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Article GNAI2 Promotes Proliferation and Decreases in Rabbit Melanocytes

Shuaishuai Hu 1, Yingying Dai 1, Shaocheng Bai 1, Bohao Zhao 1, Xinsheng Wu 1,2,* and Yang Chen 1,2

1 College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China; [email protected] (S.H.); [email protected] (Y.D.); [email protected] (S.B.); [email protected] (B.Z.); [email protected] (Y.C.) 2 Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Hangzhou 310021, China * Correspondence: [email protected]

Abstract: GNAI2 (G subunit alpha i2) is a signaling modulator or transducer, involved in several transmembrane signaling systems, that plays a vital role in the melanogenesis signaling pathway. However, whether GNAI2 regulates cell proliferation and apoptosis in rabbit melanocytes is not known. We found that GNAI2 was differentially expressed in rabbits with different coat colors using qRT-PCR and Wes assays. Furthermore, it was observed that the rabbits with black skin had the highest GNAI2 levels, and those with white skin had the lowest expression. The coding sequence of GNAI2 was successfully cloned and inserted into pcDNA3.1 and pcDNA3.1-Myc vectors. It was observed that the GNAI2 protein was mainly localized in the cytoplasm using the indirect immunofluorescence staining assay. Overexpression of GNAI2 significantly increased melanin content, promoted melanocyte proliferation, and inhibited melanocyte apoptosis. On the contrary,  the knockdown of GNAI2 using siRNA had the opposite effect. In addition, GNAI2 significantly  increased the mRNA expression levels of the melanin-related genes TYR, GPNMB, PMEL, and DCT Citation: Hu, S.; Dai, Y.; Bai, S.; Zhao, in rabbit melanocytes. The results suggested that GNAI2 regulated melanocyte development by B.; Wu, X.; Chen, Y. GNAI2 Promotes promoting melanocyte proliferation and inhibiting apoptosis. Proliferation and Decreases Apoptosis in Rabbit Melanocytes. Keywords: GNAI2; melanocyte; proliferation; apoptosis Genes 2021, 12, 1130. https://doi.org/ 10.3390/genes12081130

Academic Editor: Rajiv Kumar 1. Introduction Melanogenesis is a complex process of the synthesis and storage of melanin initiated Received: 20 May 2021 by tyrosinase, which is synthesized by melanocytes, after it enters the melanosomes [1]. Accepted: 23 July 2021 Published: 25 July 2021 Melanogenesis plays an important role in the formation of different coat colors in mam- mals. There are two distinct types of melanin pigments: eumelanin and pheomelanin,

Publisher’s Note: MDPI stays neutral the proportions of which determine the coat color of the animal [2–4]. The difference in with regard to jurisdictional claims in the eumelanin content leads to the formation of different hair types in humans, such as published maps and institutional affil- black, brown, light brown, and blond [5]. Previous studies have found that there is lower iations. eumelanin content in those with lighter skin, which is highly sensitive to UV exposure, compared to those with darker skin [6]. Melanogenesis is regulated by several signaling systems, transcription factors, and melanin-related genes [7,8]. MC1R regulates melano- genesis andt was significantly decreased in the skin of a leucistic and blind cypriniform fish (O. daqikongensis, Nemacheilidae) [9]. α-MSH plays an important role in melanogenesis Copyright: © 2021 by the authors. by regulating the proportions of pheomelanin and eumelanin via MC1R [10]. The TYR Licensee MDPI, Basel, Switzerland. This article is an open access article is essential for melanin biosynthesis in melanocytes; it was differentially expressed distributed under the terms and in black and white feather bulbs, indicating that it plays an important role in the melanin conditions of the Creative Commons formation of feather bulb color in the plumage [11]. Several studies have suggested that Attribution (CC BY) license (https:// the expression of TYR was related to the content of eumelanin; the overexpression of TYR creativecommons.org/licenses/by/ could increase eumelanin content in mammals [12]. Eumelanin content was related to TYR 4.0/).

Genes 2021, 12, 1130. https://doi.org/10.3390/genes12081130 https://www.mdpi.com/journal/genes Genes 2021, 12, 1130 2 of 10

activity. Previous studies have demonstrated that TYR activity in black bone sheep was significantly higher than that in non-black bone sheep [13]. GNAI2 ( subunit alpha i2), belonging to the G protein family, is one of the key genes involved in melanogenesis. G consist of three subunits, α, β, and γ, and are signal transducers, which connect receptors to effectors in order to regulate intracellular signaling [14]. G protein-coupled receptors (GPCRs) receive external signals that activate the G proteins to transmit the signal to further regulate the growth and development of the organisms [15]. At present, a large number of G proteins are identified, including Gs, Gt, Gi, and Go [16]. Previous research shows that GNAI2 is mostly involved in cell injury and inflammatory response [17], tumorigenesis [18], hepatic ischemia-reperfusion injury [19], etc. GNAI2 is mainly expressed in immune cells and plays an essential role in regulating cellular viability and migration [20]. In addition, previous studies have found that GNAI2 associated with melanogenesis was identified in the growth process of brindle cattle [21]. However, it remains unclear whether GNAI2 is involved in the formation of rabbits’ coat colors and regulates rabbit melanocyte proliferation and apoptosis. The purpose of the present research is to explore the molecular function of GNAI2 in melanogenesis, and melanocyte proliferation and apoptosis after GNAI2 overexpression and knockdown. The study could provide an important theoretical basis for the molecular mechanism of melanogenesis to regulate the formation of mammalian coat colors.

