Exosomal miR-320e as a Novel Potential Biomarker for Cerebral Small Vessel Disease

Figures & data

Figure 1 Characteristics of plasma-derived exosomes. (A) Representative exosome image identified by a TEM (scale bar: 100 nm). (B) Nanoparticle tracking analysis (NTA) of exosomes showed they have a diameter of 30–150 nm. (C) Western blotting identified three exosome-specific markers (CD9, CD63, and TSG101).

Table 1 Demographic Data of CSVD Patients and Controls

Characteristics	Control (80)	CSVD (150)	p
Age	62.09±13.23	61.77±10.11	0.982*
Gender (Male/Female)	36 (45.0%)	71 (47.3%)	0.735^†
Ethnicity (Han, %)	100%	100%	1^†
Hypertension (N, %)	46 (57.5%)	116 (77.3%)	0.002^†
Diabetes mellitus (N, %)	17 (21.2%)	49 (32.7%)	0.077^†
Smoking (N, %)	11 (13.8%)	45 (30%)	0.017^†
Drinking (N, %)	12 (15%)	13 (9%)	0.266^†
TG	1.59±0.89	1.50±1.02	0.516*
TC	4.43±1.07	4.31±1.03	0.421*
HDL	1.30±0.40	1.29±0.41	0.880*
LDL	2.65±0.72	2.53±0.83	0.253*
Fibrinogen	2.50 (2.50–2.79)	2.73 (2.33–3.17)	0.109^‡
tHcy	11.31 (9.85–12.96)	11.57 (10.10–14.85)	0.217^‡
Glucose	5.18 (4.63–5.76)	5.24 (4.67–6.35)	0.332^‡

Notes: Significant variables (p<0.05) are bolded. *Continuous variables were compared by Student’s t-test. ^†Categorical data were compared by the Chi-square test. ^‡Continuous variables were compared by Mann–Whitney U-test.

Abbreviations: TG, triglyceride; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; tHcy, total homocysteine; CSVD, cerebral small vessel disease.

Table 2 The Screened Differentially Expressed miRNAs in the Exosomal Samples of Patients with CSVD

MiRNA	Fold Change	P value	Regulation
hsa-miR-320e	−1.601026847	0.00174	Down
hsa-mir-8075	−1.710063621	0.00176	Down
hsa-mir-576	1.549797261	0.00284	Up
hsa-mir-6776	−1.59366401	0.00408	Down
hsa-miR-302a-5p	1.511890338	0.00466	Up
hsa-miR-668-5p	−2.106565099	0.0048	Down
hsa-mir-4722	−1.507980215	0.00494	Down
hsa-miR-3619-5p	−2.021634935	0.00976	Down
hsa-miR-3141	−1.515834765	0.01204	Down
hsa-miR-455-3p	−1.51110876	0.01326	Down
hsa-miR-8075	−1.654637289	0.0148	Down
hsa-miR-498	−2.050939593	0.01934	Down
hsa-miR-6732-5p	−1.672066817	0.0231	Down
hsa-miR-320d	−1.539778146	0.04335	Down

Figure 2 Analysis of DE miRNAs. (A) The volcano plot depicted the DE miRNAs, upregulated miRNAs and downregulated miRNAs are shown as red dots and green dots, respectively. (B) Heat maps of DE miRNAs meeting |fold change|≥1.5.

Figure 3 Enrichment of target gene function of DE miRNAs. (A) Top 20 enriched GO terms including biological processes (BP), cellular components (CC), and molecular functions (MF). (B) Top 20 enriched KEGG pathways were visualized on a bubble diagram. The size of the circle shows the number of genes, and the color of the circle corresponds to the size of the P-value.

