Differentially expressed microRNAs in peripheral blood mononuclear cells of non‐segmental vitiligo and their clinical significance

Abstract Background Vitiligo is a frequent acquired depigmentation skin disease due to a loss of melanocytes. This study sought to characterize the expression pattern of microRNA (miRNA) in the peripheral blood mononuclear cells (PBMCs) of non‐segmental vitiligo (NSV) patients. We also screened for molecular markers that can be used to evaluate the clinical stages of NSV. Methods The miRNA expression profile in the PBMCs of four patients with progressive NSV and four healthy controls was determined using high‐throughput RNA sequencing. The divergently expressed miRNA was verified via qRT‐PCR in 26 progression, 26 stable NSV, and 26 healthy controls. Results Our findings posited that 323 miRNAs were differentially expressed in the PBMCs of NSV patients. The top 10 up‐regulated miRNAs in patients were hsa‐miR‐335‐5p, hsa‐miR‐20a‐5p, hsa‐miR‐514a‐3p, hsa‐miR‐144‐5p, hsa‐miR‐450b‐5p, hsa‐miR‐369‐3p, hsa‐miR‐101‐3p, hsa‐miR‐142‐5p, hsa‐miR‐19b‐3p, and hsa‐miR‐340‐5p. The top 10 down‐regulated miRNAs in patients were hsa‐miR‐4443, hsa‐miR‐1248, hsa‐miR‐6859‐3p, hsa‐miR‐668‐3p, hsa‐miR‐7704, hsa‐miR‐323a‐5p, hsa‐miR‐1237‐3p, hsa‐miR‐3127‐3p, hsa‐miR‐6735‐3p, and hsa‐miR‐127‐3p. The expressions of hsa‐miR‐20a‐5p in PBMCs of progressive and stable NSV were remarkably elevated relative to the healthy controls. In the characteristics curve analysis of hsa‐miR‐20a‐5p for differentiating progressive and stable NSV from normal subjects in PBMCs, the area under curve (AUC) was 0.92 and 0.81. Compared with patients in stable NSV, the hsa‐miR‐20a‐5p was markedly increased in PBMCs of progressive NSV patients, and the AUC was 0.81. Conclusion Our results showed that divergently expressed miRNAs contribute to the pathogenesis of NSV and that hsa‐miR‐20a‐5p can be applied as a biosignature for stage assessment in PBMCs of patients with NSV.


| INTRODUC TI ON
Vitiligo constitutes a frequent acquired depigmentation skin disease due to a loss of melanocytes. It occurs worldwide with an estimated prevalence of 0.2% in community-hinged studies and 1.8% in hospital-centered studies. 1 It is a complex disease caused by the interaction of genetic, immune abnormalities, and psycho-psychological factors. 2 The clinical manifestations are depigmentation spots of different sizes and shapes in the skin and mucosa. Vitiligo is easily diagnosed but difficult to treat. It affects the physical beauty and torture to patients, thereby reducing their quality of life. 3 The origin of the degeneration of epidermal melanocytes is intricate and remains unclear. Diverse theories have been proposed: One of the theories suggests that melanocytes destroy themselves through autoimmune mechanism (mainly cellular immunity). 4 Peripheral blood mononuclear cells (PBMCs) are composed of lymphocytes (T, B lymphocytes), NK cells, monocytes, and dendritic cells in the blood circulation. These cells form an important part of the immune system because many immune cells migrate from the blood circulation to the skin. In addition, PBMCs in patients with vitiligo over-produce proinflammatory cytokines consisting of interleukin (IL)-1b, IL-8, IL-6, and the tumor necrosis factor (TNF)-α. 5 The cytokines infiltrate around the vitiligo lesions, indicating that PBMCs are closely related to the pathogenesis of vitiligo. However, its specific pathogenesis is still unclear.
In recent years, genome-wide microRNA (miRNA) studies have been extensively used to elucidate the genetic and immune-correlated pathogenesis of vitiligo. miRNAs constitute a kind of non-coding RNA (ncRNA) approximately 23 nucleotides long. They are present in eukaryotic organisms, and control post-transcriptional gene expression by repressing translation or enhancing the degeneration of mRNA in the cytoplasm. 6 With the discovery of gene chips and RNA sequencing technology, researchers have realized the pivotal function of miRNAs in the modulation of human gene expression. It has been shown that miRNAs were associated with complex diseases, and may become biosignatures for the diagnosis and therapy of complex diseases. 7 So far, reports have revealed that miRNAs were abnormally expressed in skin tissue, serum, and PBMCs of vitiligo. For example, Shi and coworkers have found that miR-16, miR-19b, and miR-720 were effective serological markers to differentiate NSV from healthy peoples. 8 However, studies on miRNA expression profiles and functional analysis in PBMCs of patients with NSV at different stages have not been reported.
The primary purpose of this research was to analyze the differential miRNA expression profiles between the PBMCs of patients with progressive NSV and healthy individuals. The potential functions of different miRNAs in the pathogenesis of vitiligo were analyzed to provide a theoretical foundation for further research.

