   |
|
BASIC RESEARCH |
|
| Year : 2022 | Volume
: 67
| Issue : 6 | Page : 705-714 |
|
| Differential expression and in Silico functional analysis of plasma micrornas in the pathogenesis of non-segmental vitiligo |
|
|
Suzan Demir Pektas1, Murat Kara1, Gürsoy Doğan1, Mehmet Bilgehan Pektaş2, Mehmet Cengiz Baloğlu3, Gökhan Sadi4
1 From the Department of Dermatology, Faculty of Medicine, Muğla Sıtkı Koçman University, Mugla, Turkey
2 Department of Pharmacology, Faculty of Medicine, Afyonkarahisar Health Sciences University, Kastamonu, Turkey
3 Department of Genetics and Bioengineering, Engineering Faculty, Kastamonu University, Kastamonu, Turkey
4 Department of Biology, K. Ö. Science Faculty, Karamanoğlu Mehmetbey University, Karaman, Turkey
| Date of Web Publication | 23-Feb-2023 |
Correspondence Address:
Gökhan Sadi
Department of Biology, K. Ö. Science Faculty, Karamanoğlu Mehmetbey University, 70100, Karaman
Turkey
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ijd.ijd_383_21
|
|
 Abstract | | |
Vitiligo is a disease characterized by acquired depigmentation, white macules, and patches on the skin due to the dysfunction of epidermal melanocytes. In this study, we attempt to profile the microRNA (miRNA) expression patterns and predict the potential targets, assessing the biological functions of differentially expressed miRNAs in the blood of generalized vitiligo patients. Peripheral blood samples were taken from all participants, and the expression levels of 89 identified miRNAs were analyzed with real-time quantitative polymerase chain reaction (PCR). The results indicated significant upregulation of six miRNAs and downregulation of 19 miRNAs in the plasma of vitiligo patients. The top three upregulated miRNAs were hsa-miR-451a, hsa-miR-25-3p, and hsa-miR-19a-3p, and the top three downregulated miRNAs were hsa-miR-146a-5p, hsa-miR-940, and hsa-miR-142-3p. Moreover, the miRNA expression profiles of patients with Type 3 and Type 4 phototypes were substantially different in such a way that the patients with Type 3 phototype would be more prone to the emergence of melanoma and cancer. While significant variations in the expression patterns of miRNAs in male and female vitiligo patients were demonstrated, miR-let-7i-5p, miR-19a-3p, miR-25-3p, and miR-451a were commonly upregulated, and miR-142-3p and miR-146a-5p were commonly repressed in both sexes. This study may shed light on the roles of differentially expressed miRNAs in vitiligo patients by examining the miRNA expression patterns and the combined effects of miRNA and their predicted targets.
Keywords: Functional Enrichment, generalized vitiligo, KEGG analysis, miRNA profile
How to cite this article:
Pektas SD, Kara M, Doğan G, Pektaş MB, Baloğlu MC, Sadi G. Differential expression and in Silico functional analysis of plasma micrornas in the pathogenesis of non-segmental vitiligo. Indian J Dermatol 2022;67:705-14 |
How to cite this URL:
Pektas SD, Kara M, Doğan G, Pektaş MB, Baloğlu MC, Sadi G. Differential expression and in Silico functional analysis of plasma micrornas in the pathogenesis of non-segmental vitiligo. Indian J Dermatol [serial online] 2022 [cited 2023 Mar 29];67:705-14. Available from: https://www.e-ijd.org/text.asp?2022/67/6/705/370295 |
| Introduction | |  |
Vitiligo is a skin pigmentation disorder that originates after the destruction of functional melanocyte cells. Although its origin is still unknown, an association between vitiligo, autoimmune pathogenesis, and many systemic diseases has been demonstrated.[1–3] Its estimated prevalence is near 1% and may appear at any time, affecting the quality of life.[4] Vitiligo can be classified as non-segmental and segmental types. Non-segmental vitiligo (NSV), the most typical form of this unpredictable disease, is characterized by symmetrical and bilateral white patches. Different clinical subtypes have been described, including generalized, acrofacial, and universalis types, all with a bilateral distribution.
