Serum microRNA-30c-5p and microRNA-373 expressions as potential biomarkers for Parkinson's disease

Abstract

This study aimed to confirm the use of microRNA (miR)-30c-5p and miR-373 as the potential biomarkers for Parkinson's disease (PD). In this study, 148 patients with PD admitted to our hospital were enrolled as the research group (RG) and 126 physically examined healthy individuals as the control group (CG) between December 2017 and June 2018. Serum miR-30c-5p and miR-373 expressions in both groups were determined by quantitative real-time polymerase chain reaction. Furthermore, the correlation of their expressions with PD severity was determined by Spearman's correlation analysis. Serum miR-30c-5p and miR-373 expressions were higher in RG than in CG (both P < 0.001). Their expressions were not related to smoking history and diabetes mellitus history in RG (both P>0.001) but were related to sex, age, coronary heart disease history, depression history, and Hoehn–Yahr score (all P < 0.05). Moreover, the relative expressions of serum miR-30c-5p and miR-373 were positively correlated with PD severity (P < 0.05). MiR-30c-5p and miR-373 may be involved in PD development and progression and can be adopted as potential biomarkers for PD.

KEYWORDS:

• MiR-30c-5p
• miR-373
• Parkinson’s disease
• quantitative real-time polymerase chain reaction

Background

Parkinson's disease (PD) is a disabling disease characterized by degenerative changes in the central nervous system, and it is primarily caused by the loss of dopaminergic neurons in the substantia nigra pars compacta (Alessi and Sammler 2018). PD can cause cognitive disorder and limb disability, leading to the development of psychosomatic diseases. Therefore, PD has become a crucial challenge in modern medicine and the modern society (Demirci et al. 2017; Kouchaki et al. 2018). Despite the continuous advancement in diagnosis and treatment technology, the clinical efficacy of treatments for PD remains unsatisfactory. Therefore, the detection and treatment of PD as early as possible are necessary to improve its prognosis and ease social burden (Wang et al. 2016; Yang et al. 2017). PD can be classified into the following stages according to its severity: early, midterm, and advanced. The higher its severity, the worse its prognosis; hence, identifying appropriate evaluation indexes to judge PD severity for the determination prognosis is crucial (Gao et al. 2018). According to the growing understanding of PD pathogenesis, a good biomarker is beneficial to diagnose and evaluate PD (Wei et al. 2018).

MicroRNAs (miRs) are a class of endogenous, noncoding, single-stranded RNAs (Mehta and Baltimore 2016; Peng and Croce 2016). MiRs are related to inflammatory response, endocrine changes, and pathological process of tumor and other diseases, and relevant miRs regulate the expressions of other messenger RNAs (mRNAs) and proteins by binding to their target gene mRNA (Alarcón et al. 2015; Chadly et al. 2018). According to the study by Patil et al. (Patil et al. 2019), because the methods currently used for the clinical diagnosis of PD may be inaccurate, the identification and verification of molecular biomarkers such as circulating miRs will improve the accuracy of PD diagnosis. They found that miRs can supplement and improve the accuracy of the existing methods used for PD diagnosis and can help improve clinical diagnosis and treatment of PD. Researchers have confirmed that miR-30c-5p is closely related to tumor progression, bacterial infection, and inflammatory response, and recent studies have suggested that it is a potential biomarker for multisystem atrophy (Zhong et al. 2014; Marques et al. 2017). Related studies have confirmed that miR-373 can serve as a tumor suppressor in brain tumor (Wei et al. 2016). However, at present, the expression and clinical value of miR-30c-5p and miR-373 in patients with PD remain poorly understood. Therefore, we analyzed serum miR-30c-5p and miR-373 expressions in patients with PD to determine their clinical value and short-term prognosis, thereby providing a new theoretical basis for the diagnosis and treatment of PD in molecular biology.

Material and methods

Data collection

In this study, we enrolled a total of 148 patients with PD admitted to our hospital as the research group (RG) and 126 physically examined healthy individuals as the control group (CG) from December 2017 and June 2018. RG comprised 86 males and 62 females, with a mean age of 71.36 ± 3.61 years, whereas CG comprised 74 males and 52 females, with a mean age of 65.45 ± 3.42 years. We included patients who were diagnosed with PD according to the criteria for PD diagnosis (Berg et al. 2013) and who had normal hepatic and renal function but no malignant tumor. Those who were undergoing or had undergone treatment were excluded. This study was approved by the Ethics Committee of the China–Japan Friendship Hospital, and all patients signed informed consent forms before the start of the study.

