Identification of the ceRNA networks in α-MSH-induced melanogenesis of melanocytes

Results

Differentially expressed lncRNAs, circRNAs, and mRNAs in α-MSH-treated melanocytes

To induce the melanogenesis in human melanocytes, we treated human skin primary melanocytes (MCs) with different concentrations of α-MSH (0, 50, 100, 150 nM). The qRT-PCR results showed that 150 nM α-MSH increased the expression of melanogenesis-related genes [16, 17] (MITF, TYR, Tyrosinase Related Protein 1 (TYRP1), Dopachrome Tautomerase (DCT), RAB27A (Member RAS Oncogene Family), and Myosin VA (MYO5A), Figure 1A) most significantly, so we chose 150 nM α-MSH for the subsequent experiments. Then, ceRNA microarray analysis was performed to identify the differentially expressed lncRNAs, circRNAs, and mRNAs between normal control (NC, 0 nM α-MSH) and 150 nM α-MSH treated MCs. We found that 2026 lncRNAs, 3256 circRNAs, and 933 mRNAs changed by more than two-fold (Figure 1B–1D). Specifically, 11 lncRNAs were upregulated and 9 lncRNAs were downregulated by 5-fold (normalized signal value > 2) in α-MSH-treated MCs, respectively (Table 1). Furthermore, 27 circRNAs were upregulated and 22 circRNAs were downregulated by 5-fold (normalized signal value > 1.5) in α-MSH-treated MCs, respectively (Table 2). Besides, GO and KEGG pathway enrichment analysis was performed to reveal the possible involvement of significant differentially expressed mRNAs in the α-MSH-treated MCs (Supplementary Figure 1). The melanosome/GO (TYR, ATP6V1B2, ERP29, CCT4, RAN, HSPA8, RAB38, ANXA2, RPN1, SEC22B, TYROBP, and ATP1B3), and melanogenesis/pathway (EDNRB, GNAO1, FZD7, POMC, TYR, CTNNB1, FZD4, CALM2, and WNT3) indicated that these genes were significantly enriched in melanogenesis. Combined with other studies [18–20], we selected the upregulated mRNAs (TYR, SOX6, CTNNB1, IL6, FZD4, POMC, WNT3, CALM2, FZD7, MAPK11, RAB38) and the downregulated mRNAs (EDNRB, GNAO1, TYROBP) which were involved in α-MSH-induced melanogenesis (Table 3).

Figure 1. Differentially expressed lncRNAs, circRNAs, and mRNAs in α-MSH-treated melanocytes. (A) qRT-PCR was used to detect the expression of melanogenesis related genes (MITF, TYR, TYRP1, DCT, RAB27A, and MYO5A) in α-MSH-induced melanocytes. (B–D) Scatter plot of normalized expression levels for differentially expressed lncRNAs, circRNAs, and mRNAs, respectively. Red dots: upregulated lncRNAs, circRNAs, and mRNAs; Blue dots : downregulated lncRNAs, circRNAs, and mRNAs.

Table 1. Greater than 5-fold differentially expressed lncRNAs (normalized signal value > 2).

Upregulated lncRNAs			Downregulated lncRNAs
LncRNA	Fold change (α-MSH/NC)		LncRNA	Fold change (α-MSH/NC)
ENST00000532071	10.10		NR_104161	0.19
NR_040100	9.40		ENST00000414633	0.19
ENST00000316807	8.73		ENST00000599817	0.18
ENST00000606533	6.86		lnc-ELAVL2-1:2	0.17
ENST00000474979	6.59		lnc-RBMY1J-6:1	0.16
lnc-CYP4F22-1:1	6.30		lnc-KIF20A-1:1	0.14
ENST00000587528	6.23		lnc-TRIM37-1:2	0.17
NR_031650	6.14		NR_104003	0.08
lnc-LNPEP-2:1	6.02		lnc-BTBD19-1:1	0.08
ENST00000530955	5.40
ENST00000533322	5.04

Table 2. Greater than 5-fold differentially expressed circRNAs (normalized signal value > 1.5).

