BACKGROUND As a novel class of noncoding RNAs, circRNAs have been recently identified to regulate tumorigenesis and aggressiveness. However, the function of circRNAs in colorectal cancer (CRC) metastasis remains unclear. We aimed to identify circRNAs that are upregulated in CRC tissues from patients and study their function in CRC metastasis. METHODS We compared six pairs of CRC tissues and their matched adjacent non-tumor tissues by using circRNA microarray. We first evaluated the expression of circPTK2 (hsa_circ_0005273) in fresh tissues from CRC tumors and corresponding adjacent tissues by qPCR analysis. CircPTK2 expression levels in the tissue microarray with 5 years of survival information were determined by RNA-ISH analysis. Meanwhile, the expression levels of circulating circPTK2 were further analyzed according to the patients' clinical features. We analyzed cell apoptosis, colony formation, migration, and invasion in CRC cells. To further elucidate the effect of circPTK2 in CRC metastasis, we also conducted a colon cancer hepatic and pulmonary metastasis experiment. We used RNA biotin-labeled pull down and mass spectrometry to identify the target of circPTK2. We established a PDTX model to evaluate the effect of shRNA specifically targeting circPTK2 on tumor metastasis. RESULTS We identified a novel circRNA, circPTK2, which is back-spliced of three exons (exons 27, 28 and 29) of PTK2 by using circRNA microarray, bioinformatics and functional studies. CircPTK2 was elevated in CRC tissues and positively associated with tumor growth and metastasis. CRC patients with increased circPTK2 expression were positively correlated with poorer survival rates. Furthermore, our studies showed that circPTK2 could promote EMT of CRC cells in vitro and in vivo by binding to vimentin protein on sites Ser38, Ser55 and Ser82. We further demonstrated the interaction of circPTK2 and vimentin mediated the regulation of CRC by knockdown or overexpression of vimentin. In addition, we revealed that tail vein injection of shRNA specifically targeting circPTK2 blunt tumor metastasis in a patient-derived CRC xenograft model. CONCLUSIONS Collectively, these results demonstrate that circPTK2 exerts critical roles in CRC growth and metastasis and may serve as a potential therapeutic target for CRC metastasis, and also a promising biomarker for early diagnosis of metastasis.

中文翻译：

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CircPTK2 (hsa_circ_0005273) 作为转移性结直肠癌的新型治疗靶点。


背景作为一类新型非编码RNA，circRNA最近被鉴定可调节肿瘤发生和侵袭性。然而，circRNA在结直肠癌（CRC）转移中的功能仍不清楚。我们的目的是鉴定患者 CRC 组织中上调的 circRNA，并研究它们在 CRC 转移中的功能。方法我们使用 circRNA 微阵列比较了六对 CRC 组织及其匹配的邻近非肿瘤组织。我们首先通过 qPCR 分析评估了 CRC 肿瘤新鲜组织和相应邻近组织中 circPTK2 (hsa_circ_0005273) 的表达。通过 RNA-ISH 分析确定具有 5 年生存信息的组织微阵列中的 CircPTK2 表达水平。同时，根据患者的临床特征进一步分析循环circPTK2的表达水平。我们分析了 CRC 细胞的细胞凋亡、集落形成、迁移和侵袭。为了进一步阐明circPTK2在CRC转移中的作用，我们还进行了结肠癌肝和肺转移实验。我们使用 RNA 生物素标记的 Pull down 和质谱法来鉴定 circPTK2 的靶标。我们建立了PDTX模型来评估特异性靶向circPTK2的shRNA对肿瘤转移的影响。结果我们通过circRNA微阵列、生物信息学和功能研究，鉴定了一种新型circRNA circPTK2，它是PTK2的三个外显子（外显子27、28和29）的反向剪接。 CircPTK2 在 CRC 组织中升高，并与肿瘤生长和转移呈正相关。 circPTK2 表达增加的 CRC 患者与较差的生存率呈正相关。 此外，我们的研究表明，circPTK2可以通过与波形蛋白Ser38、Ser55和Ser82位点结合在体外和体内促进CRC细胞的EMT。我们进一步证明了 circPTK2 和波形蛋白的相互作用通过敲低或过度表达波形蛋白来介导 CRC 的调节。此外，我们发现尾静脉注射特异性靶向 circPTK2 的 shRNA 可在患者来源的 CRC 异种移植模型中钝化肿瘤转移。结论总的来说，这些结果表明circPTK2在CRC生长和转移中发挥关键作用，可能作为CRC转移的潜在治疗靶点，也是转移早期诊断的有前途的生物标志物。