2. Materials and Methods 2.1. Ethics Statement This study was carried out in accordance with the recommendations of Jiangsu Ad- ministration Rule of Laboratory Animals and strictly followed Institutional Animal Care and Maintenance protocols.

2.2. Experimental Animals A total of 18 adult Rex rabbits of different coat colors (black, white, chinchilla, brown, gray, and gray-yellow) were provided by Zhejiang Yuyao Xinnong Rabbit Industry Co., Ltd (Yuyao, Zhejiang, China). Three biological rabbits of each of the coat colors were used. The dorsal skin of Rex rabbits (1.5 cm × 1.5 cm) was collected for RNA and protein extraction to determine the GNAI2 expression in the different coat colors.

2.3. Overexpression Vector Construction The coding sequence (CDS) of GNAI2 (NCBI Reference Sequence: XM_008260677.2) was cloned using rabbit melanocyte cDNA as a template using ClonExpress II One Step cloning kit (Vazyme, Nanjing, China). The GNAI2 CDS was inserted into pcDNA3.1 and pcDNA3.1-Myc vectors using restriction enzymes EcoRI and XbaI. The primers used are shown in Table1.

Table 1. The primer sequences of GNAI2 CDS.

Primers Sequence (50→30) GNAI2-F tagtccagtgtggtggaattcGCCACCATGGGCTGCACGGTGAGC GNAI2-R ttgttcgaagggccctctagaGAAGAGGCCGCAGTCCTTCA

2.4. Cell Culture and Transfection Melanocyte culture was established using the method described by Chen et al. [22]. An adult black rabbit was used to isolate the melanocytes. A tissue sample of approximately 1.5 cm × 1.5 cm was excised from the dorsal skin of the rabbit. Rabbit melanocytes were cultured in Melanocyte Medium-2 (ScienCell, San Diego, CA, USA) supplemented with 1% PMA-free melanocyte growth supplement, 0.5% fetal bovine serum (FBS), and 0.5% penicillin/streptomycin solution (ScienCell, San Diego, CA, USA). Melanocytes were ◦ transferred to 24-well plates and incubated at 37 C with 5% CO2. When the culture was Genes 2021, 12, 1130 3 of 10

70–90% confluent, overexpression and knockdown experiments were performed using 1 µg GNAI2 plasmid and 1 µL siRNAs (Table2), respectively. Both were diluted in 25 µL Opti-MEM™ medium (Gibco, Carlsbad, CA, USA) and 2 µL diluted Lipofectamine™ 3000 (Lipofectamine™ 3000 diluted in 25 µL Opti-MEM™ medium) was added to both the mixtures. These mixtures were incubated for 10-15 min at room temperature and were then added to cultured melanocytes separately, and the cells were incubated at 37 ◦C with 5% CO2 for 48 h.

Table 2. Primer sequences of the GNAI2 siRNAs.

Primers Sequence (50 to 30) Forward: UUCUCCGAACGUGUCACGUTT Negative Control Reverse: ACGUGACACGUUCGGAGAATT Forward: GCAACCUGCAGAUUGACUUTT siRNA-GNAI2-415 Reverse: AAGUCAAUCUGCAGGUUGCTT Forward: GCAUCGCACAGAGUGACUATT siRNA-GNAI2-634 Reverse: UAGUCACUCUGUGCGAUGCTT Forward: CCUGCACUUCAAGAUGUUUTT siRNA-GNAI2-731 Reverse: AAACAUCUUGAAGUGCAGGTT

2.5. Indirect Immunofluorescence Staining Assay The cells transfected with pcDNA3.1-Myc-GNAI2 plasmid were washed in phosphate- buffered saline (PBS) (HyClone, Logan, UT, USA) three times lightly and fixed at room tem- perature for 20 min using cold (4%) paraformaldehyde. Then, the cells were washed again using PBS and treated with 0.2% Triton X-100 (Solarbio, Beijing, China). After 10 min, goat serum was used to block the cells for 30 min. Then, the cells were incubated using the MYC tag monoclonal antibody (Proteintech, Wuhan, China) at 4 ◦C overnight. Subsequently, Cy3-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Proteintech, Wuhan, China) was added and the cells were incubated at room temperature for 2 h in dark. DAPI staining solution was added to the cells and they were incubated for 10 min. The cells were observed using a fluorescence inverted microscope.

2.6. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis Total RNA from the rabbit melanocytes was isolated using RNAsimple Total RNA kit (Tiangen Biotech Co., Ltd., Beijing, China). Total RNA was reverse-transcribed using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China). Quantitative real-time PCR was performed using ChamQTM SYBR® qPCR Master Mix (Vazyme, Nanjing, China) in QuantStudio®5 (Applied Biosystems; Thermo Fisher Scientific, Foster City, CA, USA). The relative mRNA expression was calculated using the 2−∆∆Ct method [23]. The primers used are shown in Table3.

Table 3. qRT-PCR primer sequences.