Main reagents and instruments

The following reagents and instruments were used: TRIzol reagent (GENMED Co., United States), quantitative real-time polymerase chain reaction (qRT-PCR) kit and minScript reverse transcription kit, HBS-1096A enzyme microplate reader (Shanghai Precision Instruments Co., Ltd., China), and qRT-PCR instrument (Thermo Fisher Company, United States). The primer sequences of miR-30c-5p, miR-373, and internal reference (U6) as well as miR negative control were all designed by Shanghai GenePharma Company (Table 1).

Table 1. Primer sequences of miR-30c-5p, miR-373, and internal reference (U6).

Group	Upstream primer	Downstream primer
miR-30c-5p	5′-UGUAAACAUCCUACACUCUCAGC-3′	5′-CCAGTGCAGGGTCCGAGGT-3′
miR-373	5′-CGCTAGCCAGTCGACATCAC-3′	5′-GCATCCAGACCTACATGCA-3′
U6	5′-CTCGCTTCGGCAGCACA-3′	5′-AACGCTTCACGAATTTGCGT-3′

Determination of serum miR-30c-5p and miR-373 expressions

Within 24 h after patient admission, we extracted an elbow venous blood sample (5 ml) from each subject and collected it in anticoagulant-free vacuum blood collection tubes. The sampled blood was centrifuged at 3500 g for 15 min to collect serum, which was then stored in an Eppendorf tube in a low-temperature refrigerator at −80°C for subsequent analysis. Serum miR-30c-5p and miR-373 expressions in both groups were determined using qRT-PCR. Total RNA was extracted from the collected serum according to the operating instructions of the TRIzol reagent and dissolved in 20 µl diethyl pyrocarbonate water. Next, analysis was completed by reverse transcription using a reverse transcription kit using a total reaction volume of 15 µl. Relative quantification of the target genes was performed using the 2^−△△Ct method.

Statistical analysis

Data were analyzed statistically using SPSS 17.0. Enumeration data are expressed as the number of cases/percentage [n (%)] and were compared using the χ² test between the groups. Measurement data are expressed as mean ± standard deviation, and they were compared using t-test or F test between the groups. Furthermore, receiver operating characteristic curves were constructed, and their correlation was analyzed using Spearman's correlation analysis. In addition, P-values of <0.05 indicated statistically significant difference.

Results

General clinical data of patients

General clinical data such as age, sex, body mass index, smoking history, drinking history, hypertension history, diabetes mellitus history, and depression history were compared between the two groups, and no significant differences were observed (all P > 0.05; Table 2).

Table 2. General clinical data of patients.

Group	RG (n = 148)	CG (n = 126)	t/F/χ^2	P-value
Sex			0.076	0.782
Male	116	97
Female	32	29
Age (years)			1.812	0.404
≤65	25	17
≤85	75	74
>85	48	35
Smoking history			0.381	0.537
Yes	85	77
None	63	49
Drinking history			0.038	0.844
Yes	51	42
No	97	84
Hypertension history			1.396	0.237
Yes	116	91
No	32	35
Diabetes mellitus history			1.057	0.304
Yes	57	41
No	91	85
Tardive dyskinesia			–	–
Yes	117	–
No	31	–
Postural instability and gait disorder			–	–
Yes	119	–
No	29	–
Hoehn–Yahr score			–	–
Early stage (Stages I and II)	76	–
Midterm stage (Stage III)	59	–
Advanced stage (Stages IV and V)	13	–
MMSE score			–	–
Mild (≥27)	57	–
Moderate (20–26)	76	–
Severe (≤9)	15	–
MADRS score			–	–
Mild (≤21)	41	–
Moderate (22–30)	69	–
Severe (≥30)	38	–

miR-30c-5p and miR-373 expressions

Serum miR-30c-5p and miR-373 expressions were higher in RG than in CG (1.95 ± 0.24 vs. 0.42 ± 0.15 and 1.82 ± 0.33 vs. 1.01 ± 0.29, respectively; both P < 0.001; Table 3 and Figures 1 and 2).

Figure 1. The relative expression of serum miR-30c-5p was significantly higher in the research group than in the control group (P < 0.001).

Note: *** indicates P < 0.001.

Figure 2. The relative expression of serum miR-373 was significantly higher in the research group than in the control group (P < 0.001).

Note: *** indicates P < 0.001.

Table 3. Serum miR-30c-5p and miR-373 expressions in the two groups.

