Li Lei, Wang Zhihai, Lu Tao, Li Yanshi, Pan Min, Yu Dan, Hu Guohua
Department of Otolaryngology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People's Republic of China.
Onco Targets Ther. 2021 Feb 25;14:1387-1399. doi: 10.2147/OTT.S292287. eCollection 2021.
The purpose of this study is to investigate the expression and functional role of Annexin (ANXA1) in lymph node (LN) metastasis of hypopharyngeal carcinoma (HSCC).
Differentially expressed genes in tissue from HSCC with or without LN metastasis were obtained from a previous RNA sequencing experiment. The presence of LN metastasis is determined by pathological diagnosis after neck dissection. ANXA1 expression was detected by qRT-PCR and Western blotting. Immunohistochemistry was used to detect the expression of ANXA1 in 74 cases of HSCC and normal control tissues. We also evaluated the clinical significance of ANXA1 in HSCC. Differentially expressed genes related to ANXA1 were analyzed using bioinformatic tools, and potential mechanisms of action of ANXA1 were assessed using in vitro experiments. In these in vitro experiments, cell proliferation was detected by CCK8 staining, and colony formation, migration and invasion were assessed using Transwell assays, and apoptosis as well as cell cycle status were quantified by flow cytometry.
ANXA1 was significantly downregulated in HSCC with LN metastasis. The survival rate of patients with low ANXA1 expression was significantly worse than that of patients with high ANXA1 expression (p<0.05). Silencing ANXA1 in cell culture experiments promoted the proliferation, migration and invasion of FaDu cells, inhibited apoptosis, and increased the proportion of cells in S phase. We furthermore found that the mRNA expression of ANXA1 was positively correlated with Yap1 expression (p<0.0001). Our in vitro experiments showed that ANXA1 regulates the expression of Yap1, and over-expression of Yap1 could reverse the effect of ANXA1 silencing on cancer cell progression.
Our findings suggest that ANXA1 is a putative LN metastasis suppressor gene in tumor, which may suppress the LN metastasis of HSCC by regulating the expression of Yap1.
本研究旨在探讨膜联蛋白（ANXA1）在下咽癌（HSCC）淋巴结（LN）转移中的表达及功能作用。
通过先前的RNA测序实验获得有或无LN转移的HSCC组织中差异表达的基因。LN转移的存在通过颈部清扫术后的病理诊断确定。采用qRT-PCR和蛋白质印迹法检测ANXA1表达。免疫组织化学用于检测74例HSCC及正常对照组织中ANXA1的表达。我们还评估了ANXA1在HSCC中的临床意义。使用生物信息学工具分析与ANXA1相关的差异表达基因，并通过体外实验评估ANXA1的潜在作用机制。在这些体外实验中，通过CCK8染色检测细胞增殖，使用Transwell实验评估集落形成、迁移和侵袭，并通过流式细胞术定量凋亡以及细胞周期状态。
ANXA1在伴有LN转移的HSCC中显著下调。ANXA1低表达患者的生存率显著低于ANXA1高表达患者（p<0.05）。细胞培养实验中沉默ANXA1可促进FaDu细胞的增殖、迁移和侵袭，抑制凋亡，并增加S期细胞比例。我们还发现ANXA1的mRNA表达与Yap1表达呈正相关（p<0.0001）。我们的体外实验表明，ANXA1调节Yap1的表达，Yap1的过表达可逆转ANXA1沉默对癌细胞进展的影响。
我们的研究结果表明，ANXA1是肿瘤中一种假定的LN转移抑制基因，其可能通过调节Yap1的表达来抑制HSCC的LN转移。