Upregulated circRNAs			Downregulated circRNAs
circRNA	Fold change α-MSH/NC)		circRNA	Fold change (α-MSH/NC)
circ_0031794	15.47		circ_0015226	0.20
circ_0035909	12.34		circ_0007726	0.19
circ_0074400	8.74		circ_0050401	0.19
circ_0066944	8.64		circ_0004126	0.19
circ_0030056	7.56		circ_0058630	0.18
circ_0054509	7.39		circ_0083902	0.17
circ_0015211	7.37		circ_0088002	0.17
circ_0054701	6.47		circ_0079872	0.17
circ_0068095	6.40		circ_0068584	0.17
circ_0037543	6.27		circ_0001808	0.15
circ_0040155	6.15		circ_0021740	0.15
circ_0084506	5.95		circ_0073019	0.15
circ_0002626	5.87		circ_0008626	0.15
circ_0083993	5.72		circ_0031728	0.14
circ_0025748	5.70		circ_0053546	0.14
circ_0069815	5.69		circ_0014138	0.13
circ_0053692	5.52		circ_0055832	0.13
circ_0067366	5.49		circ_0047279	0.12
circ_0059675	5.45		circ_0077787	0.10
circ_0091223	5.41		circ_0072730	0.10
circ_0031463	5.41		circ_0073464	0.09
circ_0053508	5.40		circ_0030604	0.07
circ_0020240	5.32
circ_0074258	5.15
circ_0058701	5.13
circ_0002563_T_L	5.06
circ_0026068	5.05

Identification of the lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA network in α-MSH-induced melanogenesis

The miRanda database was used to predict the potential targeted miRNAs of the screened differentially expressed lncRNAs and circRNAs with more than 5-fold change. TargetScan databases were used to predict potential target miRNAs of differentially expressed mRNAs focused on melanogenesis. Then, predicted miRNAs of lncRNAs were screened by the condition of Tot Score ≧160, while the numbers of predicted miRNAs of lnc-BTBD19-1:1, ENST00000414633, lnc-LNPEP-2:1, lnc-RBMY1J-6:1, lnc-TRIM37-1:2, NR_031650 were few, so the top ten predicted miRNAs of these lncRNAs were screened (Figure 2A and Supplementary Table 3); the top five high-binding sites miRNAs of circRNAs were selected (Figure 2B, Supplementary Table 4); and the top 40 predicted miRNAs of mRNAs were chosen based on the score (Figure 2C and Supplementary Table 5). Furthermore, the ceRNA network of lncRNA-miRNA-mRNA (Figure 2D and Supplementary Table 6) and circRNA-miRNA-mRNA (Figure 2E and Supplementary Table 7) were constructed by Cyto Vision 3.7.0 software.

Figure 2. The lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA network in α-MSH-treated melanocytes. (A) Potential miRNA targets (Tot Score ≥160 or top ten Tot Score) of the differentially expressed lncRNAs with 5-fold change. (B) Potential miRNA targets (Top five high-binding sites) of the differentially expressed circRNAs with 5-fold change. (C) Potential miRNA targets (top 40 predicted miRNAs based on the score) of the differentially expressed mRNAs related to melanogenesis. (D, E) The lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA network involved in melanogenesis. Purple square: upregulated lncRNAs; Blue square: downregulated lncRNAs; Blue hexagon: upregulated circRNAs; Green six square: downregulated circRNAs; Red circle: upregulated mRNAs; Green circle: downregulated mRNAs; Orange diamond: miRNAs.

Table 3. Identification of differentially expressed mRNAs related to melanogenesis by GO and KEGG pathway analysis.

