Independent validation of circulating microRNAs as biomarkers in a case-control study of adolescents with type 1 diabetes for more than 8 years. PLoS One
Swolin-Eide D, Pundziute Lyckå A, Forsander G, Novak D, Grillari J, Diendorfer AB, et al.
PLoS One 21(2):e0343117. https://doi.org/10.1371/journal.
Abstract
MicroRNAs (miRNAs) are epigenetic regulators of gene activity. Analysis of circulat-
ing miRNAs enables minimal-invasive studies of disease mechanisms and identifica-
tion of novel disease biomarkers.
The aim of this case-control validation study was
to investigate previously identified circulating miRNAs in Swedish adolescents with
long-duration (8.0–16.5 years) type 1 diabetes (T1D), and healthy matched controls
to confirm their utility as biomarker candidates to diagnose and monitor the pro-
gression of T1D. Quantitative PCR analysis of 23 previously reported miRNAs was
performed in 24 T1D and 24 control individuals. Body composition was assessed by
dual-energy X-ray absorptiometry and peripheral quantitative computed tomography.
Prospectively collected clinical data were retrieved from the Swedish diabetes quality
registry. The selected miRNAs showed higher variability in both male and female
T1D groups compared to controls. Statistical analysis confirmed differences for 12
miRNAs in comparison with controls, including miR-223-3p and miR-135a-5p, which
previously were reported to be associated with T1D. MiR-34a-5p and miR-210-3p
were positively associated with T1D duration and HbA1c (average from the last
year), respectively. In conclusion, 12 previously reported miRNAs showed consistent
differential expression between individuals with T1D and controls. Among these were
miR-223-3p and miR-135a-5p, which are associated with cardiovascular/inflamma-
tory disease and cancer, respectively.
These findings suggest potential clinical utility
of circulating miRNAs for T1D diagnosis and disease monitoring, although extended
validation of the identified miRNA biomarkers in larger, independent cohorts is
required to establish the necessary scientific evidence for clinical translation.
Utdrag ur artikeln
Discussion
This case-control validation study of previously explored miRNAs [10], confirmed that miR-135a-5p and miR-223-3p are
up-regulated in long-duration T1D in adolescents in comparison with healthy controls. In addition, six other miRNAs were
differently regulated (adjusted p-value <0.05) in comparison with control individuals, of which miR-210-3p and miR-34a-5p
showed significant associations with HbA1c levels (average from last year) and diabetes duration, respectively. Four
additional miRNAs displayed adjusted p-values between 0.05 and 0.1 and therefore have slightly higher probability for
false-discoveries. Therefore, the discussion of results will focus on miRNAs with the lowest adjusted p-values.
Several studies have identified miR-135a as a treatment target in diabetic nephropathy for renal fibrosis [25,26]. The
observed upregulation of miR-135a in this validation study, as well as in our previous case-control discovery study [10],
suggests a regulatory mechanism for miR-135a that could influence the development of renal fibrosis in T1D already
during adolescence. MiR-135a is also of importance for reprogramming acinar cells into insulin producing cells [27], which
implies a therapeutic potential for miR-135a in diabetes.
The higher cancer incidence among individuals with diabetes has been recognized for many decades [28,29]. A 2021
epidemiological analysis, comprising 313,907 matched individuals with and without diabetes, concluded a decline in
vascular complications and that cancer now is the leading cause of diabetes-related death [30]. Well-powered studies
have confirmed the link between cancer and cancer-related mortality in T1D, supporting further investigation of miRNAs
as biomarkers and therapeutic targets [31,32]. Indeed, dysregulation of miR-135a can result in various forms of cancer,
and it has been reported that miR-135a mediates cell proliferation and cancer progression through the MAPK and JAK2/
STAT3 signaling pathways [33]. Several studies have suggested targeting miR-135a in cancer-related therapy to improve
the outcome for individuals with cancer [34].
As recently reported, the plasma level of miR-223 was significantly higher in T1D compared to controls in our discovery
study [10], which could be confirmed with this larger cohort of adolescents with T1D. The transcription of miR-223 is very
high in hematopoietic cells, and is associated with autoimmunity, T1D, T2D, inflammation, obesity and diabetic microvas-
cular complications [24,35]. The review by Gangwar et al. [36] demonstrated that miR-223 is associated with hypertension
and atherosclerotic disease. A review on miR-223 and cancer development concluded that miR-223 has multiple estab-
lished roles in hematopoiesis, inflammation, and several cancer types, where miR-223 functions as either an oncogenic or
oncosuppressive miR [37].
