基于miR-29c/FBXO31轴研究铁线莲总皂苷对食管癌细胞化疗耐药的作用机制

Mechanism of Total Saponins of Clematis on Chemotherapy Resistance of Esophageal Cancer Cells Based on miR-29c/FBXO31 Axis

目的:探讨铁线莲总皂苷对食管癌(EC)Eca109/cDDP细胞化疗耐药性的影响及可能作用机制。方法:采用CCK-8法检测细胞增殖能力;流式细胞术检测细胞凋亡水平;通过Transwell小室检测细胞迁移及侵袭能力;双荧光素酶报告基因试验验证靶向关系;RT-qPCR检测miR-29c及Fbox31 mRNA相对表达;Western blot技术检测FBXO31、P38丝裂原活化蛋白激酶(P38MAPK)、p-P38MAPK、天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)及Cleaved Caspase-3(C-caspase-3)蛋白相对表达。结果:Eca109/cDDP细胞存活率随铁线莲总皂苷作用浓度增大而明显降低(P<0.05);10μg/mL的铁线莲总皂苷联合0.3μg/mL的顺铂可明显提高Eca109/cDDP细胞凋亡率,明显抑制细胞迁移及侵袭(P<0.05);抑制miR-29c表达后,细胞存活率、Fbox31 mRNA及蛋白相对表达上调,细胞凋亡率明显降低,miR-29c相对表达和p-P38MAPK、C-caspase-3蛋白相对表达下调,细胞迁移及侵袭数明显增加(P<0.05);双荧光素酶基因试验检测结果显示Fbxo31为miR-29c的靶基因。结论:铁线莲总皂苷可提高EC细胞的化疗耐药抗性,降低癌细胞存活率,促进凋亡并抑制迁移及侵袭,其作用机制可能与上调miR-29c表达,抑制FBXO31表达进而激活促癌细胞凋亡程序有关。

Objective:To investigate the effect and underlying mechanism of total saponins of clematis(TSC)on the chemotherapy resistance of esophageal cancer(EC)Eca109/cDDP cells.Methods:Cell proliferation ability was detected by CCK-8 assay,and the apoptosis level was detected by flow cytometry.Cell migration and invasion abilities were evaluated through Transwell chamber experiments.The target relationship was verified by dual-luciferase reporter gene experiments,and the relative expression levels of miR-29c and Fbox31 mRNA were detected by RT-qPCR.Western blot technology was used to measure the relative protein expression of FBXO31,p38 mitogen-activated protein kinase(p38MAPK),p-p38MAPK,caspase-3,and cleaved Caspase-3(C-Caspase-3).Results:The survival rate of Eca109/cDDP cells decreased significantly with increasing concentrations of TSC(P<0.05).TSC at 10μg/mL with 0.3μg/mL cisplatin significantly increased the apoptosis rate of Eca109/cDDP cells and markedly inhibited cell migration and invasion(P<0.05).After inhibiting miR-29c expression,cell survival rate,and Fbox31 mRNA and protein relative expression were upregulated,while apoptosis rate,miR-29c relative expression,p-P38MAPK,and C-Caspase-3 protein relative expression were downregulated,and cell migration and invasion significantly increased(P<0.05).Dual-luciferase reporter gene experiments showed that Fbox31 was the target gene of miR-29c.Conclusion:TSC can enhance the chemotherapy resistance of EC cells,reduce cancer cell survival rate,promote apoptosis,and inhibit migration and invasion.Its mechanism of action may be related to upregulating miR-29c expression,inhibiting Fbox31 expression,and thereby activating the pro-apoptotic program in cancer cells.