Gene	Fold change (α-MSH/NC)	Gene	Fold change (α-MSH/NC)
SOX6	10.16	EDNRB	0.45
CTNNB1	3.53	GNAO1	0.33
IL6	2.73	TYROBP	0.14
FZD4	2.72
WNT3	2.47
CALM2	2.43
FZD7	2.32
MAPK11	2.24
TYR	2.18
POMC	2.06
RAB38	2.04

Identification of the quaternary ceRNA network in α-MSH-treated melanocytes

Since lncRNAs, circRNAs, and mRNAs can interact with miRNAs through the same MREs, there may exist quaternary relative network among them. Venn diagram analysis was used to screen the potential quaternary ceRNA network among the above screened lncRNAs (the corresponding 303 predicted miRNAs), circRNAs (the corresponding 192 predicted miRNAs) and melanogenesis-related mRNAs (the whole predicted miRNAs) by binding the same miRNAs (Supplementary Figure 2). Because the screened lncRNAs, circRNAs and TYROBP did not bind to the same miRNA, it did not included in the Supplementary Figures 2 and 3. Using Cyto Vision 3.7.0 for cross-linking and quaternary ceRNA network construction revealed that lncRNAs (ENST00000606533 and ENST00000532071), circ_0091223 and TYR mRNA was supposed to competitively bound with miR-1291; ENST00000606533, circ_0031728, and TYR mRNA was supposed to competitively bound with miR-4530 (Figure 3A). Other melanogenesis related mRNAs (CTNNB1, IL6, CALM2, POMC, EDNRB, FZD7, WNT3, GNAO1, MAPK11, RAB38, FZD4, and SOX6) involved in the quaternary ceRNA networks with miRNAs and above screened lncRNAs, circRNAs were presented in Figure 3B–3M.

Figure 3. Identification of the quaternary ceRNA network in α-MSH-treated melanocytes. (A–M) Cyto Vision 3.7.0 was used to construct the quaternary ceRNA network focused on screened melanogenesis related mRNAs (TYR, CTNNB1, IL6, CALM2, POMC, EDNRB, FZD7, WNT3, GNAO1, MAPK11, RAB38, FZD4, SOX6). Blue square: lncRNAs; Dark green square: circRNAs; Red square: upregulated mRNAs; Green square: downregulated mRNAs; Orange square: miRNAs.

α-MSH treatment induced ENST00000606533 and circ_0091223 expression but inhibited miR-1291 expression

As TYR is the key enzyme melanogenesis, we focused on the quaternary ceRNA network aimed at TYR. qRT-PCR results showed that ENST00000606533 and circ_0091223 significantly increased in α-MSH treated MCs compared with the NC MCs (Figure 4A, 4B), which was in accordance with the data from the microarray. While the expression of ENST00000532071 and circ_0031728 had no obvious change (Figure 4A, 4B). Besides, the expression of miR-1291 was significantly inhibited in α-MSH-treated MCs, while miR-4530 only had a slight decrease (Figure 4C). We further constructed luciferase reporters containing wild type and mutated putative binding sites of ENST00000606533 or circ_0091223 transcripts (Figure 4E, 4F), respectively. Luciferase reporter assays showed that the luciferase activities of ENST00000606533 or circ_0091223 wild type reporter were significantly reduced when transfected with miR-1291 mimics compared with control reporter or mutated luciferase reporter (Figure 4D). Moreover, the binding site of miR-1291 with TYR predicted by TargetScan software possessed the same site with which miR-1291 binding to ENST00000606533 or circ_0091223 (Figure 4G), so we did not further verify the binding relationship between miR-1291 and TYR by luciferase assay. These results confirmed that ENST00000606533 and circ_0091223 competitively bound to miR-1291, to affect the bind of miR-1291 with TYR as we predicted.

Figure 4. α-MSH-treatment induced ENST00000606533 and circ_0091223 expression but inhibits miR-1291 expression. (A–C) ENST00000606533, ENST00000532071, circ_0091223, circ_0031728, miR-1291, and miR-4530 levels were detected by qRT-PCR in MCs after 150 nM α-MSH treatment. (D) Luciferase reporter assay was used to detect the binding of miR-1291 to circ_0091223 and ENST00000606533. (E, F) Schematic model for wild type (WT) or mutant (mut) transcripts of ENST00000606533 or circ_0091223 luciferase reporters. (G) The binding site of miR-1291 with TYR predicted by TargetScan software.

