内质网驻留蛋白 44 通过与三磷酸柠檬酸裂解酶相互作用促进鼻咽癌的恶性表型。
ER resident protein 44 promotes malignant phenotype in nasopharyngeal carcinoma through the interaction with ATP citrate lyase.
机构信息
Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China.
Institute of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China.
出版信息
J Transl Med. 2021 Feb 16;19(1):77. doi: 10.1186/s12967-020-02694-1.
BACKGROUND
Nasopharyngeal carcinoma (NPC) is one of the most common malignancy in head and neck. With the development of treatments, the prognosis has improved these years, but metastasis is still the main cause of treatment failure. The endoplasmic reticulum (ER) resident protein 44 is a UPR-induced ER protein of the protein disulphide isomerase (PDI) family. This study investigated the role of ERp44 in NPC progression.
METHODS
Firstly, immunohistochemistry, western blot and qRT-PCR were used to investigate the expression of ERp44 in NPC samples and cell lines. We analyzed 44 NPC samples for ERp44 expression and investigated the association between its expression level with clinicopathologic parameters. Then we took CCK8, Transwell migration assay and used the zebrafish model to access the role of ERp44 on the malignant phenotype in NPC cells. Secondly, we used co-IP to gain the proteins that interact with ERp44 and took proteomic analysis. Furthermore, we successfully constructed the mutant variants of ERp44 and found the interaction domain with ATP citrate lyase(ACLY). Lastly, we subcutaneously injected NPC cells into nude mice and took immunohistochemistry to exam the expression of ACLY and ERp44. Then we used western blot to detect the expression level of epithelial-mesenchymal transition (EMT) markers.
RESULTS
In the present study, we found ERp44 was elevated in NPC tissues and correlated with clinical stages and survive state of the patients. In vitro, the downregulation of ERp44 in NPC cells (CNE2, 5-8F) could suppress cells proliferation and migration. After that, we recognized that ACLY might be a potential target that could interact with ERp44. We further constructed the mutant variants of ERp44 and found the interaction domain with ACLY. The promotion of ERp44 on cell migration could be inhibited when ACLY was knocked down. More importantly, we also observed that the interaction of ERp44 with ACLY, especially the thioredoxin region in ERp44 play a vital role in regulating EMT. Lastly, we found ERp44 was positively correlated with the expression of ACLY and could promote NPC cells growth in nude mice.
CONCLUSION
Our data indicated that ERp44 participates in promoting NPC progression through the interaction with ACLY and regulation of EMT.
背景
鼻咽癌（NPC）是头颈部最常见的恶性肿瘤之一。随着治疗方法的发展，近年来预后有所改善，但转移仍然是治疗失败的主要原因。内质网（ER）驻留蛋白 44 是蛋白二硫键异构酶（PDI）家族中一种 UPR 诱导的 ER 蛋白。本研究探讨了 ERp44 在 NPC 进展中的作用。
方法
首先，我们使用免疫组织化学、western blot 和 qRT-PCR 来检测 NPC 样本和细胞系中 ERp44 的表达。我们分析了 44 例 NPC 样本的 ERp44 表达情况，并研究了其表达水平与临床病理参数之间的关系。然后，我们使用 CCK8、Transwell 迁移实验和斑马鱼模型来评估 ERp44 对 NPC 细胞恶性表型的作用。其次，我们使用 co-IP 获得与 ERp44 相互作用的蛋白质，并进行蛋白质组学分析。此外，我们成功构建了 ERp44 的突变变体，并发现了与 ATP 柠檬酸裂解酶（ACLY）相互作用的结构域。最后，我们将 NPC 细胞皮下注射到裸鼠体内，并用免疫组织化学检测 ACLY 和 ERp44 的表达。然后，我们使用 western blot 检测上皮-间充质转化（EMT）标志物的表达水平。
结果
在本研究中，我们发现 ERp44 在 NPC 组织中上调，并与患者的临床分期和生存状态相关。在体外，NPC 细胞（CNE2、5-8F）中 ERp44 的下调可抑制细胞增殖和迁移。之后，我们发现 ACLY 可能是与 ERp44 相互作用的潜在靶点。我们进一步构建了 ERp44 的突变变体，并发现了与 ACLY 相互作用的结构域。当 ACLY 被敲低时，ERp44 对细胞迁移的促进作用可以被抑制。更重要的是，我们还观察到 ERp44 与 ACLY 的相互作用，特别是 ERp44 中的硫氧还蛋白结构域，在调节 EMT 中起着重要作用。最后，我们发现 ERp44 与 ACLY 的表达呈正相关，并能促进 NPC 细胞在裸鼠体内的生长。
结论
我们的数据表明，ERp44 通过与 ACLY 的相互作用和 EMT 的调节参与促进 NPC 的进展。
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