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Integrated analysis of tyrosine-induced MiRNA and mRNA expression profiles in melanocytes reveals the regulatory role of miR-1560-3p in melanin deposition in Xichuan black-bone chickens

BMC Genomics volume 26, Article number: 348 (2025) Cite this article

Abstract

Background

Tyrosine is a prerequisite for melanin biosynthesis and plays a crucial role in the growth and development of melanocytes, but the underlying mechanism is still unclear. In our previous research, we added 10− 9-10− 6 mol/L tyrosine to the melanocytes of black-bone chickens and found that 10− 6 mol/L tyrosine significantly increased the tyrosinase content in melanocytes.

Methods

In this study, melanocytes from Xichuan black-bone chickens were used as research objects, 10− 6 mol/L tyrosine was added for transcriptome sequencing. By analyzing miRNA and mRNA expression profiles, the miRNA-mRNA network was constructed, the targeting relationship was demonstrated by double luciferase reporting experiments, and the influence of tyrosine-mediated miRNA-mRNA network on melanin deposition was verified by constructing overexpression and interference vectors.

Results

We found that tyrosine promoted the proliferation and migration of melanocytes, and expression profile analysis identified 57 differentially expressed mRNAs (DEGs) and 19 differentially expressed miRNAs (DEMs). Fifty miRNA‒mRNA target gene pairs were identified via coexpression network analysis of the DEGs and the DEMs that were predicted to target various genes. Notably, VIP gene was reported to be involved in the development and deposition of melanoma cells. The binding of VIP to miR-1560-3p was further validated by a dual-luciferase reporter assay. In addition, test confirmed that miR-1560-3p inhibited the proliferation and migration of melanocytes and reduced the tyrosinase content. In conclusion, we found that tyrosine may affects melanin deposition in Xichuan black-bone chickens by affecting the miR-1560-3p-VIP axis. The results of this study provide experimental evidence for elucidating the mechanism of tyrosine in melanin deposition in black-bone chickens, and may serve as a reference for future investigations.

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Introduction

Black-bone chickens are widely valued for their medicinal value. The contents of amino acids, trace elements, carnosine, melanin and other substances in the body of black-bone chickens are high, but the fat and cholesterol contents are relatively low [1]. Compared with standard chickens, black-bone chickens can be used for disease prevention and control [2]. Studies have shown that black-bone chicken products can promote human immunity and the antioxidant capacity [3]. Moreover, black-bone chicken products have good therapeutic effects on diabetes, anemia, menstrual diseases and postpartum complications [2, 4]. Black-bone chicken egg products are rich in various essential nutrients needed by the human body, which can not only improve hemoglobin and red blood cells but also regulate metabolic and endocrine functions [5]. The deposition of melanin determines the degree of blackness of black-bone chickens. Therefore, studying the molecular regulatory mechanism of melanin deposition is highly important.

Increasing evidence shows that microRNAs (miRNAs) regulate melanin deposition in mammals and that tyramine not only acts as a substrate and cofactor but also plays an important regulatory role in melanin formation [6,7,8]. Researches show that transfected miR-21a-5p into mouse melanocytes and demonstrated its function in melanin production by targeting Sox5, which inhibited melanin production in mouse melanocytes [9]. Researches show that demonstrated that miRNA-25 regulates pigmentation by targeting the transcription factor MITF in alpaca skin melanocytes [10]. Similarly, researches show that demonstrated that miR-27-3p inhibits melanin production by targeting Wnt3a protein expression [11]. However, the molecular mechanism of tyrosine-mediated melanin deposition in chickens remains poorly understood. The aim of this study was to investigate the biological function and regulatory mechanism of tyrosine-mediated miRNAs in melanin deposition in chickens.

In our previous studies, we have confirmed that the addition of exogenous tyrosine promotes the proliferation of melanocytes. Moreover, the changes in cytoplasmic and intracellular tyrosinase content were most pronounced in the group treated with 10− 6 mol/L tyrosinase [12, 13]. Therefore, we conducted transcriptomic sequencing of untreated melanocytes and melanocytes treated with exogenous tyrosine (10− 6 mol/L), analyzed the expression profiles of miRNAs and mRNAs in melanocytes, and further studied the tyrosine-mediated regulatory network and function of miRNA-mRNA. In conclusion, This study provides reference data for elucidating the molecular mechanisms by which tyrosine regulates melanin deposition.

Results

Transmission electron microscopic results

Transmission electron microscopy (Fig. 1) revealed the presence of vesicles and melanosomes in melanocytes. Melanosomes were at different developmental stages, and different degrees of melanin deposition in melanosomes were observed, indicating that chicken melanocytes were successfully isolated.

