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。
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.
作者
朱海涛
毛慧兰
陶爽
王文锐
陈昌杰
杨清玲
ZHU Haitao;MAO Huilan;TAO Shuang;WANG Wenrui;CHEN Changjie;YANG Qingling(Anhui Provincial Key Laboratory of Cancer Translational Medicine,Bengbu Medical College,Bengbu 233000,China;Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis,Bengbu Medical College,Bengbu 233000,China;Department of Biotechnology,Bengbu Medical College,Bengbu 233000,China;Department of Biochemistry and Molecular Biology,Bengbu Medical College,Bengbu 233000,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2022年第10期1476-1485,共10页
Journal of Southern Medical University
基金
安徽省教育厅自然科学重大项目(KJ2019ZD28)
蚌埠医学院面上项目攀登计划(2020bypd005)
蚌埠医学院研究生科研创新计划(Byycx21001)。
作者简介
朱海涛,在读硕士研究生,E-mail:heathzhu37@163.com;通信作者:杨清玲,教授,硕士生导师,E-mail:yqlmimi@163.com。
