Department of Tumor Biological Treatment, the Third Affiliated Hospital of Soochow University, Changzhou, 213003, People's Republic of China.
Jiangsu Engineering Research Center for Tumor Immunotherapy, Changzhou, 213003, People's Republic of China.
Mol Cancer. 2019 Mar 29;18(1):47. doi: 10.1186/s12943-019-1010-6.
It has been well established that circular RNAs (circRNAs) play an important regulatory role during tumor progression. Recent studies have indicated that even though circRNAs generally regulate gene expression through miRNA sponges, they may encode small peptides in tumor pathogenesis. However, it remains largely unexplored whether circRNAs are involved in the tumorigenesis of colon cancer (CC).
The expression profiles of circRNAs in CC tissues were assessed by circRNA microarray. Quantitative real-time PCR, RNase R digestion assay and tissue microarray were used to confirm the existence and expression pattern of circPPP1R12A. The subcellular distribution of circPPP1R12A was analyzed by nuclear mass separation assay and fluorescence in situ hybridization (FISH). SDS-PAGE and LC/MS were employed to evaluate the protein-coding ability of circPPP1R12A. CC cells were stably transfected with lentivirus approach, and cell proliferation, migration and invasion, as well as tumorigenesis and metastasis in nude mice were assessed to clarify the functional roles of circPPP1R12A and its encoded protein circPPP1R12A-73aa. RNA-sequencing and Western blotting analysis were furthered employed to identify the critical signaling pathway regulated by circPPP1R12A-73aa.
We firstly screened the expression profiles of human circRNAs in CC tissues and found that the expression of hsa_circ_0000423 (termed as circPPP1R12A) was significantly increased in CC tissues. We also found that circPPP1R12A was mostly localized in the cytoplasm of CC cells. Kaplan-Meier analysis showed that patients with higher levels of circPPP1R12A had a significantly shorter overall survival. By gain- and loss-of-function approaches, the results suggested that circPPP1R12A played a critical role in proliferation, migration and invasion of CC cells. Furthermore, we showed that circPPP1R12A carried an open reading frame (ORF), which encoded a functional protein (termed as circPPP1R12A-73aa). Next, we found that PPP1R12A-C, not circPPP1R12A, promoted the proliferation, migration and invasion abilities of CC in vitro and in vivo. Finally, we identified that circPPP1R12A-73aa promoted the growth and metastasis of CC via activating Hippo-YAP signaling pathway. In addition, the YAP specific inhibitor Peptide 17 dramatically alleviated the promotive effect of circPPP1R12A-73aa on CC cells.
In the present study, we illustrated the coding-potential of circRNA circPPP1R12A in the progression of CC. Moreover, we identified that circPPP1R12A-73aa promoted the tumor pathogenesis and metastasis of CC via activating Hippo-YAP signaling pathway. Our findings might provide valuable insights into the development of novel potential therapeutic targets for CC.
已经证实环状 RNA（circRNAs）在肿瘤进展过程中发挥重要的调控作用。最近的研究表明，尽管 circRNAs 通常通过 miRNA 海绵调控基因表达，但它们在肿瘤发病机制中可能编码小肽。然而，circRNAs 是否参与结肠癌（CC）的发生仍在很大程度上尚未得到探索。
通过 circRNA 微阵列评估 CC 组织中 circRNAs 的表达谱。采用定量实时 PCR、RNase R 消化实验和组织微阵列验证 circPPP1R12A 的存在和表达模式。通过核质量分离实验和荧光原位杂交（FISH）分析 circPPP1R12A 的亚细胞分布。采用 SDS-PAGE 和 LC/MS 评估 circPPP1R12A 的蛋白编码能力。通过慢病毒转染方法稳定转染 CC 细胞，评估细胞增殖、迁移和侵袭以及裸鼠肿瘤发生和转移，以阐明 circPPP1R12A 及其编码蛋白 circPPP1R12A-73aa 的功能作用。进一步采用 RNA 测序和 Western blot 分析鉴定由 circPPP1R12A-73aa 调控的关键信号通路。
我们首先筛选了 CC 组织中 circRNAs 的表达谱，发现 hsa_circ_0000423（称为 circPPP1R12A）在 CC 组织中的表达显著增加。我们还发现 circPPP1R12A 主要定位于 CC 细胞的细胞质中。Kaplan-Meier 分析表明，circPPP1R12A 水平较高的患者总生存期明显缩短。通过增益和缺失功能方法，结果表明 circPPP1R12A 在 CC 细胞的增殖、迁移和侵袭中发挥关键作用。此外，我们发现 circPPP1R12A 带有一个开放阅读框（ORF），该 ORF 编码一个功能性蛋白（称为 circPPP1R12A-73aa）。接下来，我们发现 PPP1R12A-C，而不是 circPPP1R12A，促进了 CC 在体外和体内的增殖、迁移和侵袭能力。最后，我们发现 circPPP1R12A-73aa 通过激活 Hippo-YAP 信号通路促进 CC 的生长和转移。此外，YAP 特异性抑制剂 Peptide 17 显著减轻了 circPPP1R12A-73aa 对 CC 细胞的促进作用。
本研究阐明了 circRNA circPPP1R12A 在 CC 进展中的编码潜力。此外，我们发现 circPPP1R12A-73aa 通过激活 Hippo-YAP 信号通路促进 CC 的肿瘤发生和转移。我们的研究结果可能为 CC 的新型潜在治疗靶点的开发提供有价值的见解。
