Non-small cell lung cancer (NSCLC) is the most common type of human lung cancers, which has diverse pathological features. Although many signaling pathways and therapeutic targets have been defined to play important roles in NSCLC, limiting efficacies have been achieved. Bioinformatics methods were used to identify differential long non-coding RNA expression in NSCLC. Real-time RT-PCR experiments were used to examine the expression pattern of lncRNA PKMYT1AR, miR-485-5p. Both in vitro and in vivo functional assays were performed to investigate the functional role of PKMYT1AR/miR-485-5p/PKMYT1 axis on regulating cell proliferation, migration and tumor growth. Dual luciferase reporter assay, fluorescent in situ hybridization (FISH), immunoblot, co-immunoprecipitation experiments were used to verify the molecular mechanism. Here, we identify a human-specific long non-coding RNA (lncRNA, ENST00000595422), termed PKMYT1AR (PKMYT1 associated lncRNA), that is induced in NSCLC by Yin Yang 1 (YY1) factor, especially in cancerous cell lines (H358, H1975, H1299, H1650, A549 and SPC-A1) compared to that in normal human bronchial epithelium cell line (BEAS-2B). We show that PKMYT1AR high expression correlates with worse clinical outcome, and knockdown of PKMYT1AR inhibits tumor cell proliferation, migration and xenograft tumor formation abilities. Bioinformatic analysis and a luciferase assay demonstrate that PKMYT1AR directly interacts with miR-485-5p to attenuate the inhibitory role on its downstream oncogenic factor PKMYT1 (the protein kinase, membrane-associated tyrosine/threonine 1) in NSCLC. Furthermore, we uncover that miR-485-5p is downregulated in both cancerous cell lines and peripheral blood serum isolated from NSCLC patients compared to reciprocal control groups. Consistently, forced expression of miR-485-5p inhibits the proliferation and migration abilities of tumor cells. Moreover, we provide evidence showing that PKMYT1AR targeting antisense oligonucleotide (ASO) dramatically inhibit tumor growth in vivo. Mechanistic study shows that PKMYT1AR/ miR-485-5p /PKMYT1 axis promotes cancer stem cells (CSCs) maintenance in NSCLC via inhibiting β-TrCP1 mediated ubiquitin degradation of β-catenin proteins, which in turn causes enhanced tumorigenesis. Our findings reveal the critical role of PKMYT1AR/miR-485-5p /PKMYT1 axis during NSCLC progression, which could be used as novel therapeutic targets in the future.

中文翻译：

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LncRNA PKMYT1AR通过激活Wnt信号通路促进非小细胞肺癌干细胞维持

非小细胞肺癌（NSCLC）是人类最常见的肺癌类型，具有多种病理特征。尽管许多信号通路和治疗靶点已被定义为在 NSCLC 中发挥重要作用，但已实现了有限的疗效。生物信息学方法用于鉴定 NSCLC 中差异的长链非编码 RNA 表达。实时 RT-PCR 实验用于检查 lncRNA PKMYT1AR、miR-485-5p 的表达模式。进行体外和体内功能测定以研究 PKMYT1AR/miR-485-5p/PKMYT1 轴在调节细胞增殖、迁移和肿瘤生长中的功能作用。双荧光素酶报告基因测定、荧光原位杂交（FISH）、免疫印迹、免疫共沉淀实验用于验证分子机制。这里，我们鉴定了一种人类特异性长非编码 RNA（lncRNA，ENST00000595422），称为 PKMYT1AR（PKMYT1 相关 lncRNA），它在 NSCLC 中由阴阳 1（YY1）因子诱导，特别是在癌细胞系（H358、H1975、H1299 、H1650、A549 和 SPC-A1) 与正常人支气管上皮细胞系 (BEAS-2B) 中的比较。我们表明 PKMYT1AR 高表达与较差的临床结果相关，并且 PKMYT1AR 的敲低抑制肿瘤细胞增殖、迁移和异种移植肿瘤形成能力。生物信息学分析和荧光素酶测定表明，PKMYT1AR 直接与 miR-485-5p 相互作用，以减弱其下游致癌因子 PKMYT1（蛋白激酶，膜相关酪氨酸/苏氨酸 1）在 NSCLC 中的抑制作用。此外，我们发现与相互对照组相比，miR-485-5p 在从 NSCLC 患者分离的癌细胞系和外周血血清中均下调。一致地，miR-485-5p的强制表达抑制肿瘤细胞的增殖和迁移能力。此外，我们提供的证据表明 PKMYT1AR 靶向反义寡核苷酸 (ASO) 在体内显着抑制肿瘤生长。机制研究表明，PKMYT1AR/miR-485-5p/PKMYT1 轴通过抑制 β-TrCP1 介导的 β-catenin 泛素降解促进 NSCLC 中癌症干细胞 (CSC) 的维持，进而导致肿瘤发生增强。我们的研究结果揭示了 PKMYT1AR/miR-485-5p/PKMYT1 轴在 NSCLC 进展过程中的关键作用，未来可用作新的治疗靶点。