| Subject information and sample collection
Fifty-six patients, as well as 30 age-and sex-corresponding healthy control individuals, were recruited in this study at our hospital from July 2019 to May 2020. Based on clinical symptoms, they were divided into progressive NSV (among four cases were used for RNA-seq) and stable NSV. The progressive vitiligo patients had enlarged lesions with new lesions forming within 6 months and with a Vitiligo European Task Force (VETF) transmission score of +1 to +5. The stable vitiligo patients had no increase in size within 6 months and had a VETF score of −5 to 0 which is considered stable vitiligo. 9,10 The protocols used in this study were ratified by the ethics review committee. All the subjects signed a written informed consent. All subjects were not accompanied by other organic, autoimmune, and infectious diseases. The patients were not systematically treated with glucocorticoids, immunosuppressants, photosensitizers, and ultraviolet rays within 1 month.

| PBMC isolation and RNA extraction
Peripheral venous blood (5 mL) was collected from patients with progressive NSV, stable NSV, and healthy controls. The PBMCs were isolated from the human peripheral blood lymphocyte using the density-gradient centrifugation method and maintained at −80°C awaiting RNA purification. We employed the TRIzol Kit (Life Technologies) in purifying the total RNA from the PBMCs as outlined in the protocol of the manufacturer. The RNA quality was inspected by a ND-1000 NanoDrop (Thermo Fisher) and Agilent 2200 Bioanalyzer (Agilent Technologies).
Ltd. The 2−△△ct method was used to quantitatively analyze the results.

| Structure of miRNA-mRNA network
miRNA target genes were predicted by miRTarBase, miRDB, TargetScan, and miRWalk. The final predicted target genes were acquired by three software (any three of the four mentioned above). Cytoscape 3.6.1 was used to construct miRNA and predict target gene network.

| Statistical analysis
All data were counted by GraphPad Prism version 7.0. We used Student's t test to analyze normally distributed measurement data between two arms. Comparisons among multiple study arms were F I G U R E 1 Expression pattern of miRNAs in progressive non-segmental vitiligo (P-NSV) and healthy controls (HC) as determined by RNA-seq. A, The scatter plot shows the correlation analysis between all samples. The greater the correlation coefficient and the darker the color (blue squares, pale blue squares, and white squares), the higher the relationships of samples. B, Volcano diagram shows divergently expressed miRNAs between the two arms. Red dot represents up-regulation, and green dot represents down-regulation

| Expression profile of miRNAs in PBMCs of NSV
The sequencing data were normalized and compared between the two arms. The Pearson correlation coefficient (r) between progressive NSV (P-NSV) and healthy controls (HC) is shown in

| Verification of the divergently expressed miRNAs
qRT-PCR was employed to inspect the expression level of seven divergently expressed miRNAs (relatively plentiful, |log2(Fold_ Change)| > 2 and P < .001) in the progressive NSV, stable NSV, and healthy individuals. Results showed that the expressions of hsa-miR-20a-5p, hsa-miR-335-5p, and hsa-miR-340-5p in PBMCs of progressive and stable NSV were markedly higher relative to the control arm (P < .001). Relative to the stable phase NSV patients, hsa-miR-20a-5p was increased in PBMC progressive NSV patients remarkably ( Figure 5).