Group	RG (n = 148)	CG (n = 126)	t	P-value
miR-30c-5p	1.95 ± 0.24	0.42 ± 0.15	61.98	<0.001
miR-373	1.82 ± 0.33	1.01 ± 0.29	21.4	<0.001

Serum miR-30c-5p expression and clinicopathological characteristics of patients with PD

Serum miR-30c-5p expression was not related to smoking history and diabetes mellitus history in RG (both P > 0.001), but it was associated with sex; age; history of coronary heart disease, tardive dyskinesia, postural instability, or gait disorder; and Hoehn–Yahr score, mini-mental state examination (MMSE), and Montgomery–Åsberg Depression Rating Scale (MADRS) scores (all P < 0.05; Table 4).

Table 4 Analysis of serum miR-30c-5p expression and clinicopathological characteristics of patients with PD.

Group	RG (n = 148)	miR-30c-5p	t/F/χ^2	P-value
Sex			114.1	<0.001
Male	116	1.96 ± 0.41
Female	32	1.85 ± 0.36
Age (years)			119.7	<0.001
≤65	25	0.79 ± 0.24
≤85	75	1.66 ± 0.37
>85	48	2.07 ± 0.32
Smoking history			0.304	0.762
Yes	85	1.95 ± 0.24
No	63	1.94 ± 0.18
CHD history			3.176	0.002
Yes	116	2.03 ± 0.27
No	32	1.85 ± 0.33
Diabetes mellitus history			0.699	0.388
Yes	57	1.96 ± 0.28
No	91	1.94 ± 0.32
Tardive dyskinesia			13.490	<0.001
Yes	117	2.23 ± 0.46
No	31	1.06 ± 0.28
Postural instability and gait disorder			15.310	<0.001
Yes	119	2.18 ± 0.31
No	29	1.22 ± 0.27
Hoehn–Yahr score			174.600	<0.001
Early stage (Stages I and II)	76	0.81 ± 0.26
Midterm stage (Stage III)	59	1.54 ± 0.37
Advanced stage (Stages IV and V)	13	2.26 ± 0.23
MMSE score			294.5	<0.001
Mild (≥27)	57	0.74 ± 0.31
Moderate (20–26)	76	1.83 ± 0.28
Severe (≤9)	15	2.34 ± 0.32
MADRS score			215.400	<0.001
Mild (≤21)	41	0.81 ± 0.26
Moderate (22–30)	69	1.54 ± 0.37
Severe (≥30)	38	2.26 ± 0.23

Serum miR-373 expression and clinicopathological characteristics of patients with PD

Serum miR-373 expression was not related to smoking history and diabetes mellitus history in RG (both P > 0.001), but it was associated with sex; age; history of coronary heart disease, tardive dyskinesia, postural instability, or gait disorder; and Hoehn–Yahr, MMSE, and MADRS scores (all P < 0.05; Table 5).

Table 5. Serum miR-373 expression and clinicopathological characteristics of patients with PD.

Group	RG (n = 148)	miR-373	t/F	P-value
Sex			5.063	<0.001
Male	116	1.92 ± 0.24
Female	32	1.61 ± 0.38
Age (years)			78.01	<0.001
≤65	25	1.20 ± 0.31
≤85	75	1.75 ± 0.29
>85	48	2.13 ± 0.32
Smoking history			1.123	0.263
Yes	85	1.84 ± 0.25
No	63	1.79 ± 0.29
CHD history			2.426	0.017
Yes	116	1.86 ± 0.47
No	32	1.64 ± 0.39
Diabetes mellitus history			0.392	0.7
Yes	57	1.83 ± 0.32
No	91	1.81 ± 0.29
Tardive dyskinesia			8.651	<0.001
Yes	117	2.11 ± 0.35
No	31	1.54 ± 0.21
Postural instability and gait disorder			13.240	<0.001
Yes	119	2.21 ± 0.28
No	29	1.42 ± 0.32
Hoehn–Yahr score			174.600	<0.001
Early stage (Stages I and II)	76	0.81 ± 0.26
Midterm stage (Stage III)	59	1.54 ± 0.37
Advanced stage (Stages IV and V)	13	2.26 ± 0.23
MMSE score			279.800	<0.001
Mild (≥27)	57	0.66 ± 0.32
Moderate (20–26)	76	1.73 ± 0.27
Severe (≤9)	15	2.14 ± 0.28
MADRS score			254.500	<0.001
Mild (≤21)	41	0.73 ± 0.27
Moderate (22–30)	69	1.68 ± 0.31
Severe (≥30)	38	2.25 ± 0.33

Correlation of serum miR-30c-5p expression with PD severity

The mean serum miR-30c-5p expression in patients with early, midterm, or advanced PD was 0.81 ± 0.26, 1.54 ± 0.37, and 2.26 ± 0.23, respectively. Serum miR-30c-5p expression was significantly upregulated in patients with midterm or advanced PD compared with that in patients with early PD (both P < 0.05), and the relative expression of serum miR-30c-5p increased as PD aggravated. Early, midterm, and advanced PD stages were set to 1, 2, and 3, respectively. Then, the correlation of the relative expression of serum miR-30c-5p with PD severity was analyzed by Spearman's correlation analysis; subsequently, a positive correlation was found (r = 0.839, P < 0.001; Figure 3).

