The experimental animals were obtained from Linquan Comprehensive Experimental Station of Anhui Agricultural University. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Anhui Agricultural University. The Institutional of Animal Care and Use Committee of Anhui Agricultural University approved all animal surgeries (Approval no. AHAUXMSQ2023065).

The goat melanocytes used in this study were established in our laboratory and maintained as previously described [21]. In the present research, we investigated the role of circRNA18_46222157_46248185 (named circRNA18) on melanogenesis at the cellular level. The research was divided into seven experiments. Each experiment had a completely randomized design, and was conducted in triplicate. Experiment 1 was designed to elucidate the expression pattern of circRNA18 in goat skins and melanocytes, including location, expression trend, etc. Experiment 2 soughted to unveil the effect of circRNA18 on melanogenesis by overexpression of siRNA-circRNA18 plasmid and negative control (NC) plasmid in melanocytes. Experiment 3 aimed to delineate the regulatory interplay between circRNA18 and specific miRNAs (miR-211 and miR-7-5p) through luciferase assay. Experiment 4 was conducted to investigate the effects of miR-211 on melanogenesis by overexpression of miR-211 plasmid in melanocytes. Experiment 5 aimed to uncover the regulatory relationship between miR-211 and E1A-binding protein p300 (EP300) in melanocytes through luciferase assay. Experiment 6 was devised to explore the impact of EP300 on melanogenesis by overexpression of siRNA-EP300 plasmid in melanocytes. Experiment 7 aimed to elucidate the molecular mechanism by which circRNA18 inhibits melanin production through the investigation of overexpressing mimics-circRNA18 plasmid.

Experimental animals were obtained from the Linquan comprehensive experimental station of Anhui Agricultural University. Skin samples were obtained from the back skin (0.8×0.8 cm) of three 3-month-old Boer goats. The goats were provided adequate drinking water and feed during the experiment to maintain good health. Total example RNAs were extracted using the TRIzol method, and the total sample RNAs were marked and stored at −80°C.

Circular RNAs have the potential to function as miRNA sponges, thereby impacting miRNA activity in the modulation of mRNA expression [14,23]. In this study, we predicted the potential target miRNAs of circNA18 using the TargetScanVert and miRDB databases. miRNAs can regulate gene expression by binding it’s 3′-untranslated regions (UTR) [22,23]. In this study, we predicted the potential target genes of miRNA (miR-211) using the TargetScanVert and miRDB databases.

Total RNA was extracted from goat skin samples and melanocytes using the TRIzol reagent, and cDNA was synthesized using a high volume cDNA reverse transcription kit (TransGen, Beijing, China). The cDNA amplification was conducted utilizing the PCR system (Thermo Fisher, Waltham, MA, USA) with specific primers (Table 1). The expression levels of genes (miRNA and circRNA) were normalized to universal U6 primer (U6) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively (n = 3, mean±standard deviation [SD]). Relative expression levels of genes were calculated using the 2^−ΔΔCt method.

In order to explore the functions of circRNA18 in melanocytes, siRNA-circRNA18 vectors and the NC vectors were chemically synthesized (Beijing Genomics institution, Shenzhen, China). The precursor sequence of potential target miR-211 was obtained from the miRBase database (http://www.mirbase.org) and chemically synthesized, then inserted into the pcDNA6.2-GW/EmGFPmiR vector (Invitrogen, Carlsbad, CA, USA). A segment of cDNA fragment of circRNA18, encompassing binding sites for miR-211, was amplified and subsequently subjected to mutation via chemical synthesis. These amplified fragments were cloned into the pmirGL0 Dual-Luciferase miRNA target vector (Promega, Madison, WI, USA) to generate the pmirGL0-circRNA18-wt and pmirGL0-circRNA18-mut plasmids. Additionally, a mimics-circRNA18 plasmid, with binding sites for miR-211, and a NC plasmid were also developed.