MicroRNAs (miRNAs) are short, highly conserved, non-coding RNAs consisting of 19–25 nucleotides. They have critical roles in post-transcriptional gene regulatory mechanisms of various biological processes, and abnormalities occurring in miRNA transcription have been associated with various diseases, including vitiligo.[5–7] For example, abnormalities in miR-155 expression in vitiligo patients were related to the inhibition of the melanogenesis pathway,[8] and increased miR-421 and miR-421 expression levels were associated with endoplasmic reticulum stress, which is a risk factor for vitiligo.[9] miR-211, which is related to oxidative phosphorylation in human melanocytes, was repressed in vitiligo patients, leading to oxidative stress.[10] miR-16, miR-19b, and miR-720 levels were increased significantly in the serum of NSV patients; however, serum miR-574-3p was associated with the severity of the disease.[11] Upregulation of miR-135a, miR-183, miR-30a-3p, and miR-487 levels in depigmented regions of the skin of vitiligo patients was related to the onset and progression of the disease.[12] Although these differentially expressed miRNAs have been discovered in vitiligo, only a few targets and role in the pathogenesis of the disease have been identified. It would be essential to identify the miRNAs involved in the pathogenesis of vitiligo and understand the molecular regulatory pathways to develop miRNA-based diagnostic and therapeutic strategies. Therefore, this study was designed to detect the differentially expressed miRNAs in the plasma samples of generalized vitiligo patients and provide some descriptions for the biological functions of the predicted target genes.
| Materials and Methods | | |
Peripheral blood specimens were obtained from the Department of Dermatology, Muğla Sıtkı Koçman University, after taking the written consent of all patients. The experimental protocols described in this study were approved by the ethics review committee of Muğla Sıtkı Koçman University (date 02/24/2016 and number 04/III) and were prospectively conducted in compliance with the guidelines. Whole blood samples (3 ml) from a total of 31 NSV patients (aged 20–50 years) were collected into tubes containing ethylenediaminetetraacetic acid (EDTA) under aseptic conditions. A total of 31 age–gender–phototype-matched healthy volunteers (control group) who had undergone systematic health diagnosis and were defined as not having first-degree, second-degree, or third-degree relatives with vitiligo (20–56 years of age) or any other autoimmune disease were studied. The patients had not been systematically treated with glucocorticoids, immunosuppressants, photosensitizers, and ultraviolet rays within the last 1 month. According to the Fitzpatrick skin phototype classification, the skin phototype of the patient and control groups was determined.
miRNA-enriched total RNAs were isolated from the 200-μl plasma samples using miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany), as described in the manufacturer's protocol. After isolation, the quality of the total RNAs was determined by Qubit 4.0 fluorometer (Thermo Scientific, Waltham, USA). Then, total RNAs were reverse transcribed to cDNA using a commercial cDNA synthesis kit (miScript II RT Kit; Qiagen, Germany). Briefly, 4 μl of 5x miScript HiSpec buffer, 2 μl of 10x miScript Nucleics Mix, and 2 μl of miScript Reverse Transcriptase were mixed with 9 μl of RNA samples and the total volume was adjusted to 20 μl with RNase-free water. Then, the samples were incubated at 37°C for 60 min for reverse transcription and at 95°C for 5 min to inactivate miScript Reverse Transcriptase.
Human 96-well miRNome miScript miRNA PCR Array (Qiagen, Waltham, USA) was utilized to profile plasma miRNA expression patterns of both control and vitiligo patients using miScript SYBR Green PCR Kit (Qiagen) with a Biomark Real-Time PCR (Fluidigm, San Francisco, USA) instrument. Accordingly, 12.5 μl of 2x QuantiTect SYBR Green PCR Master Mix, 2.5 μl of 10x miScript Universal Primer, and 9 μl of RNase-free water were mixed with 1 μl of cDNA (fivefold diluted), and then this mixture was added to each well of the array plate. Following brief centrifugation, quatitative Polymerase Chain Reaction (qPCR) was performed as follows: initial denaturation at 95°C for 15 min, denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 70°C for 30 s with 40 repeated thermal cycles, measuring the green fluorescence at the end of each extension step. The specificity of PCR products was confirmed using melt analysis.