Figure 3. The relative expression of serum miR-30c-5p was positively correlated with Parkinson's disease severity (r = 0.839, P < 0.001).

Correlation of serum miR-373 expression with PD severity

Serum miR-373 expression in patients with early, midterm, or advanced PD was 1.56 ± 0.27, 1.75 ± 0.31, or 2.08 ± 0.29, respectively. Compared with that in patients with early PD, serum miR-373 expression in patients with midterm or advanced PD was significantly upregulated (both P < 0.05), and the relative expression of serum miR-373 increased as PD aggravated. Likewise, early, midterm, and advanced PD were set to 1, 2, and 3, respectively. Using Spearman's correlation analysis, we found a positive correlation between the relative expression of serum miR-373 and PD severity (r = 0.710, P < 0.001; Figure 4).

Figure 4. The relative expression of serum miR-373 was positively correlated with Parkinson's disease severity (r = 0.710, P < 0.001).

Discussion

PD is a degenerative disease of the nervous system, and its incidence continues to increase globally, posing a severe threat to human health (Torra et al. 2018; Uysal-Cantürk et al. 2018; van Wijk et al. 2018). Early intervention and control of PD are the key to improving the quality of life of affected patients; therefore, researchers are constantly searching for more sensitive and accurate diagnostic methods for PD (Kam et al. 2018; Lionnet et al. 2018). The clinical manifestation of early PD lacks specificity, and the traditional diagnostic and treatment methods for PD are defective; hence, its prognosis is not optimistic. Therefore, investigating biomarkers that are strongly linked to the diagnosis and prognosis of PD is crucial (Gupta et al. 2018).

MiRs are a class of endogenous, noncoding, single-stranded RNAs with tumor-promoting or tumor-suppressing function (Valentino et al. 2017). Abnormal miR expression is closely related to immunoregulation, and miRs play an important role in regulating the biological functions of the cells of the immune system (Turchinovich et al. 2016). According to some studies, miRs are related to changes in the substantia nigra pars compacta in patients with PD, and miRs are closely related to the mechanisms of neuroinflammation and nutrition (Wang et al. 2015; Chen et al. 2016; Yao et al. 2018). Some studies have reported that serum miR-30c-5p expression in patients with PD was significantly upregulated compared with that in normal individuals (Vallelunga et al. 2019). In the present study, we first analyzed the differences in serum miR-30c-5p and miR-373 expressions between patients with PD and healthy individuals, and we found that serum miR-30c-5p and miR-373 expression were significantly higher in patients with PD than in healthy individuals. By analyzing the relationship between serum miR-30c-5p and miR-373 expressions and the clinicopathological characteristics of patients with PD, we found that their expressions were related to history of tardive dyskinesia, postural instability, and gait disorder as well as Hoehn–Yahr, MMSE, and MADRS scores. However, only few studies have focused on the effects and clinical significance of the changes in serum miR-30c-5p and miR-373 expressions on PD progression and prognosis. Therefore, further studies and attention are required. In a follow-up study, the correlation of serum miR-30c-5p and miR-373 expressions in patients with PD can be further explored to improve the study results. According to our results, we hypothesized that miR-30c-5p and miR-373 play important roles in evaluating PD severity.

In this study, we investigated serum miR-30c-5p and miR-373 expressions in patients with PD and their expression in those with different PD severities; however, certain deficiencies exist. For example, the regulatory effect of changes in serum miR-30c-5p and miR-373 expressions on related leukocytes in patients with PD is not analyzed more specifically, and the biological function of the two factors is not explained further. Moreover, the clinical routine inflammatory factors of miR-30c-5p, miR-373, and PD were not analyzed, which had a certain influence on the improvement of the study design. Therefore, we will refer to the latest research in real time in the later stage, add corresponding research schemes, conduct more in-depth analysis and research, and make up for the design defects to constantly improve related research.

In conclusion, serum miR-30c-5p and miR-373 expressions were upregulated in patients with PD, and they may be involved in the occurrence and development of PD. Therefore, they can be used as potential serum biomarkers for the assessment of progressive brain changes in patients with PD.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data used to support the findings of this study are available from the corresponding author upon request.

Additional information

Funding

This work was supported by the China–Japan Friendship Hospital under grant number 2018-2-QN-34.