Circulating exosomal microRNA profiles in migraine patients receiving acupuncture treatment: A placebo-controlled clinical trial

Abstract

Background: Acupuncture has a long history of being used in Chinese medicine for the treatment of migraine. However, molecular biomarkers for diagnosis and prognosis of migraine and its treatment are lacking. This study aimed to explore whether acupuncture could regulate differentially expressed exosomal miRNAs between patients with migraine without aura (MWoA) and healthy controls (HCs) and to identify diagnostic biomarkers that helped differentiate MWoA patients from HCs and identify prognostic biomarkers that helped to predict the effect of acupuncture.

Methods: Here, we isolated serum exosomes from patients with MWoA and HCs before and after true and sham acupuncture treatment. Then, small RNA sequencing and bioinformatics analysis were performed to screen out key miRNAs specifically responding to acupuncture treatment. Pearson's correlation analysis was used to evaluate the correlation between miRNAs and clinical phenotypes. Finally, we applied a machine learning method to identify diagnostic biomarkers of MWoA patients and identify prognostic biomarkers that helped to predict the effect of acupuncture.

Results: Small RNA sequencing identified 68 upregulated and 104 downregulated miRNAs in MWoA patients compared to those in HCs. Further, we identified eight upregulated and four downregulated miRNAs in migraine patients after true acupuncture treatment (trAMWoA), but not in the sham acupuncture treatment (shAMWoA) or HC group. Among them, has-miR-378a-5p was positively correlated with time unable to work, study, or do housework due to migraine (p < 0.05), whereas has-miR-605-3p was negatively correlated with the restrictive subscale of the migraine-specific quality of life questionnaire (MSQ) (p < 0.05). We then evaluated the diagnostic and prognostic potential of these 12 miRNAs in patients with MWoA. The combination of serum levels of exosomal has-miR-369-5p, has-miR-145-5p, and has-miR-5,010-3p could serve as diagnostic and prognostic biomarkers for MWoA patients following acupuncture treatment.

Conclusion: This is the first study on the serum exosomal miRNA profiles of migraineurs before and after acupuncture treatment. Our results improve our understanding of the molecular functions of miRNAs in MWoA. More importantly, they expand our view of evaluating the clinical outcomes of migraine patients treated with acupuncture, using exosomal RNA markers.

Clinical trial registration: Chinese Clinical Trial Registry, ChiCTR2000034417, July 2020.

Keywords: acupuncture; biomarker; exosome; miRNA; migraine.

Figure 1

Study outline, sample workflow and clinical outcomes before and after 4-week treatment. (A) The detailed process of screening, enrollment, randomization, and treatment. (B) Workflow from sample collection to exosome identification, small RNA sequencing and bioinformatics analysis. (C) Clinical outcomes of trAMWoA and shAMWoA in baseline and after 4-week treatment. * represents p < 0.05, ** represents p < 0.01. HCs, healthy controls; trAMWoA, migraine without aura patients receiving true acupuncture treatment; shAMWoA, migraine without aura patients receiving sham acupuncture treatment; TA, true acupuncture; SA, sham acupuncture; NTA, nanoparticle tracking analysis; TEM, transmission electron microscopy; WB, western blotting; VAS, visual analogue scale; Headache Impact Test-6 scale, HIT-6; MSQ, migraine-specific quality of life questionnaire; MWoA, migraine without aura.

Figure 2

Characterization of exosomes and miRNAs identified in HC, trAMWoA and shAMWoA before and after treatment. (A) TEM results of exosomes from MWoA patients and HC. (B) The mean size of exosomes of MWoA patients and HC participants by NTA. (C) WB revealed CD9 and TSG101 proteins in exosome samples. (D) miRNAs identified in HC and MWoA patients. (E) miRNAs identified in HC, trAMWoA and shAMWoA before and after 4-week treatment. MWoA, migraine without aura patients; HC, healthy control; TA, true acupuncture; SA, sham acupuncture.

Figure 3

Volcano plots and Venn diagram of DEmiRs in MWoA patients and HC volunteers before and after treatment. (A) Volcano plot of DEmiRs between migraine patients and HC. (B) Volcano plot of DEmiRs in migraine patients before and after TA treatment. (C) Volcano plot of DEmiRs in HC before and after TA treatment. (D) Volcano plot of DEmiRs in migraine patients before and after SA treatment. (E) Venn diagram of miRNAs up-regulated in HC after TA, up-regulated in MWoA patients after TA, up-regulated in MWoA patients after SA and down-regulated in MWoA patients compared to HC. (F) Venn diagram of miRNAs down-regulated in HC after TA, down-regulated in MWoA patients after TA, down-regulated in MWoA patients after SA and up-regulated in MWoA patients compared to HC. MWoA, migraine without aura patients; HC, healthy control; TA, true acupuncture; SA, sham acupuncture; FDR, false discovery rate; log2FC, log2 fold change; CMP, comparison.

Figure 4

Potential diagnostic and prognostic biomarkers for MWoA patients. (A) The importance of these 12 miRNAs in separating MWoA patients and healthy controls. (B) The importance of these 12 miRNAs in predicting TA treatment for MWoA patients. (C) The changes in expression level of these 5 miRNAs after TA and SA treatment. (D) ROC curve of RF classifier for diagnosis of MWoA and ROC curve of RF classifier for prognosis of TA treatment. HC, healthy controls; trAMWoA, migraine without aura patients receiving true acupuncture treatment; shAMWoA, migraine without aura patients receiving sham acupuncture treatment; ROC curve, receiver operating characteristic curve; RF, random forest; TA, true acupuncture; MWoA, migraine without aura patients.

Figure 5

miRNA sequencing results and qPCR results of hsa-miR-369-5p, hsa-miR-4,732-5p, hsa-miR-550a-3-5p, and hsa-mir-145-5p. MWoA, migraine without aura; HC, healthy control; trAMWoA, migraine without aura patients receiving true acupuncture treatment; log2 FC, log2 fold change; log2 RNE, log2 relative normalized expression. * represents p < 0.05.