Primers Sequence (50→30) Product Length (bp) Forward: ACGACTCAGCCGCCTAC GNAI2 119 Reverse: GTGCGTCTCCACGATCC Forward: CTCTTCTTGTTGCTGTGGG TYR 156 Reverse: GCTGAGTAGGTTAGGGTTTTC DCT Forward: ATTCTGCTGCCAATGACCC 154 (TYRP2) Reverse: AACGGCACCATGTTATACCTG Forward: GTCAGCACCCAGCTTGTCA PMEL 130 Reverse: GCTTCATTAGTCTGCGCCTGT Forward: TCCAGATTGCAGAAGTCCCGAT GPNMB 173 Reverse: GCAGCTCTCAGTCTCGTCCA Forward: CACCAGGGCTGCTTTTAACTCT GAPDH 141 Reverse: CTTCCCGTTCTCAGCCTTGACC Genes 2021, 12, 1130 4 of 10

2.7. Protein Preparation and Western Blotting Rabbit melanocyte lysates were obtained using cell lysis buffer (Beyotime, Shanghai, China) containing 1% phenylmethanesulfonylfluoride (PMSF). Protein concentration was estimated using the BCA protein assay kit (Beyotime, Shanghai, China). Protein assay was performed using Wes’s automated Western blotting system (Wes assay) [24] according to the manufacturer’s instructions. GAPDH monoclonal antibodies and GNAI2 monoclonal antibodies (Proteintech, Wuhan, China) were used.

2.8. Melanin Content Measurement GNAI2 overexpression and knockdown experiments were performed using melanocytes. Cells were collected 72 h after transfection. The cells were lysed using 1 M NaOH and the lysate was incubated at 80 ◦C for 1 h. Optical density (OD) was measured at 475 nm using Infinite M200 PRO (Tecan, Männedorf, Switzerland) spectrophotometer.

2.9. Cell Proliferation Assay Transfected cells were harvested and seeded into 96-well plates equally. Cell pro- liferation was detected at 0, 24, 48, and 72 h using Cell Counting Kit-8 assay (Vazyme, Nanjing, China).

2.10. Apoptosis Assay Melanocytes were transfected in 6-well plates. After 48 h, cells were harvested. Apoptosis assay was performed in order to detect the level of apoptosis using an Annexin V-FITC apoptosis detection kit (Vazyme, Nanjing, China). Later, the cells were sorted using a fluorescence-activated cell sorter (FACSAria SORP flow cytometer, Becton Dickinson, San Jose, CA, USA).

2.11. Statistical Analysis All statistical analyses were carried out using SPSS version 25 (SPSS Inc., Chicago, IL, USA). One-way ANOVA was employed to analyze significant differences among the study groups. All values are presented as mean ± standard deviation (SD).

3. Results 3.1. GNAI2 Was Differentially Expressed in the Skin of Rabbits with Different Coat Colors GNAI2 was successfully cloned, and comprised 1068 bp coding 355 amino acids (Figure1A). To determine whether GNAI2 was involved in the formation of rabbit coat colors, the expression levels of GNAI2 were detected in the skin of rabbits of different coat colors. It was found that the mRNA expression of GNAI2 in the black skin was significantly higher than that in the other skins (p < 0.05), and the lowest expression was detected in the white skin (Figure1B). Similarly, GNAI2 protein levels were different in the skins of Genes 2021, 12, x FOR PEER REVIEW 5 of 11 rabbits of different coat colors. The highest GNAI2 expression was found in the black skin, and the white skin had the lowest (Figure1C).

Figure 1. Cont.

159 160 Figure 1. The expression levels of GNAI2 in the skin of rabbits of different coat colors: (A) the 161 rabbit GNAI2 cDNA was successfully cloned. M, DL2000 DNA marker; lane 1, the coding 162 sequence (CDS) of GNAI2; (B) the GNAI2 mRNA levels in the skin of rabbits of different coat 163 colors were determined. Samples were in triplicates and the relative expression levels of the genes 164 were determined using GAPDH as an internal control and the 2−ΔΔCt method. Duncan’s Multiple 165 Range Test was employed to compare the differences across groups. Small letters indicate 166 significant differences among groups (p < 0.05); (C) the GNAI2 protein expression in the rabbit 167 skin was measured using the Wes assay. Differential expression of GNAI2 in Rex rabbits with 168 different coat colors was calculated using the relative expression ratio of GNAI2/GAPDH. BL, 169 black; CH, chinchilla; BR, brown; GR, gray; GY, gray-yellow; WH, white.

170 3.2. GNAI2 Protein Was Mainly Localized in the Cytoplasm of Melanocytes 171 The subcellular localization of the GNAI2 protein was predicted using the protein 172 subcellular localization prediction tool (PSORT). It was found that the GNAI2 protein was 173 mainly expressed in the cytoplasm of melanocytes. To further verify the expression of the 174 GNAI2 protein, an indirect immunofluorescence staining assay was performed. The 175 pcDNA3.1 group was used as a negative control and the pcDNA3.1-Myc group was used 176 as a positive control. It was found that the GNAI2 protein was localized in the cytoplasm 177 of melanocytes (Figure 2), which was consistent with the prediction.

