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Abstract
Background MicroRNAs (miRNAs) can regulate gene expression, controlling numerous cellular processes. Dysregulation of miRNA function is linked to various diseases, making them attractive diagnostic and therapeutic targets. Examples include hsa-miR-92a-3p, hsa-miR-126-3p, hsa-miR-143-3p, hsa-miR-145-5p and hsa-miR-204-5p, which are associated with endothelial function. Their prevalence in Sjögren’s disease (SjD) is unknown. We assessed the prevalence of these miRNAs in serum of patients with SjD, correlating levels with cardiovascular risk factors and carotid intima-media thickness (cIMT) to evaluate their utility in risk stratification.
Methods 199 patients with SjD and 100 age and sex-matched healthy controls (HC) were included in the study. Five different miRNAs (hsa-miR-92a-3p; hsa-miR-126-3p; hsa-miR143-3p; hsa-miR-145-5p; hsa-miR-204-5p) were analysed by quantitative real-time PCR. The miRNA results were compared with known clinical and disease-related parameters.
Results Four miRNAs showed significantly different expressions compared with HC. MiR-92a-3p was upregulated (p=0.025) and miR-126-3p (p=0.044), miR-143-3p (p=0.006) and miR-204-5p (p=0.009) downregulated in SjD compared with HC. The comparison between HC and SjD with/without organ involvement revealed descriptively increased miR-92a-3p levels in patients with SjD with organ involvement (p=0.087). Furthermore, miR-92a-3p levels correlated positively with cIMT as an expression of subclinical atherosclerosis (r=0.148, p=0.04).
Conclusion In conclusion, patients with SjD demonstrated differences in their expression of miRNAs linked to regulation of endothelial function. Reduction of specific miRNAs was associated with increased cardiovascular risk, suggesting a potentially protective role for these miRNAs. Furthermore, miR-92a-3p could be helpful for molecular detection of early-stage atherosclerosis and increased cardiovascular risk in SjD.
- Sjogren's Syndrome
- Atherosclerosis
- Cardiovascular Diseases
Data availability statement
Data are available upon reasonable request. Not applicable.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
MicroRNAs (miRNAs) regulate gene expression and are involved in various cellular processes, including disease-specific pathomechanisms. Specific miRNAs are associated with cardiovascular risk, but their role in Sjögren’s disease (SjD) has not been studied.
WHAT THIS STUDY ADDS
The study identified an upregulation of miR-92a-3p, which has been described previously as ‘athero-miR’, while miR-126-3p, miR-143-3p and miR-204-5p were found to be downregulated in patients with SjD. Additionally, the study demonstrated a weak, though positive correlation between miR-92a-3p levels and increased carotid intima-media thickness, suggesting a potential association with subclinical atherosclerosis.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICY
This study provides preliminary evidence that miR-92a-3p may serve as an indicator for cardiovascular risk in patients with SjD. However, further research and clinical practice are necessary to ascertain the potential benefits of miRNA analysis for cardiovascular disease management in SjD.
Introduction
MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression.1 These tiny molecules function by binding to target messenger RNAs, resulting in translation suppression or degradation of messenger RNA.1 MiRNAs are involved in various cellular processes, including development and disease-related pathomechanisms.2 3 Depending on the disease, specific miRNAs may be upregulated or downregulated, which can help to diagnose and treat several diseases.2 4
Previous studies have identified numerous miRNAs associated with increased cardiovascular risk.5–8 These include the endothelial-expressed miRNA hsa-miR-92a-3p, also known as an ‘atheromiR’ because of its atherosclerosis-promoting properties.9 10 Similarly, miRNAs hsa-miR-126-3p and hsa-miR-143-3p are endothelial miRNAs involved in vascular smooth muscle cell (VSMC) differentiation.11 12 MiRNAs hsa-miR-145-5p and hsa-miR-204-5p are known to affect VSMC proliferation and migration.13 14 For all five miRNAs, an association with the development of atherosclerosis and cardiovascular risk in general was demonstrated.
