Screening and Identification of Putative Long Non-Coding RNA in Childhood Obesity: Evaluation of Their Transcriptional Levels

Abstract

Background and Methods: Long non-coding RNAs (LncRNAs) and microRNAs are involved in the pathogenesis of obesity, a multifactorial disease that is characterized by inflammation, cardiometabolic complications, and increased cancer risk among other co-morbidities. The up/down regulation of LncRNAs and microRNAs may play an important role in this condition to identify new diagnostic/prognostic markers. The aim of the study was to identify circulating inflammatory LncRNAs in obese adolescents (n = 54) and to evaluate whether their expression behaved differently compared to normal-weight adolescents (n = 26). To have a more complete insight, the expression of some circulating miRNAs that are linked to obesity (miR-33a, miR-223, miR-142, miR-199a, miR-181a, and miR-4454) were also analyzed. Results: LncRNAs and miRNAs were extracted simultaneously from plasma samples and amplified by Real-Time PCR. Among the 86 LncRNAs that were analyzed with custom pre-designed plates, only four (RP11-347E10.1, RP11-10K16.1, LINC00657, and SNHG12) were amplified in both normal-weight and obese adolescents and only SNHG12 showed significantly lower expression compared to the normal-weight adolescents (p = 0.026). Circulating miRNAs showed a tendency to increase in obese subjects, except for miR-181a expression. LncRNAs and miRNAs correlated with some clinical and metabolic parameters. Conclusions: Our results suggest the importance of these new biomarkers to better understand the molecular mechanisms of childhood obesity and its metabolic disorder.

Keywords: LncRNAs; Real-Time PCR; biomarkers; childhood obesity; miRNAs.

Figure 1

(A) Clustergram graph: LncRNAs expression in obese in comparison with the mean expression in normal-weight adolescents. Red square: up-regulation, green square: down-regulation, black square: no regulation; black crossed square: absence of expression. Panel 1 reports 42 LncRNA and Panel 2 the remaining 45 LncRNAs for a total of 86 LncRNAs that were analyzed. (B) Clustergram of the four selected inflammatory LncRNAs (LINC00657, RP11-10K16.1, RP11-347E10.2, and SNHG12). On the columns the acronyms of the obese and normal-weight subjects, on the lines the names of the LncRNAs; green represents down-regulation, red represents up-regulation. (C) Dynamic heat-map graph of LINC00657, RP11-10K16.1, RP11-347E10.2, and SNHG12 in the obese and control group. On the column are the number of obese and normal-weight samples. Red square: down-regulation, blue square: up-regulation.

Figure 2

Heatmap comparing the percentage of samples that were expressing (A, up-regulation)/not expressing (B, down-regulation) LncRNA out of 7 normal-weight and 10 obese subjects.

Figure 3

The relative expression of LncRNAs (A) LINC00657, (B) RP11-10K16.1, (C) RP11-347E10.1, and (D) SNHG12 in the normal-weight (red bar) and the obese (cyan bar) subjects.

Figure 4

Simple regression analysis: (A) Ln SNHG12 vs. Ln RP11-10K16.1; (B) LINC00657 vs. BMI-z score; (C) LINC00657 vs. HOMA-IR; and (D) RP11-10K16.1 vs. insulin split by gender. Light red circle: female; cyan triangle: male.

Figure 5

Simple regression analysis: (A) Ln SNHG12 vs. Ln RP11-10K16.1; (B) LINC00657 vs. BMI-z score; (C) LINC00657 vs. HOMA-IR; and (D) RP11-10K16.1 vs. insulin split by obesity. Light red circle: normal-weight adolescents; cyan triangle: obese adolescents.

Figure 6

The relative expression of circulating miRNA (A) miR-445; (B) miR-199a; (C) miR-223; (D) miR-33a; (E) miR-142; and (F) miR-181a in normal-weight and obese subjects that were split by gender. Normal-weight females (red bar); normal-weight males (cyane bar); obese females (light red bar); obese males (light cyane bar).