There is growing appreciation for the importance of non-protein-coding genes in development and disease. Although much is known about microRNAs, limitations in bioinformatic analyses of RNA sequencing have precluded broad assessment of other forms of small-RNAs in humans. By analysing sequencing data from plasma-derived RNA from 40 individuals, here we identified over a thousand human extracellular RNAs including microRNAs, piwi-interacting RNA (piRNA), and small nucleolar RNAs. Using a targeted quantitative PCR with reverse transcription approach in an additional 2,763 individuals, we characterized almost 500 of the most abundant extracellular transcripts including microRNAs, piRNAs and small nucleolar RNAs. The presence in plasma of many non-microRNA small-RNAs was confirmed in an independent cohort. We present comprehensive data to demonstrate the broad and consistent detection of diverse classes of circulating non-cellular small-RNAs from a large population.

Background—: Biomarkers that predict response to cardiac resynchronization therapy (CRT) in heart failure patients with dyssynchrony (HF _DYS ) would be clinically important. Circulating extracellular microRNAs (miRNAs) have emerged as novel biomarkers that may also play important functional roles, but their relevance as markers for CRT response has not been examined. Methods and Results—: Comprehensive miRNA polymerase chain reaction arrays were used to assess baseline levels of 766 plasma miRNAs in patients undergoing clinically indicated CRT in an initial discovery set (n=12) with and without subsequent echocardiographic improvement at 6 months after CRT. Validation of candidate miRNAs in 61 additional patients confirmed that baseline plasma miR-30d was associated with CRT response (defined as an increase in left ventricular ejection fraction ≥10%). MiR-30d was enriched in coronary sinus blood and increased in late-contracting myocardium in a canine model of HF _DYS , indicating cardiac origin with maximal expression in areas of high mechanical stress. We examined the functional effects of miR-30d in cultured cardiomyocytes and determined that miR-30d is expressed in cardiomyocytes and released in vesicles in response to mechanical stress. Overexpression of miR-30d in cultured cardiomyocytes led to cardiomyocyte growth and protected against apoptosis by targeting the mitogen-associated kinase 4, a downstream effector of tumor necrosis factor. In HF _DYS patients, miR-30d plasma levels inversely correlated with high-sensitivity troponin T, a marker of myocardial necrosis. Conclusions—: Baseline plasma miR-30d level is associated with response to CRT in HF _DYS in this translational pilot study. MiR-30d increase in cardiomyocytes correlates with areas of increased wall stress in HF _DYS and is protective against deleterious tumor necrosis factor signaling.

Objective: Extracellular microRNAs (miRNAs) represent functional biomarkers for obesity and related disorders; this study investigated plasma miRNAs in insulin resistance phenotypes in obesity.Methods: One hundred seventy‐five miRNAs were analyzed in females with obesity (insulin sensitivity, n = 11; insulin resistance, n = 19; type 2 diabetes, n = 15) and without obesity (n = 12). Correlations between miRNA level and clinical parameters and levels of 15 miRNAs in a murine obesity model were investigated.Results: One hundred six miRNAs were significantly (adjusted P ≤ 0.05) different between controls and at least one obesity phenotype, including miRNAs with the following attributes: previously reported roles in obesity and altered circulating levels (e.g., miR‐122, miR‐192); known roles in obesity but no reported changes in circulating levels (e.g., miR‐378a); and no current reported role in, or association with, obesity (e.g., miR‐28‐5p, miR‐374b, miR‐32). The miRNAs in the latter group were found to be associated with extracellular vesicles. Forty‐eight miRNAs showed significant correlations with clinical parameters; stepwise regression retained let‐7b, miR‐144‐5p, miR‐34a, and miR‐532‐5p in a model predictive of insulin resistance (R² = 0.57, P = 7.5 × 10⁻⁸). Both miR‐378a and miR‐122 were perturbed in metabolically relevant tissues in a murine model of obesity.Conclusions: This study expands on the role of extracellular miRNAs in insulin‐resistant phenotypes of obesity and identifies candidate miRNAs not previously associated with obesity.

OBJECTIVE: Insulin resistance (IR) is a hallmark of obesity and metabolic disease. Circulating extracellular RNAs (ex-RNAs), stable RNA molecules in plasma, may play a role in IR, though most studies on ex-RNAs in IR are small. We sought to characterize the relationship between ex-RNAs and metabolic phenotypes in a large community-based human cohort. RESEARCH DESIGN AND METHODS: We measured circulating plasma ex-RNAs in 2,317 participants without diabetes in the Framingham Heart Study (FHS) Offspring Cohort at cycle 8 and defined associations between ex-RNAs and IR (measured by circulating insulin level). We measured association between candidate ex-RNAs and markers of adiposity. Sensitivity analyses included individuals with diabetes. In a separate cohort of 90 overweight/obese youth, we measured selected ex-RNAs and metabolites. Biology of candidate microRNAs was investigated in silico. RESULTS: The mean age of FHS participants was 65.8 years (56% female), with average BMI 27.7 kg/m2; participants in the youth cohort had a mean age of 15.5 years (60% female), with mean BMI 33.8 kg/m2. In age-, sex-, and BMI-adjusted models across 391 ex-RNAs in FHS, 18 ex-RNAs were associated with IR (of which 16 were microRNAs). miR-122 was associated with IR and regional adiposity in adults and IR in children (independent of metabolites). Pathway analysis revealed metabolic regulatory roles for miR-122, including regulation of IR pathways (AMPK, target of rapamycin signaling, and mitogen-activated protein kinase). CONCLUSIONS: These results provide translational evidence in support of an important role of ex-RNAs as novel circulating factors implicated in IR.