MicroRNA expression in paternal and cisplatin-resistant germ cell tumor cell lines.

Abstract

e15029 Background: Resistance to chemotherapy is a potentially deadly and so far poorly understood event in germ cell tumors (GCT). To improve our knowledge on resistance to cisplatin (CDDP) in GCT, we compared the constitutive miRNA gene expression pattern in three CDDP resistant and corresponding paternal GCT cell lines. Methods: Three CDDP resistant cell lines were derived via long term culture under increasing concentrations of CDDP from well established GCT lines (NT2, NCCIT, 2102EP). CDDP resistance was determined using MTT assy. Total RNA was isolated from all 6 cell lines in three independent experiments. Aliquots of individual total RNA were reverse transcribed without preamplification using the so called “Megaplex pools without preamplification protocol for miRNA expression analysis protocol”. RTQ-PCR was performed using two different 384 well low density arrays covering about 800 known miRNA species of human origin. Statistical measures (e.g. coefficient of variation, CV, and 25-75% quantiles) were computed using SAS. Results: CDDP resistance significantly increased in resistant cell lines (range 5-11 fold). In total, 72 genes showed differential expression between paternal and resistant cell lines (NT2=43, NCCIT=53, 2102EP=15), of which 73% (on average) were up- regulated (NT2=95.3%, NCCIT=62.3%, 2102EP=46.7%). One single miRNA, miR-10b, showed 2-3 fold upregulation in all three resistant cell lines. Previously, miR-10b has been associated with cancer progression. miR-371, miR-372, and miR-373 pre-373, which have been identified as oncogenes in GCT, were upregulated in NT2 and NCCIT, but not in 2102EP. Conclusions: To our knowledge, this is the first report on differential expression of miRNAs in CDDP resistent GCT cell lines. Constitutive miRNA gene expression revealed many similarities between NT2 and NCCIT cell lines, whereas 2102EP differed. No significant financial relationships to disclose.