南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (10): 1476-1485.doi: 10.12122/j.issn.1673-4254.2022.10.06

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miR-16-5p通过靶向YWHAQ调控乳腺癌耐药细胞的凋亡与迁移能力

朱海涛，毛慧兰，陶 爽，王文锐，陈昌杰，杨清玲   

  1. 蚌埠医学院癌症转化医学安徽省重点实验室，蚌埠医学院肿瘤基础研究与临床检验诊断重点实验室，生物技术教研室，生物化学与分子生物学教研室，安徽 蚌埠 233000
  • 出版日期:2022-10-20 发布日期:2022-11-01

miR-16-5p regulates apoptosis and migration of drug-resistant breast cancer cells by targeting YWHAQ

ZHU Haitao, MAO Huilan, TAO Shuang, WANG Wenrui, CHEN Changjie, YANG Qingling   

  1. Anhui Provincial Key Laboratory of Cancer Translational Medicine, Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis, Department of Biotechnology, Department of Biochemistry and Molecular Biology, Bengbu Medical College, Bengbu 233000, China
  • Online:2022-10-20 Published:2022-11-01

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摘要： 目的 探究miR-16-5p对乳腺癌紫杉醇耐药细胞生物学活性的影响及分子机制。方法 以人乳腺癌亲本细胞（SKBR-3）与乳腺癌紫杉醇耐药细胞（SKBR-3/PR）为研究对象。实验设空白对照组、阴性对照组、miR-16-5p类似物组（miR-16-5p mimics）、miR-16-5p抑制剂组（miR-16-5p inhibitor），YWHAQ干扰组（si-YWHAQ），以及联合组（miR-16-5p mimics+si-YWHAQ）。利用qRT-PCR检测乳腺癌组织与癌旁组织、SKBR-3与SKBR-3/PR间miR-16-5p的表达水平。使用生信数据对miR-16-5p的靶基因做预测，双荧光素酶实验验证靶向结合。免疫印迹检测细胞YWHAQ、Bcl-2和Bax表达。CCK-8和Transwell检测细胞增殖迁移能力。流式细胞术检测耐药细胞周期凋亡改变。结果 qRT-PCR显示乳腺癌组织中miR-16-5p水平低于癌旁组织（-1.19±1.90 vs 1.59±1.76，P<0.01）。生物信息预测YWHAQ是miR-16-5p的靶基因，荧光素酶实验证实miR-16-5p能够靶向调节YWHAQ（P<0.01）。相对于 SKBR-3 细胞，SKBR-3/PR 细胞中 miR-16-5p 表达水平降低（P<0.01），YWHAQ 表达水平增高（P<0.05）。Western blot 结果显示 miR-16-5p 类似物能够抑制 YWHAQ 表达，miR-16-5p 抑制剂能够促进 YWHAQ 表达（P<0.01）。相对于对照组，miR-16-5p类似物能够将耐药细胞阻滞在G0/G1期（[ 55.61±1.99）% vs（43.06±1.53）%，P<0.01]，抑制细胞增殖和迁移能力（P<0.01），促进细胞凋亡 （[ 10.37±0.23）% vs（3.81±0.88）%，P<0.01]，YWHAQ干扰组细胞迁移能力下降，凋亡率升高，miR-16-5p类似物组与YWHAQ干扰组细胞的Bax蛋白表达增加，YWHAQ与Bcl-2蛋白水平降低。相对于miR-16-5p 类似物组，联合组细胞迁移能力被抑制（75.75±29.85 vs 181.11±11.71，P<0.01），凋亡率显著升高（[ 24.20±2.43）% vs （14.10±4.47）%，P<0.01]。结论 miR-16-5p能够通过靶向调节YWHAQ调控Bcl-2/Bax的表达，从而改变乳腺癌紫杉醇耐药细胞的生物学活性。

关键词: 乳腺癌；耐药；miR-16-5p；YWHAQ

Abstract: Objective To examine the role of miR-16-5p in regulating biological behaviors of paclitaxel-resistant breast cancer cells and its molecular mechanism. Methods The expression of miR-16-5p was examined in 13 pairs of breast cancer and adjacent tissues and in parental SKBR-3 cells and paclitaxel-resistant SKBR-3/PR cells using qRT-PCR. The target genes of miR-16-5p were predicted by bioinformatic analysis, and their targeted binding was tested using luciferase assay. The cells were transfected with a miR-16-5p mimics, a miR-16-5p inhibitor, a specific siRNA targeting YWHAQ (si-YWHAQ), or both the miR-16-5p mimics and si-YWHAQ, and the changes in cellular expressions of YWHAQ, Bcl-2 and Bax were detected using Western blot. The changes in proliferation and migration of the cells were evaluated with CCK-8 assay and Transwell assay, and the cell cycle changes and cell apoptosis were analyzed with flow cytometry. Results The expression of miR-16-5p was significantly lower in breast cancer tissues than in paired adjacent tissues (P<0.01). Bioinformatic analysis predicted that YWHAQ was the target gene of miR-16-5p, which was confirmed by luciferase assay. Compared with parental SKBR-3 cells, SKBR-3/PR cells showed a lowered level of miR-16-5p expression and an increased expression of YWHAQ. Transfection with the miR-16-5p mimics significantly inhibited YWHAQ expression (P<0.01), while miR-16-5p inhibitor promoted YWHAQ expression in SKBR-3/PR cells (P<0.01). The miR-16-5p mimics caused cell cycle arrest in G0/G1 phase (P<0.0l), suppressed proliferation and migration, and increased apoptosis rate of SKBR-3/PR cells (P<0.0l). Knocking down YWHAQ also reduced the migration ability of SKBR-3/PR cells and increased cell apoptosis rate. Transfection with either miR-16-5p mimics or si-YWHAQ resulted in increased Bax expression and lowered expressions of YWHAQ and Bcl-2 in the cells. The cells transfected with both miR-16-5p mimics and si-YWHAQ showed obviously suppressed cell migration (P<0.01) and significantly increased apoptosis rate (P<0.01). Conclusion miR-16-5p can modulate the expressions of Bcl- 2 and Bax by targeted regulation of YWHAQ to modify the biological behaviors of paclitaxel-resistant breast cancer cells.

Key words: breast cancer; drug resistance; miR-16-5p; YWHAQ