Institute of Genomic Medicine, Wenzhou Medical University, 268 Xueyuan Road, Wenzhou, 325000, Zhejiang, People's Republic of China.
Key Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of ZheJiang Province, Wenzhou, 325000, Zhejiang, People's Republic of China.
J Exp Clin Cancer Res. 2019 Jun 11;38(1):249. doi: 10.1186/s13046-019-1263-3.
Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause of cancer-related death worldwide. Increasing evidence indicates that the deregulation of long noncoding RNAs (lncRNAs) contributes to tumor initiation and progression; however, little is known about the biological role of cancer susceptibility candidate 9 (CASC9) in CRC.
Novel lncRNAs potentially involved in CRC tumorigenesis were identified from datasets downloaded from The Cancer LncRNome Atlas and The Atlas of Noncoding RNAs in Cancer. The CRC cell lines HCT-116, HCT-116 p53, SW620, SW480, HT-29, LoVo, LS-174T, and RKO were used. Colony-formation, MTS, cell-cycle, apoptosis, and in-vivo tumorigenesis assays were used to determine the role of CASC9 in CRC cell growth in vitro and in vivo. Potential interaction between CASC9 and cleavage and polyadenylation specificity factor subunit 3 (CPSF3) was evaluated using RNA immunoprecipitation and RNA-protein pull-down assays. RNA-sequencing was performed to analyze gene expression following CASC9 knockdown. RT-qPCR, western blotting, and mRNA decay assays were performed to study the mechanisms involved.
CASC9 was frequently upregulated in CRC, which was correlated with advanced TNM stage, and higher CASC9 levels were associated with poor patient outcomes. Knockdown of CASC9 inhibited growth and promoted apoptosis in CRC cells, whereas ectopic CASC9 expression promoted cell growth in vitro and in vivo. We demonstrated that CPSF3 is a CASC9-interacting protein, and knockdown of CPSF3 mimicked the effects of CASC9 knockdown in CRC cells. Furthermore, we found that CASC9 exerts its oncogenic activity by modulating TGFβ2 mRNA stability and upregulating the levels of TGFβ2 and TERT, resulting in an increase in phosphorylated SMAD3 and activation of TGF-β signaling, and enhanced TERT complex function in CRC cells. Finally, CPSF3 was significantly upregulated in CRC tissues as compared with adjacent or non-adjacent normal colon tissues, and CASC9, CPSF3, and TGFβ2 levels in human CRC tissues were positively correlated.
CASC9 is a promising prognostic predictor for patients with CRC and the CASC9-CPSF3-TGFβ2 axis is a potential therapeutic target for CRC treatment.
结直肠癌（CRC）是全球第三大常见癌症，也是癌症相关死亡的第二大主要原因。越来越多的证据表明，长非编码 RNA（lncRNA）的失调有助于肿瘤的发生和发展；然而，关于癌症易感性候选基因 9（CASC9）在 CRC 中的生物学作用知之甚少。
从下载自癌症 lncRNA 图谱和癌症非编码 RNA 图谱的数据集中确定了可能参与 CRC 肿瘤发生的新型 lncRNA。使用 HCT-116、HCT-116 p53、SW620、SW480、HT-29、LoVo、LS-174T 和 RKO 结肠癌细胞系进行实验。采用集落形成、MTS、细胞周期、凋亡和体内肿瘤发生实验，在体外和体内确定 CASC9 在 CRC 细胞生长中的作用。使用 RNA 免疫沉淀和 RNA-蛋白下拉实验评估 CASC9 与切割和多聚腺苷酸化特异性因子亚基 3（CPSF3）之间的潜在相互作用。进行 RNA 测序以分析 CASC9 敲低后基因表达的变化。进行 RT-qPCR、western blot 和 mRNA 降解实验以研究相关机制。
CASC9 在 CRC 中频繁上调，与晚期 TNM 分期相关，较高的 CASC9 水平与患者预后不良相关。CASC9 敲低抑制 CRC 细胞生长并促进细胞凋亡，而过表达 CASC9 则促进体外和体内细胞生长。我们证明 CPSF3 是 CASC9 的相互作用蛋白，CPSF3 敲低可模拟 CRC 细胞中 CASC9 敲低的作用。此外，我们发现 CASC9 通过调节 TGFβ2 mRNA 稳定性并上调 TGFβ2 和 TERT 的水平发挥致癌活性，导致磷酸化 SMAD3 增加和 TGF-β 信号通路激活，并增强 CRC 细胞中的 TERT 复合物功能。最后，与相邻或非相邻正常结肠组织相比，CRC 组织中 CPSF3 显著上调，并且人 CRC 组织中的 CASC9、CPSF3 和 TGFβ2 水平呈正相关。
CASC9 是 CRC 患者有希望的预后预测因子，CASC9-CPSF3-TGFβ2 轴可能是 CRC 治疗的潜在治疗靶点。
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