Human miRNome miScript miRNA PCR Array includes the primers for the following mature miRNAs: hsa-let-7d-5p, hsa-let-7i-5p, hsa-miR-106a-3p, hsa-miR-106b-5p, hsa-miR-10a-3p, hsa-miR-1185-1-3p, hsa-miR-122-3p, hsa-miR-125a-3p, hsa-miR-125b-1-3p, hsa-miR-125b-1-3p, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-126-5p, hsa-miR-1266-5p, hsa-miR-128-3p, hsa-miR-1290, hsa-miR-133a-3p, hsa-miR-135a-3p, hsa-miR-136-3p, hsa-miR-139-5p, hsa-miR-1-3p, hsa-miR-142-3p, hsa-miR-146a-3p, hsa-miR-146a-5p, hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-150-3p, hsa-miR-151a-5p, hsa-miR-155-5p, hsa-miR-16-1-3p, hsa-miR-181a-5p, hsa-miR-183-3p, hsa-miR-186-3p, hsa-miR-191-3p, hsa-miR-192-3p, hsa-miR-193b-3p, hsa-miR-195-3p, hsa-miR-196a-3p, hsa-miR-19a-3p, hsa-miR-200a-3p, hsa-miR-200b-3p, hsa-miR-200c-3p, hsa-miR-203b-3p, hsa-miR-204-3p, hsa-mir-205-5p, hsa-miR-206, hsa-miR-210-5p, hsa-miR-21-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-223-3p, hsa-miR-22-3p, hsa-miR-24-3p, hsa-miR-25-3p, hsa-miR-26a-1-3p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-296-3p, hsa-miR-29a-3p, hsa-miR-30a-3p, hsa-miR-30b-3p, hsa-miR-30c-1-3p, hsa-miR-30d-3p, hsa-miR-31-5p, hsa-miR-328-3p, hsa-miR-342-3p, hsa-miR-369-3p, hsa-miR-375, hsa-miR-378a-3p, hsa-miR-383-5p, hsa-miR-410-3p, hsa-miR-424-5p, hsa-miR-425-3p, hsa-miR-451a, hsa-miR-483-5p, hsa-miR-487a-3p, hsa-miR-498, hsa-miR-503-5p, hsa-miR-551a, hsa-miR-574-3p, hsa-miR-590-5p, hsa-miR-663a, hsa-miR-720, hsa-miR-7-2-3p, hsa-miR-93-3p, hsa-miR-9-3p, hsa-miR-940, hsa-miR-99a-5p. In silico analysis of genes identified for this purpose was carried out according to the appropriate procedure.[13]
Statistical analysis
The relative expression of miRNAs with respect to snRNA (RNU6B) was calculated with web-based RT2 Profiler Data Analysis Software (Qiagen, USA) using miScript miRNA PCR Array data analysis tool. Once raw threshold cycle (CT) data has been obtained, the tool automatically performs all fold-change calculations using the ΔΔCT method of relative quantification, and Student's t-test was utilized to determine significant differences at P < 0.05. Venn diagrams were used for the representation of common up- and downregulated miRNAs between groups. Heat map analysis exhibiting the hierarchical clustering of miRNAs differentially expressed between vitiligo patients and non-vitiligo controls was performed using the Pearson correlation coefficient.
| Results | | |
Clinical features of control and patient groups
The age distribution, skin phototype, disease progression, symptom history, and the presence of poliosis, leukotrichi, and halo nevus of the sample groups are summarized in [Table 1]. Accordingly, the age and gender of the control and vitiligo groups had almost similar distribution; but unlike the vitiligo group, type 3 skin phototypes were higher in the control group. It is also obvious that the majority of vitiligo patients had a disease history greater than 6 months. Also, some patients had halo nevus, leukotrichi, and poliosis as side effects.
Altered plasma miRNA expression profiles of generalized vitiligo patients compared to the control group
Expression patterns of 89 pre-identified miRNAs were evaluated with human miRNome miScript miRNA PCR Array in both control and generalized vitiligo patients without subdividing the other variables such as gender, duration of disease, and skin phototypes, and the changes in expression values were calculated. The list of significantly regulated miRNAs is given in [Table 2] with fold-change (Log2FC) values and significance levels. Additionally, the number of target proteins of analyzed miRNAs was inspected with the latest version of TargetScan (v7.1; targetscan.org),[14] there by providing a valuable resource for placing the miRNAs into gene regulatory networks, which are also given in this table.  | Table 2: Changes in miRNA expression profiles of generalized vitiligo patients with Log2FC, P values, and the number of putative target proteins possibly regulated by respective miRNAs
Click here to view |
According to data, six plasma miRNAs were upregulated, while almost three times higher (19 miRNAs) number of miRNAs were downregulated in vitiligo patients (P < 0.05). The top three upregulated miRNAs were hsa-miR-451a, hsa-miR-25-3p, and hsa-miR-19a-3p and the top three downregulated miRNAs were hsa-miR-146a-5p, hsa-miR-940, and hsa-miR-142-3p. Hierarchical clustering of the analyzed miRNAs was also conducted according to fold-change values [Figure 1]. Additionally, when the patients with generalized vitiligo for more than 6 months were compared to the patient group whose disease duration was less than 6 months, hsa-miR-128-3p, hsa-miR-126-5p, and hsa-miR-122-3p expression was found to be upregulated (P < 0.05). No statistically significant relationship was found between micro-RNA expression and other accompanying disorders in the patients. | Figure 1: Hierarchical clustering of the analyzed miRNAs in the control and vitiligo groups. The red color indicates upregulation and the yellow color indicates downregulation, with different intensities representing the fold-change values miRNA = microRNA
Click here to view |
Functional enrichment of target proteins of differentially expressed miRNAs in vitiligo patients
We first queried targetscan.org to identify the predicted targets of the individual miRNAs and used Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis[15] to identify the protein pathways targeted by differentially expressed miRNAs. The results revealed the top pathways targeted by the differentially expressed miRNAs are the melanoma pathway with 11 targeted proteins and the PI3K-Akt and mitogen activated protein kinase (MAPK) signaling pathways with seven and six targeted proteins, respectively. Other top targeted pathways and the number of targeted proteins are summarized in [Figure 2]. cAMP and forkhead box protein 3 (Foxo3a) signaling, epidermal growth factor receptor (EGFR) pathway, miRNAs in cancer, and hypoxia inducible factor (HIF)-1 signaling are also targeted by differentially expressed miRNAs in generalized vitiligo patients. | Figure 2: Functional classification of differentially expressed miRNAs in the plasma of generalized vitiligo patients. The numbers of targeted proteins with differentially expressed miRNAs are summarized. One protein could be classified under more than one biological process. miRNA = microRNA