Confirming the quaternary ceRNA network regulating TYR

To further confirm that ENST00000606533 and circ_0091223 can serve as ceRNAs to regulate TYR expression, we downregulated the expression of ENST00000606533 and circ_0091223 by siRNAs. The results showed that separate siRNA had poor interference effect on the expression of ENST00000606533, but mixed siRNAs could significantly inhibit the expression of ENST00000606533 (Figure 5A), so we chose mixed siRNAs in the subsequent experiments. si-ENST00000606533 mix could decrease the expression of circ_0091223 and TYR mRNA/protein level, while increase the expression of miR-1291 (Figure 5A, 5G). si-circ_0091223 2 could decrease the expression of ENST00000606533 and TYR mRNA/protein level, while increase the expression of miR-1291 (Figure 5B, 5G). We further upregulated the miR-1291 expression in MCs by transferring miR-1291 mimics. The results presented that miR-1291 mimics significantly decreased the expression of ENST00000606533, circ_0091223, and TYR mRNA/protein level (Figure 5C, 5G). Furthermore, we co-transfected the MCs with si-ENST00000606533 mix and miR-1291 inhibitor for 48 h and found that the miR-1291 inhibitor could reverse the inhibitory effect of si-ENST00000606533 mix on the expression of circ_0091223, ENST00000606533, miR-1291 and TYR (Figure 5E). Also, the inhibitory effect of si-circ_0091223 2 on the expression of circ_0091223, ENST00000606533, miR-1291 and TYR could be reversed by co-transfected with miR-1291 inhibitor (Figure 5F). The transfection efficiency of the miR-1291 inhibitor was showed in Supplementary Figure 3. And the si-ENST00000606533 mix, si-circ_0091223 2, and miR-1291 mimics could reverse the effect of α-MSH on ENST00000606533, circ_0091223, miR-1291, and TYR mRNA/protein level (Figure 5D, 5G). Moreover, the Masson-Fontana melanin staining showed that si-ENST00000606533 mix, si-circ_0091223 2, and miR-1291 mimics could inhibit the melanin production, and reverse the effect of α-MSH on melanin production (Figure 5H). These results indicated that ENST00000606533 and circ_0091223 could compete for binding to miR-1291 to upregulate TYR expression, forming the quaternary ceRNA network, then stimulating melanogenesis.

Figure 5. Confirmation of the quaternary ceRNA network regulating TYR. (A) After transfection with siRNAs of ENST00000606533 in MCs for 48 h, the transfection efficiency of si-ENST00000606533 mix (mixing the three siRNAs) was most significant, and the expression of circ_0091223, miR-1291, and TYR were detected by transfection with si-ENST00000606533 mix. (B) After transfection with siRNAs of circ_0091223 for 48 h, the transfection efficiency of si-circ_0091223 2 was most significant, and the expression of ENST00000606533, miR-1291, and TYR were detected by transfection with si-circ_0091223 2. (C) After transfection with miR-1291 mimics for 48 h, the transfection efficiency, the expression of ENST00000606533, circ_0091223, and TYR were detected. (D) After treated with α-MSH, the MCs were transfected with si-ENST00000606533 mix, or si-circ_0091223 2, or miR-1291 mimics, and the expression of ENST00000606533, circ_0091223, miR-1291, and TYR were detected. (E) After co-transfection with siRNAs of ENST00000606533 and miRNA_1291 inhibitor for 48 h, the expression of ENST00000606533, circ_0091223, miR-1291, and TYR were detected. (F) After co-transfection with siRNA of circ_0091223 and miR-1291 inhibitor for 48 h, the expression of ENST00000606533, circ_0091223, miR-1291, and TYR were detected. (G) After treated with α-MSH and/or si-ENST00000606533 mix, or si-circ_0091223 2, or miR-1291 mimics, the protein level of TYR were detected. (H) After treated with α-MSH and/or si-ENST00000606533 mix, or si-circ_0091223 2, or miR-1291 mimics, the Masson-Fontana melanin staining were detected.