Fig. 1
figure 1

Transmission electron microscopy of melanocytes. The arrows indicate melanosomes

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Effects of tyrosine on the proliferation, migration and tyrosinase synthesis of melanocytes

The CCK-8 results revealed that tyrosine significantly promoted melanocyte proliferation (P < 0.01, Fig. 2A). Moreover, flow cytometry revealed that tyrosine significantly increased the number of S-phase cells and promoted the progression of the melanocyte cycle (P < 0.05, Fig. 2B&C). Tyrosine significantly increased the expression levels of the melanocyte proliferation-related genes MITF, TYR, TYRP-1, TYRP-2, and MC1R and the cell cycle-related genes CDK1, PCNA, and CCND1 (P < 0.05, Fig. 2D&E). Melanocyte migration plays an important role in the deposition of melanin. In this study, qRT‒PCR was used to identify changes in the expression levels of melanocyte migration-related genes, and the results revealed that tyrosine can increase the expression levels of the MLPH, Rab27a and Myosin Va genes (P < 0.05, Fig. 2F), suggesting that tyrosine promotes melanocyte migration. Tyrosinase is the rate-limiting enzyme of melanin synthesis, and the content of this enzyme is proportional to the synthesis of melanin. After tyrosine was added, the tyrosinase content in melanocytes was determined via enzyme-linked immunosorbent assays (ELISAs). The results revealed that tyrosine significantly increased the content of tyrosinase in melanocytes (P < 0.05, Fig. 2G), indicating that the model was successfully constructed and could be used for further sequencing analysis.

Fig. 2
figure 2

Effects of tyrosine on the proliferation, migration and tyrosinase synthesis of melanocytes. (A) The effect of tyrosine on melanocyte proliferation was determined via a CCK-8 assay. (B, C) The effect of tyrosine on the melanocyte cycle was determined by flow cytometry. (D) Effects of tyrosine on melanocyte proliferation-related genes. (E) Effects of tyrosine on melanocyte cycle-related genes. (F) Effect of tyrosine on melanocyte migration-related genes. (G) Effect of tyrosine on the tyrosinase content in melanocytes. * indicates P < 0.05, ** indicates P < 0.01

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Analysis of mRNA expression differences

A total of 14,706 genes were identified in the mRNA expression profile. Compared with those in the control group, the expression of 66 genes in the tyrosine treatment group significantly changed, including 57 DEGs (35 genes were significantly downregulated and 22 genes were significantly upregulated) (Fig. 3A&B and Additional file 6). Through gene clustering, the two groups of DEGs were found to be well separated, and the DEGs in the same group of samples were well clustered, indicating good sample repeatability (Fig. 3C). Enrichment analysis revealed that the DEGs were enriched mainly in biological processes and were significantly enriched in ion transport, arachidonic acid secretion and transport, and inhibition of G protein-coupled receptor phosphorylation (Fig. 3D). KEGG enrichment analysis revealed that the DEGs were enriched mainly in environmental information processing and metabolic processes, including neural active ligand–receptor interactions, the phosphatidylinositol signaling system, and the calcium signaling pathway. The enriched pathways included linoleic acid metabolism, α-linolenic acid metabolism, arachidonic acid metabolism, and inositol phosphate metabolism (Fig. 3E).

Fig. 3
figure 3

Analysis of mRNA expression differences. (A) Volcano map of DEGs. The red dots indicate upregulated genes, the blue dots indicate downregulated genes, and the gray dots indicate genes that were not significantly differentially expressed. (B) Coexpressed DEGs between the control group and the tyrosine treatment group. (C) DEG clustering. (D) GO enrichment analysis bubble map. (E) KEGG enrichment analysis bubble map

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Analysis of differences in MiRNA expression

By performing miRNA-seq on melanocytes with and without tyrosine treatment, we obtained 30,891,775 and 30,891,775 reads, respectively. We obtained 25,957,145, 41,155,097, 24,548,489, 32,866,390 and 32,023,083 original reads. After splice removal and low-quality sequence removal, clean reads with nucleotide lengths ≥ 18 nt were obtained, and the proportion of clean reads was greater than 92% (Additional file 1). Moreover, clean reads with sequence lengths of 18 nt to 25 nt were statistically analyzed and found to be distributed mainly between 21 nt and 24 nt (Fig. 4A). The deduplicated sequences were compared with the precursor and mature sequences downloaded from miRBase and annotated (Additional file 2). According to the volcano map, 19 genes showed significant changes after the addition of tyrosine: 10 downregulated miRNAs and 9 upregulated miRNAs (Fig. 4B and Additional file 5). In the cluster analysis, miRNAs whose expression was highly correlated between samples were classified into one category. Through cluster analysis, we found that the DEMs of the tyrosine-supplemented group and the group without tyrosine added could be well distinguished, whereas the DEMs of the samples of the same treatment group were clustered together. There was a high degree of similarity between the groups (Fig. 4C). To evaluate the potential regulatory role of miRNAs, we performed GO enrichment analysis of DEM target genes, and the results revealed that 148, 153 and 1,036 GO were significantly enriched in the cellular component (CC), molecular function (MF) and biological process (BP), respectively. The DEM target genes in CC were involved mainly in intracellular organelles, the Golgi apparatus and the endoplasmic reticulum. In MF, the DEM target genes were involved mainly in purine/ribonucleoside binding and tricarboxylic acid, guanase activity. In the BP, the DEM target genes were involved mainly in the transport of nitrogen compounds, proteins and peptides (Fig. 4D). In the KEGG enrichment analysis, the DEM target genes were enriched mainly in cellular metabolic processes, including amino sugar and nucleotide sugar metabolism, unsaturated fatty acid biosynthesis, cysteine and methionine metabolism, and fatty acid elongation. Moreover, the DEM target genes were significantly enriched in endocytosis, the cell cycle, the ErbB signaling pathway, the apelin signaling pathway, endoplasmic reticulum protein processing, the insulin signaling pathway, steroid biosynthesis and other pathways (Fig. 4E).

