Functional analysis of miR-375, a candidate of tumor suppressor miRNA in gastric cancer

在本研究中，我们发现miRNA-375(miR-375)的表达下调程度最高，其在胃癌细胞中的异位表达通过caspase介导的凋亡途径显著降低了细胞的存活率。有趣的是，我们发现miR-375的表达抑制了作为miR-375的直接靶点的PDK1的表达，随后抑制了Akt的磷酸化。进一步的基因表达芯片分析表明，在miR-375转染的细胞中，一个有效的抗凋亡基因14-3-3zeta在mRNA和蛋白质水平上都显著下调。在14-3-3zeta的3‘非翻译区，含有miR-375结合序列的荧光素酶报告的活性被miR-375的异位表达抑制，表明miR-375针对14-3-3zeta的3’非翻译区。此外，在胃癌细胞中，PDK1或14-3-3zeta的敲除都能诱导caspase的激活，这在miR-375细胞中也可以观察到，提示miR-375可能至少部分地通过下调PDK1和14-3-3zeta的表达而发挥其促凋亡作用。综上所述，我们认为miR-375是胃癌中的候选抑癌基因miRNA。

In this study, we found that miRNA-375(miR-375) was the most downregulated and that its ectopic expression in gastric carcinoma cells markedly reduced cell viability via the caspase-mediated apoptosis pathway. Interestingly, we found that expression of miR-375 inhibited expression of PDK1, which is a direct target of miR-375, followed by suppression of Akt phosphorylation. Further analysis by gene expression microarray revealed that 14-3-3zeta, a potent antiapoptotic gene, was significantly downregulated at both the mRNA and protein levels in cells transfected with miR-375. The activity of a luciferase reporter containing the miR-375 binding sequence at the 3' untranslated region(UTR) of 14-3-3zeta mRNA was repressed by the ectopic expression of miR-375, suggesting that miR-375 targets the 3' UTR of 14-3-3zeta. In addition, knockdown of either PDK1 or 14-3-3zeta in gastric carcinoma cells induced caspase activation, which was also observed in miR-375-transfected cells, suggesting that miR-375 may exert its proapoptotic function, at least in part, through the downregulation of PDK1 and 14-3-3zeta. Taken together, we propose that miR-375 is a candidate tumor suppressor miRNA in gastric carcinoma