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159 160 FigureFigure 1. 1. TheThe expression expression levels levels of GNAI2 of GNAI2 in the in skin the of skin rabbits of rabbits of different of different coat colors: coat (A colors:) the (A) the rabbit 161 rabbitGNAI2 GNAI2 cDNA cDNA was was successfully successfully cloned. cloned. M, M, DL2000 DL2000 DNADNA marker; lane lane 1, 1, the the coding coding sequence (CDS) 162 sequence (CDS) of GNAI2; (B) the GNAI2 mRNA levels in the skin of rabbits of different coat of GNAI2; (B) the GNAI2 mRNA levels in the skin of rabbits of different coat colors were determined. 163 colors were determined. Samples were in triplicates and the relative expression levels of the genes 164 wereSamples determined were inusing triplicates GAPDH and as an the internal relative control expression and the levels2−ΔΔCt method. of the genesDuncan’s were Multiple determined using − 165 RangeGAPDH Test as was an employed internal control to compare and the differe 2 ∆∆Ctncesmethod. across groups. Duncan’s Small Multiple letters indicate Range Test was employed 166 significantto compare differences the differences among acrossgroups groups.(p < 0.05); Small (C) the letters GNAI2 indicate protein significant expression differencesin the rabbit among groups 167 skin(p < was 0.05); measured (C) the using GNAI2 the proteinWes assay. expression Differential in expression the rabbit of skin GNAI2 was in measured Rex rabbits using with the Wes assay. 168 different coat colors was calculated using the relative expression ratio of GNAI2/GAPDH. BL, 169 black;Differential CH, chinchilla; expression BR, brown; of GNAI2 GR, gray; in Rex GY, rabbits gray-yellow; with different WH, white. coat colors was calculated using the relative expression ratio of GNAI2/GAPDH. BL, black; CH, chinchilla; BR, brown; GR, gray; GY, 170 3.2.gray-yellow; GNAI2 Protein WH, white. Was Mainly Localized in the Cytoplasm of Melanocytes 171 The subcellular localization of the GNAI2 protein was predicted using the protein 172 subcellular3.2. GNAI2 localization Protein Was predic Mainlytion tool Localized (PSORT). in the It was Cytoplasm found that of Melanocytes the GNAI2 protein was 173 mainlyThe expressed subcellular in the localizationcytoplasm of melanocyte of the GNAI2s. To proteinfurther verify was predictedthe expression using of the the protein 174 GNAI2subcellular protein, localization an indirect prediction immunofluoresc tool (PSORT).ence staining It was assay found was thatperformed. the GNAI2 The protein 175 pcDNA3.1 group was used as a negative control and the pcDNA3.1-Myc group was used was mainly expressed in the cytoplasm of melanocytes. To further verify the expression 176 as a positive control. It was found that the GNAI2 protein was localized in the cytoplasm 177 ofof melanocytes the GNAI2 (Figure protein, 2), which an indirect was consistent immunofluorescence with the prediction. staining assay was performed. The pcDNA3.1 group was used as a negative control and the pcDNA3.1-Myc group was

Genes 2021, 12, x FOR PEER REVIEWused as a positive control. It was found that the GNAI26 of protein 11 was localized in the cytoplasm of melanocytes (Figure2), which was consistent with the prediction.

178 179 Figure 2. The localization of GNAI2 protein in melanocytes (400X). The subcellular localization of 180 FigureGNAI2 in melanocytes 2. The localization was determined of using GNAI2 an indirect protein immunofluorescence in melanocytes staining assay. (400X). The The subcellular localization 181 ofpcDNA3.1 GNAI2 group in was melanocytes used as a negative was control determined and the pcDNA3.1-Myc using an group indirect was used immunofluorescence as a staining assay. 182 positive control. The pcDNA3.1 group was used as a negative control and the pcDNA3.1-Myc group was used as a 183 positive3.3. GNAI2 control. Promoted Melanogenesis 184 Our results suggested that GNAI2 is involved in the formation of coat colors in rabbits. 185 To confirm its role in melanogenesis, melanin content was measured when GNAI2 was 186 overexpressed and knocked down in melanocytes. We found that GNAI2 mRNA and 187 protein expression increased when GNAI2 was overexpressed (p < 0.01, Figure 3A,B). 188 Similarly, melanin content also increased significantly (p < 0.01, Figure 3C). GNAI2 189 knockdown was performed using siRNAs. The three different siRNAs used decreased 190 GNAI2 expression significantly (p < 0.01), but siRNA-634 showed the greatest effect 191 (Figure 3D). When GNAI2 was downregulated using siRNA-634, GNAI2 protein 192 expression (Figure 3E) and melanin content significantly decreased (p < 0.01, Figure 3F).

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3.3. GNAI2 Promoted Melanogenesis Our results suggested that GNAI2 is involved in the formation of coat colors in rabbits. To confirm its role in melanogenesis, melanin content was measured when GNAI2 was overexpressed and knocked down in melanocytes. We found that GNAI2 mRNA and pro- tein expression increased when GNAI2 was overexpressed (p < 0.01, Figure3A,B). Similarly, melanin content also increased significantly (p < 0.01, Figure3C). GNAI2 knockdown was performed using siRNAs. The three different siRNAs used decreased GNAI2 expression Genes 2021, 12, x FOR PEER REVIEW significantly (p < 0.01), but siRNA-634 showed the greatest effect (Figure3D). When GNAI27 of 11 was downregulated using siRNA-634, GNAI2 protein expression (Figure3E) and melanin content significantly decreased (p < 0.01, Figure3F).

