Department of Pathology, The Fourth People's Hospital of Hengshui.
Department of General Surgery, The Fourth People's Hospital of Hengshui.
Biol Pharm Bull. 2021 Jun 1;44(6):861-868. doi: 10.1248/bpb.b21-00163. Epub 2021 Apr 8.
MicroRNA-221 (miRNA-221) is upregulated in several malignant tumors and is associated with poor patient prognosis. Therefore, the present study aimed to investigate the role and underlying mechanism of miRNA-221 in doxorubicin (DOX) resistance in osteosarcoma cells. We constructed DOX-resistant Saos-2/DOX cells and treated them with DOX. Cell viability was determined by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were transfected with either miRNA-221 mimic or miRNA-221 inhibitor; quantitative (q)RT-PCR was performed to detect the expression of miRNA-221. Flow cytometry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) staining were used to detect cell apoptosis. The immunofluorescence method was also used to detect cell signal transduction and activator of transcription 3 (Stat3) protein expression distribution. In addition, Western blotting was used to detect changes in the expression of each protein. We found that miRNA-221 was upregulated in Saos-2/DOX cells. Moreover, the miRNA-221 mimic induced DOX resistance in Saos-2 cells, whereas the miRNA-221 inhibitor enhanced DOX sensitivity in Saos-2/DOX cells. The miRNA-221 mimic upregulated the expression of phosphorylated-Stat3, P-glycoprotein (P-gp), and B-cell lymphoma-2 (Bcl-2) proteins in Saos-2 cells and induced the entry of Stat3 into the nucleus, whereas the miRNA-221 inhibitor exerted the opposite effect. Pretreatment with the Stat3 chemical inhibitor, STAT3-IN-3, significantly inhibited the upregulation of P-gp and Bcl-2 protein expression induced by the miRNA-221 mimic in Saos-2 cells; it also caused the Saos-2 cells to overcome DOX resistance induced by the miRNA-221 mimic. Thus, miRNA-221 increased the expression of P-gp and Bcl-2 by activating the Stat3 pathway to promote DOX resistance in osteosarcoma cells, indicating a potential use of miRNA-221 in osteosarcoma treatment.
微小 RNA-221 (miRNA-221) 在多种恶性肿瘤中上调，与患者预后不良相关。因此，本研究旨在探讨 miRNA-221 在骨肉瘤细胞多柔比星（DOX）耐药中的作用及其潜在机制。我们构建了 DOX 耐药的 Saos-2/DOX 细胞，并对其进行 DOX 处理。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法测定细胞活力。用 miRNA-221 模拟物或 miRNA-221 抑制剂转染细胞；通过定量（q）RT-PCR 检测 miRNA-221 的表达。通过流式细胞术和末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸-地高辛缺口末端标记（TUNEL）染色检测细胞凋亡。还采用免疫荧光法检测细胞信号转导和转录激活因子 3（Stat3）蛋白表达分布。此外，采用 Western blot 检测各蛋白表达变化。结果发现，miRNA-221 在 Saos-2/DOX 细胞中上调。此外，miRNA-221 模拟物诱导 Saos-2 细胞产生 DOX 耐药，而 miRNA-221 抑制剂增强 Saos-2/DOX 细胞对 DOX 的敏感性。miRNA-221 模拟物上调 Saos-2 细胞中磷酸化 Stat3、P-糖蛋白（P-gp）和 B 细胞淋巴瘤-2（Bcl-2）蛋白的表达，并诱导 Stat3 进入细胞核，而 miRNA-221 抑制剂则产生相反的作用。用 Stat3 化学抑制剂 STAT3-IN-3 预处理可显著抑制 miRNA-221 模拟物诱导的 Saos-2 细胞中 P-gp 和 Bcl-2 蛋白表达上调；还使 Saos-2 细胞克服 miRNA-221 模拟物诱导的 DOX 耐药。因此，miRNA-221 通过激活 Stat3 通路增加 P-gp 和 Bcl-2 的表达，从而促进骨肉瘤细胞对 DOX 的耐药性，表明 miRNA-221 可能用于骨肉瘤的治疗。