Goat melanocytes were cultured in melanin basal medium (ScienCell Research Laboratories, Carlsbad, CA, USA). HEK 293T cells were cultured in Dulbecco’s modified eagle medium (Gibco, New York, NY, USA) supplemented with 10% fetal bovine serum (Kangyuan, Shanghai, China). The cells employed in this investigation were transfected using LipofectamineTM 2000 (Invitrogen, USA).

Counted the melanocytes, and the melanocytes were added to 90 μL of Triton X-100 (1%) and shaken for 5 min. Thereafter, 5 μL of 0.2% L-dopa was added and the cells were incubated at 37°C for 30 min. Optical density (OD) was measured at 490 nm using a microplate analyzer (Biotek, Winooski, VT, USA), and tyrosinase activity (%) was calculated according to the manufacturer’s protocol.

Melanin production was analyzed using a total alkali-soluble melanin (ASM) assay. Melanocytes were collected and counted, rinsed three times with phosphate-buffered saline (PBS) (1×), and lysed in 1 mL of NaOH (1 mol/L) at 37°C for 45 min. The OD was measured at 475 nm and normalized against the cell number. Melanin production (%) was calculated according to manufacturer’s protocol.

HEK 293T cells were co-transfected with 3 μg of pmirGL0-circRNA18-wt or pmir-GL0-circRNA18-mut together with an miR-211 plasmid. Cells were lysed after 48 h using the lysis buffer supplied with the Dual-Luciferase Assay Kit (Beyotime, Shanghai, China), and firefly and Renilla luciferase activities were measured. Luciferase activity (%) was calculated based on OD measurements.

The goat melanocytes were inoculated on slides, fixed in 4% paraformaldehyde for 30 min at 4°C, and treated with proteinase K (0.04 μg/mL; Roche Applied Science, Shanghai, China). The samples were washed with PBS (0.1 mol/L, pH = 7.4) and hybridized with circRNA 18_46222157_46248185 probe (5′-ACACCTACAATACCTCAACGACTATTACT GCCTCTT-3′; Bio-High, Shijiazhuang, China) at 37°C overnight. The slides hybridized with the probe were washed in saline sodium citrate solution, and incubated with mouse alkaline phosphatase (ALP)-conjugated anti-digoxigenin antibody (1:1,000; Bio-High, China) for 2.5 h at 37°C. The ALP reaction was performed using 3,3′-diaminobenzidine as the substrate.

Data were analyzed using GraphPad Prism software (version 8.0). Data, including tyrosinase activity, melanin production, luciferase activity, and gene abundance, were determined using Student’s t-test. Data were arcsine-transformed prior to statistical analysis, and differences among means were assessed using the least significant difference method. A single replicate was used as the experimental unit. Unless indicated, the results were expressed as the mean±SD, and values were considered statistically significant at the p<0.05 level.

Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that circRNA18 expression level in skin with white hair was significantly higher (p<0.01) than that with brown hair (Figure 1A), and the expression of cicrcRNA18 was not affected by RNA enzyme treatment (Figure 1B). The PCR results showed no positive signal in the DNA of melanocytes using specific primers, but a specific positive signal was detected in the cDNA (Figure 1C). The results of the circRNA expression profile showed distinct expression patterns of circRNA18 in diverse tissues of male (Figure 1D) and female (Figure 1E) goats, and they were highly abundant in the spleen and liver.

The in situ hybridization results revealed that circRNA18 was expressed in the basal layer of the skin (Figure 2A) and hair follicles (Figure 2B), where melanocytes reside. Moreover, we observed ubiquitous expression of circRNA18 in the cytoplasm of melanocytes (Figure 2C).