193 194 FigureFigure 3. 3.GNAI2GNAI2 promoted promoted melanogenesis: melanogenesis: (A) GNAI2 (A) GNAI2 mRNA mRNA expression expression levels in levels GNAI2- in GNAI2- 195 overexpressingoverexpressing melanocytes melanocytes were were detected using a qRT-PCR assay;assay; ( B(B)) the the GNAI2 GNAI2 protein protein expression 196 expressionwas determined was determined using Wes assayusing afterWes GNAI2assay afte wasr GNAI2 overexpressed was overexpressed in melanocytes; in melanocytes; (C) the melanin (C con-) 197 thetent melanin was measured content was in GNAI2-overexpressing measured in GNAI2-over melanocytesexpressing using melanocytes NaOH lysates; using (NaOHD) GNAI2 lysates; mRNA 198 (D) GNAI2 mRNA expression levels were determined in GNAI2-silenced melanocytes using a expression levels were determined in GNAI2-silenced melanocytes using a qRT-PCR; (E) the GNAI2 199 qRT-PCR; (E) the GNAI2 protein levels were determined using Wes assay after GNAI2 was protein levels were determined using Wes assay after GNAI2 was knocked down in melanocytes; 200 knocked down in melanocytes; (F) the melanin content was measured in GNAI2-silenced 201 melanocytes(F) the melanin using content NaOH was lysates. measured **, p in < GNAI2-silenced0.01. melanocytes using NaOH lysates. **, p < 0.01. 3.4. GNAI2 Improved Melanocyte Proliferation and Apoptosis 202 3.4. GNAI2 Improved Melanocyte Proliferation and Apoptosis To further elucidate the regulatory effect of GNAI2 on melanocytes, cell proliferation 203 andTo apoptosis further wereelucidate estimated the regulatory using a CCK-8 effect of assay GNAI2 and on fluorescence-activated melanocytes, cell proliferation cell sorting, 204 andrespectively. apoptosis Wewere found estimated that GNAI2 using promoteda CCK-8 melanocyteassay and fluorescence-activated proliferation and inhibited cell 205 sorting,apoptosis respectively. when GNAI2 We was found overexpressed that GNAI2 (Figure promoted4A,B). Conversely,melanocyte melanocyte proliferation prolifer- and 206 inhibitedation was apoptosis inhibited andwhen cell GNAI2 apoptosis was was overexpressed promoted when (Figure GNAI2 4A,B). was downregulated Conversely, 207 melanocyte(Figure4C,D). proliferation The results was demonstrated inhibited and that cell GNAI2 apoptosis promoted was melanocytepromoted when proliferation GNAI2 208 wasand downregulated decreased apoptosis. (Figure 4C,D). The results demonstrated that GNAI2 promoted 209 melanocyte proliferation and decreased apoptosis.

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210 211Figure 4.FigureGNAI2 4. GNAI2 regulated regulated melanocyte melanocyte proliferation proliferation andand apoptosis: (A ()A melanocyte) melanocyte proliferation proliferation was estimated was estimated using using 212CCK-8 assayCCK-8 at assay 24, 48, at 24, and 48, 72 and h 72 in h GNAI2-overexpressing in GNAI2-overexpressing melanocytes; (B) ( Bmelanocyte) melanocyte apoptosis apoptosis was detected was detected in GNAI2- in GNAI2- 213overexpressing overexpressing melanocytes melanocytes and and cellular cellular apoptosis apoptosis raterate was calculated; calculated; (C) ( Cmelanocyte) melanocyte proliferation proliferation was estimated was estimated in in 214 GNAI2-silenced melanocytes at 24, 48, and 72 h using the CCK-8 assay; (D) cellular apoptosis of GNAI2-silenced 215GNAI2-silenced melanocytes melanocytes was measured at 24, and 48, cellular and 72 apoptosis h using rate the CCK-8was calculated. assay; (SamplesD) cellular were apoptosis in triplicates. of GNAI2-silenced**, p < 0.01. melanocytes was measured and cellular apoptosis rate was calculated. Samples were in triplicates. **, p < 0.01. 216 3.5. GNAI2 Overexpression and Knockdown Regulated the Expression of Melanin‐ 217 3.5.Related GNAI2 Genes Overexpression and Knockdown Regulated the Expression of Melanin-Related Genes 218 To check whether GNAI2 has an important effect on the melanogenesis pathway, the 219 expressionTo check of whether melanin-related GNAI2 key has genes an wa importants measured effect after GNAI2 on the overexpression melanogenesis and pathway, 220 the expressionknockdown in of melanocytes. melanin-related The results key showed genes that was the measured mRNA levels after of the GNAI2 related overexpression genes 221 andTYR, knockdown DCT, GPNMB, in melanocytes. and PMEL The were results significantly showed upregulated that the mRNA following levels GNAI2 of the related 222 genesoverexpression TYR, DCT, ( GPNMB,p < 0.01, Figure and PMEL 5A). Furthermor were significantlye, the knockdown upregulated of GNAI2 following using GNAI2 223 overexpressionsiRNA-634, which (p < 0.01,showed Figure the highest5A). Furthermore, effect, significantly the knockdown decreased the of mRNA GNAI2 levels using of siRNA- 224 634,the which melanin-related showed the genes highest (p < effect,0.01, Figure significantly 5B). The decreasedresults indicated the mRNAthat GNAI2 levels of the 225 Genes 2021, 12, x FOR PEERpromoted REVIEW the expression of melanin-related genes. 9 of 11 melanin-related genes (p < 0.01, Figure5B). The results indicated that GNAI2 promoted the expression of melanin-related genes.

