Association of Long Non-Coding RNA Malat1 with
Serum Levels of Interleukin-1 Beta and Vitamin D
in Patients with Ischemic Stroke
Mahnaz Bayat 1, Reza Tabrizi 2, 3, Mohammad Saied Salehi 1, Najmeh Karimi 1, 4, Moosa Rahimi 5, Etrat Hooshmandi 1,
Niloufar Razavi Moosavi 1, Nima Fadakar 1, 4, Afshin Borhani-Haghighi 1
1 Clinical Neurology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
2 Noncommunicable Diseases Research Center, Fasa University of Medical Science, Fasa, Iran
3 USERN Oce, Fasa University of Medical Sciences, Fasa, Iran
⁴ Department of Neurology, Shiraz University of Medical Sciences, Shiraz, Iran
⁵ Laboratory of Basic Sciences, Mohammad Rasul Allah Research Tower, Shiraz University of Medical Sciences, Shiraz, Iran
GMJ.2023;12:e2457
www.gmj.ir
Correspondence to:
Afshin Borhani Haghighi, MD, Clinical Neurology Re-
search Center, Shiraz University of Medical Sciences,
Shiraz, Iran
Telephone Number:+98-713-6281572
Email Address:neuro.ab@gmail.com
Received 2022-04-24
Revised 2022-05-10
Accepted 2022-05-15
Abstract
Background:Previous studies have demonstrated the strong association of inammatory cyto-
kines and vitamin D (VitD) deciency and ischemic stroke (IS) pathogenesis. Due to the nega-
tive correlation between long non-coding RNA (lncRNA) Malat1 and pro-inammatory factors
we decided to investigate the associations between Malat1 expression with serum interleukin-1β
(IL-1β), and VitD levels in IS patients. Materials and Methods:In this cross-sectional study,
63 IS patients were included. We used enzyme-linked immunosorbent assays to evaluate the
serum levels of VitD and IL-1β. Malat1 expression was evaluated by the real-time polymerase
chain reaction test. The associations between Malat1expression with VitD and IL-1β were ana-
lysed with linear regression (Stepwise model) and Pearson’s correlation analysis. Results: The
Malat1 expression was inversely correlated with stroke severity (r=-0.25, P=0.043). Stepwise
regression analysis showed a signicant positive relationship between VitD level and Malat1
expression (Beta=0.28, P=0.02), and also showed a non-signicant negative relationship be-
tween IL-1β and stroke severity. VitD level showed a positive Pearson correlation with Malat1
(r=0.28, P=0.023) and a negative correlation with IL-1β (r=-0.29, P=0.018) while it could not
detect a signicantly negative correlation with stroke severity. Conclusion: For the rst time
the associations between Malat1 expression with IL-1β and VitD in IS patients was analyzed.
We found a signicant positive relationship between VitD and Malat1. This correlation needs
to be investigated with a larger sample size to achieve a strong and reliable association between
VitD and Malat1.[GMJ.2023;12:e2457] DOI:10.31661/gmj.v12i0.2457
Keywords:Long Non-coding RNA; Malat1; Interleukin-1 Beta; Vitamin D; Ischemic Stroke
Introduction
After heart disease and cancer, stroke is
the leading cause of death.Stroke with a
history of long-term or permanent post-stroke
disability has received extensive clinical re-
search attention. Inammation plays a major
role in various stages of ischemic stroke. A
novel therapeutic target for ischemic brain
cells is represented by the neuroinammato-
ry triangle entailing bursts of reactive oxygen
species (ROS), inammatory cytokines re-
lease, and disruption of the blood-brain bar-
rier (BBB) [1].
GMJ
Copyright© 2021, Galen Medical Journal.
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Email:info@gmj.ir
Bayat M, et al. Association of Malat1 with IL-1β and VitD in Ischemic Stroke
2GMJ.2023;12:e2457
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Optimal management of IS requires rapid as-
sessment of stroke severity using ideal bio-
markers within the rst hours after stroke. The
ideal properties of a stroke biomarker are non
or minimally invasive, quick, cost-eective,
and without interfering with acute therapies
[2, 3].
long non-coding RNAs (lncRNAs) are a class
of RNA transcripts with more than 200 nu-
cleotides that rarely aect protein encoding,
while it has shown essential roles in signal-
ing pathways and gene regulation related to
diseases. lncRNA, metastasis-associated lung
adenocarcinoma transcript 1 (Malat1), has
shown a regulatory role in the pathology of
ischemic stroke.
