Purpose

Growing evidence indicates that aberrant expression of microRNAs contributes to tumor development. However, the biological role of microRNA-4490 (miR-4490) in gastric cancer (GC) remains to be clarified.

Methods

To explore the function of miR-4490 in GC, we performed colony formation, EdU incorporation, qRT-PCR, Western blotting, in situ hybridization (ISH), immunohistochemistry (IHC), flow cytometry, ChIP and dual-luciferase reporter assays. In addition, the growth, migration and invasion capacities of GC cells were evaluated.

Results

We found that miR-4490 was significantly downregulated in primary GC samples and in GC-derived cell lines compared with normal controls, and that this expression level was negatively correlated with GC malignancy. Exogenous miR-4490 expression not only reduced cell cycle progression and proliferation, but also significantly inhibited GC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, we found that miR-4490 directly targets USP22, which mediates inhibition of GC cell proliferation and EMT-induced metastasis in vitro and in vivo. Moreover, we found through luciferase and ChIP assays that transcription factor POU2F1 can directly bind to POU2F1 binding sites within the miR-4490 and USP22 promoters and, by doing so, modulate their transcription. Spearman’s correlation analysis revealed a positive correlation between USP22 and POU2F1 expression and negative correlations between miR-4490 and USP22 as well as miR-4490 and POU2F1 expression in primary GC tissues.

Conclusion

Based on our results we conclude that miR-4490 acts as a tumor suppressor, and that the POU2F1/miR-4490/USP22 axis plays an important role in the regulation of growth, invasion and EMT of GC cells.

中文翻译：

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POU2F1/miR-4490/USP22 轴调节胃癌的细胞增殖和转移。

目的

越来越多的证据表明，microRNA 的异常表达有助于肿瘤的发展。然而，microRNA-4490 (miR-4490) 在胃癌 (GC) 中的生物学作用仍有待阐明。

方法

为了探索 miR-4490 在 GC 中的功能，我们进行了集落形成、EdU 掺入、qRT-PCR、蛋白质印迹、原位杂交 (ISH)、免疫组织化学 (IHC)、流式细胞术、ChIP 和双荧光素酶报告基因检测。此外，还评估了 GC 细胞的生长、迁移和侵袭能力。

结果

我们发现，与正常对照相比，miR-4490 在原代 GC 样本和 GC 衍生细胞系中显着下调，并且该表达水平与 GC 恶性程度呈负相关。外源性 miR-4490 表达不仅减少细胞周期进程和增殖，而且在体外显着抑制 GC 细胞迁移、侵袭和上皮-间质转化 (EMT)。从机制上讲，我们发现 miR-4490 直接靶向 USP22，后者在体外和体内介导 GC 细胞增殖和 EMT 诱导的转移的抑制。此外，我们通过荧光素酶和 ChIP 测定发现转录因子 POU2F1 可以直接结合 miR-4490 和 USP22 启动子内的 POU2F1 结合位点，并通过这样做来调节它们的转录。

结论

根据我们的结果，我们得出结论，miR-4490 充当肿瘤抑制因子，POU2F1/miR-4490/USP22 轴在 GC 细胞的生长、侵袭和 EMT 的调节中起重要作用。