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.
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。
Keywords: YWHAQ; breast cancer; drug resistance; miR-16-5p.