The miRNAs mentioned above associated with cardiovascular diseases have never been studied in patients with Sjögren’s disease (SjD). SjD is a common autoimmune connective tissue disease, with a worldwide prevalence of 1:2000 and is defined by lymphocytic inflammation of exocrine glands leading to xerophthalmia and/or xerostomia.15 In more than 50% extraglandular manifestations, like interstitial lung diseases or neurological manifestations, occur.16–18 A number of studies have demonstrated an increased cardiovascular risk for patients with SjD. However, the risk seems lower than that observed in patients with other autoimmune inflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus.19 20 Conversely, there are also a small number of studies that have yet to show a direct link between SjD and cardiovascular events.21 Based on the cohort used in this study, we were already able to demonstrate an increased cardiovascular risk in patients with SjD.22 They had significantly thicker carotid intima-media thickness (cIMT) and therefore more subclinical atherosclerosis than healthy subjects.22
Cardiovascular risk factors like arterial hypertension are more common among patients with SjD,23 but no biomarker is known yet to help stratifying cardiovascular risk for these patients. Therefore, we aimed to evaluate the five miRNAs known to be associated with atherosclerosis in the serum of patients with SjD, correlate them with cardiovascular parameters and investigate their suitability for cardiovascular risk stratification.
Methods
Patient data
For the evaluation of the miRNAs, we used a prospective monocentric cohort including 199 patients with SjD and 100 age-matched and sex-matched healthy controls (HC). This very well-characterised cohort has been described previously in a study demonstrating an increased risk of subclinical atherosclerosis in patients with SjD.22 Patients were enrolled during their regular visits to the rheumatology outpatient clinic or the interdisciplinary outpatient infusion clinic of the Hannover Medical School between September 2021 and March 2022. The HC were recruited in a 2:1 ratio via multimedia call.
All patients with SjD fulfilled the 2016 American College of Rheumatology/European Alliance of Associations for Rheumatology criteria and were symptomatic for at least 5 years at the time of the study.24 The SjD group comprised only patients with SjD with no other systemic autoimmune or inflammatory diseases or overlap syndromes. A structured and standardised questionnaire was completed for each study participant during an interview. Organ involvement encompassed manifestations in the lungs, kidneys, skin and liver, in addition to vasculitis, myositis and involvement of the peripheral and central nervous systems. The following exclusion criteria were defined for all participants: known manifest atherosclerotic terminal diseases, for example, myocardial infarction or stroke, cancer diseases in the past 5 years or currently existing pregnancy. For a more detailed description of our cohort, we refer to the study by Zehrfeld et al.22
The Carotid ultrasound was performed using GE LOGIQ P9 and an optimised preset profile for the 10 MHz linear transducer. cIMT was manually measured three times at the posterior arterial wall one centimetre proximal to the carotid bulb by two independent angiologists in a blinded fashion after the physical examination, following the Mannheim Consensus recommendations.25 The mean was calculated and considered pathological in age-adjusted gradations according to Chambless et al.26
All participants gave written informed consent. The study was approved by the local authorities (Institutional Review Board of Hannover Medical School approval (8179_BO_S_2018)) and conducted in accordance with the Declaration of Helsinki.27
RNA Isolation
Blood was collected from all 299 participants in a serum monovette. The blood was then centrifuged at 2000g for 10 min at room temperature to separate the serum from the cellular components of the blood. Next, the serum was transferred to RNAse/DNAse clean tubes and frozen at −80°C. The serum tubes were first thawed on ice to isolate RNA. Subsequently, according to the manufacturer’s instructions, 30 µl of RNA solution was prepared from each 200 µl of serum using the miRNeasy Serum/Plasma Advanced Kit from Qiagen. During RNA isolation, Caenorhabditis elegans miR-39 (c.elegans) was added as a spike-in control to normalise the results.
