Department of Head and Neck Surgery, Affiliated Tumor Hospital of Nantong University/Nantong Tumor Hospital, Nantong, Jiangsu, China.
Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China.
J Gene Med. 2024 Jan;26(1):e3654. doi: 10.1002/jgm.3654.
The present study aimed to explore the biological role and underlying mechanism of the long non-coding RNA actin filament-associated protein 1-antisense RNA1 (lncRNA AFAP1-AS1) in the progression of tongue squamous cell carcinoma (TSCC).
A quantitative reverse transcriptase-PCR (RT-qPCR) was conducted to assess relative levels of the miR-133a-5p, lncRNAs AFAP1-AS1 and zinc finger family member 2 (ZIC2) in TSCC cell lines and specimens, whereas ZIC2 protein levels were measured using western blotting. After modifying the levels of expression of lncRNA AFP1-AS1, miR-133a-5p and ZIC2 using lentivirus or plasmid transfection, we examined AKT/epithelial-mesenchymal transition signaling pathway alterations, in vivo carcinogenesis of TSCC in nude mice and in vitro malignant phenotypes. A dual-luciferase reporter assay was conducted to confirm the targeting relationship between ZIC2 and miR-133a-5p, as well as between miR-133a-5p and lncRNA AFAP1-AS1. Based on The Cancer Genome Atlas (TCGA) database, we additionally validated AFP1-AS1. The potential biological pathway for AFP1-AS1 was investigated using gene set enrichment analysis (GSEA). We also evaluated the clinical diagnostic capacities of AFP1-AS1 and clustered the most potential biomarkers with the Mfuzz expression pattern. Finally, we also made relevant drug predictions for AFP1-AS1.
In TSCC cell lines and specimens, lncRNA AFAP1-AS1 was upregulated. ZIC2 was upregulated in TSCC cells as a result of lncRNA AFAP1-AS1 overexpression, which also promoted TSCC cell migration, invasion, viability, and proliferation. Via the microRNA sponge effect, it was found that lncRNA AFAP1-AS1 could upregulate ZIC2 by competitively inhibiting miR-133a-5p. Interestingly, knockdown of ZIC2 reversed the biological roles of lncRNA AFAP1-AS1 with respect to inducing malignant phenotypes in TSCC cells. In addition, in vivo overexpression of lncRNA AFAP1-AS1 triggered subcutaneous tumor growth in nude mice implanted with TSCC cells and upregulated ZIC2 in the tumors. The TCGA database findings revealed that AFAP1-AS1 was significantly upregulated in TSCC specimens and had good clinical diagnostic value. The results of GSEA showed that peroxisome proliferator-activated receptor signaling pathway was significantly correlated with low expression of AFP1-AS1. Finally, the results of drug prediction indicated that the group with high AFAP1-AS1 expression was more sensitive to docetaxel, AZD4547, AZD7762 and nilotinib.
The upregulation of lncRNA AFAP1-AS1, which increases TSCC cell viability, migration, proliferation and invasion via the AFAP1-AS1/miR-133a-5p/ZIC2 axis, aids in the progression of TSCC.
本研究旨在探讨长链非编码 RNA 细丝相关蛋白 1 反义 RNA1（lncRNA AFAP1-AS1）在舌鳞状细胞癌（TSCC）进展中的生物学作用和潜在机制。
采用实时定量逆转录 PCR（RT-qPCR）检测 TSCC 细胞系和标本中 miR-133a-5p、lncRNA AFAP1-AS1 和锌指家族成员 2（ZIC2）的相对水平，并用 Western blot 检测 ZIC2 蛋白水平。通过慢病毒或质粒转染改变 lncRNA AFP1-AS1、miR-133a-5p 和 ZIC2 的表达水平后，我们在裸鼠体内观察 TSCC 的致癌作用以及体外恶性表型的变化。采用双荧光素酶报告基因实验证实 ZIC2 与 miR-133a-5p 以及 miR-133a-5p 与 lncRNA AFAP1-AS1 之间的靶向关系。基于癌症基因组图谱（TCGA）数据库，我们进一步验证了 AFP1-AS1。采用基因集富集分析（GSEA）研究了 AFP1-AS1 的潜在生物学途径。我们还使用 Mfuzz 表达模式对 AFP1-AS1 进行了聚类，并评估了最有潜力的生物标志物的临床诊断能力。最后，我们还对 AFP1-AS1 进行了相关药物预测。
在 TSCC 细胞系和标本中，lncRNA AFAP1-AS1 呈上调表达。lncRNA AFAP1-AS1 过表达导致 ZIC2 在 TSCC 细胞中上调，进而促进 TSCC 细胞迁移、侵袭、活力和增殖。通过 microRNA 海绵效应发现，lncRNA AFAP1-AS1 通过竞争性抑制 miR-133a-5p 而上调 ZIC2。有趣的是，ZIC2 的敲低逆转了 lncRNA AFAP1-AS1 诱导 TSCC 细胞恶性表型的生物学作用。此外，体内过表达 lncRNA AFAP1-AS1 可触发裸鼠皮下接种 TSCC 细胞后的肿瘤生长，并在肿瘤中上调 ZIC2。TCGA 数据库的研究结果表明，AFAP1-AS1 在 TSCC 标本中明显上调，具有良好的临床诊断价值。GSEA 的结果表明，过氧化物酶体增殖物激活受体信号通路与 AFP1-AS1 的低表达显著相关。最后，药物预测的结果表明，高 AFP1-AS1 表达组对多西他赛、AZD4547、AZD7762 和尼洛替尼更敏感。
上调 lncRNA AFAP1-AS1 通过 AFAP1-AS1/miR-133a-5p/ZIC2 轴增加 TSCC 细胞活力、迁移、增殖和侵袭，有助于 TSCC 的进展。