Previous studies have revealed that Malat1 ln-
cRNA could be a potential biomarker for the
diagnosis as well as prognosis of atheroscle-
rotic cardiovascular disease [4], cancer [5-8],
multiple sclerosis [9], and sepsis [10]. Dif-
ferent experimental and clinical research has
shown the anti-inammatory and anti-apop-
totic roles of Malat1 in the brain [11-13].
The downregulation of lncRNA Malat1 in IS
patients has been reported in previous studies
[12, 14]. Ren et al. demonstrated that Malat1
expression was inversely associated with the
National Institutes of Health Stroke Scale
(NIHSS) score and the expression of pro-in-
ammatory factors (including TNF-α, CRP,
IL-6, IL-22, and IL-8) on the rst day after
stroke [12]. Fathy et al.
in 2021 also reported the downregulation of
Malat1 in IS patients with a negative asso-
ciation with stroke severity [14] The soluble
glycoproteins that are produced by microglia,
astrocytes, endothelial cells, and neurons in
response to damaged brain tissue are cyto-
kines. Cytokines clearly play a key role in the
pathophysiology of stroke, and a high ratio of
pro-inammatory to anti-inammatory cyto-
kines is associated with larger infarct volume
and poorer functional outcomes in IS patients
[15].
Eleven isoforms have been observed in the
IL-1 family, out of which, IL-1β plays a strong
pro-inammatory role in ischemic injury and
other neurodegenerative diseases [16, 17].
IL-1β mediates brain injury through multiple
mechanisms following ischemic insult [1]. A
complex of IL-1β with transmembrane recep-
tors triggers intracellular signaling pathways
including the NF-kB, the c-Jun N-terminal ki-
nases (JNKs), and the p38 mitogen-activated
protein kinase (P32 MAPKs). The nal eects
of these signaling pathways are displayed in
the form of more inammatory damage in the
brain [18-20].
IL-1β antagonist limited excitotoxicity in
damaged brain tissues of rats [21]. Wang et al
found a negative association between serum
levels of vitamin D and IL-6 in IS patients
[22]. According to the previous research, in-
terleukin-1β was signicantly increased in
the peripheral blood of IS patients compared
to the control group [23-25]. Recently, an im-
portant relationship between the pathogenesis
of neurodegenerative disorders and Vitamin D
deciency has been reported [26-30]. Vitamin
D has shown a key adjusting role in inam-
mation and immune response [31], membrane
antioxidant activity [32], and also in synaptic
plasticity [33].
It has been revealed that serum vitamin D
levels in patients with stroke is lower than in
healthy subjects which were inversely cor-
related with stroke severity and functional
outcome [34, 22].
Vitamin D3 supplementation reduces pro-in-
ammatory mediators and brain damage in
stroke individuals [22, 35, 36] and ischemic
animals [37]. The severity of Vitamin D de-
ciency could predict the risk of stroke [38],
mortality [39], and poor functional outcome
[40].
The signicant associations between VitD
level, Malat1 expression, IL-1β, and NIHSS
within the rst hours after stroke will provide
a better and deeper understanding of the role
of Malat1 expression and the protective role
of VitD in stroke pathogenesis. Dierent stud-
ies reported the role of cytokines, dierent ln-
cRNAs, and vitamin deciency role in brain
damage, but the associations between them
have rarely been investigated after stroke.
Due to the high prevalence of vitamin D de-
ciency among the Iranian population, which
is one of the major public health issues [41],
this study initiated a longitudinal analysis
to assess the associations between lncRNA
Malat1 with VitD, and serum levels of IL-1β
in the peripheral blood of IS patients for the
rst time.
Association of Malat1 with IL-1β and VitD in Ischemic Stroke Bayat M, et al.
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Material and methods
Participants
This cross-sectional study was performed in
Shiraz Namazi Hospital from August 2020 to
2021. Sixty-three patients who were hospital-
ized within the rst 24 hours after IS were in-
cluded in the study.
Ischemic stroke in patients 18 years and older,
was diagnosed by a neurologist and conrmed
by brain non-contrast computed tomography
(CT), or diusion-weighted magnetic reso-
nance imaging. Ischemic stroke is an acute
neurologic disorder lasting more than 24
hours [42].