To investigate the function of circRNA18 in goat melanocytes, we constructed an interference vector (siRNA-circRNA18). qRT-PCR results showed that the expression level of circRNA18 was significantly reduced (p<0.001) upon transfection with siRNA-circRNA18 compared to that of NC group (Figure 3A). Downexpression of circRNA18 in melanocytes significantly promoted (p<0.0001) MITF, TYR, TYRP1, and TYRP2 expression (Figure 3B). Furthermore, we found that the downregulation of circRNA18 in melanocytes significantly increased (p<0.05) tyrosinase activity (Figure 3C) and significantly promoted (p<0.01) melanin production (Figure 3D).

Bioinformatics analysis unveiled the presence of putative binding sites for circRNA18 and miRNAs (miR-211 and miR-7-5p) (Figure 4A). qRT-PCR results showed that downregulation of circRNA18 significantly promoted (p<0.001) miR-211 expression (Figure 4B), however, there was no change in miR-7-5p expression (Figure 4C). Therefore, miR-211 is a potential tracking candidate. The luciferase assay results revealed that luciferase activity was significantly decreased (p<0.01) in 293T cells transfected with miR-211 and circRNA18-wt compared with that in 293T cells transfected with miR-211 and pmirGL0 dual-luciferase blank plasmid (Figure 4D). However, it was unchanged in 293T cells transfected with miR-211 and circRNA18-mut compared with the 293T cells transfected with miR-211 and pmirGL0 Dual-Luciferase blank plasmid (Figure 4E).

Mimics-circRNA18_46222157_46248185 (named mimic-circRNA18) plasmids and NC plasmids were transfected into melanocytes to determine the molecular mechanism by which circRNA18 inhibits melanogenesis. The results revealed that the overexpression of mimic-circRNA18 plasmid in melanocytes markedly decreased (p<0.0001) the expression of miR-211 (Figure 5A). qRT-PCR results showed that the expression level of miR-211 in goat skin with brown hair was significantly higher (p<0.01) than that with white hair (Figure 5B). Functional analysis showed that the overexpression of miR-211 in melanocytes (Figure 5C) significantly promoted (p<0.0001) MITF, TYR, TYRP1, and TYRP2 expression (Figure 5D). Furthermore, the overexpression of miR-211 in melanocytes significantly promoted (p<0.001) tyrosinase activity (Figure 5E), and markedly increased (p< 0.01) melanin production (Figure 5F).

Bioinformatic analysis revealed that the 3′ UTR of EP300 has two putative miR-211 binding sites (Figure 6A) and that such putative binding sites are conserved among goat, mice, ovis and bos (Figure 6B). The luciferase assay results revealed that miR-211 expression significantly reduced (p<0.001) the luciferase activity associated with EP300-wt compared to that of the NC group (Figure 6C). However, it was unchanged in 293T cells transfected with miR-211 and EP300-mut compared with the 293T cells transfected with miR-211 and pmirGL0 Dual-Luciferase blank plasmid (Figure 6D). The qRT-PCR results showed that the overexpression of miR-211 in goat melanocytes significantly inhibited (p<0.001) EP300 expression (Figure 6E).

We constructed interference vectors to investigate the functions of EP300 in melanocytes, and the results showed that the expression level of EP300 were significantly reduced (p<0.0001) upon transfection with siRNA-EP300 (Figure 7A). The downregulation of EP300 in melanocytes significantly increased the transcript levels of MITF (p<0.001), TYR (p<0.01), TYRP1 (p<0.0001), and TYRP2 (p<0.0001) (Figure 7B). Moreover, the downregulation of EP300 in melanocytes significantly promoted (p<0.01) tyrosinase activity (Figure 7C), and significantly increased (p<0.05) melanin production (Figure 7D).

qRT-PCR was employed to ascertain the molecular mechanisms underlying the inhibition of melanogenesis by circRNA18 via reduction of miR-211 expression. The results showed that the overexpression of the mimics-circRNA18 in goat melanocytes significantly promoted (p<0.001) EP300 expression (Figure 8A), significantly inhibited (p<0.001) tyrosinase activity (Figure 8B), and significantly decreased (p<0.001) melanin production (Figure 8C).