226 227 Figure 5. GNAI2Figure affected 5. GNAI2 the expression affected the of the expression downstream of the melanin-related downstream genes: melanin-related (A) qRT-PCR genes: was performed (A) qRT-PCR to detect was 228 the mRNAperformed expression tolevels detect of themelanin-related mRNA expression genes in levels GNAI2-overexpressing of melanin-related melanocytes; genes in GNAI2-overexpressing (B) the expression of 229 melanin-relatedmelanocytes; genes was (B determined) the expression using ofa melanin-relatedqRT-PCR after GNAI2 genes downregulation was determined in using melanocytes. a qRT-PCR *, p after< 0.05; GNAI2 **, p < 230 0.01. downregulation in melanocytes. *, p < 0.05; **, p < 0.01. 231 4. Discussion 232 In the current study, it was demonstrated that the differential expression of GNAI2 233 was involved in the different coat colors of Rex rabbits. The greatest expression of GNAI2 234 was observed in the black skin, and the least expression was detected in the white skin. In 235 addition, GNAI2 overexpression significantly promoted melanin production. Therefore, 236 the results suggested that GNAI2 plays an important role in the formation of different 237 coat colors in rabbits by regulating melanogenesis. Previous research demonstrated that 238 MITF-M mRNA levels were the lowest when the C57BL/6J black mice overexpressed miR- 239 137, and the overexpression of MITF-M increased the melanin content [25]. Melanin 240 content in the skin of Rex rabbits of different coat colors was distinct, and the highest 241 content was observed in those having black skin and the lowest content in those having 242 white skin [26]. Some studies also found that Slc7a11 mRNA and protein levels were 243 different in the skin of rabbits of different coat colors, and the rabbits that had black skin 244 had significantly higher levels than those that had other coat colors [22]. Similarly, the 245 expression pattern of KIT, a key proto-oncogene, was consistent with the Slc7a11 246 expression [27], and the difference among coat colors of Liaoning Cashmere goats could 247 be explained by mutations in KIT [28]. These findings may provide reasonable evidence 248 to suggest the importance of different genes in regulating the coat color in mammals. 249 Cell proliferation and apoptosis are the basic phenomena that help to maintain the 250 number of cells in the body during the development and regeneration of the organism 251 and/or its tissues. Melanocyte proliferation and apoptosis could be regulated by some key 252 genes of the melanogenesis pathway. Previous studies indicated that Wnt5a acted as a 253 negative regulatory gene and inhibited mouse melanocyte proliferation, and, thereby, 254 melanogenesis [29]. Skin melanocyte proliferation was regulated by Sox10 by activating 255 the DNA replication licensing factor MCM5 [30]. MicroRNA-143-5p regulated alpaca 256 melanocyte migration and proliferation and melanogenesis [31]. In addition, Opsin 3 257 (OPN3), belonging to the G protein-coupled receptor superfamily, played a vital role in 258 cell survival. Upon downregulation, it induced apoptosis of the human epidermal 259 melanocytes through calcium-dependent G protein-coupled signaling and mitochondrial 260 pathways [32]. The results from the current study demonstrated that GNAI2 promoted 261 melanocyte proliferation when it was upregulated in melanocytes. Furthermore, 262 apoptosis in the melanocytes increased when GNAI2 was knocked down. 263 GNAI2 played an important role in melanogenesis. It regulated the expression of 264 melanin-related genes and affected melanin synthesis. TYR and DCT are important 265 regulators of melanogenesis. Our results showed that the expression levels of melanin- 266 related genes TYR, DCT, GPNMB, and PMEL were significantly increased upon GNAI2 267 overexpression. On the contrary, the expression levels of these genes were downregulated

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4. Discussion In the current study, it was demonstrated that the differential expression of GNAI2 was involved in the different coat colors of Rex rabbits. The greatest expression of GNAI2 was observed in the black skin, and the least expression was detected in the white skin. In addition, GNAI2 overexpression significantly promoted melanin production. Therefore, the results suggested that GNAI2 plays an important role in the formation of different coat colors in rabbits by regulating melanogenesis. Previous research demonstrated that MITF- M mRNA levels were the lowest when the C57BL/6J black mice overexpressed miR-137, and the overexpression of MITF-M increased the melanin content [25]. Melanin content in the skin of Rex rabbits of different coat colors was distinct, and the highest content was observed in those having black skin and the lowest content in those having white skin [26]. Some studies also found that Slc7a11 mRNA and protein levels were different in the skin of rabbits of different coat colors, and the rabbits that had black skin had significantly higher levels than those that had other coat colors [22]. Similarly, the expression pattern of KIT, a key proto-oncogene, was consistent with the Slc7a11 expression [27], and the difference among coat colors of Liaoning Cashmere goats could be explained by mutations in KIT [28]. These findings may provide reasonable evidence to suggest the importance of different genes in regulating the coat color in mammals. Cell proliferation and apoptosis are the basic phenomena that help to maintain the number of cells in the body during the development and regeneration of the organism and/or its tissues. Melanocyte proliferation and apoptosis could be regulated by some key genes of the melanogenesis pathway. Previous studies indicated that Wnt5a acted as a negative regulatory gene and inhibited mouse melanocyte proliferation, and, thereby, melanogenesis [29]. Skin melanocyte proliferation was regulated by Sox10 by activating the DNA replication licensing factor MCM5 [30]. MicroRNA-143-5p regulated alpaca melanocyte migration and proliferation and melanogenesis [31]. In addition, Opsin 3 (OPN3), belonging to the G protein-coupled receptor superfamily, played a vital role in cell survival. Upon downregulation, it induced apoptosis of the human epidermal melanocytes through calcium-dependent G protein-coupled signaling and mitochondrial pathways [32]. The results from the current study demonstrated that GNAI2 promoted melanocyte proliferation when it was upregulated in melanocytes. Furthermore, apoptosis in the melanocytes increased when GNAI2 was knocked down. GNAI2 played an important role in melanogenesis. It regulated the expression of melanin-related genes and affected melanin synthesis. TYR and DCT are important regula- tors of melanogenesis. Our results showed that the expression levels of melanin-related genes TYR, DCT, GPNMB, and PMEL were significantly increased upon GNAI2 over- expression. On the contrary, the expression levels of these genes were downregulated after GNAI2 was knocked down. These results suggested that GNAI2 promoted melanin synthesis by maintaining the expression of melanin-related genes.

