Department of Genetics, Faculty of Veterinary Medicine, Uludag University, Bursa, Turkey.
Department of Urology, School of Medicine, Acibadem University, Istanbul, Turkey.
World J Urol. 2019 Jun;37(6):1117-1126. doi: 10.1007/s00345-018-2501-6. Epub 2018 Sep 22.
To investigate the expression profiles of 86 miRNAs in paclitaxel-resistant prostate cancer cell lines and to identify the genes that have a role in the development of drug resistance.
Three prostate cancer cell lines, androgen-dependent VCaP, androgen-independent PC-3 and DU-145, were used to obtain paclitaxel-resistant cells by progressively increasing the concentration of paclitaxel in the culture medium. Viability assays with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium and sulforhodamine B were used to assess the cell resistance level and cytotoxic effects of paclitaxel treatment. Total RNA was isolated from both prostate cancer cell lines and their resistant versions, and cDNA samples were reverse transcribed from total RNA. Selected target genes of miRNAs that showed differences in expression and were estimated to be effective on drug resistance mechanism were analyzed with western blot analysis.
Expression study of 86 miRNAs by RT-PCR demonstrated that several of the miRNAs were expressed at different levels in paclitaxel-resistant cells compared to wild-type cells. Moreover, the expression profiles of these miRNAs varied among different prostate cancer cell line types, with 13 miRNAs being up-regulated in the resistant cells. Among these, miR-200b-3p, miR-34b-3p and miR-375 exhibited a marked up-regulation. Further, miR-100-5p showed a prominent increase in paclitaxel-resistant VCaP-R and DU145-R cells. Western blot and RT-PCR studies showed that only the LARP1 and CCND1 genes were over-expressed up to 2-5 times in all paclitaxel-resistant cell lines compared to the other investigated genes.
In this study, the three paclitaxel-resistant prostate cancer cell lines examined showed remarkably different miRNA expression profiles.
研究 86 种 miRNA 在紫杉醇耐药前列腺癌细胞系中的表达谱，并鉴定与耐药性发展相关的基因。
使用雄激素依赖性 VCaP、雄激素非依赖性 PC-3 和 DU-145 三种前列腺癌细胞系，通过逐渐增加培养基中紫杉醇的浓度来获得紫杉醇耐药细胞。采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑和磺基罗丹明 B 的细胞活力测定来评估细胞耐药水平和紫杉醇处理的细胞毒性作用。从前列腺癌细胞系及其耐药版本中分离总 RNA，并从总 RNA 反转录 cDNA 样品。用 Western blot 分析分析差异表达且估计对耐药机制有效的 miRNA 的选定靶基因。
RT-PCR 对 86 种 miRNA 的表达研究表明，与野生型细胞相比，紫杉醇耐药细胞中几种 miRNA 的表达水平不同。此外，这些 miRNA 的表达谱在不同的前列腺癌细胞系类型中存在差异，有 13 种 miRNA 在耐药细胞中上调。其中，miR-200b-3p、miR-34b-3p 和 miR-375 表现出明显上调。此外，miR-100-5p 在紫杉醇耐药 VCaP-R 和 DU145-R 细胞中显著增加。Western blot 和 RT-PCR 研究表明，与其他研究基因相比，仅 LARP1 和 CCND1 基因在所有紫杉醇耐药细胞系中的表达上调至 2-5 倍。
在这项研究中，三种被研究的紫杉醇耐药前列腺癌细胞系表现出明显不同的 miRNA 表达谱。
