Junzhen Zhang

Identification of a novel microRNA important for melanogenesis in alpaca (Vicugna pacos)

The molecular mechanisms underlying the formation of coat colors in animals are poorly understood. Recent studies have demonstrated that microRNA play important roles in the control of melanogenesis and coat color in mammals. In a previous study, we characterized the miRNA expression profiles in alpaca skin with brown and white coat color and identified a novel miRNA (named lpa-miR-nov-66) that is expressed significantly higher in white skin compared to brown skin. The present study was conducted to determine the functional roles of this novel miRNA in the regulation of melanogenesis in alpaca melanocytes. lpa-miR-nov-66 is predicted to target the soluble guanylate cyclase (sGC) gene based on presence of a binding site in the sGC coding sequence (CDS). Overexpression of lpa-miR-nov-66 in alpaca melanocyes upregulated the expression of sGC both at the mRNA and protein level. Overexpression of lpa-miR-nov-66 in melanocyes also resulted in decreased expression of key melanogenic genes including tyrosinase (TYR), tyrosinase related protein 1 (TYRP1), and microphthalmia transcription factor (MITF). Our ELISA assays showed increased cyclic guanosine monophosphate (cGMP) but decreased cyclic adenosine monophosphate (cAMP) production in melanocytes overexpressing lpa-miR-nov-66. In addition, overexpression of lpa-miR-nov-66 also reduced melanin production in cultured melanocytes. Results support a role of lpa-miR-nov-66 in melanocytes by directly or indirectly targeting , which regulates melanogenesis via the cAMP pathway.

Role of microRNA508-3p in melanogenesis by targeting microphthalmia transcription factor in melanocytes of alpaca.

It has been demonstrated that microRNAs (miRNAs) play important roles in the control of melanogenesis and hair color in mammals. By comparing miRNA expression profiles between brown and white alpaca skin, we previously identified miR508-3p as a differentially expressed miRNA suggesting its potential role in melanogenesis and hair color formation. The present study was conducted to determine the role of miR508-3p in melanogenesis in alpaca melanocytes. In situ hybridization showed that miR508-3p is abundantly present in the cytoplasma of alpaca melanocytes. miR508-3p was predicted to target the gene encoding microphthalmia transcription factor (MITF) and a luciferase reporter assay indicated that miR508-3p regulates MITF expression by directly targeting its 3′UTR. Overexpression of miR508-3p in alpaca melanocytes down-regulated MITF expression both at the messenger RNA and protein level and resulted in decreased expression of key melanogenic genes including tyrosinase and tyrosinase-related protein 2. Overexpression of miR508-3p in melanocytes also resulted in decreased melanin production including total alkali-soluble melanogenesis, eumelanogenesis and pheomelanogenesis. Results support a functional role of miR508-3p in regulating melanogenesis in alpaca melanocytes by directly targeting MITF.

Biological characteristics of mouse skin melanocytes.

The objective of this research was to evaluate the optimal passage number according to the biological characteristics of mouse skin melanocytes from different passages. Skin punch biopsies harvested from the dorsal region of 2-day old mice were used to establish melanocyte cultures. The cells from passage 4, 7, 10 and 13 were collected and evaluated for their melanogenic activity. Histochemical staining for tyrosinase (TYR) activity and immunostaining for the melanocyte specific markers including S-100 antigen, TYR, tyrosinase related protein 1 (TYRP1), tyrosinase related protein 2 (TYRP2) and micropthalmia associated transcription factor (MITF) confirmed purity and melanogenic capacity of melanocytes from different passages, with better melanogenic activity of passage 10 and 13 cells being observed. Treatment of passage 13 melanocytes with α-melanocyte stimulating hormone (α-MSH) showed increased expression of MITF, TYR and TYRP2 mRNA. However, considering the TYR mRNA dramatically high expression which is the characteristics of melanoma cells, melanocytes from passage 10 was the optimal passage number for the further research. Our results demonstrate culture of pure populations of mouse melanocytes to at least 10 passages and illustrate the potential utility of passage 10 cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in mouse.

Functional Role of Cyclin-Dependent Kinase 5 in the Regulation of Melanogenesis and Epidermal Structure

The mammalian integumentary system plays important roles in body homeostasis, and dysfunction of melanogenesis or epidermal development may lead to a variety of skin diseases, including melanoma. Skin pigmentation in humans and coat color in fleece-producing animals are regulated by many genes. Among them, microphthalmia-associated transcription factor (MITF) and paired-box 3 (PAX3) are at the top of the cascade and regulate activities of many important melanogenic enzymes. Here, we report for the first time that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of MITF and PAX3. Cdk5 knockdown in mice causes a lightened coat color, a polarized distribution of melanin and hyperproliferation of basal keratinocytes. Reduced expression of Keratin 10 (K10) resulting from Cdk5 knockdown may be responsible for an abnormal epidermal structure. In contrast, overexpression of Cdk5 in sheep (Ovis aries) only produces brown patches on a white background, with no other observable abnormalities. Collectively, our findings show that Cdk5 has an important functional role in the regulation of melanin production and transportation and in normal development of the integumentary system.