Discussion

Melanogenesis is regulated by multiple pathways in receptor dependent or independent mechanisms, which are driven by hormonal, autocrine, paracrine, or intracrine manner; one of the most important stimulus is α-MSH [21]. α-MSH controls diverse melanocytic functions, such as proliferation, eumelanin synthesis, and cytokine production [2]. In the skin, α-MSH is present in melanocytes and keratinocytes; thus, its cutaneous effects are presumably mediated via paracrine and/or autocrine mechanisms [22]. The paracrine and autocrine α-MSH performs its melanogenic action through several pathways, including cAMP/PKA/CREB/MITF, MAPK-ERK, and the Wnt signaling pathway [23–25]. Besides, some new mechanisms are involved in α-MSH-stimulated melanogenesis. One of them is autophagic tumor suppressor UVRAG, which is required for the dynamic integrity and pigmentation of melanosomes [26]. Moreover, microRNA-141-3p and microRNA-200a-3p can suppress α-MSH-induced melanogenesis via inhibiting MITF expression [27]. Hence, the mechanisms involved in α-MSH- induced melanogenesis is worthy of further study.

Recent studies have shown that ceRNAs are involved in various diseases, especially in cancers [8]. In some skin disease, such as psoriasis [28] and atopic dermatitis [29], lncRNAs or circRNAs also can act as ceRNAs and regulate these diseases via their interaction with disease-associated mRNAs. However, the functions of lncRNAs and circRNAs in α-MSH-induced melanogenesis are poorly understood. In our study, ceRNA microarray results showed that the expression profiles of lncRNAs and circRNAs are changed in α-MSH-treated melanocytes, suggesting that lncRNAs and circRNAs participate in α-MSH-induced melanogenesis. Our study also identified several lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA networks in α-MSH treated melanocytes, which can regulated differentially expressed melanogenesis-related genes (TYR, CTNNB1, IL6, CALM2, POMC, EDNRB, FZD7, WNT3, GNAO1, MAPK11, RAB38, FZD4, SOX6, and TYROBP). Since lncRNAs, circRNAs, and mRNAs can interact with miRNAs through MREs, we wondered if there is quaternary ceRNAs crosstalk involved in melanogenesis. The results of Venn diagram analysis and Cyto software (Vision 3.7.0) revealed that 13 quaternary ceRNA networks could regulate melanogenesis-related genes that differentially expressed in α-MSH treated melanocytes (Supplementary Figure 2 and Figure 3). Of course, these predicted quaternary ceRNA crosstalk requires further experimental confirmation.

TYR is the key enzyme that catalyzes L-tyrosine into L-DOPA, which is a rate-limiting step in melanin synthesis; thus, TYR can act as a marker that evaluates the function of melanogenesis [30]. As reported, TYR is regulated by transcription factors, histone modification related proteins, ubiquitination related proteins, miRNAs, and lncRNAs. For example, MITF is the most important transcription factor to promote TYR expression [31], the histone acetyl transferase (HAT) p300/CBP can mediate H3K27 acetylation, which further regulates TYR expression through the interaction with MITF [30]. Besides, TYR protein can be ubiquitinated and degraded by proteasome complex (E1, E2, and E3) of ER [32, 33]. Moreover, miR-125b can decrease TYR expression in human melanocytes and pigmented tissue [34], LncRNA H19 and UCA1 can directly or indirectly regulate TYR expression [14, 15]. As we know, the ceRNA hypothesis proposes that lncRNAs, circRNAs, and mRNAs competing for binding the miRNAs and regulating the expression of each other, which forms a complex post-transcriptional regulatory network. Since that miRNA and lncRNA are related to TYR, does the ceRNA regulatory mechanism play a role in regulating α-MSH-induced TYR expression? Our study had identified the possible quaternary ceRNA network regulating TYR (Figure 3A). So we further confirmed the hypothesistaking TYR as an example. We firstly found that the expression of ENST00000606533, circ_0091223, and TYR was upregulated, miR-1291 was downregulated in melanocytes after α-MSH treatment. Simultaneously, we designed ENST00000606533 or circ_0091223 luciferase reporter plasmids for miR-1291, and found that miR-1291 mimics reduced the luciferase activity of ENST00000606533 and circ_0091223 luciferase reporter, respectively. Besides, the silence of ENST00000606533 and circ_0091223 could increase miR-1291 expression and decrease TYR expression. miR-1291 mimics could decrease the expression of ENST00000606533, circ_0091223, and TYR. Further rescue experiments showed that miR-1291 inhibitor significantly attenuated the effects of ENST00000606533 and circ_0091223 silencing on TYR, suggesting that ENST00000606533 and circ_0091223 may function as ceRNAs to regulate α-MSH-induced TYR by sponging miR-1291 (Figure 6). The identification of “ENST00000606533/circ_0091223- miR-1291-TYR” axis expands the understanding of the underlying mechanism of α-MSH-induced melanogenesis in melanocytes, which also provides novel research directions for skin pigmentation disease. These results also support that ENST00000606533, circ_0091223, and miR-1291 may play an important role in melanogenesis.

