Circulating microRNAs are promising novel biomarkers for drug-resistant epilepsy

Jun Wang, Lan Tan, Lin Tan, Yan Tian, Jing Ma, Chen-Chen Tan, Hui-Fu Wang, Ying Liu, Meng-Shan Tan, Teng Jiang, Jin-Tai Yu, Jun Wang, Lan Tan, Lin Tan, Yan Tian, Jing Ma, Chen-Chen Tan, Hui-Fu Wang, Ying Liu, Meng-Shan Tan, Teng Jiang, Jin-Tai Yu

Abstract

MicroRNAs (miRNAs) open up a new field for molecular diagnosis for cancer and other diseases based on their stability in serum. However, the role of circulating miRNAs in plasma/serum in epilepsy diagnosis is still unclear. The aim of this study was to evaluate whether miRNAs can be used as biomarkers for drug-resistant epilepsy. We measured the differences in serum miRNA levels between 30 drug-resistant patients and 30 drug-responsive epilepsy patients in discovery and training phases using Illumina HiSeq2000 sequencing followed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays. The selected miRNAs were then validated in 77 drug-resistant epilepsy patients, 81 drug-responsive epilepsy patients and 85 healthy controls by qRT-PCR. We found that circulating miRNAs are differentially expressed between drug-resistant group and drug-responsive group. MiR-194-5p, -301a-3p, -30b-5p, -342-5p and -4446-3p were significantly deregulated in drug-resistant group compared to drug-responsive group and control group. Among these 5 miRNAs, miR-301a-3p had the best diagnostic value for drug-resistant epilepsy with 80.5% sensitivity and 81.2% specificity, and was negatively associated with seizure severity. These provide the rationale for further confirmation studies in larger prospective cohorts and in other ethnics.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. The circulating miRNAs signatures identified…
Figure 1. The circulating miRNAs signatures identified by Illumina Hiseq2000 sequencing.
The length distribution and frequency percentages of the sequences identified in drug-resistant epilepsy samples (A) and drug-responsive epilepsy samples (B) RNA species in drug-resistant epilepsy samples (C) and drug-responsive epilepsy samples (D) and RNA read counts in drug-resistant epilepsy samples (E) and drug-responsive epilepsy samples (F).
Figure 2. Differential expression of miRNAs in…
Figure 2. Differential expression of miRNAs in drug-resistant epilepsy patients.
A Significantly downregulated miRNAs in drug-resistant epilepsy compared to drug-responsive epilepsy in secondary screening stage. B Large-scale validation of the 6 miRNAs selected from secondary screening in drug-resistant epilepsy compared to drug-responsive epilepsy and controls. The blue dots represent health control group, green dots represent drug-responsive group, and purple dots represent drug-resistant group. Expression levels of the miRNAs (LnΔCq scale at Y-axis) were normalized to spiked-in cel-miR-39. The line represents the median value. Mann-Whitney U test was used to determine statistical significance.
Figure 3. Receiver operating characteristic (ROC) curve…
Figure 3. Receiver operating characteristic (ROC) curve analysis using 5 serum miRNAs selected in large-scale validation and the miRNA panel for discriminating drug-resistant epilepsy from drug-responsive epilepsy.
AUC, area under the ROC curve.
Figure 4. Correlation between the National Hospital…
Figure 4. Correlation between the National Hospital Seizure Severity Scale (NHS3) scores and the expression level of miR-301a-3p.
Serum miR-301a-3p level was significantly associated with the NHS3 scores in drug-resistant epilepsy patients (r = 0.604, P = 6.2 × 10−9). Expression level of miR-301a-3p (LnΔCq scale at Y-axis) were normalized to spiked-in cel-miR-39.
Figure 5. Overview of the study design.
Figure 5. Overview of the study design.
qRT-PCR, quantitative reverse transcriptase polymerase chain reaction.

