Demographic information and clinical characteristics
The study recruited 30 patients with MMD (including 10 hemorrhagic MMD patients, 12 ischemic MMD patients, and 8 asymptomatic MMD patients), and 10 healthy adults with matched demographic characteristics. The detailed characteristics of the included subjects were shown in Table 1.
miRNA microarray
In analysis of 30 MMD patients compared with 10 controls, 9 miRNAs were found to be differentially expressed, with 5 up-regulated (miRNA-142-5p, miRNA-29b-3p, miRNA-424-5p, miRNA-582-5p, miRNA-6807-5p) and 4 down-regulated (miRNA-144-3p, miRNA-144-5p, miRNA-451a, miRNA-486-5p) (Table 2, Figure 1). In addition, miRNA expression was significantly different in 6 samples (4 in the MMD group and 2 in the control group). After the elimination of these 6 samples, miRNA-142-3p, miRNA-340-5p, miRNA-363-3p were also differentially expressed in MMD patients (miRNA-142-3p and miRNA-340-5p up-regulated, while miRNA-363-3p down-regulated).
In analysis of 10 hemorrhagic MMD patients compared with 10 controls, 17 miRNAs were found to be differentially expressed, with 13 up-regulated (miRNA-101-3p, miRNA-142-3p, miRNA-142-5p, miRNA-148a-3p, miRNA-29b-3p, miRNA-340-5p, miRNA-374a-5p, miRNA-424-5p, miRNA-450a-5p, miRNA-542-3p, miRNA-582-5p, miRNA-590-5p, miRNA-6807-5p) and 4 down-regulated (miRNA-144-3p, miRNA-144-5p, miRNA-451a, miRNA-486-5p). In analysis of 8 asymptomatic MMD patients compared with 10 controls, 7 miRNAs were found to be differentially expressed, with 3 up-regulated (miRNA-142-3p, miRNA-29b-3p, miRNA-6515-5p) and 4 down-regulated (miRNA-144-3p, miRNA-144-5p, miRNA-451a, miRNA-486-5p). In analysis of 12 ischemic MMD patients compared with 10 controls, 3 miRNAs were found to be differentially expressed (miRNA-29b-3p and miRNA-424-5p up-regulated, while miRNA-486-5p down-regulated) (Figure 2).
In addition, miRNA-486-5p was found to be differentially down-regulated in hemorrhagic MMD patients, compared with ischemic MMD patients. Also, miRNA-6515-5p was found to be differentially up-regulated in asymptomatic MMD patients, compared with symptomatic (hemorrhagic and ischemic) MMD patients.
Real-time PCR
The RT-PCR verification of 10 differentially expressed miRNAs was performed between 30 MMD patients and 10 controls, including miRNA-142-5p, miRNA-29b-3p, miRNA-424-5p, miRNA-582-5p, miRNA-144-3p, miRNA-451a, miRNA-486-5p, miRNA-142-3p, miRNA-340-5p, miRNA-363-3p. The probe was synthesized by Thermo Fisher Scientific, and the target sequence of the probe is shown in Table 3. Three independent experiments had strong reproducibility, and all of the 10 genes were identified successfully. However, two normal control samples had higher overall CT values than other samples, but miRNA-16 (internal reference) was relatively lower, which led to higher overall expression and affected the overall analysis results, so the two samples were removed during the final analysis.
Compared with 10 healthy adults, 7 miRNAs had differential expression in 30 MMD patients (P<0.05), of which miRNA-29b-3p (P<0.001), miRNA-142-3p (P=0.013), miRNA-340-5p (P=0.002), miRNA-582-5p (P=0.005) was up-regulated, while miRNA-363-3p (P=0.007), miRNA-451a (P<0.001), miRNA-486-5p (P=0.001) was down-regulated (Figure 3).
