Absolute quantification of cell-free microRNAs in cancer patients Absolute quantification of cell-free microRNAs in cancer patients

Skin cancer is the most common type of cancer and is classified in melanoma and non-melanoma cancers, which include basal cell, squamous cell, and Merkel cell carcinoma. Specific microRNAs are dysr...

In this review, we summarize new knowledge on long noncoding RNAs (lncRNAs) linked to cardiovascular disease, in particular to heart failure. LncRNAs are dysregulated in specific developmental and pathological conditions and regulate critical responses to stress. LncRNAs are being recognized as molecules regulating myocardial remodeling during disease, participating for instance in the regulation of cardiomyocyte hypertrophy and function and fibroblast proliferation. LncRNAs add a new layer of complexity to the regulation of the biological processes underlying normal cardiac development and myocardial remodeling during disease; they may represent targets for novel therapeutic strategies for cardiovascular diseases.

The aim of our study was to set up a simple and reliable isolation method of living ventricular cardiomyocytes (vCMs) for molecular and biological studies. A standard technique for the retrograde perfusion of an enzymatic solution was used to isolate cardiac cells from adult mouse heart. Fluorescence-activated cell sorting (FACS) on adult murine cardiac ventricle cells was performed, comparing the intrinsic autofluorescence in the FITC channel and the forward scatter (FSC) parameter in order to isolate highly fluorescent cells. The expression of cell-specific mRNAs was assessed with real-time PCR in cells sorted on the basis of their FITC and FSC characteristics. We identified two distinct subpopulations of cells harvested after retrograde perfusion of wild-type heart: FITC^high/FSC^dim and FITC^dim/FSC^high. Immunophenotyping and mRNA analysis (qPCR and RNA sequencing) revealed that only FITC^high/FSC^dim cells were highly enriched in CM markers. Genes with high expression in endothelial cells and fibroblasts were enriched in the FITC^dim/FSC^high subpopulation. With the use of tdTomato^fl/fl-α-myosin heavy chain MerCreMer^+/−mouse heart, we found that tdTomato-positive vCMs were present in the FITC^high/FSC^dim region but were only rare in the FITC^dim/FSC^high fraction. We have developed a simple and reliable method for the isolation of highly purified vCMs from the adult murine myocardium, avoiding fixation and permeabilization steps. These isolated vCMs can be used in particular for detailed molecular studies, avoiding contamination with other myocardial cell types.

Enzyme replacement therapy (ERT) is a mainstay of treatment for Anderson–Fabry disease (AFD), a pathology with negative effects on the heart and kidneys. However, no reliable biomarkers are available to monitor its efficacy. Therefore, we tested a panel of four microRNAs linked with cardiac and renal damage in order to identify a novel biomarker associated with AFD and modulated by ERT. To this end, 60 patients with a definite diagnosis of AFD and on chronic ERT, and 29 age- and sex-matched healthy individuals, were enrolled by two Italian university hospitals. Only miR-184 met both conditions: its level discriminated untreated AFD patients from healthy individuals (c-statistic = 0.7522), and it was upregulated upon ERT (P < 0.001). On multivariable analysis, miR-184 was independently and inversely associated with a higher risk of cardiac damage (odds ratio = 0.86; 95% confidence interval [CI] = 0.76–0.98; P = 0.026). Adding miR-184 to a comprehensive clinical model improved the prediction of cardiac damage in terms of global model fit, calibration, discrimination, and classification accuracy (continuous net reclassification improvement = 0.917, P < 0.001; integrated discrimination improvement [IDI] = 0.105, P = 0.017; relative IDI = 0.221, 95% CI = 0.002–0.356). Thus, miR-184 is a circulating biomarker of AFD that changes after ERT. Assessment of its level in plasma could be clinically valuable in improving the prediction of cardiac damage in AFD patients.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology...