Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

A triplet repeat expansion leading to transcriptional silencing of the FMR1 gene results in fragile X syndrome (FXS), which is a common cause of inherited intellectual disability and autism. Phenotypic variation requires personalized treatment approaches and hampers clinical trials in FXS. We searched for microRNA (miRNA) biomarkers for FXS using deep sequencing of urine and identified 28 differentially regulated miRNAs when 219 reliably identified miRNAs were compared in dizygotic twin boys who shared the same environment, but one had an FXS full mutation, and the other carried a premutation allele. The largest increase was found in miR-125a in the FXS sample, and the miR-125a levels were increased in two independent sets of urine samples from a total of 19 FXS children. Urine miR-125a levels appeared to increase with age in control subjects, but varied widely in FXS subjects. Should the results be generalized, it could suggest that two FXS subgroups existed. Predicted gene targets of the differentially regulated miRNAs are involved in molecular pathways that regulate developmental processes, homeostasis, and neuronal function. Regulation of miR-125a has been associated with type I metabotropic glutamate receptor signaling (mGluR), which has been explored as a treatment target for FXS, reinforcing the possibility that urine miR-125a may provide a novel biomarker for FXS.

Objective: This study aimed to analyze the potential association of different microRNA (miRNA) molecules with both type 2 diabetes (T2D) and obesity and determine their target genes. Methods: Quantitative PCR was used to analyze the miR-20b, miR-296, and Let-7f levels in human visceral and subcutaneous adipose tissues (ATs) in relation to obesity and T2D, miRTarBase 4.0 was used for validation of target genes, and the Protein Analysis Through Evolutionary Relationships (PANTHER) Classification System and the Database for Annotation, Visualization and Integrated Discovery (DAVID) were used to annotate the biological processes of the predicted targets. Results: In AT, miR-20b, miR-296, and Let-7f levels were significantly different between normoglycemic subjects and those with T2D. In visceral adipose tissue, miRNA levels were higher in normoglycemic/obesity samples than in T2D/obesity samples. miR-20b, miR-296, and Let-7f target genes that showed significant differences in both ATs in relation to obesity and T2D were CDKN1A, CX3CL1, HIF1A, PPP2R1B, STAT3, and VEGFA. These genes are known to be principally involved in the vascular endothelial growth factor (VEGF) and WNT pathways. Conclusions: This study provides experimental evidence of the possible correlation between AT miR-20b, miR-296, and Let-7f with obesity and T2D, which might involve vascular endothelial growth factor and WNTdependent pathways that are regulated by six different genes, suggesting a novel signaling pathway that could be important for understanding the mechanisms underlying the AT dysfunction associated with obesity and T2D.