Patients with immunosuppressive therapy,
transient ischemic attack, and severe inam-
mation were excluded from this study. Using
the NIHSS score, stroke severity is assessed
at admission, with higher scores indicating
greater severity [43].
In this study, hypertension and diabetes were
diagnosed according to dened criteria [44,
45]. The local Ethics Committee of Shiraz
University of Medical Sciences has approved
the study ethically with grant number (IR.
SUMS.REC.1398.17988). Written informed
consent was provided by all patients (or their
proxy respondents). Peripheral venous blood
samples were collected from patients 0-24
hours after the stroke.
Laboratory tests
The coagulated blood was centrifuged (3000
g, 10 minutes), and the serum was stored at
−80 °C until use. The major circulating me-
tabolite and the best indicator of vitamin D are
25(OH)D [46]. 25-OH vitamin D levels in the
serum of the patients were measured with an
enzyme-linked immunosorbent assay (ELI-
SA) Kit (Monobind Inc.®, United States).
Serum levels of IL-1β were measured using
specic ELISA kit (Kermania Pars Gen, Ker-
man, Iran) according to the manufacturer’s
instructions.
RNA extraction and real-timepolymerase
chain reaction
A total RNA extraction kit (Favorgen, Tai-
wan) was used to extract total RNA from
whole blood according to the manufacturer’s
instructions. RNA samples with the A260/
A230 and A260/A280 ratios above 1.7 were
used for cDNA synthesis by cDNA synthesis
Kit (AddBio, Korea).
We also used Quantstudio 3 Real-Time PCR
System (Applied Biosystems, Foster City,
USA) and RealQ Plus 2x Master Mix Green
Low Ampliqon, Denmark). The thermal-cy-
cling set was adjusted at 95 -10 min which
was accompanied by 40 cycles for 15 s at 95
C and 1 min at 60 C and, also in the melting
phase thermal setting as follows 15 s at 95 C,
30 s at 60 C, and 15 s at 95 C.
The following primers were used;
Malat1:
-Forward:5′-TCAGTGTTGGGG-
CAATCTT-3′
-Reverse:5′-CGTTCTTCCGCTCAAATCC-3
TATA box-binding protein (TBP, reference
gene):
-Forward:5′-CCCGAAACGCCGAATATA-
ATC-3′
-Reverse:5′-TCTGGACT-
GTTCTTCACTCTTG-3′
Using the cycle threshold (Ct), the variation of
the value in expression levels was analyzed.
The dierence Ct between TBP and Malat1
was expressed as ΔCt. Finally, 2−ΔCt was used
to dene the relative Malat1 expression levels
for every subject [47].
Statistics
The correlation between dierent clinical
and laboratory parameters was analyzed by
the Pearson correlation test. To compare the
blood level of Malat1, IL-1β, and VitD in dif-
ferent subgroups of IS patients, we used sub-
group analysis with an independent two-sam-
ple t-test.
The relationships between lncRNA Malat1,
VitD level, IL-1β, and atherosclerotic risk
factors were analyzed by a Linear regression
(stepwise model) after adjusting the important
variables.
The analyses were performed using the SPSS
Inc., Chicago, IL, USA (version 19.0) and
GraphPad Prism (version 5.01). The P<0.05
was considered statistically signicant.
Results
The expression level of Malat1 lncRNA and
the serum level of IL-1β, and VitD in IS pa-
tients
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Bayat M, et al. Association of Malat1 with IL-1β and VitD in Ischemic Stroke
63 IS patients with a mean age of 64.4±1.7
years (minimum: 28 and maximum: 90
years) were included in this study. The mean
of Malat1 in the peripheral blood of IS pa-
tients was evaluated by RT.PCR and report-
ed 3.64±0.6 (fold change). The mean level
of IL-1β and VitD in the serum of patients
were measured as 53.37±5.14 (pg/ml) and
23.01±1.4 (ng/ml) respectively (Table-1).
Pearson correlation of VitD, Malat1, IL-1β
with clinical parameters in IS patients
The Pearson correlation test showed that the
Malat1 expression had a signicant positive
correlation with VitD level (r=0.28, P=0.023)
and a signicant negative correlation with IL-
1β level in our patients (r=-0.28, P=0.027)
(Table-2).
