Deregulation of exosomal miRNAs in rheumatoid arthritis patients

Abstract

Exosomes are small-diameter endosomal vesicles secreted in all biological fluids and play biological/pathological roles in the cell. These pathological roles are played by exosome's cargo molecules through inter-cellular communication. Exosomal cargo molecules contain proteins and miRNAs. miRNAs are small non-coding RNA fragments involved in the reduction of final protein output by destabilizing or suppressing the translation of target messenger RNA (mRNA). This deregulation of the protein due to miRNAs ultimately accelerates the process of disease pathogenesis. The role of exosomal miRNAs has been investigated in different diseases and the limited number of studies have been published concerning exosomal miRNAs and rheumatoid arthritis (RA). The current study is designed to investigate the role of exosomal miRNAs (miRNA-103a-3p, miRNA-10a-5p, miRNA-204-3p, miRNA-330-3p, and miRNA-19b) in the pathogenesis of RA. Furthermore, the role of selected exosomal miRNAs in RA pathogenesis was further explored by estimating oxidative stress and histone deacetylation in RA patients. In the current study, 306 RA patients and equal numbers of age/gender-matched controls were used. The level of expression of above-mentioned exosomal miRNAs was assessed by performing qRT PCR. Deacetylation and oxidative stress assays were performed to estimate the 8-hydroxydeoxyguanosine (8-OHdG level) and histone deacetylation levels using the Enzyme-linked immunosorbent assay (ELISA). Statistical analysis indicated a significantly downregulated expression of miRNA-103a-3p (p<0.0001), miR-10a-5p (p<0.0001), miR-204-3p (p<0.0001), miR-330-3p (p<0.0001) and miR-19b (p<0.0001) in RA patients compared to controls. Significantly increased levels of 8-OHdG (p<0.0001) and histone deacetylation (p<0.0001) were observed among RA patients compared to controls. Spearman correlation showed a negative correlation between the deregulated exosomal miRNAs and increased oxidative stress and histone deacetylation in RA patients. Receiver operating characteristics (ROC) curve analysis showed a good diagnostic specificity/sensitivity of the above-mentioned exosomal miRNAs among RA patients. These analyses indicated the potential role of deregulated exosomal miRNAs in the initiation of RA by targeting oxidative stress and histone deacetylation processes.

Fig 1

Characterization of exosomes extracted from RA patients and controls (A) Determination of exosomes size using DLS (B) Characterization of exosomes using TEM.

Fig 2. Expression of exosome surface markers.

(A) Relative Expression of CD9, CD63, and CD81 in RA patients and controls. (B) Association of expression of exosome surface markers with demographic parameters of RA patients such as age, gender, and anti-CCP level. (C) Association of expression of exosome surface markers with pathological parameters of RA patients such as ESR level and CRP level and treatment. Pg/ml (picogram/mililitre).

Fig 3. Expression analysis of exosomal miRNA-103a-3p in RA patients.

(A) The relative expression level of miRNA-103a-3p in RA patients vs controls. (B) Association of relative expression of miRNA-103a-3p with demographic/pathological parameters of RA patients such as age, gender, anti-CCP level, ESR level, and CRP level. (C) ROC curve analysis of miRNA-103a-3p in RA disease. Level of significance p<0.05.

Fig 4. Expression analysis of exosomal miRNA-10a-5p in RA patients.

(A) The relative expression level of miRNA-10a-5p in RA patients vs controls. (B) Association of relative expression of miRNA-10a-5p with demographic/pathological parameters of RA patients such as age, gender, anti-CCP level, ESR level, and CRP level. (C) ROC curve analysis of miRNA-10a-5p in RA disease. Level of significance p<0.05.

Fig 5. Expression analysis of exosomal miRNA-204-3p in RA patients.

(A) The relative expression level of miRNA-204-3p in RA patients vs controls. (B) Association of relative expression of miRNA-204-3p with demographic/pathological parameters of RA patients such as age, gender, anti-CCP level, ESR level, and CRP level. (C) ROC curve analysis of miRNA-204-3p in RA disease. Level of significance p<0.05.

Fig 6. Expression analysis of exosomal miRNA-330-3p in RA patients.

(A) Relative expression level miRNA-330-3p in RA patients vs controls. (B) Association of relative expression of miRNA-330-3p with demographic/pathological parameters of RA patients such as age, gender, anti-CCP level, ESR level, and CRP level. (C) ROC curve analysis of miRNA-330-3p in RA disease. Level of significance p<0.05.

Fig 7. Expression analysis of exosomal miRNA-19b in RA patients.

(A) Relative expression level miRNA-19b in RA patients vs controls. (B) Association of relative expression of miRNA-19b with demographic/pathological parameters of RA patients such as age, gender, anti-CCP level, ESR level, and CRP level. (C) ROC curve analysis of miRNA-19b in RA disease. Level of significance p<0.05.

Fig 8. Association of treatment modalities such as methotrexate and biologics with relative expression of selected exosomal miRNA such as miRNA-103a-3p, miRNA-10a-5p, miRNA-204-3p, miRNA-330-3p, and microRNA-19b.

Level of significance p<0.05.

Fig 9. Measurement of oxidative stress and histone deacetylation in RA patients and controls.

(A) 8-OHdG level in RA patients vs controls. (B) Association of 8-OHdG with demographic/pathological parameters of RA patients such as age, gender, anti-CCP level, ESR level, and CRP level. (C) ROC curve analysis of 8-OHdG in RA disease. (D) Histone deacetylation level in RA patients vs controls. (B) Association of histone deacetylation level with demographic/pathological parameters of RA patients such as age, gender, anti-CCP level, ESR level, and CRP level. (C) ROC curve analysis of histone deacetylation in RA disease. AUC = area under the curve; Level of significance p<0.05.