5. Conclusions Our study suggested that GNAI2 was involved in the formation of the coat colors in Rex rabbits, depending upon its expression levels. GNAI2 overexpression promoted melanocyte proliferation and inhibited cell apoptosis. In all, GNAI2 played a positive role in melanogenesis.

Author Contributions: S.H. performed experiments, analyzed results, and wrote the manuscript. Y.D. analyzed the data. S.B. prepared figures and/or tables. B.Z. contributed new reagents or analytic tools. X.W. and Y.C. designed the study. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Natural Science Foundation of China, grant number 32072724, China Agriculture Research System of MOF and MARA, grant number CARS-43-A-1, and the Postgraduate Research & Practice Innovation Program of Jiangsu Province, grant number KYCX20_2988. Genes 2021, 12, 1130 9 of 10

Institutional Review Board Statement: The experimental procedures in this study was approved by the Animal Care and Use Committee at Yangzhou University (Yangzhou, China, 8 May 2021, No. 202102006). Informed Consent Statement: Not applicable. Data Availability Statement: All data generated or analyzed during this study are available from the corresponding author on reasonable request. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Videira, I.F.D.S.; Moura, D.F.L.; Magina, S. Mechanisms regulating melanogenesis. An. Bras. Dermatol. 2013, 88, 76–83. [CrossRef] 2. Naysmith, L.; Waterston, K.; Ha, T.; Flanagan, N.; Bisset, Y.; Ray, A.; Wakamatsu, K.; Ito, S.; Rees, J.L. Quantitative measures of the effect of the melanocortin 1 receptor on human pigmentary status. J. Investig. Dermatol. 2004, 122, 423–428. [CrossRef] 3. Slominski, A.; Tobin, D.J.; Shibahara, S.; Wortsman, J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol. Rev. 2004, 84, 1155–1228. [CrossRef][PubMed] 4. Ito, S.; Wakamatsu, K.; Ozeki, H. Chemical analysis of melanins and its application to the study of the regulation of melanogenesis. Pigment Cell Res. 2000, 13 (Suppl. S8), 103–109. [CrossRef] 5. Ito, S.; Wakamatsu, K. Quantitative Analysis of Eumelanin and Pheomelanin in Humans, Mice, and Other Animals: A Compara- tive Review. Pigment. Cell Res. 2003, 16, 523–531. [CrossRef] 6. Del Bino, S.; Ito, S.; Sok, J.; Nakanishi, Y.; Bastien, P.; Wakamatsu, K.; Bernerd, F. Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio. Pigment. Cell Melanoma Res. 2015, 28, 707–717. [CrossRef] [PubMed] 7. Hou, L.; Panthier, J.J.; Arnheiter, H. Signaling and transcriptional regulation in the neural crest-derived melanocyte lineage: Interactions between KIT and MITF. Development 2000, 127, 5379–5389. [CrossRef] 8. D’Mello, S.A.; Finlay, G.J.; Baguley, B.C.; Askarian-Amiri, M.E. Signaling pathways in melanogenesis. Int. J. Mol. Sci. 2016, 17, 1144. [CrossRef][PubMed] 9. Liu, Z.; Wen, H.; Hailer, F.; Dong, F.; Yang, Z.; Liu, T.; Han, L.; Shi, F.; Hu, Y.; Zhou, J. Pseudogenization of Mc1r gene associated with transcriptional changes related to melanogenesis explains leucistic phenotypes in Oreonectes cavefish (Cypriniformes, Nemacheilidae). J. Zool. Syst. Evol. Res. 2019, 57, 900–909. [CrossRef] 10. Valverde, P.; Healy, E.; Jackson, I.; Rees, J.L.; Thody, A.J. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nat. Genet. 1995, 11, 328–330. [CrossRef] 11. Yu, S.; Wang, G.; Liao, J.; Tang, M. Five alternative splicing variants of the TYR gene and their different roles in melanogenesis in the Muchuan black-boned chicken. Br. Poult. Sci. 2019, 60, 8–14. [CrossRef] 12. Guibert, S.; Girardot, M.; Leveziel, H.; Julien, R.; Oulmouden, A. Pheomelanin coat colour dilution in French cattle breeds is not correlated with the TYR, TYRP1 and DCT transcription levels. Pigment Cell Res. 2004, 17, 337–345. [CrossRef] 13. Yang, S.-L.; Mao, H.-M.; Shu, W.; Deng, W.-D. Melanin traits of Yunnan black bone sheep and TYR gene polymorphism. Hereditas 2006, 28, 291–298. 14. Ross, E.M.; Gilman, A.G. Biochemical properties of hormone-sensitive adenylate cyclase. Annu. Rev. Biochem. 1980, 49, 533–564. [CrossRef] 15. Wootten, D.; Christopoulos, A.; Marti-Solano, M.; Babu, M.M.; Sexton, P.M. Mechanisms of signalling and biased agonism in G protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 2018, 19, 638–653. [CrossRef][PubMed] 16. Simon, M.I.; Strathmann, M.P.; Gautam, N.J.S. Diversity of G proteins in . Science 1991, 252, 802–808. [CrossRef] [PubMed] 17. Lu, Y.; Xi, J.; Zhang, Y.; Chen, W.; Zhang, F.; Li, C.; Wang, Z. SNHG1 Inhibits ox-LDL-Induced Inflammatory Response and Apoptosis of HUVECs via Up-Regulating GNAI2 and PCBP1. Front. Pharmacol. 2020, 11, 703. [CrossRef][PubMed] 18. Li, Z.-W.; Sun, B.; Gong, T.; Guo, S.; Zhang, J.; Wang, J.; Sugawara, A.; Jiang, M.; Yan, J.; Gurary, A.J.G. GNAI1 and GNAI3 reduce colitis-associated tumorigenesis in mice by blocking IL6 signaling and down-regulating expression of GNAI2. Gastroenterology 2019, 156, 2297–2312. [CrossRef][PubMed] 19. Sun, Q.; He, Q.; Xu, J.; Liu, Q.; Lu, Y.; Zhang, Z.; Xu, X.; Sun, B.J.T.F.J. Guanine nucleotide–binding protein G (i) α2 aggravates hepatic ischemia-reperfusion injury in mice by regulating MLK3 signaling. FASEB J. 2019, 33, 7049–7060. [CrossRef] 20. Wang, Z.; Dela Cruz, R.; Ji, F.; Guo, S.; Zhang, J.; Wang, Y.; Feng, G.S.; Birnbaumer, L.; Jiang, M.; Chu, W.M. G(i)α proteins exhibit functional differences in the activation of ERK1/2, Akt and mTORC1 by growth factors in normal and breast cancer cells. Cell Commun. Signal. CCS 2014, 12, 10. [CrossRef] 21. Jung, K.S.; Kim, S.H.; Yoon, J.T. Production, Differential Methylation of Melanin-related Epigenetic Genes during Brindle Cattle Growth. J. Anim. Health Prod. 2020, 8, 80–88. [CrossRef] 22. Chen, Y.; Hu, S.; Mu, L.; Zhao, B.; Wang, M.; Yang, N.; Bao, G.; Zhu, C.; Wu, X. Slc7a11 Modulated by POU2F1 is Involved in Pigmentation in Rabbit. Int. J. Mol. Sci. 2019, 20, 2493. [CrossRef] Genes 2021, 12, 1130 10 of 10

