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miR-21 反义寡核苷酸提高 A375 人黑色素瘤细胞对顺铂的敏感性:体外研究。

MicroRNA-21 antisense oligonucleotide improves the sensitivity of A375 human melanoma cell to Cisplatin: An in vitro study.

机构信息

Department of Plastic and Reconstructive Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, P.R. China.

出版信息

J Cell Biochem. 2018 Apr;119(4):3129-3141. doi: 10.1002/jcb.26455. Epub 2018 Jan 4.

Abstract

This study explored Cisplatin resistance effect of microRNA-21 (miR-21) antisense oligonucleotide (AS-ODN) in human melanoma A375 cell. AS-ODN was transfected in melanoma A375 cells and Cisplatin-resistant cell line A375/CDDP, and divided into the AS-ODN, nonsense oligonucleotide (NS-ODN) and normal groups. Cell ultrastructure changes were observed through transmission electron microscope. MiR-21 AS-ODN could be tested cell growth effect in different time periods by trypan blue exclusion. MiR-21 mRNA expression change was detected by quantitative fluorescence PCR. Cell apoptosis, cycle distribution and miR-21 AS-ODN effect on proliferation and Cisplatin sensitivity were tested by flow cytometry, MTT assay, TUNEL, and Clonogenic assay. Cell apoptosis was observed after transfection 24 h with the AS-ODN group, while the NS-ODN and normal group cells had no apoptotic symptoms; Compared with the normal group, the AS-ODN group began to show obvious cell growth inhibition effect after transfection 24 h lasting 72 h (all P < 0.05), but the NS-ODN group had no significant difference (P > 0.05). miR-21 mRNA expression in the AS-ODN group was obviously decreased with rising apoptosis rate (all P < 0.05) and there was no significant difference in the NS-ODN group (P > 0.05). MiR-21 AS-ODN could remarkably increase A375 cell and A375/CDDP cell sensitivity to Cisplatin (P < 0.05), while A375 cell sensitivity to Cisplatin between the NS-ODN group and the normal group had no difference. MiR-21 AS-ODN decreased IC and increased Cisplatin sensitivity for A375 cells and A375/CDDP cells, which would be a new target of melanoma treatment.

摘要

本研究探讨了 microRNA-21(miR-21)反义寡核苷酸(AS-ODN)对人黑色素瘤 A375 细胞顺铂耐药的影响。将 AS-ODN 转染入黑色素瘤 A375 细胞和顺铂耐药细胞系 A375/CDDP,并分为 AS-ODN、无意义寡核苷酸(NS-ODN)和正常组。通过透射电子显微镜观察细胞超微结构变化。用台盼蓝排斥法检测不同时间 miR-21 AS-ODN 对细胞生长的影响。用定量荧光 PCR 检测 miR-21 mRNA 表达变化。用流式细胞术、MTT 法、TUNEL 和集落形成实验检测细胞凋亡、周期分布及 miR-21 AS-ODN 对增殖和顺铂敏感性的影响。转染 24 h 后,AS-ODN 组可见细胞凋亡,NS-ODN 组和正常组细胞无凋亡表现;与正常组相比,AS-ODN 组转染 24 h 后开始出现明显的细胞生长抑制作用,持续至 72 h(均 P < 0.05),但 NS-ODN 组无明显差异(P > 0.05)。AS-ODN 组 miR-21 mRNA 表达明显降低,凋亡率升高(均 P < 0.05),NS-ODN 组无明显差异(P > 0.05)。miR-21 AS-ODN 可显著增加 A375 细胞和 A375/CDDP 细胞对顺铂的敏感性(P < 0.05),而 NS-ODN 组 A375 细胞对顺铂的敏感性与正常组无差异。miR-21 AS-ODN 降低了 A375 细胞和 A375/CDDP 细胞的 IC50 值,增加了顺铂敏感性,可能成为黑色素瘤治疗的新靶点。

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