Plasma Humanin and Non-Coding RNAs as Biomarkers of Endothelial Dysfunction in Rheumatoid Arthritis: A Pilot Study

Abstract

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disorder associated with an increased risk of cardiovascular disease (CVD), largely driven by peripheral endothelial dysfunction (ED). Humanin, a mitochondrial-derived peptide, has been suggested to play a protective role in endothelial function. However, the relationship between Humanin levels and ED in RA, as well as the interaction between Humanin and non-coding RNAs such as Long Non-Coding RNA GAS5, microRNA-21 (miR-21), and microRNA-103 (miR-103), remains unclear. Objective: This study aimed to investigate the relationship between circulating Humanin levels, non-coding RNAs (GAS5, miR-21, miR-103), and endothelial dysfunction (ED) in patients with RA. Additionally, we explored the correlation between Humanin expression and specific non-coding RNAs (GAS5, miR-21, and miR-103) to better understand their potential role in vascular health. Methods: Peripheral ED was assessed using flow-mediated pulse amplitude tonometry, with Ln-RHI values <0.51 indicating dysfunction. Humanin levels, GAS5, miR-21, and miR-103 were measured in RA patients. Univariate and multivariate analyses were conducted to determine the relationship between these biomarkers and ED. Kaplan-Meier survival analysis and ROC curve analysis were used to assess the prognostic value of Humanin. Results: Higher Humanin levels were significantly associated with better endothelial function (OR = 0.9774, p = 0.0196). Kaplan-Meier analysis demonstrated that higher Humanin levels correlated with improved survival (p < 0.0001). The non-coding RNAs (GAS5, miR-21, and miR-103) did not show significant associations with ED. Conclusions: Humanin is a potential protective biomarker for endothelial dysfunction and survival in RA patients. Further research is needed to explore the interaction between Humanin and non-coding RNAs in the context of vascular health.

Keywords: biomarkers; cardiovascular risk; endothelial dysfunction; humanin; long non-coding RNA; miRNA; rheumatoid arthritis.

Figure 1

miRNA expression in plasma and exosomes. The expression of miR-21 Panels (A) and (B) and miR-103 Panels (C) and (D) was evaluated in plasma and exosomes. mRNA levels were normalized to U6snRNA. The data are presented as the mean ± SD relative to the control (* p ≤ 0.05, ** p ≤ 0.01, and **** p ≤ 0.0001).

Figure 2

Lnc-RNA GAS5 expression in plasma (A) and (B). Expression levels were normalized to Glyceraldehyde-3-Phosphate-Dehydrogenase (GAPDH). The data are presented as the mean ± SD relative to the control (**** p ≤ 0.0001).

Figure 3

Humanin levels in plasma were determined by ELISA. Data are presented as mean ± SD relative to control (**** p ≤ 0.0001).

Figure 4

ROC curve analysis for Humanin’s predictive value for ED.

Figure 5

Kaplan–Meier survival analysis to evaluate the prognostic value of Humanin levels for survival outcomes. 0 = patients with serum Humanin concentration < 124.44pg/mL; 1 = patients with serum Humanin concentration ≥ 124.44pg/mL.

Figure 6

The Kaplan–Meier survival curve showing the survival probabilities for the two groups based on the ROC-determined Humanin levels. Group 0 (blue): Higher survival probability. Group 1 (red dashed): Lower survival probability. The number at risk at different time points is detailed in Table 5.