Department of Thoracic Surgery, Xiangya Hospital, Central South University, Changsha, 410008, Hunan Province, P. R. China.
Xiangya Lung Cancer Center, Xiangya Hospital, Central South University, Changsha, 410008, Hunan Province, P. R. China.
Cancer Gene Ther. 2023 Dec;30(12):1649-1662. doi: 10.1038/s41417-023-00670-7. Epub 2023 Oct 27.
Non-small cell lung cancer (NSCLC) is a common and lethal malignancy. The carcinogenic roles of lncRNA CALML3 antisense RNA 1 (CALML3-AS1) have been documented. However, the function and potential mechanisms of CALML3-AS1 in the progression of NSCLC need to be further explored. The molecule expression was assessed by qRT-PCR and Western blot. The subcellular localization of CALML3-AS1 was observed by fluorescence in situ hybridization (FISH). The malignant behaviors of NSCLC cells were evaluated by CCK-8, colony formation, EdU, wound healing and transwell assays. In vivo xenograft tumor and liver metastatic models were established. The molecular mechanisms were investigated by RIP, RNA pull-down and ChIP assays. The methylation level was detected by MSP. Herein, we found that CALML3-AS1 was upregulated, while butyrophilin-like 9 (BTNL9) was downregulated in NSCLC. Functionally, CALML3-AS1 depletion repressed NSCLC cell malignant phenotypes, in vivo tumor growth, and liver metastasis. Mechanistically, AlkB homolog 5 (ALKBH5) enhanced CALML3-AS1 stability via N-methyladenosine (mA) demethylation, whereas mA reader YTH domain-containing 2 (YTHDC2) destabilized CALML3-AS1. Moreover, CALML3-AS1 inhibited BTNL9 transcription and expression through the recruitment of Zeste homolog 2 (EZH2). Rescue experiments demonstrated that BTNL9 downregulation counteracted sh-CALML3-AS1-mediated antitumor effects on NSCLC. Taken together, CALML3-AS1 modulated by ALKBH5 and YTHDC2 in an mA modification dependent manner drives NSCLC progression via epigenetically repressing BTNL9.
非小细胞肺癌（NSCLC）是一种常见且致命的恶性肿瘤。已有研究证明长链非编码 RNA CALML3 反义 RNA 1（CALML3-AS1）的致癌作用。然而，CALML3-AS1 在 NSCLC 进展中的功能和潜在机制仍需进一步探索。通过 qRT-PCR 和 Western blot 评估分子表达。通过荧光原位杂交（FISH）观察 CALML3-AS1 的亚细胞定位。通过 CCK-8、集落形成、EdU、划痕愈合和 Transwell 分析评估 NSCLC 细胞的恶性行为。建立体内异种移植肿瘤和肝转移模型。通过 RIP、RNA 下拉和 ChIP 分析研究分子机制。通过 MSP 检测甲基化水平。在此，我们发现 CALML3-AS1 在 NSCLC 中上调，而丁酰胆碱结合蛋白样 9（BTNL9）下调。功能上，CALML3-AS1 耗竭抑制 NSCLC 细胞恶性表型、体内肿瘤生长和肝转移。机制上，AlkB 同源物 5（ALKBH5）通过 N6-甲基腺苷（m6A）去甲基化增强 CALML3-AS1 的稳定性，而 m6A 阅读器 YTH 结构域包含蛋白 2（YTHDC2）则使 CALML3-AS1 不稳定。此外，CALML3-AS1 通过募集 SET 域包含 2（EZH2）抑制 BTNL9 的转录和表达。挽救实验表明，BTNL9 下调抵消了 sh-CALML3-AS1 对 NSCLC 的抗肿瘤作用。总之，CALML3-AS1 通过依赖 m6A 修饰的 ALKBH5 和 YTHDC2 进行调节，通过表观遗传抑制 BTNL9 驱动 NSCLC 进展。
