Department of Orthopedics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
PLoS One. 2023 Jun 7;18(6):e0286422. doi: 10.1371/journal.pone.0286422. eCollection 2023.
Long noncoding RNAs (lncRNAs) encompass short open reading frames (sORFs) that can be translated into small peptides. Here, we investigated the encoding potential of lncRNA LINC00665 in osteosarcoma (OS) cells. Bioinformatic analyses were utilized to predict the lncRNAs with encoding potential in human U2OS cells. Protein expression was assessed by an immunoblotting or immunofluorescence method. Cell viability was assessed by cell counting Kit-8 (CCK-8). Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell migration was gauged by transwell assay. The downstream effectors of the short peptide were verified using qualitative proteome analysis after immunoprecipitation (IP) experiments. The effect of the short peptide on protein interactions were confirmed by Co-Immunoprecipitation (CoIP) assays. We found that lncRNA LINC00665 encoded an 18-amino acid (aa)-long short peptide (named LINC00665_18aa). LINC00665_18aa suppressed the viability, proliferation, and migration of human MNNG-HOS and U2OS OS cells in vitro and diminished tumor growth in vivo. Mechanistically, LINC00665_18aa impaired the transcriptional activity, nuclear localization, and phosphorylation of cAMP response element-binding protein 1 (CREB1). Moreover, LINC00665_18aa weakened the interaction between CREB1 and ribosomal protein S6 kinase A3 (RPS6KA3, RSK2). Additionally, increased expression of CREB1 reversed the inhibitory effects of LINC00665_18aa on OS cell proliferation and migration. Our findings show that the short peptide LINC00665_18aa exerts a tumor-inhibitory function in OS, providing a new basis for cancer therapeutics through the functions of the short peptides encoded by lncRNAs.
长链非编码 RNA（lncRNA）包含可以翻译成小肽的短开放阅读框（sORF）。在这里，我们研究了长链非编码 RNA LINC00665 在骨肉瘤（OS）细胞中的编码潜力。利用生物信息学分析预测人类 U2OS 细胞中具有编码潜力的 lncRNA。通过免疫印迹或免疫荧光法评估蛋白质表达。通过细胞计数试剂盒-8（CCK-8）评估细胞活力。通过 5-乙炔基-2'-脱氧尿苷（EdU）测定检测细胞增殖。通过 Transwell 测定评估细胞迁移。通过免疫沉淀（IP）实验后的定性蛋白质组分析验证短肽的下游效应子。通过 Co-Immunoprecipitation（CoIP）测定证实短肽对蛋白质相互作用的影响。我们发现 lncRNA LINC00665 编码一个 18 个氨基酸（aa）长的短肽（命名为 LINC00665_18aa）。LINC00665_18aa 在体外抑制人 MNNG-HOS 和 U2OS OS 细胞的活力、增殖和迁移，并减少体内肿瘤生长。机制上，LINC00665_18aa 损害 cAMP 反应元件结合蛋白 1（CREB1）的转录活性、核定位和磷酸化。此外，LINC00665_18aa 减弱了 CREB1 与核糖体蛋白 S6 激酶 A3（RPS6KA3，RSK2）之间的相互作用。此外，CREB1 的表达增加逆转了 LINC00665_18aa 对 OS 细胞增殖和迁移的抑制作用。我们的研究结果表明，短肽 LINC00665_18aa 在 OS 中发挥肿瘤抑制功能，通过 lncRNA 编码的短肽的功能为癌症治疗提供了新的基础。