MiR-34a-5p, a known biomarker of T2D and metabolic disease including metabolic dysfunction-associated steatotic
liver disease (a.k.a. MASLD), was found to be lower in T1D but associated with diabetes duration. MiR-34a-5p, a member
of the miR-34 family, is implicated in various cardiovascular pathologies, including myocardial infarction, heart failure, and
atherosclerosis. It modulates key processes such as apoptosis, autophagy, inflammation, senescence, and remodeling
by targeting signaling pathways like Smad4/TGF-β1, FOXO3/PUMA, Notch1/ETBR, PTEN/PI3K/SIRT1, and FOXM1/
NRF2/HO-1 [38]. In line with this, Mone et al. [39] concluded that “miR-34 may be linked to diabetes and endothelial
dysfunction”, proposing it as a potential biomarker of frailty in diabetic older adults, and highlighting its role in oxidative
stress as a promising drug target for novel therapies.
The meta-analysis by Margaritis et al. [11] reported that miR-210-3p is higher in individuals with T1D, but associated
to the average HbA1c level during the previous year. Their conclusion was, however, only based on two articles [40,41].
Osipova et al. [41], explored miR-210 in 68 individuals, aged 6–18 years, with a duration of T1D of at least 1 year (mean
5.0 years). They reported up-regulated miR-210 plasma levels in comparison with age- and sex-matched controls, which
is in contrast to our findings. Nielsen et al. [40], investigated miR-210 in serum samples from newly diagnosed children
with T1D and found up-regulated miR-210 levels. These opposing results, regarding up- or down-regulation in comparison
with the current study, could be due to differences in T1D duration (between 8.0 to 16.5 years in this cohort), or differ-
ences in age. In addition, differences in the miRNA baseline levels between serum and EDTA-plasma samples, which are
caused by miRNA release during blood clotting [42], could also contribute to such discrepancies.
The observed sex differences in circulating miRNA levels may reflect underlying biological distinctions between males
and females during adolescence, including sex-hormone effects on gene regulation and immune function. Pubertal
stage and circulating sex steroids can alter miRNA expression in multiple tissues and metabolic pathways, which may be
reflected by serum miRNA profiles.
This study was powered based on previous findings and specifically designed to validate or refute the association of
selected circulating miRNAs with T1D (Table 2). The long diabetes duration, between 8.0 to 16.5 years, in a narrow age
span with registered metabolic data is a strength in this study. The study groups were well-matched regarding age and
sex, and body composition did not differ between the groups. Strict exclusion criteria were applied, which resulted in a
homogenous group of individuals with T1D. Validated state-of-the art RT-qPCR protocols [12,13] were used for analysis
of circulating miRNAs. Known sources of methodological variability such as hemolysis, RT-qPCR inhibition, and lack of
homogenous RNA recovery [43] were successfully controlled, which resulted in high-quality data with low analytical vari-
ability suitable for assessing miRNA variability in the context of T1D.
The present study also has limitations. A larger study group, including a wider age span and individuals with obesity,
coeliac disease and hypothyroidism, i.e., conditions that frequently co-occur with T1D, would have been desirable to
increase the generalizability to the target patient population. Considering that the research topic of TID and miRNAs is
novel, with specific knowledge gaps and challenges, there might be yet unidentified miRNAs not included in this study.
Conclusions
In this clinical case-control validation study, involving a well-matched cohort of young individuals with long-standing T1D
(between 8.0 to 16.5 years), we demonstrate that the plasma levels of miR-223-3p and miR-135a-5p are elevated. Given
the established links of miR-223-3p to cardiovascular and inflammatory disease, and miR-135a-5p to cancer, these
findings suggest that both miRNAs may serve as promising biomarkers for the early identification of patients at increased
risk of diabetes-related complications and malignancies.
Future larger-scale, prospective studies incorporating a broader
validation of the proposed miRNA biomarkers in independent study populations is still needed to generate sufficient scien-
tific evidence for their use in clinical practice. Particular attention should be paid to minimizing pre-analytical and analyt-
ical variability in sample collection and processing to ensure the reliability of miRNA measurements in broader clinical
applications.
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