Figure 6. The schematic diagram of the ceRNA mechanism in α-MSH-induced melanogenesis. ENST00000606533 and circ_0091223 may function as ceRNAs to regulate TYR expression by sponging miR-1291, thus promoting α-MSH-induced melanogenesis in melanocytes.

The mechanism of α-MSH promoting ENST00000606533 and circ_0091223 expression in melanocytes is unclear. At present, the expression of lncRNAs and circRNAs are mainly regulated at three different levels: epigenetic regulation (DNA methylation, histone modification, chromatin remodeling and RNA modification), transcriptional regulation (the interaction with transcription factors and single nucleotide polymorphism), and post-transcriptional regulation (the interaction with miRNAs, RNA-binding proteins, and general splicing factors) [35, 36]. α-MSH can promote melanogenesis via binding to its receptor and driving downstream signaling pathways, especially cAMP/PKA signaling pathway. Multiple studies have shown that cAMP/PKA pathway can not only induce the expression of some transcription factors, such as Signal Transducer And Activator Of Transcription 5B (STAT5B), Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3), c-Fos, and CREB [37, 38], but also drive the epigenetic regulation and post-transcriptional regulation. For example, PKA can phosphorylate histone demethylases PHF2, which can bind and demethylate DNA-binding protein ARID5B, thus affecting the expression of downstream genes [39]. Besides, cAMP/PKA pathway can promote apoptosis through the post-transcriptional regulation such as lysosomal, ribosomal, Golgi-related functions and RNA splicing [40–42]. Hence, α-MSH may increase the expression of ENST00000606533 and circ_0091223 through one of above mechanism, which needs further investigation in the follow-up study. There are some limitations in this study. For example, our study is based on in vitro experiments and lacking in vivo evidence, such as clinical specimens and animal experiments. We have confirmed that the ceRNA regulatory network can regulate TYR in α-MSH treated melanocytes, but other melanogenesis-related genes included in the networks are not explored, which might be investigated in the future.

Author Contributions

Qinghai Zeng, Jing Chen, Jinhua Huang, and Ling Jiang designed the research; Ling Jiang, Li Lei, Yujie Ouyang, Yan Long, Hui Li, Si Li, Lun Yang, and Yan Yang performed the experiments; Yibo Hu made the surgery of circumcision to acquire the foreskin tissues; Ling Jiang and Yibo Hu organized the pictures of the article; Hong Xiang and Lihua Huang designed and analyzed the primers sequences; Ling Jiang and Qinghai Zeng wrote the paper; Qinghai Zeng, Jing Chen, and Rong Xiao revised the manuscript.

Acknowledgments

We would like to thank our teachers who have provided helpful suggestions. We would also like to thank the adolescent males who were circumcised in the Department of Urology Surgery, Third Xiangya Hospital, Central South University for providing the the prepuce.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

This study was funded by the National Natural Science Foundation of China (No. 81703101), the New Xiangya Talent Projects of the Third Xiangya Hospital of Central South University (No. JY201623 and No. 20170301), the Natural Science Foundation of Hunan Province (No. 2018JJ3788 and No. 2018JJ3793), the Health and Family Planning Commission of Hunan Province (No. C2019173), and the Fundamental Research Funds for the Central Universities of Central South University (No. 2019zzts364).

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