Click here to view |
Our results indicated an indirect association of vitiligo with melanoma, since most of the target proteins of differentially expressed miRNAs function in regulating melanoma, cancer, and cancer-related pathways such as MAPK, cAMP, and PI3K/Akt. The target proteins of differentially expressed miRNAs in the melanoma pathway according to the KEGG pathway analysis have been demonstrated in [Figure 3]. | Figure 3: Melanoma and related pathway proteins in the KEGG database. The proteins targeted with differentially expressed miRNAs in vitiligo patients have been demonstrated in red color. KEGG = Kyoto Encyclopedia of Genes and Genomes, miRNA = microRNA
Click here to view |
Altered expression levels of miRNAs in vitiligo patients with Type 3 and Type 4 phototypes
We analyzed the difference in the miRNA expression profile of patients with Type 3 and Type 4 phototypes and found interesting differences. On analysis of control and vitiligo patients with Type 3 phototype, 50 miRNAs out of 89 were found to be significantly different in such a way that 44 miRNAs were downregulated and six miRNAs were upregulated. The top four suppressed miRNAs (Log2FC >2.0) were hsa-miR-142-3p, hsa-miR-151a-5p, hsa-miR-940, and hsa-miR-146a-5p and the top four induced miRNAs (Log2FC >1.5) were hsa-miR-139-5p hsa-miR-19a-3p, hsa-miR-25-3p, and hsa-miR-451a. A similar comparison was also conducted based on skin Type 4 phototype; only hsa-miR-146a-5p was suppressed, but three (hsa-miR-25-3p, hsa-miR-378a-3p, hsa-miR-296-3p) were induced significantly. Among the significantly regulated miRNAs, hsa-miR-146a-5p was commonly downregulated, while hsa-miR-25-3p was similarly upregulated in both phototypes. On the other hand, while hsa-miR-296-3p was suppressed in Type 3 patients, it was augmented in the Type 4 vitiligo group. The changes in both hsa-miR-146a-5p and hsa-miR-25-3p were also validated in overall comparison, and they could be important as a biomarker and a contributing factor for the pathology of vitiligo.
Altered expression levels of miRNAs in vitiligo patients depending on sex
Various circumstances ranging from physiological processes to therapeutic responses alter with sex, and a comprehensive analysis of sex difference in differentially expressed miRNAs has been performed in vitiligo patients, the results of which are summarized in [Figure 4]. Accordingly, we identified nine and seven significantly upregulated miRNAs in male and female vitiligo patients, respectively. Among these, miR-let-7i-5p, miR-19a-3p, miR-25-3p, and miR-451a were commonly upregulated in both sexes [Figure 4]a. Additionally, 40 miRNAs in males and three miRNAs in females were suppressed [Figure 4]b, reflecting gender-specific repression of a variety of miRNAs. miR-142-3p and miR-146a-5p were the commonly repressed ones, and they could serve as important contributors for vitiligo pathogenesis, together with commonly upregulated miRNAs. | Figure 4: Venn diagrams for differentially upregulated (a) and downregulated (b) miRNAs in male and female vitiligo patients. The number of miRNAs unique to each group is shown inside the circle; the number of miRNAs changed in two groups is shown in the shaded overlapping areas. miRNA = microRNA
Click here to view |
Heat map analysis exhibiting the hierarchical clustering of 89 miRNAs in plasma from male and female patients with NSV and healthy plasma from control individuals has been demonstrated in [Figure 5]. | Figure 5: The hierarchical clustering of the miRNA expression profile of males and female samples. Red and green colors indicate relative expression levels corresponding to the scale bar shown at the bottom. Green color indicates reduced expression; red color indicates increased expression. miRNA = microRNA
Click here to view |
| Discussion | | |
Small non-coding RNAs play important functions in various cellular and biological processes such as cell proliferation and differentiation,[16],[17] growth and development,[18] and metabolic homeostasis.[19] Their circulating forms have been identified in various body fluids like the serum, plasma, saliva, and urine, with high stability.[20],[21] Passive leakage from the damaged cells due to chronic inflammation, cell apoptosis, or necrosis and active secretion via exosomes released by almost all cell types under both normal and pathological conditions have been proposed as the sources of such extracellular miRNAs.[22] Vitiligo is a depigmentation disease where the functions of the melanocytes are disordered, causing white macules on the skin, and the precise mechanisms for the pathogenesis of vitiligo have remained elusive. It has been reported that many factors such as autoimmunity, cytotoxic metabolites, neural and genetic theories have been proposed to explain the mechanisms of pigmentation loss. In addition to melanocyte dysfunction, keratinocyte alteration plays a role; keratinocytes in the depigmented epidermis are more vulnerable to apoptosis and produce lower amounts of melanogenic mediators than normal skin.[8] Here, we have attempted to study the alterations in the expression pattern of plasma miRNAs of generalized vitiligo patients and to analyze further the potential target proteins and pathways that are regulated in response to these altered miRNA molecules.
