showed that the expression of miRNA-340 was significantly lower in GC tissues than in the adjacent tissues (P < 0.05). MTT and EdU cell proliferation assays showed that miRNA-340 overexpression inhibited the proliferation of GC cells in vitro. In the nude mouse models, the proliferation of GC cells transfected with miRNA-340 inhibitor was obviously enhanced. Bioinformatics analysis suggested that miRNA-340 had 21 target genes with 3 or more conserved sites, and these genes were involved in tumorigenesis and invasion. The top 10 circRNAs were selected as the most powerful sponge circRNAs interacting with miRNA-340.
Conclusions: miRNA-340 may play the role of a tumor suppressor in tumorigenesis and progression. Overexpression of miRNA-340 suppress the proliferation of GC cells, suggesting its involvement in the development of GC along with multiple circRNAs.
目的: 了解miRNA-340在胃癌发生中的可能机制，在线预测与其相互作用的circRNAs及其下游靶基因和参与的信号通路。
方法: 利用芯片筛选胃癌细胞株中差异表达miRNA；利用Real-time RT-PCR，检测miRNA-340在21对胃癌组织与癌旁正常组织中的表达水平；利用MTT及EdU细胞增殖实验，检测miRNA-340对正常胃上皮细胞HFE145与胃癌细胞BGC-823的体外增殖能力；利用裸鼠成瘤实验检测miRNA-340在体内成瘤的能力。随后，通过在线软件Targetscan、David数据库及Starbase分别对miRNA-340的下游的靶基因、相关信号通路及相互作用的circRNAs进行生物信息分析。
结果: 与正常胃上皮细胞HFE145相比较，miRNA-340在胃癌细胞株中的表达水平显著下调；Real-time RT-PCR结果显示，miRNA-340在胃癌组织中的表达明显低于正常粘膜组织（P < 0.048）；体内体外实验表明miRNA-340对细胞的体外增殖能力具有一定的抑制作用；生物信息分析显示，预测的miRNA-340相互作用的下游靶基因中，筛选出21个具有3个及以上的保守结合部位的靶基因，其中多个靶基因均参与肿瘤的发生及侵袭；相互作用的circRNAs中筛选出分值前10个circRNAs作为与miRNA-340可能作用最强的sponge circRNAs。
结论: miRNA-340在肿瘤发生发展中可能起到了抑癌基因的作用，对胃癌细胞的增殖具有一定的抑制作用；miRNA-340与多个circRNAs可能存在相互作用，共同调控下游靶基因参与胃癌的发生发展。
Keywords: bioinformatics; circular RNA; gastric cancer; miRNA-340; signal pathways.