circCDYL2, Overexpressed in Highly Migratory Colorectal Cancer Cells, Promotes Migration by Binding to Ezrin.
circCDYL2, Overexpressed in Highly Migratory Colorectal Cancer Cells, Promotes Migration by Binding to Ezrin.
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circCDYL2 在高度迁移的结直肠癌细胞中过表达，通过与 Ezrin 结合促进迁移
DOI：
10.3389/fonc.2021.716073
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发表时间：
2021
影响因子：
4.7
通讯作者：
Li X
中科院分区：
文献类型：
--
作者：
Li X;Wang J;Long H;Lin W;Wang H;Chen Y;Yuan Q;Li X
结直肠癌（Colorectal cancer，CRC）是世界范围内最常见的恶性肿瘤之一，死亡率高，尤其是由于转移。然而，没有临床上可用的治疗CRC转移的策略。探讨结直肠癌转移的机制是提高结直肠癌转移治疗水平的关键。方法本研究采用重复transwell迁移实验构建了高迁移性大肠癌细胞亚系H-RKO，并通过高通量RNA测序鉴定了参与大肠癌迁移的circRNA。通过RT-qPCR验证上调的circRNA以鉴定最升高的circRNA。通过RT-qPCR在40对CRC组织和4个CRC细胞系中评估该circRNA（circCDYL 2）的表达。进行Transwell迁移和伤口愈合测定以验证circCDYL 2在细胞迁移中的功能。使用PCR证实circCDYL 2的细胞分布。RNA pulldown和RNA免疫沉淀用于证实circCDYL 2和Ezrin之间的相互作用。使用Western印迹、免疫组织化学和拯救实验来确定circCDYL 2在调节Ezrin蛋白表达和AKT磷酸化中的作用。结果在候选circRNA中，circCDYL 2在H-RKO细胞中的过表达率最高。此外，circCDYL 2表达在CRC组织和细胞系中升高。功能获得和丧失测定表明circCDYL 2增强CRC细胞的迁移。circCDYL 2位于CRC细胞的细胞质中，并与Ezrin相互作用以上调其蛋白水平，导致AKT磷酸化。Ezrin敲低消除了由circCDYL 2过表达诱导的CRC细胞迁移。结论我们的研究首次证明circCDYL 2通过结合Ezrin并激活AKT通路促进CRC迁移。CircCDYL 2代表用于预防CRC转移的潜在治疗靶标。
Background Colorectal cancer (CRC) is one of the most common malignancies with high mortality worldwide, particularly due to metastasis. However, there are no clinically available strategies for treating CRC metastasis. Exploring the mechanisms underlying CRC metastasis is the key to improve the treatment of CRC with metastasis. Methods In this study, we generated the highly migratory CRC cell subline H-RKO using a repeated transwell migration assay to identify circRNAs involved in CRC migration by high-throughput RNA sequencing. Upregulated circRNAs were validated by RT-qPCR to identify the most elevated circRNA. The expression of this circRNA (circCDYL2) was evaluated in 40 pairs of CRC tissues and four CRC cell lines by RT-qPCR. Transwell migration and wound healing assays were performed to verify the function of circCDYL2 in cell migration. The cellular distribution of circCDYL2 was confirmed using PCR. RNA pulldown and RNA immunoprecipitation were used to confirm the interaction between circCDYL2 and Ezrin. Western blotting, immunohistochemistry, and rescue experiments were used to determine the role of circCDYL2 in regulating Ezrin protein expression and AKT phosphorylation. Results Among the candidate circRNAs, circCDYL2 was the highest overexpressed circRNA in H-RKO compared to parental N-RKO cells. Furthermore, circCDYL2 expression was elevated in CRC tissues and cell lines. Gain- and loss-of-function assays indicated that circCDYL2 enhanced the migration of CRC cells. circCDYL2 was located in the cytoplasm of CRC cells and interacted with Ezrin to upregulate its protein levels, resulting in AKT phosphorylation. Ezrin knockdown abrogated the CRC cell migration induced by circCDYL2 overexpression. Conclusions Our study demonstrated for the first time that circCDYL2 promotes CRC migration by binding Ezrin and activating the AKT pathway. CircCDYL2 represents a potential therapeutic target for preventing CRC metastasis.
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