Fig. 4
figure 4

miRNA expression profile analysis. (A) Sequence length distribution diagram. The large figure shows the distribution of total reads with different lengths before weight removal, whereas the small figure at the upper right shows the distribution of unique reads with different lengths after weight removal. (B) Volcano map of DEMs. The red dots represent upregulated genes, the blue dots represent downregulated genes, and the gray dots represent nonsignificant DEGs. (C) Cluster map of DEMs. (D) Bubble map of GO enrichment analysis of DEM target genes. (E) Bubble map of KEGG enrichment analysis of DEM target genes

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Screening and validation of mRNAs and MiRNAs

The DEM target genes were predicted via the miRanda, miRDB and TargetScan online websites, and the intersection of the target genes predicted via these websites revealed 6,825 predicted target genes. The intersection of DEM target genes and differentially expressed mRNAs was obtained via a Venn diagram. A total of 21 coexpressed genes were found (Fig. 5A). Fifty miRNA‒mRNA target gene pairs were generated via Cytoscape for 17 DEMs and 21 DEGs (Fig. 5B). To verify the accuracy of the miRNA-seq data, we randomly selected 9 DEMs. The genes gga-miR-12225-3p, gga-miR-12-1-5p and gga-miR-1560-3p were significantly downregulated, and the genes gga-miR-1667-3p, gga-miR-32-5p, gga-miR-2184a-3p, gga-miR-29b-3p, gga-miR-3583, and gga-miR-23b-3p were significantly upregulated. The sequencing results were verified via qRT‒PCR with U6 as the internal reference gene. The results revealed that the expression trend of the miRNAs detected via qRT‒PCR was consistent with that detected via miRNA‒seq, indicating that the sequencing results were accurate and reliable (Fig. 5C). To verify the accuracy of the mRNA-seq data, we randomly selected 9 DEGs, 5 downregulated genes (RASD1, IL18R1, TRPM8, MTMR7, and CDH8) and 4 upregulated genes (SLC46A3, FGF12, VIP, and SEMA5A), to verify the accuracy of the sequencing results. The qRT‒PCR results revealed that the upregulated and downregulated DEGs were consistent with the high-throughput sequencing results. Nine DEGs, 5 downregulated genes (RASD1, IL18R1, TRPM8, MTMR7, and CDH8) and 4 upregulated genes (SLC46A3, FGF12, VIP, and SEMA5A), were randomly selected to verify the accuracy of the sequencing results. The qRT‒PCR results revealed that the trend of upregulated and downregulated DEGs was consistent with the high-throughput sequencing results (Fig. 5D).

Fig. 5
figure 5

miRNA and mRNA screening and validation. (A, B) Differentially expressed miRNA‒mRNA coexpression network. (C) Validation of the miRNA sequencing results. (D) Verification of the mRNA sequencing results. * indicates P < 0.05, ** indicates P < 0.01

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Verification of the targeting relationship between gga-miR-1560-3p and VIP

Through the prediction of DEM target genes and the analysis of the differentially expressed mRNA coexpression network, a potential targeting relationship between the gga-miR-1560-3p and VIP genes was identified, and tyrosine may regulate melanin deposition via the gga-miR-1560-3P-VIP network. The potential relationship between gga-miR-1560-3p and VIP was identified through online prediction via miRanda, miRDB and TargetScan. The gga-miR-1560-3p seed sequence has complementary base pairing sites with 115 nt-122 nt of the VIP gene (Fig. 6A), and the two combine to form a complex with a double-strand free energy of -19.9 kcal/mol (Fig. 6B). According to the sequence information of the chicken VIP gene in NCBI, the full-length CDS of the VIP gene was selected. Specific primer information is provided (Additional file 3). A dual-luciferase reporter gene assay was used to verify the targeting relationship between gga-miR-1560-3p and the VIP gene, and the results are shown in Fig. 6C. After gga-miR-1560-3p was cotransfected with the VIP 3’UTR wild-type overexpression vector, compared with those of the other three groups, the fluorescence activity of psiCHECK2 was significantly decreased (P < 0.01). psiCHECK2-VIP-3’UTR-WT and mimic NC, psiCHECK2-VIP-3’UTR-MUT and gga-miR-1560-3p.mimics, psiCHECK2-VIP-3’UTR-MUT and mimics were used. There was no significant difference from the NC group after cotransfection (P > 0.05).