23. Schmittgen, T.D.; Livak, K.J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 2008, 3, 1101–1108. [CrossRef] 24. Harris, V.M. Protein Detection by Simple Western™ Analysis. In Western Blotting; Humana Press: New York, NY, USA, 2015; Volume 1312, pp. 465–468. 25. Chen, T.; Zhao, B.; Liu, Y.; Wang, R.; Yang, Y.; Yang, L.; Dong, C. MITF-M regulates melanogenesis in mouse melanocytes. J. Dermatol. Sci. 2018, 90, 253–262. [CrossRef][PubMed] 26. Hu, S.; Zhai, P.; Chen, Y.; Zhao, B.; Yang, N.; Wang, M.; Xiao, Y.; Bao, G.; Wu, X. Morphological Characterization and Patterns for Melanin Pigmentation in Rex Rabbit. Biochem. Genet. 2019, 57, 734–744. [CrossRef] 27. Hu, S.; Chen, Y.; Zhao, B.; Yang, N.; Chen, S.; Shen, J.; Bao, G.; Wu, X. KIT is involved in melanocyte proliferation, apoptosis and melanogenesis in the Rex Rabbit. PeerJ 2020, 8, e9402. [CrossRef][PubMed] 28. Li, J.; Chen, W.; Wu, S.; Ma, T.; Jiang, H.; Zhang, Q. Differential expression of MC1R gene in Liaoning Cashmere goats with different coat colors. Anim. Biotechnol. 2019, 30, 273–278. [CrossRef][PubMed] 29. Zhang, J.; Li, Y.; Wu, Y.; Yang, T.; Yang, K.; Wang, R.; Yang, J.; Guo, H. Wnt5a inhibits the proliferation and melanogenesis of melanocytes. Int. J. Med Sci. 2013, 10, 699–706. [CrossRef] 30. Su, Z.; Zheng, X.; Zhang, X.; Wang, Y.; Zhu, S.; Lu, F.; Qu, J.; Hou, L.J.J. Sox10 regulates skin melanocyte proliferation by activating the DNA replication licensing factor MCM5. J. Dermatol. Sci. 2017, 85, 216–225. [CrossRef] 31. Ji, K.; Zhang, P.; Zhang, J.; Fan, R.; Liu, Y.; Yang, S.; Hu, S.; Liu, X.; Dong, C. MicroRNA 143-5p regulates alpaca melanocyte migration, proliferation and melanogenesis. Exp. Dermatol. 2018, 27, 166–171. [CrossRef] 32. Wang, Y.; Lan, Y.; Lu, H. Opsin3 Downregulation Induces Apoptosis of Human Epidermal Melanocytes via Mitochondrial Pathway. Photochem. Photobiol. 2020, 96, 83–93. [CrossRef][PubMed]