Liu Chunhua, Wang Lijing, Jiang Qingping, Zhang Junyi, Zhu Litong, Lin Li, Jiang Huiping, Lin Dan, Xiao Yanyi, Fang Weiyi, Guo Suiqun
Department of Obstetrics and Gynecology, The Third Affiliated Hospital, Southern Medical University, Guangzhou, China.
Department of Pathology, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Front Oncol. 2019 Apr 11;9:211. doi: 10.3389/fonc.2019.00211. eCollection 2019.
Our previous work determined the correlation between high nuclear expression of hepatoma-derived growth factor (HDGF) and clinicopathological data of endometrial cancer (EC); however, the modulatory mechanisms and biological role of HDGF in EC have not been reported. Lentiviral particles carrying human HDGF short hairpin RNA (shHDGF-1, -2, and -3) vector and plasmids for HDGF, DDX5, and β-catenin expression were, respectively introduced into EC cells to evaluate the effects and molecular mechanisms underlying EC cell proliferation, migration, invasion, and metastasis. Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting were used to determine HDGF and DDX5 expression. Co-immunoprecipitation (co-IP), mass spectrometry, and an immunofluorescence co-localization study were conducted to explore the relationship between HDGF, DDX5, and β-catenin. Immunohistochemistry was used to analyze the clinical associations between HDGF and DDX5 in EC. Knocking down HDGF expression significantly decreased EC cellular proliferation, migration, invasion , as well as tumorigenesis and metastasis . Conversely, HDGF overexpression reversed these effects. Stable knockdown-based HDGF suppression activated the PI3K/AKT signaling pathway, along with downstream β-catenin-mediated cell cycle and epithelial-mesenchymal transition signaling. Furthermore, co-IP combined with mass spectrometry and an immunofluorescence co-localization study indicated that HDGF interacts with DDX5, whereas β-catenin was associated with DDX5 but not HDGF. Overexpression of DDX5 reversed the suppression of shHDGF. Immunohistochemistry analysis showed that high expression of DDX5 constituted an unfavorable factor with respect to the clinicopathological characteristics of EC tissues and that HDGF and DDX5 high expression (HDGF+/DDX5+) led to a worse prognosis for patients with EC ( < 0.001). In addition, we found that the expression of HDGF and DDX5 was positively correlated in EC tissues ( = 0.475, < 0.001). Our results provide novel evidence that HDGF interacts with DDX5 and promotes the progression of EC through the induction of β-catenin.
我们之前的研究确定了肝癌衍生生长因子（HDGF）的高核表达与子宫内膜癌（EC）临床病理数据之间的相关性；然而，HDGF在EC中的调节机制和生物学作用尚未见报道。分别将携带人HDGF短发夹RNA（shHDGF-1、-2和-3）载体的慢病毒颗粒以及用于HDGF、DDX5和β-连环蛋白表达的质粒导入EC细胞，以评估其对EC细胞增殖、迁移、侵袭和转移的影响及分子机制。采用定量实时逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测HDGF和DDX5的表达。通过免疫共沉淀（co-IP）、质谱分析和免疫荧光共定位研究来探究HDGF、DDX5和β-连环蛋白之间的关系。采用免疫组织化学方法分析HDGF和DDX5在EC中的临床相关性。敲低HDGF表达可显著降低EC细胞的增殖、迁移、侵袭以及肿瘤发生和转移。相反，HDGF过表达可逆转这些作用。基于稳定敲低的HDGF抑制激活了PI3K/AKT信号通路以及下游β-连环蛋白介导的细胞周期和上皮-间质转化信号通路。此外，co-IP结合质谱分析和免疫荧光共定位研究表明，HDGF与DDX5相互作用，而β-连环蛋白与DDX5相关，但与HDGF无关。DDX5过表达可逆转shHDGF的抑制作用。免疫组织化学分析显示，DDX5高表达是EC组织临床病理特征的不利因素，且HDGF和DDX5高表达（HDGF+/DDX5+）导致EC患者预后更差（P<0.001）。此外，我们发现EC组织中HDGF和DDX5的表达呈正相关（r=0.475，P<0.001）。我们的研究结果提供了新的证据，表明HDGF与DDX5相互作用，并通过诱导β-连环蛋白促进EC的进展。