Compared with 10 healthy adults, 9 miRNAs had differential expression in 10 hemorrhagic MMD patients (P<0.05), of which miRNA-29b-3p (P=0.002), miRNA-142-3p (P=0.004), miRNA-142-5p (P=0.047), miRNA-340-5p (P<0.001), miRNA-582-5p (P=0.003) were up-regulated, while miRNA-451a (P=0.001), miRNA-486-5p (P=0.035), miRNA-144-3p (P=0.009), miRNA-363-3p (P=0.002) were down-regulated. Compared with 10 healthy adults, 6 miRNAs had differential expression in 8 asymptomatic MMD patients (P<0.05), of which miRNA-29b-3p (P=0.004), miRNA-142-3p (P=0.001), miRNA-340-5p (P=0.009), miRNA-582-5p (P=0.005) were up-regulated, while miRNA-363-3p (P=0.015), miRNA-451a (P=0.009) were down-regulated. Compared with 10 healthy adults, 4 miRNAs had differential expression in 12 ischemic MMD patients (P<0.05), of which miRNA-29b-3p (P=0.006), miRNA-142-5p (P=0.035), miRNA-340-5p (P=0.030) were up-regulated, while miRNA-451a (P=0.030) was down-regulated.
In addition, the miRNA-142-3p was up-regulated in hemorrhagic MMD patients and miRNA-340-5p was up-regulated in hemorrhagic and the asymptomatic MMD patients compared with ischemic MMD patients (P < 0.05). The miRNA-486-5p was down-regulated in hemorrhagic and asymptomatic MMD patients, but there was no statistically significant difference (P > 0.05). There was no significant differential expression between the hemorrhagic and the asymptomatic MMD patients.
Target gene prediction and bioinformatics analysis
We used the TargetScan target gene prediction program to predict the potential target genes of differentially expressed miRNAs by microarray and RT-PCR. According to the 7 differentially expressed miRNAs in peripheral leukocytes of MMD patients, a total of 3728 target genes were predicted. Of the 3728 target genes, the biological processes and signal transduction pathways that may be involved in the enrichment analysis are carried out through GO and KEGG. The associated biological processes and signal pathways were screened (according to the criteria: P value ≤ 0.05, number of target genes ≥ 10) and sequenced according to the enrichment degree, and the top 30 biological processes and signal pathways are shown in Figure 4. Both the GO and KEGG analysis suggested that the Wnt signaling pathway may be involved in the pathogenesis of MMD (Figure 4, Figure 5).
Table 1
Demographic and clinical characteristics of the subjects
ID
|
Subtypes
|
Gender
|
Age (years)
|
Suzuki stage
|
Left
|
Right
|
HM_01
|
Hemorrhagic MMD
|
Female
|
32
|
4
|
4
|
HM_02
|
Hemorrhagic MMD
|
Female
|
43
|
2
|
3
|
HM_03
|
Hemorrhagic MMD
|
Female
|
24
|
3
|
3
|
HM_04
|
Hemorrhagic MMD
|
Female
|
49
|
6
|
6
|
HM_05
|
Hemorrhagic MMD
|
Male
|
49
|
6
|
3
|
HM_06
