Molecular Changes in Circulating microRNAs’ Expression and Oxidative Stress in Adults with Mild Cognitive Impairment: A Biochemical and Molecular Study

Figures & data

Table 1 Assessment of Cognitive Performance According to Petersen’s Criteria

Criteria of MCI	Control Group (n=80)	MCI Group (n=70)
Memory complaint by participant or family (Yes/No)	No	Yes
Normal activities of daily living (20 items; score <26)	>26	<26
Normal general cognitive function (A: MMSE score between 20 and 27 [cutoff points for illiterate (≤ 20), primary school (≤23) and secondary school and above (≤ 27)]; B: MoCA score <26)	MMSE:> 27 MoCA: >26	MMSE: ≤27 MoCA: <26
Objective impairment scores	>1.5 SD	<1.5 SD
The Clinical Dementia Rating scale	No	0–0.5

Table 2 The Demographics and Baseline Characteristics of Participants

Parameters	Control Group	MCI Group
N	80 (53.33%)	70 (46.66%)
Male/Female	50/30	40/30
Age (years)	65.3 ± 3.5	64.9 ± 4.1
BMI (kg/m^2)	19.9± 2.6	24.1± 3.6 ^b
Waist (cm)	77.3± 9.3	98.1 ± 6.3 ^b
Hips (cm)	86.9 ± 7.5	92.6 ± 18.3 ^b
WHR	0.89±0.11	1.4±0.16 ^b
Education (years)	10.2 ± 0.8	5.3 ± 0.6^b
Lifestyle factors, %  Working  Exercising regularly	96.5 88.4	89.5^a 76.1^a

Notes: Values are expressed as mean ±SD; Significance at p <0.05. ^ap <0.01, ^bp <0.001.

Table 3 Neuropsychological Test Scores and the Cognitive Status of the Participants (n=15; Mean ±SD)

Parameters	Control Group	MCI Group	t	p-Value
MMSE	28.4 ± 3.3	21.3 ± 2.5	12.8	<0.001
MoCA	22.7±2.9	27.9±5.4	18.9	<0.001
ADL	22.1±3.7	28.6±10.3	19.7	<0.001
IADL	8.3±0.2	6.9±0.3	15.3	<0.001
Wechsler Memory Scale – Revised-				<0.001
VPA	10.3±2.8	6.9±3.1	12.5
VR	12.8±4.3	8.5±2.7	10.4
IM	7.2±2.9	4.5±3.1	11.9
PR	17.0±3.9	12.9±2.9	16.9
Wechsler Adult Intelligence scale – Chinese revision				<0.001
S	19.6±3.8	12.8±5.8	18.9
R	14.1±3.7	8.1±2.8	16.9
DSC	38.2±8.6	24.2±10.3	12.8
PC	10.4±1.9	9.4±1.3	15.8
BDs	79.3±24.3	89.8±31.6	14.7
V	17.2±3.7	14.5±4.1	8.5

Note: Values are expressed as mean ±SD.

Abbreviations: MMSE, Mini-Mental State Examination; MoCA, Montreal Cognitive Assessment; ADL, activity of daily living; IADL, instrumental activities of daily living; VPA, verbal paired associates; VR, visual reproduction; IM, immediate memory; PR, picture recall; S, similarities; R, arithmetic; DSC, digit symbol-coding; PC, picture completion; BDs, block design(s); V, vocabulary.

Table 4 Correlations Between DPP4 Activities, BDNF, SIRT-1, Celluar Oxidative Stress, Apoptosis, and Relative Expression of miRNAs vs Cognitive Parameters

Characteristics	Cognitive Function (n=70)
	MMSE^a		MoCA^a		ADL^a		Memory Scores^a
	r	P	r	P	r	P	r	P
DPP4 activity (nmol/min/mL)	−0.320	<0.001	−0.291	< 0.001	−0.31	<0.001	−0.24	<0.001
BDNF (ng/mL)	0.192	<0.001	0.23	<0.001	0.18	<0.001	0.15	<0.001
SIRT1(ng/mL)	0.31	<0.001	0.45	<0.001	0.27	<0.001	0.34	<0.001
NO	0.35	<0.001	0.28	<0.001	0.29	<0.001	0.47	<0.001
MDA	0.23	<0.001	0.36	<0.001	0.51	<0.001	0.14	<0.001
CAT	−0.32	<0.001	−0.39	<0.001	−0.25	<0.001	−0.31	<0.001
SOD	−0.54	0.001	−0.48	<0.001	−0.37	<0.001	−0.43	<0.001
Bax	0.21	<0.001	0.18	<0.001	0.26	<0.001	0.26	<0.001
Bcl-2	−0.45	<0.001	−0.49	<0.001	−0.53	<0.001	−0.78	<0.001
Caspase-3	0.48	<0.001	0.21	<0.001	0.23	<0.001	0.19	<0.001
miR-124a	0.251	<0.001	0.362	<0.001	0.182	<0.001	0.135	<0.001
miR-483-5p	0.314	<0.001	0.217	<0.001	0.321	<0.001	0.158	<0.001
miR-142-3p	−0.242	<0.001	−0.342	<0.001	−0.124	<0.001	−0.314	<0.001
miR-125b	−0.254	<0.001	−0.227	<0.001	−0.351	<0.001	−0.318	<0.001

Note: ^aP-value determined by partial correlation analysis with respect to the DPP4 activity, BDNF, SIRT-1, cellular oxidative stress, apoptosis, and relative expression of miRNAs adjusted for age, BMI, gender, education level, exercise levels, and working.

