MicroRNA downregulation is frequent in malignant pleural mesothelioma (MPM), but the mechanisms responsible for loss of miR-15/16 and miR-193a are yet to be elucidated and were investigated in this study. Copy Number Variation (CNV) of microRNA-coding genes was analyzed in MPM cells by digital droplet PCR (ddPCR) and revealed heterozygous loss of miR-193a and miR-15a/16-1, but no change in miR-15b/16-2. Epigenetic control of microRNA expression was inferred following decitabine and Trichostatin A (TSA) treatment which did not substantially affect microRNA expression. Knockdown of c-Myc expression led to upregulation of SMC4 , miR-15b and 16, and to a lesser extent DLEU2 and miR-15a, whereas c-Myc overexpression repressed microRNA expression. Chromatin immunoprecipitation (ChIP) assays confirmed the interaction of c-Myc with the DLEU2 and SMC4 promoters. Tumor microRNA expression was determined in samples from MPM patients, with samples of pleura from cardiac surgery patients used as controls. In tumor samples, a strong correlation was observed between the expression of miR-15b and 16 (R 2 =0.793), but not miR-15a and 16. Our data suggest that in MPM, the downregulation of miR-15/16 is due to transcriptional repression by c-Myc, primarily via control of the miR-15b/16-2 locus, while miR-193a-3p loss is due to genomic deletion.
Aim: Aberrant microRNA expression is a common event in cancer drug resistance, however its involvement in malignant pleural mesothelioma (MPM) drug resistance is largely unexplored. We aimed to investigate the contribution of microRNAs to the resistance to drugs commonly used in the treatment of MPM. Methods: Drug resistant MPM cell lines were generated by treatment with cisplatin, gemcitabine or vinorelbine. Expression of microRNAs was quantified using RT-qPCR. Apoptosis and drug sensitivity assays were carried out following transfection with microRNA mimics or BCL2 siRNAs combined with drugs. Results: Expression of miR-15a, miR-16 and miR-34a was downregulated in MPM cells with acquired drug resistance. Transfection with miR-15a or miR-16 mimics reversed the resistance to cisplatin, gemcitabine or vinorelbine, whereas miR-34a reversed cisplatin and vinorelbine resistance only. Similarly, in parental cell lines, miR-15a or miR-16 mimics sensitised cells to all drugs, whereas miR-34a increased response to cisplatin and vinorelbine. Increased microRNA expression increased drug-induced apoptosis and caused BCL2 mRNA and protein reduction. RNAi-mediated knockdown of BCL2 partly recapitulated the increase in drug sensitivity in cisplatin and vinorelbine treated cells. Conclusion: Drug-resistant MPM cell lines exhibited reduced expression of tumour suppressor microRNAs. Increasing tumour suppressor of microRNA expression sensitised both drug resistant and parental cell lines to chemotherapeutic
ObjectiveMicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR.ResultsThe custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients.
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