Plasma Small RNA profiling reveals a predictive three-miRNAs signature for early beta cell dysfunction across glucose tolerance stages

Abstract Aims/hypothesis Type 2 diabetes mellitus (T2D) is a multifactorial disease marked by progressive beta cell dysfunction; however, no reliable, easily measurable and non-invasive circulating biomarkers are currently available to track this decline. Since microRNAs (miRNAs) tightly regulate beta cell physiology and are secreted and found in blood, we investigated whether specific circulating miRNAs may reflect in vivo beta cell function in living donors. Methods We conducted a cross-sectional study on two independent cohorts: a discovery cohort composed by 78 individuals with different glucose tolerance [23 with normal glucose tolerance (NGT), 22 with impaired glucose tolerance (IGT), and 33 with T2D] and a validation cohort composed by 158 subjects [71 non-diabetic (ND) and 87 with T2D] who were administered an oral glucose tolerance test (OGTT) and/or a mixed meal test (MMT) with measurement of glucose, insulin and C-peptide. Plasma RNA was profiled by small RNA sequencing and differences between glucose tolerance groups were analysed using DESeq2. Linear regression analyses were used to associate miRNA abundance to clinical and metabolic measures. Droplet Digital PCR (ddPCR) was used to validate selected miRNAs. A LASSO model was used to select a set of features predictive of beta cell function. Results We identified eleven differentially expressed miRNAs across the three glucose tolerance groups of the discovery cohort. Integrated analyses pinpointed a three-miRNAs signature (miR-34a-5p, miR-1306-5p and miR-335-5p) each significantly correlated with beta cell rate sensitivity (RS), an early indicator of insulin secretory impairment. Incorporating this panel with age and 1-hour post-load glucose into a LASSO regression model enabled accurate prediction of RS (Spearman ρ=0.43, p<0.05). Model performance was confirmed in the validation cohort of 71 ND and 87 T2D subjects (Spearman ρ=0.23, p<0.05). To streamline its clinical use, we substituted fasting glucose for 1-hour post-load glucose while retaining miRNA levels and age; this adaptation preserved predictive power (Spearman ρ=0.46, p<0.05 discovery; ρ=0.15, p=0.06 validation). Conclusion/interpretation This three-miRNAs signature combined with simple clinical parameters constitutes a promising, non-invasive biomarker for early beta cell dysfunction and T2D progression. Research in context What is already known about this subject? Beta cell dysfunction is a key event in Type 2 Diabetes (T2D) pathogenesis Circulating microRNAs (miRNAs) are biomarkers linked to T2D onset and T2D-associated metabolic abnormalities What is the key question? Can specific circulating miRNAs serve as reliable biomarkers of beta cell dysfunction across different stages of glucose tolerance and Type 2 Diabetes (T2D)? What are the new findings? Small RNA profiling of plasma samples from deep metabolically characterized NGT, IGT, and T2D subjects revealed circulating miRNAs significantly associated with in-vivo beta cell function, reflecting key mechanisms underlying pancreatic beta cell physiology A specific set of three circulating miRNAs- miR-34a-5p, miR-1306-5p and miR-335-5p - resulted linked to beta cell dysfunction This three-miRNA signature can predict early alterations in insulin secretion, representing a promising non-invasive biomarker for the early identification of individuals at risk of developing T2D How might this impact on clinical practice in the foreseeable future? Integrating circulating miRNA profiling with routine baseline clinical parameters could provide a simpler, non-invasive, and more accessible method to identify individuals at risk of beta cell dysfunction and T2D. Implementing miRNA-based diagnostics could facilitate personalized monitoring of disease progression. Graphical Abstract