Fig. 6
figure 6

Verification of the targeting relationship between gga-miR-1560-3p and VIP. (A) Complementary base pairing site of the gga-miR-1560-3p seed sequence and VIP gene; (B) stem ring structure of the gga-miR-1560-3p and VIP genes; (C) results of the double-luciferase reporter gene assay

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Tyrosine-mediated gga-miR-1560-3p targeting of VIP

To further study the regulatory effect of gga-miR-1560-3p on the VIP gene, we detected changes in the expression of the VIP gene after the overexpression of gga-miR-1560-3p. The results revealed that gga-miR-1560-3p was significantly increased after overexpression (P < 0.01), and the expression was more than 1200 times greater than that of the mimic NC group, whereas for the target gene VIP, gga-miR-1560-3p expression was significantly decreased after overexpression (P < 0.01) (Fig. 7A). Moreover, we detected changes in the VIP gene after gga-miR-1560-3p interference. The results revealed that gga-miR-1560-3p interference significantly decreased the expression (P < 0.01), and the interference rate was above 90%. After interference with gga-miR-1560-3p, the expression of the VIP gene increased (P > 0.05) (Fig. 7B), further confirming the targeting relationship between gga-miR-1560-3p and the VIP gene. The effects of gga-miR-1560-3p overexpression and interference on melanocyte proliferation were determined via a CCK-8 assay. The results revealed that the overexpression of gga-miR-1560-3p significantly inhibited the proliferation of melanocytes (P < 0.01). Conversely, interference with gga-miR-1560-3p significantly promoted the proliferation of melanocytes (P < 0.05) (Fig. 7C). After gga-miR-1560-3p overexpression, the levels of the melanocyte proliferation-related genes MITF and TYR were significantly decreased (P < 0.01), but the expression levels of the TYRP2 and MC1R genes were not significantly changed (P > 0.05). Interference with gga-miR-1560-3p significantly promoted the expression of the MITF, TYR, TYRP-2 and MC1R genes (P < 0.05) (Fig. 7D). The effect of gga-miR-1560-3p on the melanocyte cycle was determined via gga-miR-1560-3p overexpression and interference assays. We found that the overexpression of gga-miR-1560-3p significantly inhibited the expression of CDK1, PCNA and CCND1 (P < 0.05). Interference with gga-miR-1560-3p significantly promoted the expression of CDK1, PCNA, and CCND1 (P < 0.05) (Fig. 7E). We examined the effects of gga-miR-1560-3p on melanocyte migration via gga-miR-1560-3p overexpression and interference assays. We found that the overexpression of gga-miR-1560-3p significantly inhibited the expression of MLPH and Rab27a (P < 0.01). Interference with gga-miR-1560-3p significantly promoted the expression of MLPH, Rab27a and myosin Va (P < 0.05) (Fig. 7F). To further verify whether tyrosine regulates melanin deposition by regulating gga-miR-1560-3p, we cotransfected gga-miR-1560-3p mimics and tyrosine. Compared with the control, gga-miR-1560-3p overexpression significantly inhibited the synthesis of tyrosinase (P < 0.01). Compared with the gga-miR-1560-3p and tyrosine cotreatment groups, the gga-miR-1560-3p overexpression group showed significantly increased tyrosinase synthesis (P < 0.01) (Fig. 7G), indicating that tyrosine restored the inhibitory effect of gga-miR-1560-3p on tyrosinase synthesis. To further verify whether VIP is regulated by tyrosine and thus affects the deposition of melanin, we cotreated cells with pcDNA3.1-VIP-3xFlag and a tyrosine-overexpressing plasmid vector. Compared with the control group, the VIP overexpression group showed significantly increased synthesis of tyrosinase (P < 0.01), and compared with the VIP and tyrosine cotreatment groups, the VIP overexpression group showed significantly increased synthesis of tyrosinase (P < 0.05) (Fig. 7H), indicating that tyrosine promoted the synthesis of tyrosinase by VIP.

Fig. 7
figure 7

Analysis of tyrosine-mediated gga-miR-1560-3p targeting of VIP function. (A) gga-miR-1560-3p overexpression efficacy and determination of the influence of gga-miR-1560-3p overexpression on VIP genes. (B) gga-miR-1560-3p interference efficacy and determination of the effect of gga-miR-1560-3p interference on the VIP gene (C) The effects of overexpression and interference of gga-miR-1560-3p on melanocyte proliferation determined by CCK-8 assays. (D) The effect of gga-miR-1560-3p on melanocyte proliferation-related genes (E) Effects of gga-miR-1560-3p on melanocyte cycle-related genes (F) Effects of gga-miR-1560-3p on melanocyte migration-related genes (G) Effects of gga-miR-1560-3p and tyrosine cotreatment on the content of tyrosinase in melanocytes (H) Effect of VIP and tyrosine cotreatment on tyrosinase content in melanocytes * indicates P < 0.05, ** indicates P < 0.01

