Yuan Hongfan, Ren Qianwen, Du Yantao, Ma Yuwan, Gu Liankun, Zhou Jing, Tian Wei, Deng Dajun
Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Cancer Etiology, Peking University Cancer Hospital and Institute, Beijing, 100142, China.
The Department of Medical Oncology, Sichuan Cancer Hospital and Institute, Affiliated Cancer Hospital of University of Electronic and Technology of China, Chengdu, 610042, China.
Cell Death Discov. 2023 Jul 3;9(1):220. doi: 10.1038/s41420-023-01510-1.
The MIR663AHG gene encodes both miR663AHG and miR663a. While miR663a contributes to the defense of host cells against inflammation and inhibits colon cancer development, the biological function of lncRNA miR663AHG has not been previously reported. In this study, the subcellular localization of lncRNA miR663AHG was determined by RNA-FISH. miR663AHG and miR663a were measured by qRT-PCR. The effects of miR663AHG on the growth and metastasis of colon cancer cells were investigated in vitro and in vivo. CRISPR/Cas9, RNA pulldown, and other biological assays were used to explore the underlying mechanism of miR663AHG. We found that miR663AHG was mainly distributed in the nucleus of Caco2 and HCT116 cells and the cytoplasm of SW480 cells. The expression level of miR663AHG was positively correlated with the level of miR663a (r = 0.179, P = 0.015) and significantly downregulated in colon cancer tissues relative to paired normal tissues from 119 patients (P < 0.008). Colon cancers with low miR663AHG expression were associated with advanced pTNM stage (P = 0.021), lymph metastasis (P = 0.041), and shorter overall survival (hazard ratio = 2.026; P = 0.021). Experimentally, miR663AHG inhibited colon cancer cell proliferation, migration, and invasion. The growth of xenografts from RKO cells overexpressing miR663AHG was slower than that of xenografts from vector control cells in BALB/c nude mice (P = 0.007). Interestingly, either RNA-interfering or resveratrol-inducing expression changes of miR663AHG or miR663a can trigger negative feedback regulation of transcription of the MIR663AHG gene. Mechanistically, miR663AHG could bind to miR663a and its precursor pre-miR663a, and prevent the degradation of miR663a target mRNAs. Disruption of the negative feedback by knockout of the MIR663AHG promoter, exon-1, and pri-miR663A-coding sequence entirely blocked these effects of miR663AHG, which was restored in cells transfected with miR663a expression vector in rescue experiment. In conclusion, miR663AHG functions as a tumor suppressor that inhibits the development of colon cancer through its cis-binding to miR663a/pre-miR663a. The cross talk between miR663AHG and miR663a expression may play dominant roles in maintaining the functions of miR663AHG in colon cancer development.
MIR663AHG基因同时编码miR663AHG和miR663a。虽然miR663a有助于宿主细胞抵御炎症并抑制结肠癌发展，但lncRNA miR663AHG的生物学功能此前尚未见报道。在本研究中，通过RNA荧光原位杂交（RNA-FISH）确定lncRNA miR663AHG的亚细胞定位。采用qRT-PCR检测miR663AHG和miR663a。在体外和体内研究miR663AHG对结肠癌细胞生长和转移的影响。利用CRISPR/Cas9、RNA下拉实验及其他生物学实验探究miR663AHG的潜在机制。我们发现，miR663AHG主要分布于Caco2和HCT116细胞的细胞核以及SW480细胞的细胞质中。miR663AHG的表达水平与miR663a的水平呈正相关（r = 0.179，P = 0.015），且相对于119例患者配对的正常组织，其在结肠癌组织中显著下调（P < 0.008）。miR663AHG低表达的结肠癌与晚期pTNM分期（P = 0.021）、淋巴结转移（P = 0.041）及较短的总生存期相关（风险比 = 2.026；P = 0.021）。实验表明，miR663AHG抑制结肠癌细胞的增殖、迁移和侵袭。在BALB/c裸鼠中，过表达miR663AHG的RKO细胞异种移植瘤的生长比载体对照细胞的异种移植瘤慢（P = 0.007）。有趣的是，RNA干扰或白藜芦醇诱导miR663AHG或miR663a表达变化均可触发MIR663AHG基因转录的负反馈调节。机制上，miR663AHG可与miR663a及其前体pre-miR663a结合，并阻止miR663a靶mRNA的降解。敲除MIR663AHG启动子、外显子1和pri-miR663A编码序列破坏负反馈，完全阻断了miR663AHG的这些作用，在拯救实验中转染miR663a表达载体的细胞中这些作用得以恢复。总之，miR663AHG作为一种肿瘤抑制因子，通过其与miR663a/pre-miR663a的顺式结合抑制结肠癌发展。miR663AHG和miR663a表达之间的相互作用可能在维持miR663AHG在结肠癌发展中的功能方面起主导作用。