Reverse transcription and real-time PCR-based amplification of miRNAs
The isolated RNA was then transcribed into complementary DNA. This was carried out using the TaqMan microRNA Reverse Transcription Kits (Applied Biosystems) for hsa-miR-92a-3p (Assay ID: 000431), hsa-miR-126-3p (Assay ID: 002228), hsa-miR-143-3p (Assay ID: 002249), hsa-miR-145-5p (Assay ID: 002278), hsa-miR-204-5p (Assay ID: 000508) and cel-miR-39 (control; Assay ID: 000200) primers according to the manufacturer’s instructions. The primer sequences can be found in online supplemental data 1. The miRNAs were then amplified by qRT-PCR using TaqMan microRNA assays (Applied Biosystems). To avoid potential inaccuracies in the PCR, a replicate analysis was performed for all samples and each miRNA. If the two values differed significantly, the measurement was repeated.
Supplemental material
Statistics and graphical illustration
Dichotomous variables were tested using χ2 or Fisher’s exact test. Normal distribution of metric variables was tested using the Shapiro-Wilk and Kolmogorov-Smirnov tests. Normally distributed metric variables were then compared using a Student’s t-test, and non-normally distributed variables were compared using a Mann-Whitney U-test. Kruskal-Wallis test was used for multiple comparison tests of miRNAs between HC and different SjD subgroups. Hierarchical linear models were used for analysis due to the hierarchical data structure (two cIMT points per person: left and right).
Results are presented as mean±SD for illustrative presentation.
Values beyond the mean±3 SDs were identified as outliers. These values exhibited a markedly elevated c.elegans CT value, indicating an ineffective RNA isolation process and therefore did not meet quality standards. Consequently, they were excluded from further analysis. A maximum of four outliers were identified per miRNA.
All statistical analyses were performed with Statistical Package for the Social Science, V.28.0 (IBM SPSS), R V.4.2.1 with the R-Studio IDE and GraphPad Prism 9.0.1 (GraphPad). Statistical significance was defined by a p value <0.05. Figures were created using GraphPad Prism V.9.0.1 (GraphPad) and R V.4.2.1 with the R-Studio IDE.
Results
The n=199 SjD cohort and the n=100 age-matched and gender-matched control cohort included within the study showed no significant differences in terms of demographic characteristics or cardiovascular risk factors, apart from a positive family history of cardiovascular events (online supplemental data 2). This was significantly more common in patients with SjD.
Supplemental material
The comparison of miRNA levels between patients with SjD and HCs shows that four out of five miRNAs are differentially expressed (figure 1). A comparison of the exact miRNA levels is provided in table 1. MiR92a-3p is significantly upregulated in patients with SjD (12.2 (10.5–13.3) vs 11.5 (9.9–12.9); p=0.025), whereas miR126-3p is significantly downregulated (11.4 (10.1–12.9) vs 12.0 (10.9–13.5); p=0.044). The levels of miR143-3p and miR204-5p are also significantly reduced in patients with SjD compared with HC (7.2 (6.0–8.8) vs 8.0 (6.2–10.5); p=0.006 and 5.1 (3.8–6.7) vs 6.0 (4.4–8.0); p=0.009). No significant differences were found for miR145-5p (6.7 (5.5–8.1) vs 7.1 (5.3–8.7); p=0.669).
Comparison of miRNA levels in healthy controls (HC) and patients with Sjögren’s disease (SjD). The line shows the median, the surrounding box displays the IQR and the whiskers mark the minimum/maximum. a=normalised to c. elegans.
Overview of microRNAs (miRNA) levels in patients with Sjögren’s disease (SjD) and healthy controls (HC)
Comparing patients with SjD with and without organ involvement, miR-92a-3p was found to be descriptively increased in patients with SjD with organ involvement (12.3 (11.2–13.8) vs 11.7 (10.1–13.2); p=0.087). For the remaining miRNAs 126-3p, 143-3p, 145-5p and 204-5p, no differences were found between these subgroups (figure 2).
Comparison of MicroRNAs (miRNAs) in healthy controls (HC), patients with Sjögren’s disease (SjD) with and without organ involvement. The line shows the median, the surrounding box displays the IQR and the whiskers mark the minimum/maximum. a=normalised to c. elegans.
The correlation between cIMT and the relative expression of miR-92a-3p for all 199 patients with SjD (r=0.148, p=0.040, R2=0.009) is visualised in figure 3 and underlines a significant positive correlation between the two parameters can be seen: increasing serum miR-92a-3p levels are associated with higher cIMT values. Additionally, a median split was calculated, as illustrated in online supplemental data 3, which yielded consistent results.