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Discussion

Black-bone chickens constitute an integral part of quality chicken breeds in China. In recent years, black-bone chicken products have been shown to effectively scavenge free radicals and antioxidation, delaying aging and improving body immunity [2, 14]. The deposition of melanin directly affects the quality and efficacy of black-bone chicken products. Tyrosinase is a glycoprotein located on the membrane of melanosomes with endosomal, transmembrane and cytoplasmic domains [15, 16]. According to relevant studies, tyrosine increases blood pressure [17] and anti-inflammatory effects [18] and has also been reported to affect poultry tonic immobilization and consumption [19]. In addition, tyrosine is the classic factor affecting melanin deposition [20, 21]. The color of feathers and skin is largely determined by melanocytes, whose main function is to produce melanin [22]. Tyrosinases are essential for the functional development of melanocytes, and their expression and activity determine the rate and yield of melanin synthesis [23,24,25,26].

miRNAs are a class of small noncoding RNAs that negatively regulate gene expression mainly at the mRNA level and are powerful regulators of various cell activities [7, 27]. miRNAs play a regulatory role mainly through binding to target genes. In this study, RNA-seq was performed on melanocytes with and without tyrosine, and different miRNAs and mRNAs were screened out [7, 28, 29]. The differentially expressed mRNAs were significantly enriched in ion transport, the calcium signaling pathway, arachidonic acid secretion and transport, and phosphatidylinositol signaling. Studies have shown that UV exposure can promote the secretion of arachidonic acid from melanocytes, which in turn increases the number of DOPA-positive melanocytes in the skin of mice. Researches show that reported that arachidonic acid promoted the formation of melanocytes in guinea pigs [30]. Recent studies have also shown that the transcription factor Nrf2 negatively regulates melanin production by regulating PI3K/Akt signaling. Phosphatidylinositol diphosphate (PI 3,5) regulates the activity of TPC2 ion channel proteins to control melanosome membrane potential, pH and pigmentation, suggesting that phosphatidylinositol signaling plays an important role in melanin formation [31, 32]. GO and KEGG enrichment analyses revealed that the DEM target genes were significantly enriched in the Golgi apparatus, endoplasmic reticulum, endoplasmic reticulum protein processing, the insulin signaling pathway, steroid biosynthesis and other processes. Proteins of the tyrosine gene family are synthesized in the Golgi apparatus of melanocytes and transported from the early endosome to the melanosome, where they catalyze the synthesis of melanin [33]. Studies have shown that SMER28 or 4-phenylbutyric acid (PBA) can relieve endoplasmic reticulum stress and partially reverse the reduction in melanosis in mice [34]. Moreover, researches show that reported that the inhibition of the 26 S protease complex leads to the expansion of the endoplasmic reticulum of melanocytes, thus affecting the output of tyrosinase and the expression of tyrosinase in the endoplasmic reticulum of melanocytes [35]. Moreover, The insulin was found to reduce the proliferation and tyrosinase synthesis of mouse melanoma cells [36].

In existing research, transcriptomic sequencing has been performed on different tissues, such as chicken pectoral muscles [37], eyelids [38], skin [39], hair follicles [40], and bursa of Fabricius [41], and a series of genes related to melanin deposition have been identified. The expression level of melanin synthesis-related genes determines phenotypic changes, such as skin, hair, and eye color, in animals [42]. Moreover, through transcriptomic sequencing analysis, TRPM8 inhibits the expression of prostaglandin E2 (PGE2) in keratinocytes, whereas PEG2 stimulates the elongation of melanocyte dendrites and affects skin pigmentation [43, 44]. In this study, TRPM8 was significantly downregulated after the addition of tyrosine, indicating that tyrosine may participate in the ion exchange of melanocytes by regulating TRPM8, thus affecting the synthesis of melanin; however, its specific mechanism needs to be further studied. We also found through our transcriptomic sequencing that gga-miR-212-5p, gga-miR-29b-3p, gga-miR-23b-3p, gga-miR-221-3p and gga-miRNA-32-5p may play potential regulatory roles in tyrosine-mediated melanin deposition. Studies have shown that COX-2 is a direct target gene of miR-212-5p [45]. Moreover, COX-2 interference inhibited the generation of tyrosinase and melanin in mouse melanoma cells and inhibited the expression of MITF, TYRP1 and TYRP2 [46]. These results demonstrated the potential regulatory effect of miR-212-5p on melanin deposition. miR-29b-3p has been shown to play a regulatory role in alleviating UV irradiation-induced photoaging of skin fibroblasts [47]. SOX9 has been shown to be a target gene of miR-29b-3p [48]. SOX9, a member of the SOX family, can regulate the MITF, TYRP2 and TYR promoters, resulting in increased expression of melanin-producing proteins and ultimately stimulating pigmentation [49]. miR-23b-3p has been reported to be involved in the occurrence and treatment of liver cancer, cervical cancer, osteosarcoma and other diseases, and the targeting relationships among peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), tyrosine kinase receptor (c-MET) and miR-23b-3p have been confirmed [50, 51]. Studies have shown that miR-221-3p, a potential biomarker of psoriasis, is involved in the inflammatory response of keratinocytes [52], and miR-221-3p can also promote the proliferation and migration of mouse Schwann cells [53], which are the prerequisite cells of melanocytes. Therefore, miR-221-3p may play an important role in regulating melanin production. Studies have shown that miR-32-5p is involved in the PI3K/Akt pathway and plays an important regulatory role, such as inducing multidrug resistance in hepatocellular carcinoma [54]. The PI3K/Akt pathway has been proven to be a classic pathway for melanin formation. Therefore, miR-32-5p may play a role in melanocytes through the PI3K/Akt pathway, thereby affecting melanin synthesis. In this study, we revealed a potential targeting relationship between the differentially expressed gga-miR-1560-3p and the DEG VIP, miR-1560-3p has rarely been reported. Interestingly, researches show that treated B16F10 mouse melanoma cells with VIP to significantly increase the melanin content, tyrosinase activity and gene expression of tyrosinase and MITF [55]. At the cellular level, VIP stimulates cell proliferation modestly and melanogenesis pronouncedly in growing chick embryonic RPE cultures [56].