References

    1. Moshe S. L., Perucca E., Ryvlin P. & Tomson T. Epilepsy: new advances. Lancet doi:10.1016/S0140-6736(14)60456-6 (2014).
    1. Devinsky O. Patients with Refractory Seizures. N. Engl. J. Med. 340, 1565–1570 (1993).
    1. Kwan P. & Brodie M. J. Early identification of refractory epilepsy. N. Engl. J. Med. 342, 314–319 (2000).
    1. Chen X. et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 18, 997–1006 (2008).
    1. ElSharawy A. et al. Genome-wide miRNA signatures of human longevity. Aging. Cell 11, 607–616 (2012).
    1. Mitchell P. S. et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc. Natl. Acad. Sci. USA 105, 10513–10518 (2008).
    1. Cardo L. F. et al. Profile of microRNAs in the plasma of Parkinson’s disease patients and healthy controls. J. Neurol. 260, 1420–1422 (2013).
    1. Gandhi R. et al. Circulating microRNAs as biomarkers for disease staging in multiple sclerosis. Ann. Neurol. 73, 729–740 (2013).
    1. Tan L. et al. Circulating miR-125b as a biomarker of Alzheimer’s disease. J. Neurol. Sci. 336, 52–56 (2013).
    1. Song Y. J. et al. Temporal lobe epilepsy induces differential expression of hippocampal miRNAs including let-7e and miR-23a/b. Brain Res. 1387, 134–140 (2011).
    1. Hu K. et al. MicroRNA expression profile of the hippocampus in a rat model of temporal lobe epilepsy and miR-34a-targeted neuroprotection against hippocampal neurone cell apoptosis post-status epilepticus. BMC Neurosci. 13, 115 (2012).
    1. Kan A. A. et al. Genome-wide microRNA profiling of human temporal lobe epilepsy identifies modulators of the immune response. Cell. Mol. Life Sci. 69, 3127–3145 (2012).
    1. McKiernan R. C. et al. Reduced mature microRNA levels in association with dicer loss in human temporal lobe epilepsy with hippocampal sclerosis. PLoS One 7, e35921 (2012).
    1. Bot A. M., Debski K. J. & Lukasiuk K. Alterations in miRNA levels in the dentate gyrus in epileptic rats. PLoS One 8, e76051 (2013).
    1. Gorter J. A. et al. Hippocampal subregion-specific microRNA expression during epileptogenesis in experimental temporal lobe epilepsy. Neurobiol. Dis. 62, 508–520 (2014).
    1. Li M. M. et al. Genome-wide microRNA expression profiles in hippocampus of rats with chronic temporal lobe epilepsy. Sci. Rep. 4, 4734 (2014).
    1. Sun Z. et al. Genome-wide microRNA profiling of rat hippocampus after status epilepticus induced by amygdala stimulation identifies modulators of neuronal apoptosis. PLoS One 8, e78375 (2013).
    1. Henshall D. C. MicroRNA and epilepsy: profiling, functions and potential clinical applications. Curr Opin Neurol 27, 199–205 (2014).
    1. Iyer A. et al. MicroRNA-146a: a key regulator of astrocyte-mediated inflammatory response. PLoS One 7, e44789 (2012).
    1. Tan L. et al. Genome-wide serum microRNA expression profiling identifies serum biomarkers for Alzheimer’s disease. J. Alzheimers. Dis. 40, 1017–1027 (2014).
    1. Proper E. A. et al. Distribution of glutamate transporters in the hippocampus of patients with pharmaco-resistant temporal lobe epilepsy. Brain 125, 32–43 (2002).
    1. Cross J. H., Boyd S. G., Gordon I., Harper A. & Neville B. G. Ictal cerebral perfusion related to EEG in drug resistant focal epilepsy of childhood. J. Neurol. Neurosurg. Psychiatry. 62, 377–384 (1997).
    1. Holmes G. L. EEG abnormalities as a biomarker for cognitive comorbidities in pharmacoresistant epilepsy. Epilepsia 54 Suppl 2, 60–62 (2013).
    1. Aronica E. et al. Expression pattern of miR-146a, an inflammation-associated microRNA, in experimental and human temporal lobe epilepsy. Eur. J. Neurosci. 31, 1100–1107 (2010).
    1. Nudelman A. S. et al. Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo. Hippocampus 20, 492–498 (2010).
    1. Liu D. Z. et al. Brain and blood microRNA expression profiling of ischemic stroke, intracerebral hemorrhage, and kainate seizures. J. Cereb. Blood Flow Metab. 30, 92–101 (2010).
    1. Wieser H. G. & Epilepsy I. C. o. N. o. ILAE Commission Report. Mesial temporal lobe epilepsy with hippocampal sclerosis. Epilepsia 45, 695–714 (2004).
    1. Lu Z. et al. miR-301a as an NF-kappaB activator in pancreatic cancer cells. EMBO J. 30, 57–67 (2011).
    1. Kumar P. et al. Circulating miRNA biomarkers for Alzheimer’s disease. PLoS One 8, e69807 (2013).
    1. Kosik K. S. The neuronal microRNA system. Nat. Rev. Neurosci. 7, 911–920 (2006).
    1. Wayman G. A. et al. An activity-regulated microRNA controls dendritic plasticity by down-regulating p250GAP. Proc. Natl. Acad. Sci. USA 105, 9093–9098 (2008).
    1. Edbauer D. et al. Regulation of synaptic structure and function by FMRP-associated microRNAs miR-125b and miR-132. Neuron 65, 373–384 (2010).
    1. Gaughwin P., Ciesla M., Yang H., Lim B. & Brundin P. Stage-specific modulation of cortical neuronal development by Mmu-miR-134. Cereb. Cortex. 21, 1857–1869 (2011).
    1. Tao J. et al. Deletion of astroglial Dicer causes non-cell-autonomous neuronal dysfunction and degeneration. J. Neurosci. 31, 8306–8319 (2011).
    1. O’Donoghue M. F., Duncan J. S. & Sander J. W. The National Hospital Seizure Severity Scale: a further development of the Chalfont Seizure Severity Scale. Epilepsia 37, 563–571 (1996).
    1. Engel J. Jr. A proposed diagnostic scheme for people with epileptic seizures and with epilepsy: report of the ILAE Task Force on Classification and Terminology. Epilepsia 42, 796–803 (2001).
    1. Kwan P. et al. Definition of drug resistant epilepsy: consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies. Epilepsia 51, 1069–1077 (2010).
    1. Yu S. et al. Circulating microRNA profiles as potential biomarkers for diagnosis of papillary thyroid carcinoma. J Clin Endocrinol Metab 97, 2084–2092 (2012).
    1. Yang C. et al. Identification of seven serum microRNAs from a genome-wide serum microRNA expression profile as potential noninvasive biomarkers for malignant astrocytomas. Int J Cancer 132, 116–127 (2013).
    1. McDonald J. S., Milosevic D., Reddi H. V., Grebe S. K. & Algeciras-Schimnich A. Analysis of circulating microRNA: preanalytical and analytical challenges. Clin. Chem. 57, 833–840 (2011).
    1. Livak K. J. & Schmittgen T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001).

Source: PubMed

Sponsor e collaboratori

Condizioni mediche

Interventi farmacologici

3
Sottoscrivi