The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection

Our plain-English summary of The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection, by P. Winata, M. Williams, E. McGowan, et al (BMC Research Notes, 2017).

The study

• Authors: P. Winata, M. Williams, E. McGowan, N. Nassif, N. van Zandwijk, G. Reid
• Journal: BMC Research Notes
• Published: 2017;10(1):600
• Cited by: 6 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Objective: MicroRNAs 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.

Results: The 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.

In plain English

This laboratory study did not look at asbestos exposure or at patients. It tested whether a microRNA-measuring method, stem-loop RT-qPCR, could detect lab-made microRNA 'mimics' in RNA extracted from cancer cells. These mimics are the microRNA component of TargomiRs, an experimental mesothelioma treatment. The authors used two human malignant pleural mesothelioma cell lines, MSTO-211H and VMC23. With purified lab-made RNA, the tests mostly detected only their intended target. However, one mimic, conmiR-15/107.2, also reacted with the primer meant for the other mimic, conmiR-15/107.4. With RNA taken from whole cells, the tests gave signals for both mimics even in cells that had never received them. They also gave non-specific signals in cells given the other mimic. The authors attributed the signals in untreated cells to related natural microRNAs. They did not know which ones, but thought the miR-15/16 family likely contributed in part. They judged that even full delivery of the trial dose would be unlikely to show up above this background. They concluded that future studies would likely need a sequencing-based method. The authors noted that the exact cause was unclear and that they compared only one natural microRNA, miR-16.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) experiment in cancer cell lines, testing how specific a microRNA detection method (stem-loop RT-qPCR with hydrolysis-probe TaqMan assays) is
• Who was studied: Two human malignant pleural mesothelioma (MPM) cell lines, MSTO-211H and VMC23, plus synthetic RNA templates; no human participants
• Exposure: No asbestos exposure was studied. Cells were transfected with miR-16, the consensus mimics conmiR-15/107.2 or conmiR-15/107.4, or a negative control at 5 nM, and were harvested after 48 hours
• Main results:
  • Using synthetic RNA templates, the three assays amplified their own targets with efficiencies of 87.3% for hsa-miR-16, 81.1% for conmiR-15/107.2 and 75.3% for conmiR-15/107.4
  • With synthetic templates, the conmiR-15/107.2 template cross-reacted with the RT primer specific for conmiR-15/107.4. For miR-16 and conmiR-15/107.4, only the matching primer and assay combination gave discernible amplification
  • In total RNA from untransfected cells, which contained only natural miR-16, signals appeared with a Cq of less than 30 when cDNA made with the conmiR RT primers was amplified
  • In untransfected cells, the conmiR-15/107.2-specific primer and assay gave a Cq value of 30.5, although these cells had not received that mimic
  • In RNA from cells transfected with conmiR-15/107.4, a non-specific signal was detected with the conmiR-15/107.2 primer and assay
  • The authors attributed detection of the mimics in untransfected cell lines to non-specific reverse transcription and amplification of related natural (endogenous) microRNAs
  • The specific natural microRNAs responsible were unknown, but the authors considered it likely that miR-15/16 family members contributed at least in part
  • Given this background signal, the authors judged that even complete delivery of the trial's maximum tolerated dose to a tumour would be unlikely to be detectable in a tumour biopsy
• The authors' conclusion: The authors concluded that the RT-qPCR approach could distinguish the mimic sequences when using synthetic RNA but had difficulty doing so in total RNA from cells, and that future studies of microRNA delivery will likely need a sequencing-based approach
• Limitations the authors state:
  • The RT step appeared responsible for the cross-reactivity, but the exact mechanism was unclear and would require cloning and sequencing of the qPCR products
  • Only miR-16 was used as the natural microRNA for comparison; other family members, especially miR-15a and miR-15b, could also contribute to cross-reactivity

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: STEM, Clinical Trial, Pleural Mesothelioma, EGFR, Mesothelioma.

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• A Significant Metabolic and Radiological Response after a Novel Targeted MicroRNA-based Treatment Approach in Malignant Pleural Mesothelioma — S.C. Kao, M. Fulham, et al. (2015)
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