In conclusion, In this study, the targeting relationship and binding site between gga-miR-1560-3p and VIP were predicted and confirmed by dual Luciferase. After overexpression of miR-1560-3p, the expression level of VIP was significantly decreased. Through overexpression and interference tests, it was further confirmed that miR-1560-3p inhibited the proliferation and migration of melanocytes. Reduce tyrosinase content. These results suggest that tyrosine may promote melanin deposition through the gga-miR-1560-3p-VIP network. However, this study still has some limitations, although it has been confirmed that tyrosine may promote melanin deposition through the gga-miR-1560-3p-VIP network. But it has not been tested at the living level.

Conclusion

We performed tyrosine functional verification and RNA-seq on both tyrosine-containing and no tyrosine-containing melanocytes, followed by GO and KEGG enrichment analysis and coexpression network analysis of differentially expressed mRNAs and miRNA target genes. The gga-miR-1560-3p-VIP network, a key network of tyrosine-mediated melanin deposition, was identified. Through overexpression and interference experiments, the specific mechanism by which the gga-miR-1560-3p-VIP network affect melanin deposition was further explored, In addition, test confirmed that miR-1560-3p inhibited the proliferation and migration of melanocytes and reduced the tyrosinase content. In conclusion, we found that tyrosine may affects melanin deposition in Xichuan black-bone chickens by affecting the miR-1560-3p-VIP axis. The results of this study provide experimental evidence for elucidating the mechanism of tyrosine in melanin deposition in black-bone chickens, and may serve as a reference for future investigations.

Materials and methods

Ethics statement

In this study, the eggs of Xichuan black-bone chicken, a local chicken breed in China, were selected as the experimental animal samples from the Animal Research Center of Henan Agricultural University, all of the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Henan Agricultural University, Zhengzhou, China, and performed in strict accordance with the guidelines of the Animal Use Committee of the Chinese Ministry of Agriculture, Beijing, China (Approval number: 11–0085). These measures are in place to safeguard animal welfare and reduce any potential for suffering.

Isolation and cultivation of melanocytes

Twenty embryonic eggs of 18 embryo age Xichuan black-bone chickens were selected, chickens were anesthetized by intravenous injection of sodium pentobarbital (40 mg/kg) at a concentration of 0.2% in the wing vein. Under deep anesthesia, these individuals were euthanized by intravenous KCL (1 ~ 2 mg/kg), and peritoneal tissue was cut under sterile conditions for the isolation and culture of melanocytes. First, the cut peritoneum was washed with PBS preheated at 37 °C to remove the oil, and the blood was stored. An appropriate amount of fresh culture medium was added, and the peritoneum was cut into fragments. The samples were digested in a 37 °C water bath for 1 h with an equal volume of protease II enzyme and trypsin (1:1 ratio). After digestion was completed, 1.5 times the volume of complete DMEM/F12 culture medium (containing 10% fetal bovine serum) was added to terminate digestion. After repeated aspiration, the samples were filtered through 100-, 200-, and 500-mesh cell sieves, transferred to a 15 mL centrifuge tube, and centrifuged at 1000 r/min for 5 min. The supernatant was discarded, the samples were aspirated and mixed with PBS, and the samples were centrifuged again at 1000 r/min for 5 min (repeated twice). After centrifugation, Medium 254 medium containing 1% human melanocyte growth additive (HMGS) was added, and the mixture was gently aspirated and mixed. The cells were inoculated in a cell culture bottle and cultured in a 5% CO2, 37 °C incubator. After 12 h, the medium was replaced. The mixture was cleaned with PBS, and Medium 254 (containing 1% HMGS) was added for further cultivation. The medium was changed every 2 days thereafter, the exogenously added tyrosine was sourced from Sigma-Aldrich Shanghai Trading Co., Ltd., China (Product No. T8566-100G).