Our data indicated the modulation of plasma miRNAs in response to vitiligo in such a way that six were upregulated, while 19 miRNAs were downregulated significantly. The top three upregulated (Log2FC > 1.5) miRNAs were hsa-miR-451a, hsa-miR-25-3p, and hsa-miR-19a-3p. Among these, hsa-miRNA-451a has been associated with cell proliferation, migration, and apoptosis[23] and hsa-miR-25 highlights the key biological processes and pathways such as angiogenesis and cell–cell adhesion.[23] Additionally, miR-19a-3p has been associated with the invasion, migration, and metastasis of cancer tissues.[24] Enhanced expression levels of these miRNAs indicate the induction of cell cycle progression, differentiation, and cell survival in generalized vitiligo patients. Our results also demonstrated significant suppression of plasma miRNAs, the top three of which are hsa-miR-146a-5p, hsa-miR-940, and hsa-miR-142-3p. Among these, miR-146a-5p has been associated with cancer metabolism and proposed as a noninvasive biomarker and is targeted therapeutically in several cancers.[25],[26] Its downregulation occurs in the development of melanoma resistance to targeted drugs in melanoma patients.[27] Additionally, miR-146a-5p is considered as an essential regulator of autoimmune diseases since its main target Foxo3a controls regulatory T cells having critical roles in avoiding autoimmunity.[28] Diverse roles of miR-940 in cell proliferation, migration, metastasis, and apoptosis in various types of cancers[29],[30] and the regulatory functions of miR-142-3p over T-cell function and autoimmunity have recently been demonstrated.[31],[32] miR-142-3p is also proposed as a potential prognostic biomarker for esophageal squamous cell carcinoma.[33]
In the literature, some studies demonstrated that the biochemical and histopathologic properties might differ in spreading versus quiescent vitiligo, but they are not well characterized.[34] Our results showed that any miRNAs studied within the scope of this study had been differentially expressed in spreading and stable vitiligo patients. However, a very recent study conducted next-generation sequencing of serum miRNAs, in which overexpression of hsa-miR-20a-5p, hsa-miR-335-5p, and hsa-miR-340-5p in vitiligo patients was confirmed, and it is speculated that the divergently expressed miRNAs might serve a significant role in the pathogenesis of vitiligo. In this study, hsa-miR-20a-5p was significantly higher in progressive vitiligo patients compared with stable vitiligo patients, and it is proposed to be associated with vitiligo activity.[35] This evidence might reflect the potential of miRNAs to become a biomarker for disease progression.
Vitiligo leads to progressive destruction of skin melanocytes, and it has been associated with cutaneous melanoma.[11],[12],[36] Studies also indicate the presence of halo nevus, hypopigmentation, or depigmentation that may occur in patients with melanoma.[37] KEGG pathway and Gene Ontology (GO) analysis revealed the top biological pathways targeted by the differentially expressed miRNAs, and the melanoma pathway with 11 targeted proteins is the most prominent one. This result indicates an indirect association of vitiligo with melanoma since many of the target proteins of differentially expressed miRNAs are functioning in the regulation of melanoma, cancer, and cancer-related pathways. Detailed analysis of the functions of the differentially expressed miRNA-associated predicted target genes also suggests that the most significantly enriched target genes are focused on signal transduction pathways such as MAPK, cAMP, and PI3K/Akt, which could directly be associated with cell proliferation. These findings support the clinical link between vitiligo and melanoma.
