Guo Shichao, Wang Guangchao, Zhao Zitong, Li Dan, Song Yongmei, Zhan Qimin
State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Laboratory of Molecular Oncology, Peking University Cancer Hospital & Institute, Beijing 100142, China.
Chin J Cancer Res. 2022 Feb 28;34(1):11-27. doi: 10.21147/j.issn.1000-9604.2022.01.02.
Cleavage and polyadenylation specific factor 6 (CPSF6) has been documented as an oncoprotein in different types of cancer. However, functions of CPSF6 have not been investigated yet in esophageal squamous cell carcinoma (ESCC). Here, we aimed to investigate the potential clinical values and biological functions of CPSF6 in ESCC.
For determining the expression level of CPSF6 in ESCC patients, we analyzed published data, performed quantitative real-time polymerase chain reaction (RT-qPCR) and immunohistochemistry assays. Kaplan-Meier curves and log-rank tests were used for survival analyses. GO and KEGG analyses were done for CPSF6-related genes. Cell proliferation, colony formation and xenograft assays were conducted to verify the effects of CPSF6 on ESCC. In addition, cell cycle and apoptosis assays were also performed to manifest the functions of CPSF6 and circCPSF6. RNA pulldown and radioimmunoprecipitation (RIP) assays were used for confirming the interaction between circCPSF6 (hsa_circ_0000417) and CPSF6 protein. The regulatory relationship between CPSF6 protein and circCPSF6 was determined by RT-qPCR.
We found that CPSF6 was upregulated in ESCC tissues and overexpression of cytoplasmic CPSF6 was associated with poor prognosis. GO and KEGG analyses suggested that CPSF6 could mainly affect cell division in ESCC. Further experiments manifested that CPSF6 promoted cell proliferation and colony formation. Xenograft assay showed that knockdown of CPSF6 significantly decreased tumor growth rate . Subsequently, we verified that depletion of CPSF6 led to cell cycle arrest and apoptosis. Finally, we validated that CPSF6, as a circRNA-binding protein, interacted with and regulated its circular isoform circCPSF6 (hsa_circ_0000417), of which depletion also resulted in cell cycle arrest and cell apoptosis in ESCC.
These findings gave us insight that overexpression of cytoplasmic CPSF6 protein is associated with poor prognosis in ESCC and CPSF6 may function as an oncoprotein, at least in part, through regulating circCPSF6 expression.
切割与聚腺苷酸化特异性因子6（CPSF6）已被证明是不同类型癌症中的一种癌蛋白。然而，CPSF6在食管鳞状细胞癌（ESCC）中的功能尚未得到研究。在此，我们旨在探讨CPSF6在ESCC中的潜在临床价值和生物学功能。
为了确定ESCC患者中CPSF6的表达水平，我们分析了已发表的数据，进行了定量实时聚合酶链反应（RT-qPCR）和免疫组织化学分析。采用Kaplan-Meier曲线和对数秩检验进行生存分析。对与CPSF6相关的基因进行了基因本体（GO）和京都基因与基因组百科全书（KEGG）分析。进行细胞增殖、集落形成和异种移植实验以验证CPSF6对ESCC的影响。此外，还进行了细胞周期和凋亡实验以阐明CPSF6和环状CPSF6（circCPSF6）的功能。采用RNA下拉和放射免疫沉淀（RIP）实验来证实环状CPSF6（hsa_circ_0000417）与CPSF6蛋白之间的相互作用。通过RT-qPCR确定CPSF6蛋白与circCPSF6之间的调控关系。
我们发现CPSF6在ESCC组织中上调，细胞质CPSF6的过表达与不良预后相关。GO和KEGG分析表明，CPSF6在ESCC中主要影响细胞分裂。进一步的实验表明，CPSF6促进细胞增殖和集落形成。异种移植实验表明，敲低CPSF6显著降低肿瘤生长速率。随后，我们证实CPSF6的缺失导致细胞周期停滞和凋亡。最后，我们验证了CPSF6作为一种环状RNA结合蛋白，与它的环状异构体circCPSF6（hsa_circ_0000417）相互作用并对其进行调控，circCPSF6的缺失也导致ESCC中的细胞周期停滞和细胞凋亡。
这些发现使我们认识到，细胞质CPSF6蛋白的过表达与ESCC的不良预后相关，并且CPSF6可能至少部分地通过调节circCPSF6的表达发挥癌蛋白的作用。