The NIHSS score has shown a signicant
negative correlation with the Malat1 level (r=-
0.25, P=0.04) and also a positive correlation
with IL-1β (r=0.58, P=0.0001). A non-signif-
icant negative correlation was also detected
between VitD level and NIHSS score (r=-
0.22, P=0.08).
Comparison of the blood level of Malat1,
IL-1β, and VitD in dierent Subgroups of IS
patients
Subgroup analysis showed that sex, hyperten-
sion, hyperlipidemia, diabetes, smoking, and
drinking did not aect Malat1 expression, IL-
1β, and VitD in peripheral blood of ischemic
stroke patients (Table-3).
The evaluation of stroke severity in IS pa-
tients revealed that the Malat1 expression
signicantly was lower (2.7±0.51 vs 5±1.3,
P=0.001) in patients with high NIHSS score
(>7), while IL-1β was higher in patients with
NIHSS 0-6 (65.3±7.7 vs 35.1±2.9, P=0.000).
Relationship between Malat1 expression with
VitD, IL-1β, and atherosclerotic risk factors
in IS patients by Linear regression (stepwise
model)
After adjusting the important variables, step-
wise regression analysis showed that VitD
level could only show a signicant positive
relation with the expression level of Malat1
(Beta=0.28, 95% condence interval (0.018-
0.231), P=0.02).
Table 1. Demographic and Clinical Characteristics
of Ischemic Stroke Patients
Characteristics IS patients (n=63)
Male, n (%) 43 (68.3%)
Female, n (%) 20 (31.7%)
Age, years 64.4±1.7
BMI, (kg/m2)26.39±0.66
Hypertension, n (%) 34 (54%)
Diabetes, n (%) 23 (36.5%)
Hyperlipidemia, n (%) 21 (33.3%)
Smoking, n (%) 10 (15.9%)
Drinking, n (%) 2 (3.2%)
TG, mg/dL 125.4±6.78
TC, mg/dL 162.04±5.349
LDL, mg/dL 98.38±4.383
HDL, mg/dL 33.8±0.9
WBC 7849.21±247.071
Hgb, (g/dL) 14.03±3.271
PLT 196301±7274
BUN, mg/dL 16.26±5.593
Cr, mg/dL 1.20±0.4
AST (U/L) 21.06±8.807
ALT (U/L) 18.52±9.959
IL1β, pg/ml 53.37±5.144
Malat1(fold change) 3.64±0.643
VitD, ng/ml23.01±1.482
Types of stroke
LAA 26 (41.2%)
SVD 20 (31.7%)
CE 10(15.8%)
UD 7(11.11%)
NIHSS at admission
≤6 25 (39.7%)
≥7 38 (60.3%)
Data were shown as mean±SEM or as n (%).
IS:Ischemic stroke; BMI: Body mass index; TG:Tri-
glyceride; TC:Total cholesterol; LDL:Low density
lipoprotein; HDL: High density lipoprotein; WBC:
White blood cell; Hgb: Hemoglobin; PLT: Platelet;
BUN: Blood urea nitrogen; Cr: Creatinine; AST:
Aspartate aminotransferase ; ALT:Alanine amino-
transferase; IL-1β: Interleukin-1β; Malat1: Metas-
tasis-associated lung adenocarcinoma transcript 1;
VitD:Vitamin D; LAA: Large artery atherosclerosis;
SVD: Small vessel disease; CE:Cardiac embolism;
UD:Undetermined; NIHSS:National institutes of
health stroke scale.
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Association of Malat1 with IL-1β and VitD in Ischemic Stroke Bayat M, et al.
Table 2:Pearson Correlation of Vitamin D (VitD), Metastasis-Associated Lung Adenocarcinoma Transcript 1
(Malat1) Interleukin-1β (IL-1β) with Clinical Parameters and Together in Ischemic Stroke (IS) Patients.