Skin type (or phototype) classifies the amount of melanin pigment in the skin, and it is a constitutional characteristic present at birth. In the Turkish population, the majority of skin has Type 3 (light brown) or Type 4 (moderate brown) characteristics, and our study group had individuals of both. Even though there is no significant correlation between vitiligo and skin phototypes, the miRNA expression profile of patients with Type 3 or Type 4 phototype was substantially different. In vitiligo patients with Type 3 phototype, 44 miRNAs were downregulated and six miRNAs were upregulated. However, only hsa-miR-146a-5p was suppressed, but three miRNAs were induced significantly in patients with Type 4 phototype. The difference in the number of differentially expressed miRNAs reflects the modulation of a higher number of biological pathways in patients with Type 3 phototype. In the literature, it has been demonstrated that specific pigmentary characteristics are associated with increased relative risks of melanoma. A meta-analysis demonstrated more than twice the higher risk for Type 1 skin compared with people with skin phototype 4 for melanoma development. In that study, 35% higher risk for skin phototype 3 compared with skin phototype 4 has been confirmed.[38],[39] Skin depigmentation, such as that occurring in vitiligo, has been associated with cutaneous melanoma. Most of the antigens recognized by melanoma-specific cytotoxic T lymphocytes (CTLs) isolated from melanoma patients are expressed by both melanoma cells and normal melanocytes, explaining why autoimmune responses against melanocytes that lead to vitiligo could also be present in melanoma patients. Autoimmune responses against such antigens occasionally get into a specific immune reaction against melanoma and into tumor regression. In the light of this information, the results of our study may suggest that individuals with lighter skin types would be more prone to the emergence of melanoma and cancer.[36]
It is presently well established that females and males have completely different immune responses, consequently displaying differing degrees of susceptibility to and severity of various diseases. Accumulating evidence for differential miRNA expression between different sexes across a variety of tissues has been identified, and therefore, the sex-biased expression of miRNAs may have practical implications. For instance, miR-29a and miR-29c involved in neuronal cell maintenance are significantly upregulated in the frontal cortex of females, but not in male mice.[40] Likewise, the miR-200 family members are also differentially expressed in female and male rats, contributing to the sexual discrepancy in brain development.[41] Prominent expression of miR-221 and let-7g in women's plasma compared to that of men has been identified and these miRNAs are considered to be sex-specific biomarkers of metabolic syndrome.[41] It is known that the incidence of vitiligo does not differ between men and women in adult and pediatric patients.[1] In our study, these differences in serum miRNA expression levels in male and female patients may be related to the evaluation of the miRNA level in serum rather than the skin, the fact that the patients were not separated as active or stable, and dynamic changes of the serum values.
Herein, we also demonstrated the variations in the expression patterns of miRNAs in male and female vitiligo patients. Nine miRNAs were significantly upregulated in males, while only seven were upregulated in females. Among these, miR-let-7i-5p, miR-19a-3p, miR-25-3p, and miR-451a were commonly upregulated in both sexes. Furthermore, 40 miRNAs in males and three miRNAs in females were repressed, showing gender-specific repression of miRNAs. miR-142-3p and miR-146a-5p were the commonly repressed ones and they could serve as important contributors for vitiligo pathogenesis, together with commonly upregulated miRNAs.
| Conclusion | | |
In conclusion, our data in this study indicate the collective roles of miRNAs in the progression of vitiligo. The differential expression levels of plasma miRNAs in various subgroups of vitiligo patients are summarized, demonstrating the role of miRNAs and targets in disease progression. The current study also provides new insights into the role of miRNAs in the treatment of vitiligo. The miRNA signatures identified in our study need to be further validated with large sample size, along with their functional validation by in vitro studies, which could be used as predictive markers in the diagnosis and treatment of vitiligo. We believe that the limitation of our research is the dynamic nature of tissues such as blood, which is also very dynamic, and they often do not represent well the molecular events on a specific site such as the skin. Therefore, the miRNA signatures identified in our study need to be further studied. Validation of these serum miRNA biomarkers in a study with a large sample size and evaluation of their dynamic changes during the NSV process and treatment in future studies are essential, as they could be used as predictive markers in the diagnosis and treatment of vitiligo.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Data availability statement
The data that support the findings of the study are available on request from the corresponding author. All authors declared this situation.
Financial support and sponsorship
This study was supported by grants from the Muğla Sıtkı Koçman University Research Fund under grant (16/084).