Figure 4 The expression of the two selective miRNAs (miR-320e, miR-668-5p) was validated by qRT-PCR. U6 snRNA was used as internal control for normalization. (A) The expression of plasma exosomal miR-320e from CSVD patients was clear downregulated (p<0.0001, Mann–Whitney test). (B) There are no significant differences in plasma exosomal miR-668-5p expression between CSVD and the control group (p=0.062, Mann–Whitney test). Violin plots showed the expression of exosomal miR-320e and miR-668-5p in patients and controls representatively. ****p < 0.0001, CSVD patients vs controls. The y-axis corresponds to miR-320e and miR-668-5p expression levels by log10 change. n = 150 in CSVD group, n = 80 in control group.

Abbreviation: ns, not statistically significant.

Table 3 Exosomal miR-320e and the Risk Factors

Items	P-value	OR	95% CI
miR-320e	<0.001	0.352	0.239–0.519
Hypertension	0.067	1.871	0.956–3.659
Smoking	0.043	2.289	1.027–5.103

Notes: Exosomal miR-320e expression together with risks (Hypertension, Smoking) were analyzed in a statistical model by multivariable logistic regression.

Abbreviations: OR, odds ratio; 95% CI, 95% confidence interval.

Figure 5 ROC curves of exosome-derived miR-320e and miR-668-5p in diagnosing CSVD.

Table 4 Univariable and Multivariable Logistic Regression Analyses Between Possible Predictors and Moderate-Severe WMH

Items	Univariable Analysis		Multivariable Analysis
	OR (95% CI)	p	aOR (95% CI)	p
Age	1.119 (1.071 to 1.170)	<0.001	1.093 (1.042 to 1.148)	<0.001
Sex, male	1.743 (0.911 to 3.338)	0.094	–	–
Hypertension	1.821 (0.839 to 3.953)	0.13	–	–
Diabetes	1.168 (0.548 to 2.335)	0.66	–	–
CHD	4.465 (1.216 to 16.386)	0.024	2.406 (0.512 to 11.314)	0.266
ALT	0.966 (0.933 to 1.000)	0.05	–	–
AST	1.009 (0.971 to 1.049)	0.641	–	–
TG	0.766 (0.524 to 1.119)	0.168	–	–
TC	0.878 (0.640 to 1.204)	0.419	–	–
HDL	1.061 (0.487 to 2.310)	0.882	–	–
LDL	0.885 (0.601 to 1.304)	0.536	–	–
Glucose	0.941 (0.819 to 1.081)	0.39	–	–
BUN	1.312 (1.035 to 1.663)	0.025	1.239 (0.894 to 1.717)	0.199
Cr	1.027 (1.003 to 1.052)	0.029	0.986 (0.951 to 1.024)	0.472
Uric	1.000 (0.996 to 1.004)	0.884	–	–
tHcy	1.311 (1.150 to 1.495)	<0.001	1.308 (1.094 to 1.562)	0.003
Fibrinogen	1.595 (0.948 to 2.685)	0.079	–	–
miR-320	0.414 (0.258 to 0.664)	<0.001	0.452 (0.258 to 0.792)	0.006

Note: Significant variables (p<0.05) are bolded.

Abbreviations: CHD, coronary heart disease; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TG, triglyceride; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; BUN, tlood urea nitrogen; Cr, creatinine; Uric, uric acid; tHcy, total homocysteine; OR, odds ratio; 95% CI, 95% confidence interval.

Figure 6 Distribution of mean values of miR-320e according to severity of white matter hyperintensity. (A) miR-320e level was negatively correlated with Fazekas score of PVWMH (p=0.0021, ANOVA), n=79 when Fazekas score=0 or 1, n=46 when Fazekas score=2, n=25 when Fazekas score=3; (B) miR-320e level was negatively correlated with Fazekas score of DWMH (p=0.0018, ANOVA), n=97 when Fazekas score=0 or 1, n=24 when Fazekas score=2, n=29 when Fazekas score=3, **P < 0.01, *P < 0.05.

Abbreviations: PVWMH, periventricular white matter hyperintensity; DWMH, deep white matter hyperintensity; ns, not statistically significant.

Figure 7 The correlation between exosomal miR-320e level and the total burden of CSVD. The scatter plot showing correlations between the two variables was calculated by Spearman correlation test (r=−0.276, p=0.001).