Figure 1 Changes in cellular oxidative stress; NO and MDA (A and B) and antioxidant enzymes; Cat and SOD the levels (C and D) in healthy control (n=80) and older adults with MCI (n=70). The results showed significant increase (p=0.001) in the levels of cellular NO and MDA in older adults with MCI compared to healthy controls (A and B). In addition, significant decrease (p=0.001) in the levels of antioxidant enzymes; CAT and SOD in older adults with MCI compared to healthy controls. Significance of the comparison was evaluated by Mann–Whitney-Wilcoxon test and sample t-test, ^{* *} p≤ 0.01, ^{* * *}p≤ 0.001.

Abbreviations: MCI, mild cognitive impairment; NO, nitric oxide; MDA, malonaldehyde, SOD, superoxide dismutase, CAT, catalase enzyme.

Figure 2 Metabolic changes in the DPP4 activity, BDNF, SIRT-1, and apoptotic genes Bcl-2, Bax, and caspase-3 in healthy control (n=80) and older adults with MCI (n=70). (A–D). The results showed that the levels of BDNF, SIRT-1 significantly reduced and the levels of DPP4 activity significantly increased in older subjects with MCI compared to healthy controls (A–C). In addition, the expression of apoptotic genes was significantly reported in all subjects. In Older adults with MCI, the expression levels of both Bax and caspase-3 genes significantly (p=0.001) increased, and the expression levels of Bcl-2 gene significantly (p=0.01) reduced in comparison with the results of healthy controls (D). Significance of the comparison was evaluated by Mann–Whitney–Wilcoxon test and sample t-test, **p≤ 0.01 ***p≤ 0.001.

Abbreviation: MCI, mild cognitive impairment.

Table 5 Correlations Between DPP4 Activities, BDNF, and SIRT-1 vs Celluar Oxidative Stress and Apoptosis in Older Adults with MCI

Characteristics	Cognitive-Related Metabolic Parameters^a
	DPP4 Activity		BDNF		SIRT1(ng/mL)
	r	P	r	P	r	P
NO	−0.128	<0.01	−0.32	<0.01	−0.56	<0.01
MDA	−0.18	<0.001	−0.23	<0.001	−0.58	<0.001
CAT	0.89	<0.001	0.76	<0.001	0.81	<0.001
SOD	0.48	<0.001	0.71	<0.001	0.56	<0.001
Bax	−0.31	<0.001	−0.43	<0.001	−0.48	<0.001
Bcl-2	0.36	<0.001	0.51	<0.001	0.42	<0.001
Caspase-3	−0.87	<0.001	−0.96	<0.001	−0.74	<0.001

Note: ^aP-value determined by partial correlation analysis with respect to the DPP4 activity, BDNF, DBR, cellular oxidative stress, and apoptosis adjusted for age, BMI, gender, education level, exercise levels, and working among older adults.

Figure 3 MicroRNAs’ differential expression profile in healthy control (n=80) and older adults with MCI (n=70). The results showed that the relative expression of miR-124a and miR-483-5p significantly increased (P=0.001), and miR-142-3p, and miR-125b significantly reduced (P=0.01) in older adults with MCI compared healthy controls. Significance of the comparison was evaluated by Mann–Whitney–Wilcoxon test and sample t-test, **p≤ 0.01, ***p≤ 0.001.

Abbreviation: MCI, mild cognitive impairment.

Table 6 Correlations Between DPP4 Activities, BDNF, SIRT-1, Celluar Oxidative Stress, and Apoptosis vs Relative Expression of miRNAs in Older Adults with MCI

Characteristics	miRNAs Relative Expression^a
	miR-124a		miR-483-5p		miR-142-3p		miR-125b
	r	P	r	P	r	P	r	P
DPP4 activity (nmol/min/mL)	0.12	<0.01	0.24	<0.002	0.38	< 0.002	0.145	<0.001
BDNF (ng/mL)	0.21	<0.01	0.31	<0.01	0.28	< 0.01	0.31	<0.001
SIRT1(ng/mL)	0.45	<0.01	0.57	<0.01	0.49	< 0.01	0.49	<0.01
NO	0.37	<0.001	0.42	<0.001	0.36	< 0.001	0.76	<0.001
MDA	0.18	<0.001	0.23	<0.001	0.42	< 0.001	0.27	<0.001
CAT	−0.51	<0.001	−0.62	<0.001	−0.54	< 0.001	−0.63	<0.001
SOD	−0.85	<0.001	−0.82	<0.001	−0.47	< 0.001	−0.84	<0.001
Bax	−0.45	<0.001	−0.36	<0.001	−0.56	< 0.001	−0.46	<0.001
Bcl-2	−0.17	<0.001	−0.42	<0.001	−0.68	< 0.001	−0.91	<0.001
Caspase-3	−0.75	<0.001	−0.53	<0.001	−0.47	< 0.001	−0.43	<0.001

Note: ^aP-value determined by partial correlation analysis with respect to the DPP4 activity, BDNF, DBR, cellular oxidative stress, and apoptosis adjusted for age, BMI, gender, education level, exercise levels, and working among older adults with MCI.