Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China.
Key Laboratory of Ningxia Minority Medicine Modernization Ministry of Education, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China.
World J Gastroenterol. 2023 Jul 21;29(27):4317-4333. doi: 10.3748/wjg.v29.i27.4317.
Gastric cancer (GC) is one of the most common cancer types worldwide, and its prevention and treatment methods have garnered much attention. As the active ingredient of licorice, 18β-glycyrrhetinic acid (18β-GRA) has a variety of pharmacological effects. The aim of this study was to explore the effective target of 18β-GRA in the treatment of GC, in order to provide effective ideas for the clinical prevention and treatment of GC.
To investigate the mechanism of 18β-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells.
Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs (miRNAs) in GC cells after 18β-GRA intervention. Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability, cell colony formation ability was detected by the clone formation assay, and flow cytometry was used to detect the cell cycle and apoptosis. A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells. Tumor tissue morphology was observed by hematoxylin and eosin staining, and microtubule-associated protein light chain 3 (LC3) expression was detected by immunohistochemistry. TransmiR, STRING, and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3 (STAT3)-related information. Expression of mRNA and was detected by quantitative polymerase chain reaction (qPCR) and the expression levels of STAT3, phosphorylated STAT3 (p-STAT3), and LC3 were detected by western blot analysis. The targeted relationship between and was detected using the dual-luciferase reporter gene system. AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label. LC3 was labeled and autophagy flow was observed under a confocal laser microscope.
The expression of miR-328-3p was significantly upregulated after 18β-GRA intervention in AGS cells ( = 4.51E-06). Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability, arrested the cell cycle in the G0/G1 phase, promoted cell apoptosis, and inhibited the growth of subcutaneous tumors in BALB/c nude mice ( < 0.01). No obvious necrosis was observed in the tumor tissue in the negative control group (no drug intervention or lentivirus transfection) and vector group (the blank vector for lentivirus transfection), and more cells were loose and necrotic in the miR-328-3p group. Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3, and STAT3 was closely related to autophagy markers such as p62. After overexpressing miR-328-3p, the expression level of mRNA was significantly decreased ( < 0.01) and p-STAT3 was downregulated ( < 0.05). The dual-luciferase reporter gene assay showed that the luciferase activity of and 3' untranslated regions of the wild-type reporter vector group was significantly decreased ( < 0.001). Overexpressed miR-328-3p combined with bafilomycin A (Baf A) was used to detect the expression of LC3 II. Compared with the vector group, the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated ( < 0.05), and compared with the Baf A group, the expression level of LC3 II in the overexpressed miR-328-3p + Baf A group was upregulated ( < 0.01). The expression of LC3 II was detected after intervention of 18β-GRA in GC cells, and the results were consistent with the results of miR-328-3p overexpression ( < 0.05). Additional studies showed that 18β-GRA promoted autophagy flow by promoting autophagosome synthesis ( < 0.001). qPCR showed that the expression of mRNA was downregulated after drug intervention ( < 0.05). Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention ( < 0.05).
18β-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.
胃癌（GC）是全球最常见的癌症类型之一，其预防和治疗方法备受关注。18β-甘草次酸（18β-GRA）作为甘草的有效成分，具有多种药理作用。本研究旨在探讨 18β-GRA 治疗 GC 的有效靶点，为 GC 的临床防治提供有效思路。
探讨 18β-GRA 抑制 GC 细胞增殖和促进自噬流的作用机制。
采用全转录组分析方法，分析筛选 18β-GRA 干预后 GC 细胞中差异表达的 microRNAs（miRNAs）。利用慢病毒转染 GC 细胞和细胞计数试剂盒-8（Cell Counting Kit-8，CCK-8）检测细胞增殖能力，克隆形成实验检测细胞集落形成能力，流式细胞术检测细胞周期和凋亡。构建 GC 细胞裸鼠移植瘤模型，验证 miR-328-3p 过表达对 GC 细胞致瘤性的影响。通过苏木精-伊红（H&E）染色观察肿瘤组织形态，免疫组织化学检测微管相关蛋白轻链 3（LC3）表达。利用 TransmiR、STRING 和 miRWalk 数据库预测 miR-328-3p 与信号转导和转录激活因子 3（signal transducer and activator of transcription 3，STAT3）相关信息。采用定量聚合酶链反应（quantitative polymerase chain reaction，qPCR）检测 mRNA 和 的表达水平，采用 Western blot 分析检测 STAT3、磷酸化 STAT3（phosphorylated STAT3，p-STAT3）和 LC3 的表达水平。利用双荧光素酶报告基因系统检测 和 之间的靶向关系。用单体红色荧光蛋白-绿色荧光蛋白-LC3 腺病毒双标记 AGS 细胞，通过共聚焦激光显微镜观察 LC3 标记和自噬流。
18β-GRA 干预 AGS 细胞后，miR-328-3p 的表达水平显著上调（ = 4.51E-06）。过表达 miR-328-3p 抑制 GC 细胞增殖和集落形成能力，将细胞周期阻滞在 G0/G1 期，促进细胞凋亡，抑制 BALB/c 裸鼠皮下肿瘤生长（ < 0.01）。阴性对照组（无药物干预或慢病毒转染）和载体组（慢病毒空载体转染）的肿瘤组织未见明显坏死，miR-328-3p 组细胞更为疏松坏死。生物信息学工具预测 miR-328-3p 与 STAT3 具有靶向关系，STAT3 与 p62 等自噬标志物密切相关。过表达 miR-328-3p 后， mRNA 的表达水平显著降低（ < 0.01），p-STAT3 下调（ < 0.05）。双荧光素酶报告基因实验显示，野生型报告载体组的 和 3'非翻译区的荧光素酶活性显著降低（ < 0.001）。过表达 miR-328-3p 联合巴弗洛霉素 A（Baf A）检测 LC3 II 的表达。与载体组相比，过表达 miR-328-3p 组 LC3 II 的表达水平下调（ < 0.05），与 Baf A 组相比，过表达 miR-328-3p + Baf A 组 LC3 II 的表达水平上调（ < 0.01）。干预 GC 细胞 18β-GRA 后检测 LC3 II 的表达，结果与 miR-328-3p 过表达的结果一致（ < 0.05）。进一步研究表明，18β-GRA 通过促进自噬体合成促进自噬流（ < 0.001）。qPCR 显示药物干预后 mRNA 的表达下调（ < 0.05）。Western blot 分析显示药物干预后 STAT3 和 p-STAT3 的表达水平显著下调（ < 0.05）。
18β-GRA 通过调节 miR-328-3p/STAT3 信号通路促进自噬体合成，抑制 GC 细胞增殖。
