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张文娟, 靳晓彩, 刘海涛, 李常娟, 赵学荣, 马立东, 乔冠恩.基于miR-96/RECK信号通路探讨南蛇藤总萜对食管鳞癌细胞增殖、侵袭的作用机制[J].湖南中医药大学学报英文版,2025,45(6):1021-1029.[Click to copy
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| 基于miR-96/RECK信号通路探讨南蛇藤总萜对食管鳞癌细胞增殖、侵袭的作用机制 |
| 张文娟,靳晓彩,刘海涛,李常娟,赵学荣,马立东,乔冠恩 |
| (邯郸市第一医院消化内科, 河北 邯郸 056000;承德医学院免疫教研室, 河北 承德 067000) |
| 摘要: |
| 目的 探讨南蛇藤总萜（TTC）对食管鳞状细胞癌（ESCC）细胞增殖和侵袭的影响，并研究其是否通过调控miR-96/回复引导半胱氨酸丰富蛋白含Kazal基元基因（RECK）通路实现。方法 选取人ESCC细胞系KYSE410为研究对象。将KYSE410细胞分为8组，每组设置3个复孔：Ctrl组（无药物处理）、L-TTC组（40 μg/mL的TTC处理24 h）、M-TTC组（80 μg/mL的TTC处理24 h）、H-TTC组（160 μg/mL的TTC处理24 h）、miR-96 NC组（转染阴性对照）、miR-96 NC+TTC组（转染阴性对照，并用160 μg/mL的TTC处理24 h）、miR-96 mimic组（转染miR-96 mimic）和miR-96 mimic+TTC组（转染miR-96 mimic，并用160 μg/mL的TTC处理24 h）。MTT检测TTC对ESCC细胞活力的影响。平板克隆实验、细胞划痕以及Transwell实验检测KYSE410细胞增殖、迁移和侵袭能力。RT-qPCR检测miR-96表达水平。Western blot检测具有Kazal基序的逆转诱导RECK、基质金属蛋白酶（MMP）-9和MMP-2蛋白表达水平。结果 KYSE410细胞经20、40、80、160 μg/mL的TTC处理后，可显著抑制其细胞活力，并呈剂量依赖性（P<0.05）。与Ctrl组相比，L-TTC组、M-TTC组和H-TTC组的细胞克隆数、穿膜细胞数和细胞迁移率及miR-96 mRNA、MMP-9、MMP-2蛋白表达水平均显著降低（P<0.05），RECK蛋白表达水平显著增加（P<0.05），呈剂量依赖性。与miR-96 NC组相比，miR-96 NC+TTC组的miR-96 mRNA表达水平、细胞克隆数、细胞迁移率和穿膜细胞数及MMP-9、MMP-2蛋白表达水平显著降低（P<0.05），RECK蛋白表达水平增加（P<0.05）。与miR-96 NC组相比，miR-96 mimic组的miR-96 mRNA表达水平、细胞克隆数、穿膜细胞数和细胞迁移率及MMP-9、MMP-2蛋白表达水平显著增加（P<0.05），RECK蛋白表达水平显著降低（P<0.05）。与miR-96 mimic组相比，miR-96 mimic+TTC组的miR-96 mRNA表达水平、细胞克隆数、穿膜细胞数和细胞迁移率及MMP-9、MMP-2蛋白表达水平显著降低（P<0.05），RECK蛋白表达水平显著增加（P<0.05）。结论 TTC通过调控miR-96/RECK信号通路，有效下调MMP-9和MMP-2的表达，进而抑制ESCC细胞的增殖、迁移及侵袭能力。 |
| 关键词: 食管鳞癌 南蛇藤总萜 miR-96 回复引导半胱氨酸丰富蛋白含Kazal基元基因 增殖 侵袭 基质金属蛋白酶 |
| DOI：10.3969/j.issn.1674-070X.2025.06.005 |
| Received:March 03, 2025 |
| 基金项目:河北省医学科学研究课题计划资助项目（20220500）。 |
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| Mechanism of action of total terpenoids from Celastrus orbiculatus on the proliferation and invasion of esophageal squamous cell carcinoma cells based on the miR-96/RECK pathway |
| ZHANG Wenjuan, JIN Xiaocai, LIU Haitao, LI Changjuan, ZHAO Xuerong, MA Lidong, QIAO Guan'en |
| (Department of Gastroenterology, The First Hospital of Handan, Handan, Hebei 056000, China;Department of Immunology, Chengde Medical University, Chengde, Hebei 067000, China) |
| Abstract: |
| Objective To investigate the effects of total terpenoids from Celastrus orbiculatus (TTC) on the proliferation and invasion of esophageal squamous cell carcinoma (ESCC) cells, and to study whether it is achieved by regulating the miR-96 / RECK pathway.Methods Human ESCC cell line KYSE410 was selected as the research object. KYSE410 cells were divided into 8 groups, each with 3 replicates: Ctrl group (no drug treatment), L-TTC group (40 μg/mL TTC treatment for 24 h), M-TTC group (80 μg/mL TTC treatment for 24 h), H-TTC group (160 μg/mL TTC