Objective: To establish a screening system for anti-metastatic small-molecule compounds based on perinucleolar compartment (PNC) prevalence in liver cancer cells and to investigate its validity. Methods: Polypyrimidine tract-binding protein (PTB) monoclonal antibody was used to measure the PNC prevalence in HepG2, HepG2M, and Huh7 cells, and wound healing assay and transwell assay were used to analyze the migration and invasion abilities of hepatoma cells. HepG2M cells were used as the model for the screening of anti-metastatic small-molecule compounds, and after the treatment with the compounds A1, A4, and E696, qPCR was used to measure the expression of metastasis-related miRNAs (miR-141 and miR-200c). A one-way analysis of variance was used for comparison of data between multiple groups. Results: PTB immunofluorescence assay showed that HepG2M cells had the highest PNC prevalence, followed by Huh7 and HepG2 cells, and PNC prevalence was positively correlated with the metastasis and invasion abilities of hepatoma cells. The PNC prevalence of HepG2M cells was reduced to 22.88% ±4.61% by A1, 14.22% ± 3.05% by A4, and 26.12% ± 4.94% by E696. Wound healing assay showed that the 48-hour scratch ratio increased from 17.70% ± 3.34% to 64.50% ± 2.65%, 83.40% ± 5.10%, and 57.20% ± 3.06% (F = 171.1, P < 0.01), respectively. Transwell assay showed that the number of invasive cells was reduced from 264.33 ± 30.50 to 104.33 ± 13.50, 58.00 ± 11.00, and 111.33 ± 19.50 (F = 59.87, P < 0.01), respectively. The anti-metastatic effect of these three compounds was positively correlated with their ability to destroy PNC. A4 upregulated the expression of miR-141 and miR-200c in a dose-dependent manner, and after HepG2M cells were treated with A4 at a concentration of 5 μM, 10 μM, or 20 μM, the level of miR-141 was increased to 3.61 ± 0.78, 8.12 ± 1.15, and 18.24 ± 2.44 folds (F = 88.01, P < 0.01), respectively, and that of miR-200c was increased to 2.82 ± 0.43, 4.82 ± 0.89, and 10.74 ± 1.22 folds (F = 87.94, P < 0.01), respectively. Conclusion: The screening system for anti-metastatic small-molecule compounds based on PNC prevalence can provide an effective technical platform for research and development of anti-metastatic drugs for liver cancer.
目的： 拟建立基于PNC（perinucleolar compartment）的抗肝癌转移小分子化合物筛选模型并验证其有效性。 方法： 采用多嘧啶序列结合蛋白(PTB)单克隆抗体检测肝癌细胞HepG2、HepG2M与Huh7中PNC比例，通过划痕实验和Transwell实验分析肝癌细胞的迁移和侵袭性。以HepG2M细胞作为抗转移小分子化合物筛选的细胞模型，化合物（A1、A4、E696）作用细胞后，qPCR检测转移相关miRNAs（miR-141和miR-200c）的表达水平。多组间数据比较采用单因素方差分析。 结果： PTB免疫荧光显示PNC比例HepG2M > Huh7 > HepG2，其PNC比例与细胞的迁移和侵袭能力呈正相关。A1、A4、E696三种化合物可使HepG2M细胞的PNC比例分别降至22.88%±4.61%、14.22%±3.05%、26.12%±4.94%；划痕实验显示48 h划痕比值由17.70%±3.34%分别增加至64.50%±2.65%、83.40%±5.10%、57.20%±3.06%（F = 171.1，P < 0.01）；Transwell实验示侵袭细胞数由（264.33±30.50）个分别减少至（104.33±13.50）个、（58.00±11.00）个、（111.33±19.50）个，F = 59.87，P < 0.01。三者抗转移效果与其PNC瓦解效力呈正相关。且A4呈剂量依赖性地上调miR-141和miR-200c的表达水平，5、10、20 μmol/L作用HepG2M细胞后，miR-141和miR-200c的相对水平分别增加至3.61±0.78、8.12±1.15、18.24±2.44（F = 88.01，P < 0.01）和2.82±0.43、4.82±0.89、10.74±1.22（F = 87.94，P < 0.01）。 结论： 基于PNC指标的抗转移小分子化合物筛选体系，可为肝癌抗转移新药的研发提供有效的技术平台。.
Keywords: Carcinoma, hepatocellular; Drug screening; MicroRNA; Neoplasm metastasis; perinucleolar compartment.