细胞外囊泡(EV)能够将货物从供体转移到受体细胞,但确切地说,货物内容是如何管制出口的,目前尚不清楚。对于miRNA货物,我们以前表明,与表达野生型KRAS的同基因结直肠癌(CRC)细胞相比,不同的miRNA子集在来自KRAS突变活性CRC细胞的EV中差异富集,miR-100是最丰富的。可以解释miR-100和其他miRNA如何差异输出到EV中的机制尚未完全阐明。这里,我们测试了N6-甲基腺苷(m6A)修饰对miRNA通过METTL3和ALKBH5(m6A修饰的作者和擦除器)耗尽向EV输出的影响,分别。虽然ALKBH5击倒的效果相当温和,METTL3水平降低导致含有m6A修饰共有序列的miRNA亚群的细胞和细胞外水平降低。从表达针对METTL3的shRNA的细胞制备的EV的功能测试表明,它们不太能够在3D中将菌落生长赋予野生型KRAS细胞,并且在很大程度上也不能赋予西妥昔单抗抗性的传播。我们的数据支持METTL3修饰对细胞miRNA水平和特定miRNA输出的作用。
Extracellular vesicles (EVs) are capable of transferring cargo from donor to recipient cells, but precisely how cargo content is regulated for export is mostly unknown. For
miRNA cargo, we previously showed that when compared to isogenic colorectal cancer (CRC) cells expressing wild-type KRAS, a distinct subset of miRNAs are differentially enriched in EVs from KRAS mutant active CRC cells, with miR-100 being one of the most enriched. The mechanisms that could explain how miR-100 and other miRNAs are differentially exported into EVs have not been fully elucidated. Here, we tested the effect of N6-methyladenosine (m6A) modification on
miRNA export into EVs by depletion of METTL3 and ALKBH5, a writer and eraser of m6A modification, respectively. While the effects of ALKBH5 knockdown were quite modest, decreased levels of METTL3 led to reduced cellular and extracellular levels of a subset of miRNAs that contain
consensus sequences for m6A modification. Functional testing of EVs prepared from cells expressing shRNAs against METTL3 showed that they were less capable of conferring colony growth in 3D to wild-type KRAS cells and were also largely incapable of conferring the spread of cetuximab resistance. Our data support a role for METTL3 modification on cellular
miRNA levels and export of specific miRNAs.