Huang Cheng-Yi, Lee Ko-Chao, Tung Shui-Yi, Huang Wen-Shin, Teng Chih-Chuan, Lee Kam-Fai, Hsieh Meng-Chiao, Kuo Hsing-Chun
Division of Colon and Rectal Surgery, Department of Surgery, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan.
Chang Gung Memorial Hospital-Kaohsiung Medical Center, Division of Colorectal Surgery, Department of Surgery, Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan.
Cancers (Basel). 2022 Jun 29;14(13):3189. doi: 10.3390/cancers14133189.
BACKGROUND/AIMS: A combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry approach was used to search for potential markers for prognosis and intervention of colorectal cancer (CRC) at different stages of lymph node metastasis (LMN). This quantitative proteomic survey aimed to investigate the LNM-associated proteins and evaluate the clinicopathological characteristics of these target proteins in CRC from stage I to stage IV.
Sixteen CRC cases were categorized into paired non-LNM and LNM groups, and two-dimensional difference gel electrophoresis and MS proteome analysis were performed. Differential protein expression between non-LNM and LNM CRC was further validated in a tissue microarray, including 40 paraffin-embedded samples by immunohistochemistry staining. Moreover, a Boyden chamber assay, flow cytometry, and shRNA were used to examine the epithelial-mesenchymal transition and mechanism invasiveness of the differentially expressed proteins in DLD-1 cells and in vivo xenograft mouse model.
Eighteen differentially expressed proteins were found between non-LNM and LNM CRC tissues. Among them, protein levels of Gelsolin (GSN) and peroxiredoxin 4 (PRDX4) were abundant in node-positive CRC. Downregulation of GSN and PRDX4 markedly suppressed migration and invasiveness and also induced cell cycle G1/S arrest in DLD-1. Mechanistically, the EGFR/RhoA/PKCα/ERK pathways are critical for transcriptional activation of histone modification of H3 lysine 4 trimethylation (H3K4me3) of GSN and PRDX4 promoters, resulting in upregulation of GSN, PRDX4, Twist-1/2, cyclinD1, proliferating cell-nuclear antigen, β-catenin, N-cadherin, and matrix metalloprotein-9.
GSN and PRDX4 are novel regulators in CRC lymph node metastasis to potentially provide new insights into the mechanism of CRC progression and serve as a biomarker for CRC diagnosis at the metastatic stage.
背景/目的：采用荧光二维差异凝胶电泳（2D-DIGE）和基质辅助激光解吸电离飞行时间质谱联用方法，寻找结直肠癌（CRC）不同淋巴结转移（LMN）阶段预后和干预的潜在标志物。这项定量蛋白质组学研究旨在调查与LMN相关的蛋白质，并评估这些靶蛋白在Ⅰ期至Ⅳ期CRC中的临床病理特征。
将16例CRC病例分为配对的无LMN组和LMN组，进行二维差异凝胶电泳和质谱蛋白质组分析。通过免疫组织化学染色，在包含40个石蜡包埋样本的组织芯片中进一步验证无LMN和有LMN的CRC之间的差异蛋白表达。此外，采用博伊登室试验、流式细胞术和短发夹RNA（shRNA）检测DLD-1细胞和体内异种移植小鼠模型中差异表达蛋白的上皮-间质转化及侵袭机制。
在无LMN和有LMN的CRC组织之间发现了18种差异表达蛋白。其中，凝溶胶蛋白（GSN）和过氧化物酶体增殖物激活受体4（PRDX4）的蛋白水平在淋巴结阳性CRC中丰富。GSN和PRDX4的下调显著抑制迁移和侵袭，并在DLD-1中诱导细胞周期G1/S期阻滞。机制上，表皮生长因子受体（EGFR）/RhoA/蛋白激酶Cα（PKCα）/细胞外信号调节激酶（ERK）通路对于GSN和PRDX4启动子组蛋白修饰H3赖氨酸4三甲基化（H3K4me3）的转录激活至关重要，导致GSN、PRDX4、Twist-1/2、细胞周期蛋白D1、增殖细胞核抗原、β-连环蛋白、N-钙黏蛋白和基质金属蛋白酶-9上调。
GSN和PRDX4是CRC淋巴结转移中的新型调节因子，可能为CRC进展机制提供新见解，并作为转移阶段CRC诊断的生物标志物。
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