F I G U R E 3
The top 10 GO enrichment terms. GO assessment was conducted on the parental genes of miRNAs with different expression levels (|log2Fold_Change| ≥ 1, P < .05). The significant threshold is P < .05, which has obtained statistically significant biological functions and pathways. GO enrichment analysis showed that changes in biological processes of differential miRNAs mainly occurred in cellular, metabolic, primary metabolic, and cellular metabolic We then assessed the possibility of hsa-miR-20a-5p as a prospective disease marker for NSV. ROC curve inspection unearthed that the PBMCs hsa-miR-20a-5p level could serve as an effective biosignature for differentiating progressive and stable NSV patients from healthy individuals with the AUC of 0.92 and 0.81 ( Figure 6).
More importantly, the PBMC hsa-miR-20a-5p level could serve as an effective marker for differentiating progressive NSV from stable NSV patients with the AUC of 0.81 ( Figure 6).

F I G U R E 4
The top 30 KEGG pathways are enriched in terms. The KEGG pathway assessment revealed that TGF-beta, mTOR, focal adhesion, and PI3K-Akt signaling cascade were significantly related to divergently expressed miRNAs in NSV ellipses) of three significantly differential miRNAs (red triangles) was drawn using Cytoscape3.6.1 (Figure 7).

| D ISCUSS I ON
Vitiligo is a common depigmented skin disease, characterized by In this study, high-throughput RNA sequencing was employed to analyze the expression pattern of miRNA between progressive NSV and healthy people. We additionally studied the relationships between abnormal expression of miRNAs and NSV activities. We found a large number of divergently expressed miRNAs, of which 223 miRNAs were markedly upmodulated and 100 downmodulated.
Previous studies have shown that the miRNAs family is a considerable part of the gene expression regulatory network. Determination of miRNAs target genes is crucial for comprehending the biological roles of miRNAs. The functional analysis of target genes is helpful to conjecture the possible function of miRNAs. 16 A single miRNA can act on many target genes to regulate gene expression, while multiple miRNAs can also adjust certain target genes. We established that divergently expressed miRNAs participated in the modulation of 43 144 target genes based on GO analysis. They are mainly involved in combined, cell periphery, and single biological processes. The KEGG pathway analysis implied that focal adhesion, 17 TGF-beta, 18 mTOR, 19 and PI3K-Akt signaling pathway were markedly contacted for divergently expressed (P < .05) miRNAs in NSV. 20 Therefore, abnormal expression of miRNAs might be participating and modulating the development of NSV.
Subsequently, we selected seven miRNAs (relatively plentiful, |log2(Fold_Change)| > 2 and P < .001) for further evaluation. The results showed that the expression of hsa-miR-20a-5p, hsa-miR-335-5p, and hsa-miR-340-5p in PBMCs of progressive and stable NSV was significantly higher than healthy controls. It is speculated that the divergently expressed miRNAs might serve a significant role in the pathogenesis of vitiligo. Further analysis revealed that hsa-miR-20a-5p was significantly increased in PBMCs of progressive NSV patients relative to the patients in stable phase NSV. This implies that hsa-miR-20a-5p was associated with vitiligo activity.
The ROC curve assessment disclosed that the PBMC hsa-miR- is a common target gene of hsa-miR-340-5p and hsa-miR-335-5p.
In conclusion, the present work provided a profile of miRNA expression profiles in PBMCs in patients with NSV. Moreover, this study found that miRNAs are involved in vitiligo activity. We demonstrate that hsa-miR-20a-5p has the potential to become a biomarker for stage assessment of NSV. Further studies should focus on investigating the exact mechanism of miRNA-mRNA network in NSV in a large sample.

ACK N OWLED G M ENTS
We are grateful to all the study participants.

E TH I C A L A PPROVA L
The study was approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University in agreement with the Declaration of Helsinki. Written informed consent was obtained from the guardians of the study subject.

DATA AVA I L A B I L I T Y S TAT E M E N T
All relevant data are included in the manuscript. The datasets used and/or analyzed during the current study are available from the corresponding author upon request.