Malat1
r P-value
IL-1β
r P-value
VitD
r P-value
Age 0.206 0.105 -0.019 0.883 0.155 0.226
BMI -0.084 0.513 0.095 0.459 -0.281 0.026
FBS -0.058 0.65 -0.033 0.797 -0.028 0.827
PLT -0.071 0.579 0.123 0.337 -0.208 0.102
WBC -0.218 0.086 0.277 0.028 -0.166 0.193
HB 0.111 0.385 -0.049 0.706 0.031 0.811
HDL 0.043 0.74 0.105 0.411 -0.043 0.741
LDL -0.049 0.705 0.063 0.626 -0.158 0.215
TC 0.01 0.938 0.066 0.606 -0.121 0.346
TG -0.158 0.216 -0.136 0.287 -0.091 0.476
ALT -0.021 0.868 0.055 0.669 0.062 0.630
AST -0.075 0.56 -0.018 0.888 -0.129 0.312
Cr 0.189 0.137 -0.215 0.09 0.067 0.6
BUN 0.077 0.549 0.011 0.934 0.31 0.013
NIHSS -0.256 *0.043 0.584 **0.0001 -0.223 0.08
Malat1 -0.28 *0.027 0.287 *0.023
IL-1β -0.28 *0.027 -0.296 *0.018
VitD 0.287 *0.023 -0.296 *0.018
BMI: Body mass index; FBS: Fast blood sugar; PLT: Platelet; WBC:Wight blood cell; HB: Hemoglobin; HDL:
High-density lipoprotein; LDL: Low-density lipoprotein; TC: Total cholesterol; TG: Triglyceride; A LT: Alanine
aminotransferase; AST: Aspartate aminotransferase; Cr: Creatinine; BUN: Blood urea nitrogen; NIHSS:
National institutes of health stroke scale; Malat1: Metastasis-associated lung adenocarcinoma transcript 1;
IL-1β: Interleukin-1β; VitD: Vitamin D. (*P<0.05, **P<0.001).
We also found a non-signicant negative cor-
relation between Malat1 expression with IL-
1β and NIHSS score.
Discussion
In this study, serum VitD levels in the periph-
eral blood of patients with IS showed a nega-
tive Pearson correlation with IL-1β and a pos-
itive correlation with the expression level of
Malat1 lncRNA. Linear regression conrmed
the signicant positive relationship between
Malat1 and VitD levels.
Patients with an NIHSS score>7 showed a
signicantly higher level of IL-1β and lower
expression of Malat1 relative to other patients
who had an NIHSS score<6 while we couldn’t
detect a signicant negative correlation be-
tween stroke severity and VitD level. We also
found a signicant negative correlation be-
tween VitD and IL-1β.
Previous studies have also demonstrated
the contribution of IL-1β or VitD decien-
cy in the progression of ischemic injury [17,
48, 26] and the anti-inammatory eect of
Malat1 following IS [11, 12]. These ndings
are consistent with our results. The downreg-
ulation of Malat1 with a signicant negative
association with stroke severity has been re-
ported in patients with IS [12, 14]. We found
signicant negative Pearson correlations (r=-
0.25, P=0.04) between Malat1 expression and
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Bayat M, et al. Association of Malat1 with IL-1β and VitD in Ischemic Stroke
Table 3: Comparison of Metastasis-Associated Lung Adenocarcinoma Transcript 1(Malat1) Interleukin-1β,
(IL-1β) Malat1, IL-1β and Vitamin D (VitD), in Dierent Subgroups of Ischemic Stroke (IS) Patients
Malat1 P-value IL-1β (pg/ml) P-value VitD (ng/ml) P-value
Sex
Male
Female
3.9 ±0.85
3.0 ±0.88
0.35 54.9 ±6.2
53.4 ±9.3
0.36 23.2 ±1.7
22.3 ±2.6
0.81
Diabetes
Positive
Negative
3.4 ±0.97
3.7 ±0.85
0.92
63.9 ±9.1
48.8 ±6.1
0.31 21 ±1.9
24 ±2
0.32
Hypertension
Positive
Negative
4 ±0.7
3.1 ±1
0.52 52.5 ±7.1
56.8 ±7.6
0.75 22.1 ±1.8
23.8 ±2.3
0.47
Hyperlipidemia
Positive
Negative
4 ±0.99
3.4 ±0.83
0.66 51.1 ±8.9
56.2 ±6.4
0.34
22.1 ±2.4
22.9 ±1.8
0.93
Smoking
Positive
Negative
3.9 ±1.4
3.5 ±0.7
0.96 46.4 ±10.7
56.0 ±5.8
0.26 25.3 ±3
22.5 ±1.6
0.73
Drinking
Positive
Negative
4.5 ±3.3
3.5 ±0.65
0.86 82.3 ±47.8
53.6 ±5.2
0.37 18.5 ±1.5
23 ±1.5
0.25
NIHSS
(admission)
≤0-6
>7
5±1.3
2.7 ±0.51
*0.001 35.1 ±2.9
65.3 ±7.7
**0.000 24.2 ±2.5
22.1 ±1.7
0.7
NIHSS:National institutes of health stroke scale; Malat1:metastasis-associated lung adenocarcinoma
transcript 1; IL-1β:Interleukin-1β; VitD:Vitamin D; (*P<0.01 and **P<0.001)
NIHSS score. Ren et al. reported a negative
correlation between Malat1 expression and
pro-inammatory factors expression (CRP,
TNF-α, IL-22, IL-6, and IL-8) in patients with
IS [12].