Identification of melanocytes via transmission electron microscopy

Melanocytes were inoculated in 6-well plates. When the cells reached 80–90% confluence, the samples were collected via transmission electron microscopy. The cell culture medium was first extracted with a pipette gun, and then, 600 µL of 2.5% glutaraldehyde fixing solution was added to each well. The mixture was fixed at room temperature for 5 min, after which the cells were gently scraped in the same direction (the mixture was not scraped repeatedly to avoid scratching the cells). All the cells were extracted into a centrifuge tube and centrifuged at 2500 r/min for 2 min. The fixing solution was discarded, the cell precipitate was collected, new glutaraldehyde fixing solution was added, and the cells were gently aspirated to suspend the cell precipitation. The samples were fixed at room temperature for 30 min in the dark and then observed via transmission electron microscopy.

CCK-8 assay

Digestion, termination, and centrifugation were performed when the cell density reached 80%~90%. The cells were resuspended in Medium 254 (containing 1% HMGS) and inoculated onto a 96-well plate. The cells were subsequently cultured in a 5% CO2 incubator at 37 °C for 24 h. Then, 100 µL of Medium 254 (containing 1% HMGS) was added to the control well, 100 µL of Medium 254 (containing 1% HMGS) containing 10− 6 mol/L tyrosine was added to the treatment well, and the medium was withdrawn after 72 h. Medium 254 containing 10% CCK-8 solution (containing 1% HMGS) was added to 100 µL per well and cultured in a CO2 incubator for 2 h, after which the OD value of each well was measured at a wavelength of 450 nm on an enzyme-labeled instrument (8 replicates per group), the CCK-8 assay was performed using a kit from Beijing Solarbio Science & Technology Co., Ltd., China (Product No. CA1210).

Flow cytometry

The cell suspension was collected and centrifuged at 1000 r/min for 5 min, and then, the cell precipitate was washed with PBS and centrifuged again. The cell precipitate was resuspended in 70% anhydrous ethanol precooled at 4 °C and fixed overnight at 4 °C. The fixed solution was discarded, the cells were washed with PBS and centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. Five hundred microliters of PI/RNase A staining working solution was added, and the mixture was incubated in the dark at room temperature for 30 min. Flow cytometry (BD Biosciences, USA) was used to assess the cell cycle, a wavelength of 488 nm was selected, and the offline data were processed and analyzed via FlowJo 7.6 software.

Determination of tyrosinase content

The determination of tyrosinase content in melanocytes was carried out via the Nanjing built kit (Cat H310-1-2, Nanjing Jiancheng Bioengineering Institute, China), with specific steps referred to in the kit.

mRNA sequencing and pathway analysis of differentially expressed genes (DEGs)

The total RNA of the cells was extracted with TRIzol (TaKaRa, Dalian, China). The RNA bands were detected by 1% agarose gel electrophoresis to determine whether the RNA was degraded. The integrity of the RNA was detected with an Agilent 2100 Bioanalyzer (Agilent, CA, USA), and then, the concentration and purity of the total RNA in the samples were determined with a micro nucleic acid protein detector. Qualified samples were selected for subsequent tests. After the ribosomal RNA was removed, the RNA fragment was purified, and two strands of cDNA were synthesized via random primers and reverse transcriptase. After library construction was completed, the Illumina HiSeq sequencing platform was used for dual-terminal sequencing of the library. Low-quality reads with splices in the sequencing data were filtered out, and the filtered reads were compared to the reference genome (Gallus_gallus. GRCg6a. dna. toplevel. fa) via TopHat2 software. HTSeq was used to statistically compare the read count value for each gene as the original expression level, and the expression level was standardized via fragments per kilobases per million fragments (FPKM). DESeq was used to perform differential analysis of gene expression and screen for DEGs under the following conditions:|log2FoldChange|>1 and P value < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted via topGO and KOBAS, respectively (the standard for significant enrichment in GO and KEGG analyses was a P value < 0.05) [57].

MiRNA array and pathway analysis of differentially expressed MiRNAs (DEMs)

After the original data connector was removed, quality pruning was performed, and the original sequence was searched with a window of 5 base lengths. When the average sequencing quality of the bases in the window was less than 20, the part starting from the forefront of the window was truncated and discarded. Statistical analyses were performed on the number of clean reads from 18 ~ 36 nt, and after deduplication, the sequence abundance was calculated to obtain unique reads. miRDeep2 software was used to compare unique reads with the reference genome. The precursor and mature sequences of chicken-derived miRNAs were obtained from miRBase, the detected miRNAs were annotated, and new miRNAs were predicted and analyzed via MIREAP. Differential expression analysis was performed on miRNAs via DESeq, and differential miRNAs with P values < 0.05 were screened [58, 59].

Differential MiRNA‒mRNA coexpression network analysis

The DEMs were predicted via miRanda, TargetScan, and miRDB, and GO and KEGG enrichment analyses were performed on the DEMs predicted via the three software programs simultaneously [60].

DF-1 cell resuscitation and culture

The DF1 cell line was frozen in liquid nitrogen and quickly thawed in a 37 °C water bath. The cells were extracted into a 15 mL centrifuge tube, and complete DMEM/F12 culture medium containing 10% fetal bovine serum was added. The mixture was centrifuged at 1000 r/min for 3 min. After the solution was discarded, the cell precipitates were collected, and complete culture medium supplemented with DMEM/F12 was added to resuspend the cells. The bacteria were inoculated in a 24-well plate and cultured at 37 °C in a 5% CO2 incubator.