Supplemental material
Scatter plot showing the correlation between carotid intima-media thickness (cIMT) (mm) and the relative expression of miR-92a-3p (normalised to c.elegans), in patients with Sjögren’s disease (n=199). Each person is represented by two dots (left and right cIMT) and a linear regression line was calculated for illustration purposes. r=0.148, p=0.040, R2=0.009.
Discussion
To our knowledge, this is the first study evaluating endothelial-associated miRNAs in patients with SjD aiming to identify patients at risk of developing atherosclerosis or other cardiovascular risk factors. It is known that patients with SjD face an increased susceptibility to cardiovascular diseases such as coronary heart disease, myocardial infarction and stroke as demonstrated by Bartoloni et al.20 28 In a recently published study of the same cohort of patients, we have already shown a significant increase in subclinical atherosclerosis in patients with SjD. A clear difference in cIMT was observed, with patients with SjD having significantly increased cIMT bilaterally. Now we can show more evidence of an altered endothelial function in patients with SjD by upregulated or downregulated miRNAs, which are known to be associated with the development of atherosclerosis.
Biomarkers are becoming increasingly important for diagnosis, individualised risk stratification and therapeutic approaches. In SjD, anti-Ro/SSA-ab and anti-La/SSB-ab are of particular clinical relevance.29 Apart from other specific antibodies, miRNAs in general represent promising biomarkers due to their stability and diversity.
Examining miRNA regulation, miR-92a-3p was found to be upregulated in patients with SjD, including those with organ involvement. Previous research has demonstrated endothelial expression of miR-92a, with increased levels linked to reduced vascular flow, ischaemia and tissue necrosis.9 In a mouse model of ischaemia, inhibiting this miRNA led to reduced tissue necrosis and significantly improved blood flow.9 While expression data from blood samples may not directly translate to the cellular level, the elevated blood-based miR-92a levels suggest more tissue damage or a greater burden of atherosclerosis. This interpretation is supported by the corresponding cIMT data, indicating that miR-92a could serve as both a risk marker for atherosclerosis and a potential therapeutic target for affected patients. This is underlined by other studies labelling miR-92a as ‘atheromiR’.10 30
In another in vivo model, inhibiting miR-92a also showed a clear improvement in endothelial function, leading to a reduction in atherosclerosis. In line with this, a study involving patients with essential hypertension revealed a notable increase in plasma miR-92a among individuals with elevated cIMT.31 This strengthens the hypothesis that miR-92a serves as an indicator of atherosclerosis in patients with SjD, especially in the carotid arteries. MiRNA-92a has also been identified as a risk marker for acute coronary syndrome. In a study by Wang et al32, elevated levels of miRNA-92a were predictive of acute coronary syndrome in patients with diabetes.32 A similar correlation, but independent of SjD, was found by Chen et al. MiRNA-92a was significantly higher in patients with asymptomatic carotid stenosis and served as an independent predictor of cerebrovascular events.33
In contrast, a study from 2010 indicated that patients with coronary artery disease (CAD) had lower plasma levels of miR-92a, although carotid arteries were not specifically considered. It is theorised that patients with severe CAD have a high uptake of miR-92a within vascular lesion, leading to reduced plasma concentrations.34 Another study on SjD found no detectable miR-92a-5p in plasma, but no correlation to cardiovascular diseases, risk factors or clinical investigation was investigated in this study.35 However, this study did not focus on miR-92a-3p, which was our primary focus. These two distinct miRNA strands can serve entirely different functions after processing from pre-miRNA, explaining why the data do not contradict each other.
Examining miR-126-3p, miR-143-3p and miR-204-5p, the levels of these miRNAs were lower in patients with SjD compared with the HC group.