We could also detect the signicant negative
correlation between Malat1 and IL-1β levels.
This result may reinforce the anti-inammato-
ry role of lncRNA Malat1 after stroke which
has been reported by previous studies [11, 49].
The protective roles of lncRNA Malat1 in
cerebrovascular diseases have been reported
through activating phosphatidylinositol 3-ki-
nase (PI3K) [50] via inhibition of pro-apop-
totic or pro-inammatory factors [51, 52].
Nowrouzi et al.reported a signicant decrease
in the level of Malat1 and CD36 in peripheral
blood mononuclear cells of participants with
vitamin D deciency which was accompanied
by a signicantly higher plasma level of IL-6,
IL-10, and IL-22 [53].
Additionally, the signicant positive rela-
tionship between Malat1 and VitD levels in
our patients may lead to the identication of
a novel mechanism for its anti-inammatory
eect in the future.
Evan et al. reported that the expression of
IL-1β, IL-6, TGF-β, IL-23a, and NADPH ox-
idase-2 was decreased after ischemic stroke
in the brains of mice supplemented with 1,25-
VitD3 and also demonstrated that expression
of the 1-α-hydroxylase as a vitamin D-activat-
ing enzyme was decreased, while expression
of 24-hydroxylase (vitamin D inactivating en-
zyme), was increased in brain and spleen after
a stroke [37].
Thus, the active form of vitamin D in the brain
may decrease after stroke.
The negative correlation between Malat1
downregulation and VitD level after stroke
leads us to the hypothesis that the reduction of
the active form of VitD in the ischemic brain
may be due to a decrease in a lncRNA such as
Malat1.
Several mechanisms have been investigated
for the anti-inammatory eects of vitamin
D, such as regulation of the immune system
[54], cytokine release [55], inhibiting nucle-
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Association of Malat1 with IL-1β and VitD in Ischemic Stroke Bayat M, et al.
ar factor kappa-B (NFκB) activity [56], and
up-regulating MKP5 [57]. It seems that the
Malat1 upregulation appears to be a promis-
ing approach to increase the anti-inammato-
ry mediated eect of VitD.
The novelty of this study relates to the correla-
tion between these parameters (Malat1, IL-1β,
and VitD) in the peripheral blood of patients
with IS during the rst 24 hours after stroke.
Understanding the strong correlation between
VitD and various cytokines or lncRNAs pro-
duced during a stroke may be important for
the medical management of stroke severity
in patients and will provide the information
about attenuation of ischemic damage, espe-
cially its anti-inammatory role.
These correlations need to be conrmed by
further studies with a larger sample size along
with full transcriptome analysis. Also, by fur-
ther research, the precise molecular mecha-
nisms of Malat1 and its correlations with VitD
and IL-1β in the pathogenesis of IS must be
investigated.
Conclusion
We evaluated the associations of Malat1 ex-
pression with VitD level, and IL-1β on the
peripheral blood of IS patients 0-24 h after
stroke onset for the rst time. A signicant
positive Pearson correlation was detected be-
tween VitD and Malat1 expression which was
conrmed by stepwise regression analysis.
However, we need to study these correlations
with a larger sample size, to reach a strong
and reliable association between Malat1 and
VitD levels in IS patients.
Acknowledgments
This work was supported by Shiraz Universi-
ty of Medical Sciences (IR.SUMS.REC.1398.
with grant agreement No. 17988).
Conict of interest
The authors declare no competing interests.
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