Vector construction

By using the seed sequence of miR-1560-3p and the principle of complementary base pairing, we identified and randomly mutated the binding site with the 3’ untranslated region (UTR) of the VIP gene. XhoI and NotI restriction endonucleases were selected to design VIP gene 3’UTR wild-type and mutant vector primers, and the psiCHECK2-VIP-3’UTR-WT and psiCHECK2-VIP-3’UTR-MUT vectors were constructed through homologous recombination. The miRNA NC and mimics were purchased from GenePharma, and the primer information is shown in Table 1.

Table 1 VIP gene 3’UTR wild and mutant type vector primer information
Full size table

Extraction of the Recombinant plasmid

The recombinant plasmid was extracted via a small and medium extraction kit for endotoxin-free plasmids (DP1118; Tiangen Biochemical Technology Co., Ltd., China). For specific steps, refer to the kit.

DF-1 cell transfection and luciferase reporter assay

The psiCHECK2-VIP-3’UTR-WT and psiCHECK2-VIP-3’UTR-MUT vectors were cotransfected with miR-1560-3p NC and miR-1560-3p mimics, respectively, via lip3000. The cells were collected for 24 h, and a multifunctional microplate reader was used to determine the sea kidney and firefly luciferase activities.

Extraction and quality detection of total RNA from cells

Total RNA was extracted via TRIzol reagent (Invitrogen, USA). The purity and amount of RNA were measured via a NanoPhotometer spectrophotometer (Implen, Munich, Germany).

Quantitative real-time PCR (qRT-PCR)

qRT‒PCR was performed via a TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) Kit (Cat RR820B, TaKaRa, Dalian, China). The qRT‒PCR gene-specific primers were designed via NCBI online software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and purchased from BiosSunya Co., Ltd. The sequence of primers is shown in Additional file 4. (Zhengzhou, China. The qRT‒PCR amplification procedure was as follows: 95 °C for 3 min; 35 cycles of 95 °C for 12 s, 60 °C for 30 s, and 72 °C for 30 s; and an extension for 10 min at 72 °C (n = 3). The expression of all chosen mRNAs was determined via the comparative CT (2-ΔΔCT) method with GAPDH as an internal reference RNA (GAPDH acts as an internal reference gene and is stably expressed in melanocytes) [61].

Statistical analysis

Statistical analysis was carried out by using Prism 8 software (GraphPad Software, Inc., USA). All the results are presented as the means ± SEMs. The significance of the differences between the two groups was assessed via a one-sample t test via SPSS 24.0 (IBM, New York, USA). P < 0.05 was considered to indicate a significant difference.

Data availability

Data availability statement: The datasets supporting the conclusions of this article are included within the article and its additional files. The transcriptome sequencing data were deposited in the NCBI SRA database (SRA accession: PRJNA1141255).

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Acknowledgements

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Funding

The Youth Fund of the National Natural Science Fund project (32102540), the University Youth Innovation Fund (30501085) and the Scientific Studio of Zhongyuan Scholars (30601985) were used.

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  1. Zhiyuan Zhang and Pengwei Zhang contributed equally to this work.

Authors and Affiliations

  1. College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, 450046, P. R. China

    Zhiyuan Zhang, Pengwei Zhang, Fumin He, Yingdong Hou, Xiaowen Geng, Ruilong Xu, Yadong Tian, Wenting Li, Guirong Sun, Ruirui Jiang, Xiaojun Liu, Ruili Han, Guoxi Li, Xiangtao Kang & Donghua Li

  2. The Shennong Laboratory, Zhengzhou, 450002, China

    Yadong Tian, Wenting Li, Guirong Sun, Ruirui Jiang, Xiaojun Liu, Ruili Han, Guoxi Li, Xiangtao Kang & Donghua Li

  3. Henan University of Animal Husbandry and Economy, Zhengzhou, 450046, China

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Contributions

“ZZ and PZ conceived the project and designed the experiments. FH, YH, XG, RX and RL performed the animal experiments and sample collection. YT, WL, GS, RJ, XL, RH, GL and XK provided valuable suggestion and comments to improve the manuscript with contributions from all other authors. DL discussed the results. All authors have read and approved the manuscript”.

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Correspondence to Donghua Li.

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All of the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Henan Agricultural University, Zhengzhou, China, and performed in strict accordance with the guidelines of the Animal Use Committee of the Chinese Ministry of Agriculture, Beijing, China (Approval number: 11–0085). All test animals in this study received informed consent from their owners to use these animals in the study.

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Zhang, Z., Zhang, P., He, F. et al. Integrated analysis of tyrosine-induced MiRNA and mRNA expression profiles in melanocytes reveals the regulatory role of miR-1560-3p in melanin deposition in Xichuan black-bone chickens. BMC Genomics 26, 348 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11543-8

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