BARX2 转录激活 PHLDA3，通过下调 PI3K/AKT 水平抑制食管鳞状细胞癌的恶性发展。

PHLDA3 activated by BARX2 transcription, suppresses the malignant development of esophageal squamous cell carcinoma by downregulating PI3K/AKT levels.

机构信息

Department of Thoracic Surgery, Quanzhou First Hospital Affiliated to Fujian Medical University, Quanzhou, China.

Department of Thoracic Surgery, Fujian Medical University Union Hospital, Quanzhou, China.

出版信息

Exp Cell Res. 2023 May 15;426(2):113567. doi: 10.1016/j.yexcr.2023.113567. Epub 2023 Mar 23.

DOI:10.1016/j.yexcr.2023.113567

PMID:36965748

Abstract

BACKGROUND

Low pleckstrin homology-like domain family A, member 3 (PHLDA3) expression has been reported to be associated with cancer specificity and disease-free survival in esophageal squamous cell carcinoma (ESCC), and was an independent predictor of postoperative recurrence. However, the specific mechanisms involved are still unclear. This paper aimed to explore the role and its mechanisms of PHLDA3 in ESCC.

MATERIALS AND METHODS

PHLDA3 and BarH-like homeobox 2 (BARX2) expressions in ESCC were predicted by Gene Expression Profiling Interactive Analysis (GEPIA) analysis and determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western Blot. Western blot detected the expression of proteins associated with migration, angiogenesis and phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway. The University of California Santa Cruz Genomics Institute (UCSC) database predicted that the relationship of BARX2 and PHLDA3 promoter and JASPAR identified the possible binding sites. Dual luciferase gene reporter verified PHLDA3 promoter activity, and the relationship of both was determined by chromatin immunoprecipitation (CHIP). Cell counting kit (CCK)-8, 5-ethynyl-2'-deoxyuridine (EDU) and colony formation were used to assess cell proliferation. Wound healing and transwell were used to detect cell migration and invasion ability. Tube formation assay was applied to assess angiogenesis. Mice were injected with transfected KYSE30 cells under the right axilla. Body weight and tumor volume and mass were recorded for each group of mice. Immunohistochemistry was performed to detect KI67 level in tumor tissues.

RESULTS

Both PHLDA3 and BARX2 were downregulated in ESCC. The upregulated PHLDA3 suppressed PI3K/AKT expression. In addition, BARX2 bound to the PHLDA3 promoter and transcriptionally activated PHLDA3. PHLDA3 overexpression inhibited ESCC cell proliferation, migration, invasion and angiogenesis, but this effect was reversed by BARX2 knockdown. In addition, BARX2 overexpression inhibited ESCC cell proliferation, migration, invasion and angiogenesis, but this effect was reversed by PHLDA3 knockdown.

CONCLUSION

PHLDA3 was transcriptionally activated by BARX2 and inhibited malignant progression of ESCC by downregulating PI3K/AKT levels.

摘要

背景

低 pleckstrin 同源样结构域家族 A，成员 3（PHLDA3）的表达与食管鳞状细胞癌（ESCC）的肿瘤特异性和无病生存有关，是术后复发的独立预测因子。然而，具体的机制尚不清楚。本文旨在探讨 PHLDA3 在 ESCC 中的作用及其机制。

材料和方法

通过基因表达谱分析交互分析（GEPIA）预测 ESCC 中 PHLDA3 和 BarH 样同源盒 2（BARX2）的表达，并通过定量实时聚合酶链反应（qRT-PCR）和 Western Blot 确定。Western blot 检测与迁移、血管生成和磷酸肌醇 3-激酶（PI3K）/蛋白激酶 B（PKB/AKT）信号通路相关的蛋白表达。加利福尼亚大学圣克鲁兹基因组研究所（UCSC）数据库预测 BARX2 和 PHLDA3 启动子的关系，JASPAR 鉴定可能的结合位点。双荧光素酶基因报告验证 PHLDA3 启动子活性，并通过染色质免疫沉淀（CHIP）确定两者之间的关系。细胞计数试剂盒（CCK-8）、5-乙炔基-2'-脱氧尿苷（EDU）和集落形成用于评估细胞增殖。划痕愈合和 Transwell 用于检测细胞迁移和侵袭能力。管形成实验用于评估血管生成。将转染的 KYSE30 细胞注射到右腋窝下的小鼠体内。记录每组小鼠的体重、肿瘤体积和质量。免疫组织化学检测肿瘤组织中 KI67 水平。

结果

PHLDA3 和 BARX2 在 ESCC 中均下调。上调的 PHLDA3 抑制了 PI3K/AKT 的表达。此外，BARX2 与 PHLDA3 启动子结合并转录激活 PHLDA3。PHLDA3 过表达抑制 ESCC 细胞增殖、迁移、侵袭和血管生成，但 BARX2 敲低可逆转这种作用。此外，BARX2 过表达抑制 ESCC 细胞增殖、迁移、侵袭和血管生成，但 PHLDA3 敲低可逆转这种作用。