|
Hemorrhagic MMD
|
Male
|
42
|
5
|
5
|
HM_07
|
Hemorrhagic MMD
|
Female
|
50
|
3
|
3
|
HM_08
|
Hemorrhagic MMD
|
Female
|
34
|
3
|
3
|
HM_09
|
Hemorrhagic MMD
|
Male
|
50
|
0
|
3
|
HM_10
|
Hemorrhagic MMD
|
Female
|
42
|
4
|
4
|
IM_01
|
Ischemic MMD
|
Male
|
34
|
3
|
3
|
IM_02
|
Ischemic MMD
|
Female
|
36
|
3
|
1
|
IM_03
|
Ischemic MMD
|
Female
|
46
|
3
|
3
|
IM_04
|
Ischemic MMD
|
Male
|
35
|
3
|
3
|
IM_05
|
Ischemic MMD
|
Female
|
30
|
4
|
3
|
IM_06
|
Ischemic MMD
|
female
|
46
|
3
|
3
|
IM_07
|
Ischemic MMD
|
Male
|
47
|
4
|
3
|
IM_08
|
Ischemic MMD
|
Female
|
21
|
2
|
3
|
IM_09
|
Ischemic MMD
|
Female
|
29
|
2
|
2
|
IM_10
|
Ischemic MMD
|
Male
|
39
|
5
|
4
|
IM_11
|
Ischemic MMD
|
Male
|
37
|
2
|
2
|
IM_12
|
Ischemic MMD
|
Male
|
39
|
2
|
2
|
AM_01
|
Asymptomatic MMD
|
Male
|
42
|
2
|
2
|
AM_02
|
Asymptomatic MMD
|
Male
|
34
|
5
|
3
|
AM_03
|
Asymptomatic MMD
|
Female
|
29
|
3
|
3
|
AM_04
|
Asymptomatic MMD
|
Female
|
38
|
3
|
3
|
AM_05
|
Asymptomatic MMD
|
Female
|
34
|
4
|
4
|
AM_06
|
Asymptomatic MMD
|
Female
|
48
|
2
|
2
|
AM_07
|
Asymptomatic MMD
|
Male
|
50
|
3
|
3
|
AM_08
|
Asymptomatic MMD
|
Female
|
33
|
6
|
5
|
C_01
|
Control
|
Male
|
42
|
-
|
-
|
C_02
|
Control
|
Male
|
35
|
-
|
-
|
C_03
|
Control
|
Male
|
35
|
-
|
-
|
C_04
|
Control
|
Male
|
34
|
-
|
-
|
C_05
|
Control
|
Female
|
34
|
-
|
-
|
C_06
|
Control
|
Female
|
32
|
-
|
-
|
C_07
|
Control
|
Female
|
33
|
-
|
-
|
C_08
|
Control
|
Female
|
35
|
-
|
-
|
C_09
|
Control
|
Female
|
31
|
-
|
-
|
C_10
|
Control
|
Female
|
33
|
-
|
-
|
HM: hemorrhagic moyamoya disease; IM: ischemic moyamoya disease; AM: asymptomatic moyamoya disease; C: controls |
Table 2
Differentially expressed miRNAs in microarray analysis between 30 MMD patients and 10 controls
Systematic Name
|
P values
|
Fold change (abs)
|
Regulation
|
miRNA-142-5p
|
0.000020
|
2.020448
|
up
|
miRNA-144-3p
|
0.000958
|
2.043367
|
down
|
miRNA-144-5p
|
0.002665
|
2.413351
|
down
|
miRNA-29b-3p
|
0.000006
|
2.420699
|
up
|
miRNA-424-5p
|
0.000043
|
2.309035
|
up
|
miRNA-451a
|
0.000068
|
2.478956
|
down
|
miRNA-486-5p
|
0.000165
|
5.521476
|
down
|
miRNA-582-5p
|
0.001293
|
2.228220
|
up
|
miRNA-6807-5p
|
0.000010
|
2.227829
|
up
|
Table 3
Target sequence of the probe in real-time PCR
miRNA
|
Target sequence of the probe
|
miRNA-16
|
UAGCAGCACGUAAAUAUUGGCG
|
miRNA-29b
|
UAGCACCAUUUGAAAUCAGUGUU
|
miRNA-142-3p
|
UGUAGUGUUUCCUACUUUAUGGA
|
miRNA-142-5p
|
CAUAAAGUAGAAAGCACUACU
|
miRNA-144
|
UACAGUAUAGAUGAUGUACU
|
miRNA-340
|
UUAUAAAGCAAUGAGACUGAUU
|
miRNA-363
|
AAUUGCACGGUAUCCAUCUGUA
|
miRNA-424
|
CAGCAGCAAUUCAUGUUUUGAA
|
miRNA-451
|
AAACCGUUACCAUUACUGAGUU
|
miRNA-486
|
UCCUGUACUGAGCUGCCCCGAG
|
miRNA-582-5p
|
UUACAGUUGUUCAACCAGUUACU
|