treatment for 24 h), miR-96 NC group (transfected with negative control), miR-96 NC+TTC group (transfected with negative control and treated with 160 μg/mL TTC for 24 h), miR-96 mimic group (transfected with miR-96 mimic), and miR-96 mimic+TTC group (transfected with miR-96 mimic and treated with 160 μg/mL TTC for 24 h). MTT assay was used to detect the effect of TTC on the viability of ESCC cells. Plate cloning assay, cell scratch assay, and Transwell assay were used to detect the proliferation, migration, and invasion abilities of KYSE410 cells. RT-qPCR was used to detect the expression level of miR-96. Western blot was used to check the protein expression levels of reversion-inducing cysteine-rich protein with Kazal motifs (RECK), matrix metalloproteinase 9 (MMP-9), and matrix metalloproteinase 2 (MMP-2).Results After KYSE410 cells were treated with 20, 40, 80, 160 μg/mL TTC, the cell viability was significantly inhibited in a dose-dependent manner (P<0.05). Compared with the Ctrl group, the number of cell clones, transmembrane cells, cell migration rate, the expression levels of miR-96 mRNA, MMP-9 and MMP-2 proteins in the L-TTC group, M-TTC group, and H-TTC group were significantly decreased (P<0.05), while the expression level of RECK protein was significantly increased (P<0.05), showing a dose-dependent effect. Compared with miR-96 NC group, miR-96 mRNA expression level, cell clone number, cell migration rate, and transmembrane cell number, MMP-9 and MMP-2 protein expression levels in miR-96 NC+TTC group were significantly decreased (P<0.05), and RECK protein expression level was increased (P<0.05). Compared with the miR-96 NC group, the expression level of miR-96 mRNA, the number of cell clones, the number of transmembrane cells, and cell migration rate, the expression levels of MMP-9 and MMP-2 protein in the miR-96 mimic group were significantly increased (P<0.05), and the expression level of RECK protein was significantly decreased (P<0.05). Compared with miR-96 mimic group, miR-96 mRNA expression level, cell clone number, transmembrane cell number, and cell migration rate, MMP-9 and MMP-2 protein expression levels in miR-96 mimic+TTC group were significantly decreased (P<0.05), and RECK protein expression level was significantly increased (P<0.05).Conclusion TTC effectively down-regulates the expression of MMP-9 and MMP-2 by regulating the miR-96/RECK signaling pathway, thereby inhibiting the proliferation, migration, and invasion of ESCC cells. |
| Key words: esophageal squamous cell carcinoma total terpenoids from Celastrus orbiculatus miR-96 recovery guide cysteine-rich protein containing Kazal motif gene proliferation invasion matrix metalloproteinase |
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