癌症干细胞在肿瘤中形成罕见的细胞群，有助于癌症患者的转移、复发和化疗耐药。环状 RNA (circRNA) 是基因表达的转录后调节因子，可吸收靶向 microRNA (miRNA) 来影响多种下游细胞过程。我们之前在一项表达谱研究中表明，circZNF800 (hsa_circ_0082096) 在源自结直肠癌 (CRC) 细胞系的富含癌症干细胞的球体中上调。球体是在悬浮球体培养中产生的。通过定量 RT-PCR 测定 ZNF800 mRNA、多能干细胞标记物和 circZNF800 水平。 RNA Pulldown 测定显示了 CircZNF800-miRNA 相互作用，并通过茎环 qRT-PCR 测定了 miRNA 水平。通过 EdU 染色和流式细胞术测试 circZNF800 对细胞增殖的影响。免疫荧光显微镜证实了干细胞标记物 CD44/CD133、Lgr5 和 SOX9 的表达。为了操纵 circZNF800 的细胞水平，通过转染体外合成和环化的 circZNF800 实现 circZNF800 过表达，并使用 CRISPR-Cas13d-circZNF800 载体系统实现敲低。异种移植裸鼠被用来证明circZNF800过表达和敲低对体内肿瘤生长的影响。CircZNF800被证明在CRC患者的晚期肿瘤组织中过表达。数据显示，circZNF800 阻碍 miR-140-3p、miR-382-5p 和 miR-579-3p 的表达，同时促进 miRNA 靶向的 ALK/ACVR1C、FZD3 和 WNT5A 的 mRNA 水平，这得到了 之间种子序列比对的支持。 circZNF800-miRNA 和 miRNA-mRNA 配对相互作用。对CRC细胞和活检组织的分析表明，circZNF800正向调节肠干细胞、多能性和癌症干细胞标志物的表达，并在体外促进CRC细胞增殖、球状体和集落形成，所有这些都是癌症干细胞特性。在异种移植小鼠中，circZNF800过表达促进了肿瘤生长，而通过在CRC肿瘤位点施用CRISPR Cas13d-circZNF800病毒颗粒来敲低circZNF800则阻碍了肿瘤生长。CircZNF800是一种致癌因子，调节癌症干细胞特性从而导致结直肠肿瘤发生，并且可用作肿瘤进展的预测标记以及用于结直肠癌治疗干预的 CRISPR Cas13d-circZNF800 敲低策略。© 2023。作者。

Cancer stem cells form a rare cell population in tumors that contributes to metastasis, recurrence and chemoresistance in cancer patients. Circular RNAs (circRNAs) are post-transcriptional regulators of gene expression that sponge targeted microRNA (miRNAs) to affect a multitude of downstream cellular processes. We previously showed in an expression profiling study that circZNF800 (hsa_circ_0082096) was up-regulated in cancer stem cell-enriched spheroids derived from colorectal cancer (CRC) cell lines.Spheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo.CircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth.CircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.© 2023. The Author(s).