Conflicts of interest
There are no conflicts of interest.
| References | | |
| 1. | Ezzedine K, Eleftheriadou V, Whitton M, Van Geel N. Vitiligo. Lancet 2015;386:74–84.  |
| 2. | Shong YK, Kim JA. Vitiligo in autoimmune thyroid disease. Thyroidology 1991;3:89–91. |
| 3. | Manga P, Elbuluk N, Orlow SJ. Recent advances in understanding vitiligo. F1000Res 2016;5:F1000 Faculty Rev-2234. doi: 10.12688/F1000RESEARCH.8976.1. |
| 4. | Lotti T, D'Erme AM. Vitiligo as a systemic disease. Clin Dermatol 2014;32:430–4. |
| 5. | Chansing K, Pakakasama S, Hongeng S, Thongmee A, Pongstaporn W. Lack of association between the MiR146a polymorphism and susceptibility to Thai childhood acute lymphoblastic leukemia. Asian Pac J Cancer Prev 2016;17:2435–8. |
| 6. | Bushati N, Cohen SM. microRNA functions. Annu Rev Cell Dev Biol 2007;23:175–205. |
| 7. | Fu G, Brkić J, Hayder H, Peng C. MicroRNAs in human placental development and pregnancy complications. Int J Mol Sci 2013;14:5519–44. |
| 8. | Šahmatova L, Tankov S, Prans E, Aab A, Hermann H, Reemann P, et al. MicroRNA-155 is dysregulated in the skin of patients with vitiligo and inhibits melanogenesis-associated genes in melanocytes and keratinocytes. Acta Derm Venereol 2016;96:742–7. |
| 9. | Sun X, Wang T, Huang B, Ruan G, Xu A. MicroRNA-421 participates in vitiligo development through regulating human melanocyte survival by targeting receptor-interacting serine/threonine kinase 1. Mol Med Rep 2020;21:858–66. |
| 10. | Sahoo A, Lee B, Boniface K, Seneschal J, Sahoo SK, Seki T, et al. MicroRNA-211 regulates oxidative phosphorylation and energy metabolism in human vitiligo. J Invest Dermatol 2017;137:1965–74. |
| 11. | Shi YL, Weiland M, Li J, Hamzavi I, Henderson M, Huggins RH, et al. MicroRNA expression profiling identifies potential serum biomarkers for non-segmental vitiligo. Pigment Cell Melanoma Res 2013;26:418–21. |
| 12. | Mansuri MS, Singh M, Dwivedi M, Laddha NC, Marfatia YS, Begum R. MicroRNA profiling reveals differentially expressed microRNA signatures from the skin of patients with non-segmental vitiligo. Br J Dermatol 2014;171:1263–7. |
| 13. | Barh D, Yiannakopoulou EC, Salawu EO, Bhattacharjee A, Chowbina S, Nalluri JJ, et al. In silico disease model: From simple networks to complex diseases. Anim Biotechnol 2020:441–60. doi: 10.1016/B978-0-12-811710-1.00020-3. |
| 14. | Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. Elife 2015;4:e05005. |
| 15. | Kanehisa M, Goto S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000;28:27–30. |
| 16. | Lu Y, Thomson JM, Wong HYF, Hammond SM, Hogan BLM. Transgenic over-expression of the microRNA miR-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells. Dev Biol 2007;310:442–53. |
| 17. | Wang Y, Baskerville S, Shenoy A, Babiarz JE, Baehner L, Blelloch R. Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat Genet 2008;40:1478–83. |
| 18. | Ebert MS, Sharp PA. Roles for MicroRNAs in conferring robustness to biological processes. Cell 2012;149:515–24. |
| 19. | Frost RJA, Olson EN. Control of glucose homeostasis and insulin sensitivity by the Let-7 family of microRNAs. Proc Natl Acad Sci U S A 2011;108:21075–80. |
| 20. | Condrat CE, Thompson DC, Barbu MG, Bugnar OL, Boboc A, Cretoiu D, et al. miRNAs as biomarkers in disease: Latest findings regarding their role in diagnosis and prognosis. Cells 2020;9:276. |
| 21. | Wang J, Chen J, Sen S. MicroRNA as biomarkers and diagnostics. J Cell Physiol 2016;231:25–30. |
| 22. | Mumford SL, Towler BP, Pashler AL, Gilleard O, Martin Y, Newbury SF. Circulating microRNA biomarkers in melanoma: Tools and challenges in personalised medicine. Biomolecules 2018;8:21. doi: 10.3390/biom8020021. |
| 23. | Su Z, Ni L, Yu W, Yu Z, Chen D, Zhang E, et al. MicroRNA-451a is associated with cell proliferation, migration and apoptosis in renal cell carcinoma. Mol Med Rep 2015;11:2248–54. |
