Department of Thoracic Surgery, First Affiliated Hospital of Xinjiang Medical University, Urumqi, 830054, Xinjiang Uygur Autonomous Region, People's Republic of China.
Clinical Medical Research Institute, First Affiliated Hospital of Xinjiang Medical University, Urumqi, 830054, Xinjiang Uygur Autonomous Region, People's Republic of China.
J Cancer Res Clin Oncol. 2018 Jun;144(6):1025-1035. doi: 10.1007/s00432-018-2625-5. Epub 2018 Mar 12.
MLL2 has been identified as one of the most frequently mutated genes in a variety of cancers including esophageal squamous cell carcinoma (ESCC). However, its clinical significance and prognostic value in ESCC has not been elucidated. In the present study, we aimed to investigate the expression and role of MLL2 in ESCC.
Immunohistochemistry (IHC) and qRT-PCR were used to examine the expression profile of MLL2. Kaplan-Meier survival analysis and univariate and multivariate Cox analyses were used to investigate the clinical and prognostic significance of MLL2 expression in Kazakh ESCC patients. Furthermore, to evaluate the biological function of MLL2 in ESCC, we applied the latest gene editing technique CRISPR/Cas9 to knockout MLL2 in ESCC cell line Eca109. MTT, colony formation, flow cytometry, scratch wound-healing and transwell migration assays were performed to investigate the effect of MLL2 on ESCC cell proliferation and migration. The correlation between MLL2 and epithelial-mesenchymal transition (EMT) was investigated by Western blot assay in vitro and IHC in ESCC tissue, respectively.
Both mRNA and protein expression levels of MLL2 were significantly overexpressed in ESCC patients. High expression of MLL2 was significantly correlated with TNM stage (P = 0.037), tumor differentiation (P = 0.032) and tumor size (P = 0.035). Kaplan-Meier survival analysis showed that patients with low MLL2 expression had a better overall survival than those with high MLL2 expression. Multivariate Cox analysis revealed that lymph node metastasis and tumor differentiation were independent prognostic factors. Knockout of MLL2 in Eca109 inhibited cell proliferation and migration ability, induced cell cycle arrest at G1 stage, but it had no significant effect on apoptosis. In addition, knockout of MLL2 could inhibit EMT by up-regulation of E-Cadherin and Smad7 as well as down-regulation of Vimentin and p-Smad2/3 in ESCC cells. In cancer tissues, the expression of E-Cadherin was negatively correlated with MLL2 expression while Vimentin expression was positively correlated with MLL2 expression.
Our results indicate that overexpression of MLL2 predicts poor clinical outcomes and facilitates ESCC tumor progression, and it may exert oncogenic role via activation of EMT. MLL2 may be used as a novel prognostic factor and therapeutic target for ESCC patients.
MLL2 已被鉴定为多种癌症（包括食管鳞状细胞癌 [ESCC]）中最常突变的基因之一。然而，其在 ESCC 中的临床意义和预后价值尚未阐明。在本研究中，我们旨在研究 MLL2 在 ESCC 中的表达和作用。
免疫组织化学（IHC）和 qRT-PCR 用于检测 MLL2 的表达谱。Kaplan-Meier 生存分析和单因素及多因素 Cox 分析用于研究哈萨克族 ESCC 患者中 MLL2 表达的临床和预后意义。此外，为了评估 MLL2 在 ESCC 中的生物学功能，我们应用最新的基因编辑技术 CRISPR/Cas9 敲除 ESCC 细胞系 Eca109 中的 MLL2。MTT、集落形成、流式细胞术、划痕愈合和 Transwell 迁移实验用于研究 MLL2 对 ESCC 细胞增殖和迁移的影响。通过 Western blot 实验在体外和 ESCC 组织中的免疫组织化学分别研究 MLL2 与上皮间质转化（EMT）的相关性。
MLL2 的 mRNA 和蛋白表达水平在 ESCC 患者中均显著上调。MLL2 高表达与 TNM 分期（P=0.037）、肿瘤分化（P=0.032）和肿瘤大小（P=0.035）显著相关。Kaplan-Meier 生存分析显示，MLL2 低表达患者的总生存率高于 MLL2 高表达患者。多因素 Cox 分析显示，淋巴结转移和肿瘤分化是独立的预后因素。Eca109 中 MLL2 的敲除抑制了细胞增殖和迁移能力，诱导细胞周期停滞在 G1 期，但对细胞凋亡没有显著影响。此外，MLL2 的敲除通过上调 E-钙黏蛋白和 Smad7 以及下调波形蛋白和 p-Smad2/3 来抑制 ESCC 细胞中的 EMT。在癌症组织中，E-钙黏蛋白的表达与 MLL2 的表达呈负相关，而波形蛋白的表达与 MLL2 的表达呈正相关。
我们的结果表明，MLL2 的过表达预示着不良的临床结局，并促进 ESCC 肿瘤的进展，它可能通过激活 EMT 发挥致癌作用。MLL2 可作为 ESCC 患者的一种新的预后因素和治疗靶点。
