Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
Mol Cancer. 2011 May 15;10:52. doi: 10.1186/1476-4598-10-52.
We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance.
Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R) showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP). Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738) known microRNA species of human origin.
Altogether 72 of 738 (9.8%) microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15) of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%). The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79%) and NCCIT-R/NCCIT (64%), and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%). Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold) in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency), as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21) were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up-regulated) and hsa-miR-99a/-100/-145 (up to 10-fold down-regulated).
Examining almost all known human micro-RNA species confirmed the miR-371-373 cluster as a promising target for explaining cisplatin resistance, potentially by counteracting wild-type P53 induced senescence or linking it with the potency to differentiate. Moreover, we describe for the first time an association of the up-regulation of micro-RNA species such as hsa-miR-512-3p/-515/-517/-518/-525 and down-regulation of hsa-miR-99a/-100/-145 with a cisplatin resistant phenotype in human germ cell tumors. Further functional analyses are warranted to gain insight into their role in drug resistance.
为了提高我们对顺铂耐药机制的理解，我们比较了三种源自亲代顺铂敏感生殖细胞瘤细胞系的顺铂耐药亚系中的 microRNA 表达模式。
与三种已建立的相对顺铂敏感的生殖细胞瘤细胞系（NTERA-2、NCCIT、2102EP）相比，三种经间歇性暴露于递增剂量顺铂后耐药性增加 2.7-11.3 倍的顺铂耐药亚系（NTERA-2-R、NCCIT-R、2102EP-R）被比较。从所有 6 种细胞系的三个独立实验中培养细胞并分离总 RNA。将 RNA 转化为 cDNA，并使用覆盖几乎所有（738 种）人类来源 microRNA 物种的 384 孔低密度阵列进行定量 RT-PCR。
总共在敏感和耐药细胞系对（NTERA-2R/NTERA-2=43、NCCIT-R/NCCIT=53、2102EP-R/2102EP=15）之间出现 738 种（9.8%）microRNA 差异表达，其中 46.7-95.3%上调（NTERA-2R/NTERA-2=95.3%、NCCIT-R/NCCIT=62.3%、2102EP-R/2102EP=46.7%）。在多个细胞系对中显示差异表达的基因数量在 NTERA-2R/NTERA-2（79%）和 NCCIT-R/NCCIT（64%）之间为 34，在这两个细胞系与 2102EP-R/2102EP 之间分别为 3 和 4（约 27%）。只有参与乳腺癌侵袭和转移的 has-miR-10b 和 has-miR-512-3p 在所有三种细胞系中似乎均上调（2-3 倍）。hsa-miR-371-373 簇（拮抗细胞衰老并与分化潜能相关）以及 hsa-miR-520c/-520h（抑制肿瘤抑制因子 p21）在两种顺铂耐药细胞系中上调了 3.9-16.3 倍。可以识别出几种缺少顺铂耐药注释的新 microRNA 物种。这些是 hsa-miR-512-3p/-515/-517/-518/-525（上调 8.1 倍）和 hsa-miR-99a/-100/-145（下调 10 倍）。
检查几乎所有已知的人类 microRNA 物种证实了 miR-371-373 簇是解释顺铂耐药的有希望的靶标，可能通过拮抗野生型 P53 诱导的衰老或将其与分化潜能联系起来。此外，我们首次描述了 microRNA 物种如 hsa-miR-512-3p/-515/-517/-518/-525 的上调和 hsa-miR-99a/-100/-145 的下调与人类生殖细胞瘤中的顺铂耐药表型之间的关联。需要进一步的功能分析来深入了解它们在耐药性中的作用。