| 24. | Nariman-saleh-fam Z, Vahed SZ, Aghaee-Bakhtiari SH, Daraei A, Saadatian Z, Kafil HS, et al. Expression pattern of miR-21, miR-25 and PTEN in peripheral blood mononuclear cells of patients with significant or insignificant coronary stenosis. Gene 2019;698:170–8. |
| 25. | Iacona JR, Lutz CS. miR-146a-5p: Expression, regulation, and functions in cancer. Wiley Interdiscip Rev RNA 2019;10:e1533. |
| 26. | Williams AE, Perry MM, Moschos SA, Larner-Svensson HM, Lindsay MA. Role of miRNA-146a in the regulation of the innate immune response and cancer. Biochem Soc Trans 2008;36:1211–5. |
| 27. | Vergani E, Dugo M, Cossa M, Frigerio S, Di Guardo L, Gallino G, et al. MiR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators. Cell Commun Signal 2020;18:156. |
| 28. | Hassan A, Neinaa YME-H, El-Bendary A, Zakaria S. MicroRNA-146a and Forkhead box protein 3 expressions in non-segmental vitiligo: An insight into disease pathogenesis. J Egypt Womens Dermatologic Soc 2019;16:105. doi: 10.4103/JEWD.JEWD_19_19. |
| 29. | Zhang H, Peng J, Lai J, Liu H, Zhang Z, Li X, et al. MiR-940 promotes malignant progression of breast cancer by regulating FOXO3. Biosci Rep 2020;40:BSR20201337. doi: 10.1042/BSR20201337. |
| 30. | Hashimoto K, Ochi H, Sunamura S, Kosaka N, Mabuchi Y, Fukuda T, et al. Cancer-secreted hsa-miR-940 induces an osteoblastic phenotype in the bone metastatic microenvironment via targeting ARHGAP1 and FAM134A. Proc Natl Acad Sci U S A 2018;115:2204–9. |
| 31. | Wang Y, Liang J, Qin H, Ge Y, Du J, Lin J, et al. Elevated expression of miR-142-3p is related to the pro-inflammatory function of monocyte-derived dendritic cells in SLE. Arthritis Res Ther 2016;18:263. |
| 32. | Dekkema GJ, Bijma T, Jellema PG, Van Den Berg A, Kroesen B-J, Stegeman CA, et al. Increased miR-142-3p expression might explain reduced regulatory T cell function in granulomatosis with polyangiitis. Front Immunol 2019;10:2170. |
| 33. | Lin RJ, Xiao DW, Liao L-D, Chen T, Xie ZF, Huang WZ, et al. MiR-142-3p as a potential prognostic biomarker for esophageal squamous cell carcinoma. J Surg Oncol 2012;105:175–82. |
| 34. | Sahni K, Parsad D. Stability in vitiligo: Is there a perfect way to predict it? J Cutan Aesthet Surg 2013;6:75-82. [ PUBMED] [Full text] |
| 35. | Zhang Z, Yang X, Liu O, Cao X, Tong J, Xie T, et al. Differentially expressed microRNAs in peripheral blood mononuclear cells of non-segmental vitiligo and their clinical significance. J Clin Lab Anal 2021;35:e23648. |
| 36. | Failla CM, Carbone ML, Fortes C, Pagnanelli G, D'atri S. Melanoma and vitiligo: In good company. Int J Mol Sci 2019;20:5731. doi: 10.3390/ijms20225731. |
| 37. | Langford MA, Al-Ghazal SK. Halo naevus or malignant melanoma: A case report. JPRAS Open 2018;16:98–9. |
| 38. | Olsen CM, Carroll HJ, Whiteman DC. Estimating the attributable fraction for melanoma: A meta-analysis of pigmentary characteristics and freckling. Int J Cancer 2010;127:2430–45. |
| 39. | Fontanillas P, Alipanahi B, Furlotte NA, Johnson M, Wilson CH, 23andMe Research team, et al. Disease risk scores for skin cancers. Nat Commun 2021;12:160. |
| 40. | Koturbash I, Zemp F, Kolb B, Kovalchuk O. Sex-specific radiation-induced microRNAome responses in the hippocampus, cerebellum and frontal cortex in a mouse model. Mutat Res 2011;722:114–8. |
| 41. | Murphy SJ, Lusardi TA, Phillips JI, Saugstad JA. Sex differences in microRNA expression during development in rat cortex. Neurochem Int 2014;77:24–32. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2] |
|
|
|
|
|
|
|
|
|
|
|
|
| Article Access Statistics | | | Viewed | 394 | | | Printed | 12 | | | Emailed | 0 | | | PDF Downloaded | 7 | | | Comments | [Add] | | |
|
|
