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Archive > Volume 30 Issue 7 > 2023,30(7):552-559. DOI:10.3872/j.issn.1007-385X.2023.07.002 Prev Next
Basic Research
miR-216b-5p mediates autophagy to reverses cisplatin resistance of esophageal cancer Eca109 cells by targeting ATG5
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Abstract:
Objective: To investigate the effect of miR-216b-5p on cisplatin (DDP) resistance in esophageal cancer Eca109 cells and its mechanism. Methods: The expression levels of miR-216b-5p in esophageal cancer cells TE-1, KYSE-150, Eca109 and drug resistant Eca109/DDP cells were detected by qPCR. The miR-216b-5p mimics, mimic NC and autophagy related protein 5 (ATG5) over-expressed plasmids were transfected into Eca109/DDP cells by liposome transfection technique. The proliferation and apoptosis of transfected Eca109/DDP cells were detected by CCK-8, EdU methods and flow cytometry, respectively. The occurrence of autophagy in each group of cells after transfection with mRFP-eGFP-LC3 lentivirus was examined by mRFP-eGFP-LC3 dual fluorescent labeling assay, and the expression of autophagy-related markers, LC3, Beclin 1 and P62, was detected by WB. The targeting relationship between miR-216b-5p and ATG5 was verified by luciferase reporter gene assay, and the expression of ATG5 was detected by Western blotting. A nude mouse Eca109/DDP cell transplanted tumor model was established to observe the effect of miR-216b-5p over-expression on the growth of transplanted tumor. Results: miR-216b-5p was lowly expressed in TE-1, KYSE-150, Eca109 and Eca109/DDP cells (all P<0.05). Over-expression of miR-216b-5p significantly inhibited the proliferation and induced apoptosis of Eca109/DDP cells (both P<0.05), reduced the number of autophagosomes in transfected cells (P<0.05), and down-regulated LC3Ⅱ/LC3Ⅰratio and Beclin 1 protein level, but up-regulated P62 protein level (all P<0.05). Luciferase reporter gene assay confirmed that miR-216b-5p negatively regulated ATG5 expression (P<0.05). Over-expression of ATG5 could significantly weaken the effects of miR-216b-5p mimic on suppressing proliferation, autophagy and inducing apoptosis of Eca109/DDP cells (all P<0.05), and reduce the expression of autophagy related protein P62, but increase the LC3Ⅱ/LC3Ⅰratio and Beclin 1 protein level (all P<0.05). The tumor bearing experiments showed that over-expression of miR-216b-5p could significantly inhibit the growth of transplanted tumors in nude mice (P<0.05).Conclusion: Over-expression of miR-216b-5p can reverse DDP resistance in Eca109/DDP cells, and the mechanism may be related to its negative regulation of autophagy-related gene ATG5 to affect cell autophagy.
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