艾迪注射液通过ADAMTS8调控β-catenin/c-Myc通路增强宫颈癌细胞放疗敏感性并抑制其迁移研究

Aidi Injection enhances radiosensitivity and inhibits migration of cervical cancer cells by regulating ADAMTS8-mediated β-catenin/c-Myc pathway

关键词:  艾迪注射液  ADAMTS8  宫颈癌  迁移  放疗敏感性

Key words:  Aidi Injection  ADAMTS8  cervical cancer  migration  radiosensitivity

摘要:

[目的] 探究艾迪注射液通过具有血小板反应蛋白1型基序8的ADAM金属肽酶（ADAMTS8）对宫颈癌细胞迁移及放疗增敏作用的影响。[方法] 1）用实时荧光定量聚合酶链反应（PCR）和蛋白质免疫印迹法检测人宫颈上皮HcerEpic、宫颈癌SiHa和HeLa细胞株中ADAMTS8 mRNA及蛋白水平。2）将SiHa细胞分别在含0、1、5、10、30和50 mg/mL浓度艾迪注射液的培养基中培养，用CCK-8法检测细胞增殖情况。3）分别给予SiHa细胞空白对照（对照组）、10 mg/mL浓度艾迪注射液（艾迪组）、10 mg/mL浓度艾迪注射液和阴性对照质粒（艾迪+si-NC组）、10 mg/mL浓度艾迪注射液和ADAMTS8沉默（艾迪+si-ADAMTS8组）处理后，用医用直线加速器分别给予0、2、4、6和8 Gy照射剂量处理，用克隆形成实验及单击多靶模型比较放疗敏感性。4）对照组、艾迪组、艾迪+si-NC组、艾迪+si-ADAMTS8组均给予4 Gy照射剂量处理，用Transwell小室法比较细胞迁移能力。[结果] 1）HcerEpic细胞株中ADAMTS8 mRNA及蛋白水平均高于SiHa和HeLa细胞株（P＜0.05）。2）艾迪注射液浓度在0~10 mg/mL，细胞增殖抑制率增幅较大；在10~50 mg/mL，细胞增殖抑制率增幅较小。3）艾迪组的ADAMTS8 mRNA及蛋白水平均高于对照组，艾迪+si-ADAMTS8组的ADAMTS8 mRNA及蛋白水平均低于艾迪+si-NC组（P＜0.05）。与对照组比较，艾迪组的放射增敏比（SER）为1.320；与艾迪+si-NC组比较，艾迪+si-ADAMTS8组的SER为0.746。照射剂量为2 Gy时，艾迪组的β-连环蛋白（β-catenin）和c-骨髓细胞瘤癌基因（c-Myc）蛋白水平均低于对照组；艾迪+si-ADAMTS8组的β-catenin和c-Myc蛋白水平均高于艾迪+si-NC组（P＜0.05）。4）艾迪组的细胞迁移数、β-catenin和c-Myc蛋白水平均低于对照组，艾迪+si-ADAMTS8组的细胞迁移数、β-catenin和c-Myc蛋白水平均高于艾迪+si-NC组（P＜0.05）。[结论] 艾迪注射液通过上调ADAMTS8抑制β-catenin/c-Myc通路，抑制宫颈癌SiHa细胞迁移，并提高其放疗敏感性；而沉默ADAMTS8则削弱了这些作用。

Abstract:

[Objective] To investigate the effect of Aidi Injection on the migration and radiosensitization of cervical cancer cells by ADAM metallopeptidase with thrombospondin type 1 motif 8（ADAMTS8）. [Methods] 1） Real-time quantitative PCR and Western blot were used to detect ADAMTS8 mRNA and protein levels in human cervical epithelial HcerEpic cells and cervical cancer SiHa and HeLa cells. 2） SiHa cells were cultured in media containing 0，1，5，10，30，and 50 mg/mL Aidi Injection，and cell proliferation was measured using the CCK-8 assay. 3） SiHa cells were divided into four groups：control（no treatment），10 mg/mL Aidi Injection（Aidi group），10 mg/mL Aidi Injection with negative control plasmid（Aidi+si-NC group），and 10 mg/mL Aidi Injection with ADAMTS8 silencing（Aidi+si-ADAMTS8 group）. These groups were irradiated with 0，2，4，6，and 8 Gy using a medical linear accelerator. Clonogenic assays and the single-hit multi-target model were used to compare radiosensitivity. 4） All groups received a fixed 4 Gy irradiation dose，and cell migration was assessed using the Transwell assay. [Results] 1） ADAMTS8 mRNA and protein levels were significantly higher in HcerEpic cells compared to SiHa and HeLa cells（P＜0.05）. 2） Aidi Injection showed a more pronounced inhibitory effect on cell proliferation at concentrations between 0 and 10 mg/mL，while the inhibitory effect slowed between 10 and 50 mg/mL. 3） The levels of ADAMTS8 mRNA and protein were significantly higher in the Aidi group than in the control group，while were significantly lower in the Aidi+si-ADAMTS8 group than in the Aidi+si-NC group（P＜0.05）. Compared to the control group，the sensitization enhancement ratio（SER） of the Aidi group was 1.320；compared to the Aidi+si-NC group，the SER of the Aidi+si-ADAMTS8 group was 0.746. At an irradiation dose of 2 Gy，the protein levels of β-catenin and c-myelocytoma oncogene（c-Myc） were significantly lower in the Aidi group than in the control group，and significantly higher in the Aidi+si-ADAMTS8 group than in the Aidi+si-NC group（P＜0.05）. 4） The number of migrating cells and the protein levels of β-catenin and c-Myc were significantly lower in the Aidi group than in the control group，while significantly higher in the Aidi+si-ADAMTS8 group than in the Aidi+si-NC group（P＜0.05）. [Conclusion] Aidi Injection upregulates ADAMTS8 to inhibit the β-catenin/c-Myc pathway，suppressing migration and enhancing the radiosensitivity of SiHa cells. Silencing ADAMTS8 weakens those effects.