Also, miR-126-3p is a miRNA associated with endothelial function and angiogenesis.11 It is described that miR-126 is linked to reduced pathological endothelial processes, particularly inflammation.36 This is coherent with our observations. While levels in unaffected HC’s appear normal, patients with SjD present lower levels, indicating more inflammation in the vascular system, which corresponds with diminished miR-126 levels.36 Our findings are consistent with the observation that elevated levels of miR-126 result in a decrease in reactive oxygen species. Consequently, these reduced levels account for the elevated oxidative stress experienced by endothelial cells, their inclination towards inflammation and the associated tendency towards atherosclerosis.37 38
Consistent with the results of Rohzkov et al (2022), miR-143-3p levels were decreased in patients with SjD. Studies demonstrated that plasma miR-143 levels were lower in patients with more extensive atherosclerosis compared with those with less plaque.12 Additionally, De-Gonzalo et al (2020) found that miR-143 inversely correlated with the severity of coronary heart disease and, consequently, with the time until a cardiovascular event in CAD. This resulted in a positive correlation between miR-143 and a lower cardiovascular risk profile.39 MiR-143 was further identified as a predictive marker for in-stent restenosis: low miR-143 levels correlated with the occurrence of in-stent restenosis in lower extremity arterial occlusive disease.40 The reduction of both miR-143 and miR-145 was observed in patients undergoing therapeutic angioplasty, leading to a significant endothelial damage.41 42 Summing up previously published data, the decreased levels of miR-143 in our patients with SjD suggest a tendency towards inflammation.
Furthermore, miR-204-5p was also found to be reduced in patients with SjD, which aligns with prepublished data and indicates an increased susceptibility to vascular inflammation. Previous studies have demonstrated that miR-204 is reduced in atherosclerotic tissue, and its overexpression has a therapeutic effect by reducing VSMC proliferation and migration.14 43
In contrast, miR-145-5p showed no significant differences between control and patients with SjD. It has been described that miR-145-5p might have a protective effect on blood vessels by decreasing smooth muscle cells proliferation.13
Despite the fact that we have analysed a large and detailed cohort, it remains a monocentric study. As this study was conducted in a university hospital, we are also affected by a selection bias due to university medicine, resulting in a higher proportion of patients with more severe manifestations of SjD. Furthermore, the control group was recruited through a multimedia call, which may not be representative of the general healthy population. Another common problem with this type of study is the fact that serum measurements provide only limited information about cellular processes. However, serum measurements are much easier to integrate into patient care and risk stratification. Further prospective studies are required to evaluate whether miRNA might have any prognostic or diagnostic value.
Several questions remain open, such as: Should we treat patients with SjD at risk preventively with statins? How do miRNA levels change over the course of disease?
Conclusion
In summary, our findings highlight that patients with SjD face a significantly elevated risk of endothelial inflammatory processes. This is evidenced by the reduced levels of endothelial-protective miRNAs (miR-126-3p, miR-143-3p and miR-204-5p). Furthermore, the miRNA miR-92a, which is associated with cellular inflammation and atherosclerosis, was increased in patients with SjD and correlated with changes in intima-media thickness. It may serve as a potential circulating risk marker for vascular involvement. Nevertheless, further large-scale investigations with precise clinical endpoints are needed to evaluate the potential of this miRNA as a proatherosclerotic biomarker.
Data availability statement
Data are available upon reasonable request. Not applicable.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by Institutional Review Board of Medical University Hannover approval (8179_BO_S_2018). Participants gave informed consent to participate in the study before taking part.
References
Supplementary materials
Supplementary Data
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Footnotes
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Contributors Conceptualisation and writing - original draft preparation: MA, NZ, AAD and DE. Methodology: NZ, MA, SB, DE and AAD. Formal analysis and investigation: MA, NZ, SB and TS. Writing - review and editing: all authors. Resources: DE, TW, TS and TT. Supervision: DE, TW, AAD, CB and TT. All authors read and approved the final manuscript. Guarantor: DE.
Funding Else Kröner Fresenius Foundation and research grants from Novartis. Novartis did not contribute to the study design, analyses or data interpretation. AAD was supported by PRACTIS—Clinician Scientist Program of Hannover Medical School, funded by the German Research Foundation (DFG, ME 3696/3-1).
Competing interests Novartis did not contribute to the study design, analyses or data interpretation.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.