Supplementary Material for: Endogenous and extracellular roles of a tumor suppressor miR-379-5p in gastric cancer

Introduction: MiRNAs play important roles in development of various cancers including gastric cancer. Exosomes are extracellular vesicles for translocating molecules. This study aims to investigate the tumor suppressive roles of miR-379-5p in gastric cancer, and to investigate the roles of exosomes in transporting miR-379-5p from intracellular to extracellular.
Methods: Fifty-three pairs of gastric cancer and non-tumor tissue samples were collected. Five cell lines were applied. Functional assays including cell proliferation, cell migration and invasion, and cell adhesion assay were performed. Targets of miR-379-5p were screened and validated by western blot. Expressions of endogenous miR-379-5p in gastric cancer cells and exosomal miR-379-5p in cell culture medium were evaluated by RT-qPCR. Medium of culturing AGS or BCG23 was applied for culturing MKN45 and HEK293T.
Results: The results indicated that miR-379-5p was significantly downregulated in gastric cancer tissue samples and cell lines. Enforced expression of miR-379-5p inhibited gastric cancer cell proliferation, migration, and invasion, while miR-379-5p mimic enhanced cell adhesion to extracellular matrix. IGF1R was a potential target of miR-379-5p in gastric cancer. Expression of miR-379-5p was dramatically higher in exosomes in cell culture medium than its endogenous expression. Exosomes from cell culture medium of AGS or BCG23 could regulate endogenous expression of miR-379-5p in HEK293T cells.
Conclusions: MiR-379-5p was significantly downregulated and it functioned as a tumor suppressor in gastric cancer. MiR-379-5p was higher expressed in exosomes of culture medium than its endogenous expression. MiR-379-5p could be translocated from cells into cell culture medium and entered certain cell types via exosomes.

引言：微小RNA（miRNAs）在包括胃癌在内的多种癌症的发生发展中发挥关键调控作用。外泌体（exosomes）是介导分子跨细胞转运的细胞外囊泡。本研究旨在探讨miR-379-5p在胃癌中的抑癌功能，同时明确外泌体在miR-379-5p从细胞内向细胞外转运过程中的作用。
方法：本研究收集了53对胃癌组织与配对非肿瘤组织样本，采用5种细胞系开展实验。依次进行细胞增殖、细胞迁移与侵袭、细胞黏附等功能学检测。通过蛋白质印迹（western blot）筛选并验证miR-379-5p的潜在靶基因。采用逆转录定量实时聚合酶链反应（RT-qPCR）检测胃癌细胞内源性miR-379-5p及细胞培养上清中外泌体miR-379-5p的表达水平。使用AGS或BCG23细胞的培养上清培养MKN45与HEK293T细胞。
结果：实验结果显示，miR-379-5p在胃癌组织样本及细胞系中显著低表达。过表达miR-379-5p可抑制胃癌细胞的增殖、迁移与侵袭能力，而miR-379-5p模拟物可增强细胞对细胞外基质的黏附活性。胰岛素样生长因子1受体（IGF1R）是miR-379-5p在胃癌中的潜在靶基因。细胞培养上清中外泌体携带的miR-379-5p表达水平显著高于其细胞内源性表达水平。AGS或BCG23细胞培养上清中的外泌体可调控HEK293T细胞内源性miR-379-5p的表达水平。
结论：miR-379-5p在胃癌中显著低表达并发挥抑癌基因功能。细胞培养上清中外泌体中的miR-379-5p表达水平高于其细胞内源性表达水平。miR-379-5p可从细胞内转运至细胞培养上清，并通过外泌体进入特定细胞类型。