Department of Otorhinolaryngology, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China.
Neoplasma. 2022 Jul;69(4):841-858. doi: 10.4149/neo_2022_220310N263. Epub 2022 May 9.
The present study aimed to investigate LINC00278 expression in laryngeal squamous cell carcinoma (LSCC) and its involvement in the process of proliferation, migration, and invasion, providing a rationale for mining potential diagnostic and therapeutic targets of LSCC. Univariate and multivariate Cox regression analyses were performed to identify optimal prognostic lncRNAs. MTS, colony formation, wound healing, and Transwell invasion assays were used to determine the effects of LINC00278 overexpression on the proliferation, migration, and invasion of cancer cells. The expressions of signaling pathway-related proteins and epithelial-mesenchymal transition (EMT) marker proteins were detected using western blot. The chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were performed to demonstrate the binding of ETS proto-oncogene 1, transcription factor (ETS1), and LINC00278 promoter region. The molecular targets of LINC00278 were identified by RNA sequencing analysis and co-expression analysis. Kaplan-Meier analysis and CIBERSORT algorithm were used to analyze survival and immune cell infiltration based on LINC00278, COL4A1, and COL4A2. Multivariate Cox regression was used to establish a six-gene prognostic model. LINC00278 expression was low in LSCC tissues, and it was significantly associated with the TNM (tumors/nodes/metastases) stage (p<0.001), lymphatic metastasis (p<0.01), and pathological differentiation (p<0.01). LINC00278 overexpression significantly reduced LSCC cell proliferation, migration, and invasion in TU686, TU177, and AMC-HN-8 cell lines. E-cadherin protein expression was increased, while N-cadherin, Vimentin, Zeb1, and Snail protein expression was decreased in the LINC00278 group, compared to the pcDNA3.1 group. Additionally, in AMC-HN-8 and FaDu cell lines, the LINC00278-treated group had significantly lower p-AKT and p-mTOR protein levels than the control group. ETS1 is a direct transcriptional regulator of the LINC00278 gene based on luciferase reporter assays and ChIP experiments. Western blot analysis demonstrated that high LINC00278 expression inhibited both ETS1 expression and phosphorylation. COL4A1/COL4A2 were identified as potential downstream targets of LINC00278. Meanwhile, the LINC00278/COL4A1/COL4A2-dominated low-risk group showed higher antigen-presenting activity and a higher immune score than the high-risk group. The findings indicated that ETS1 upregulated LINC00278 expression on the Y chromosome, which in turn inhibited LSCC growth in vivo and in vitro by inhibiting the AKT/mTOR signaling pathway via downregulation of COL4A1/COL4A2.
本研究旨在探讨 LINC00278 在喉鳞状细胞癌（LSCC）中的表达及其在增殖、迁移和侵袭过程中的作用，为挖掘 LSCC 的潜在诊断和治疗靶点提供依据。采用单因素和多因素 Cox 回归分析鉴定最佳预后的 lncRNA。通过 MTS、集落形成、划痕愈合和 Transwell 侵袭实验来确定 LINC00278 过表达对癌细胞增殖、迁移和侵袭的影响。采用 Western blot 检测信号通路相关蛋白和上皮-间充质转化（EMT）标志物蛋白的表达。采用染色质免疫沉淀（ChIP）和双荧光素酶报告基因实验证明 ETS 原癌基因 1、转录因子（ETS1）与 LINC00278 启动子区域的结合。通过 RNA 测序分析和共表达分析鉴定 LINC00278 的分子靶标。基于 LINC00278、COL4A1 和 COL4A2 的 Kaplan-Meier 分析和 CIBERSORT 算法分析生存和免疫细胞浸润。采用多因素 Cox 回归建立 6 基因预后模型。LSCC 组织中 LINC00278 表达水平较低，与 TNM（肿瘤/淋巴结/转移）分期（p<0.001）、淋巴转移（p<0.01）和病理分化（p<0.01）显著相关。在 TU686、TU177 和 AMC-HN-8 细胞系中，过表达 LINC00278 可显著降低 LSCC 细胞的增殖、迁移和侵袭。与 pcDNA3.1 组相比，LINC00278 组 E-钙黏蛋白蛋白表达增加，而 N-钙黏蛋白、波形蛋白、Zeb1 和 Snail 蛋白表达减少。此外，在 AMC-HN-8 和 FaDu 细胞系中，LINC00278 处理组的 p-AKT 和 p-mTOR 蛋白水平明显低于对照组。荧光素酶报告基因实验和 ChIP 实验表明，ETS1 是 LINC00278 基因的直接转录调节剂。Western blot 分析表明，高表达 LINC00278 抑制 ETS1 的表达和磷酸化。COL4A1/COL4A2 被鉴定为 LINC00278 的潜在下游靶标。同时，LINC00278/COL4A1/COL4A2 主导的低危组表现出比高危组更高的抗原呈递活性和更高的免疫评分。这些发现表明，ETS1 在 Y 染色体上上调 LINC00278 的表达，通过下调 COL4A1/COL4A2 抑制 AKT/mTOR 信号通路，从而抑制 LSCC 的体内和体外生长。