Effect of different dietary energy on collagen accumulation in skeletal muscle of ram lambs.

Tenderness is one of the most appreciated characteristics of meat quality. The objective of this trial was to investigate the effect of different energy diets on collagen deposition and meat tenderness. Twelve one-half Dorper × one-half small thin-tailed sheep crossed ram lambs (20 ± 0.5 kg of BW) were randomly selected and divided into 2 groups in a completely randomized design. Animals were offered identical diets at 100 or 65% of ad libitum intake. Lambs were euthanized when BW in the ad libitum group reached 35 kg, and the semitendinosus (ST) muscle were sampled. The results showed that Warner-Bratzler shear force (WBSF) was significantly increased when lambs were fed an energy-restricted diet ( < 0.05). Masson trichrome stain and hydroxyproline assay demonstrated increased collagen content in ST muscle of feed restriction lambs. Both and mRNA contents were significantly increased when lambs were fed an energy-restricted diet ( < 0.05), whereas no difference for mRNA expression was observed ( > 0.05). Expression of α () was greater in the feed restriction group ( < 0.01), and no differences were observed for both () and () mRNA contents ( > 0.05). In addition, 1, 2, 9, and 13 (, , , and ) did not change with the feed restriction, whereas both 1 and 2 ( and ) were increased. Feed restriction did not alter TGF-β and SMAD protein contents, but phosphor-p38 protein content was elevated. In summary, feed restriction enhanced collagen accumulation in ST muscle, which may negatively affect the lamb tenderness, and was associated with the upregulated p38 signaling pathway.

The role of sex-determining region Y-box 6 in melanogenesis in alpaca melanocytes

BACKGROUND Sex-determining region Y-box (SOX) proteins function as transcriptional regulators. The derivation of melanocytes from nerve crest cells has been reported to depend on SOX proteins, including SOX10 and SOX5. Whether SOX6 is expressed and has a functional role in melanocytes is unknown. OBJECTIVE We aimed to study the effect of transcription factor SOX6 on melanogenesis in alpaca melanocytes. METHODS We verified the role of SOX6 in melanogenesis by overexpressing and inhibiting SOX6 in melanocytes. Co-immunoprecipitation (co-IP) experiments were performed to further explore the function of SOX6 in melanogenesis and its mechanism of melanin production. We found that SOX6 interacted with cyclin-dependent kinase （CDK5）, β-catenin, and Cyclin D1. RESULTS Bioinformatics analysis suggested that SOX6 has a phosphorylation site for CDK5, which regulates melanogenesis, suggesting that SOX6 might play a role in melanogenesis. Co-IP experiments indicated that SOX6 interacted with CDK5, β-catenin, and Cyclin D1. Quantitative real-time polymerase chain reaction and western blot analyses of SOX6-overexpressing melanocytes revealed increased mRNA and protein expression of Cyclin D1, CDK5, microphthalmia transcription factor (MITF), tyrosinase (TYR), tyrosine related protein-1 (TYRP1), and dopachrome-tautomerase (DCT), whereas β-catenin levels decreased in SOX6-overexpressing melanocytes. The opposite results were observed upon SOX6 knockdown. The melanin content was significantly increased or decreased, respectively, by SOX6 overexpression or knockdown. CONCLUSION Our results suggest that SOX6 might enhance melanogenesis by binding with β-catenin to increase Cyclin D1 and MITF expression.

MicroRNA 143-5p regulates alpaca melanocyte migration, proliferation and melanogenesis

microRNAs (miRNAs) have been shown to be closely involved in the control of melanogenesis and hair colour in mammals. Previous data also indicate that miR‐143 regulates cell growth in melanoma. Here, we aimed to investigate the role of miR‐143‐5p in alpaca melanocytes. We found that miR‐143‐5p was highly expressed in the cytoplasm of alpaca melanocytes as demonstrated by an in situ hybridization assay. Prediction analysis revealed that miR‐143‐5p could regulate TGF‐β‐activated kinase 1 (TAK1) expression, which we confirmed by luciferase reporter assay, indicating that miR‐143‐5p controls TAK1 expression by directly targeting its 3′ untranslated region (UTR). miR‐143‐5p overexpression decreased TAK1 expression, which led to increased melanocyte migration and proliferation, and downregulation of microphthalmia‐associated transcription factor (MITF), which regulates melanin production. These results support a functional role for miR‐143‐5p in regulating alpaca melanocyte migration, proliferation and melanogenesis through direct targeting of TAK1.

Cyclin-dependent kinase 5 regulates MAPK/ERK signaling in the skin of mice

Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase that has been shown to play important roles in many tissues except the nervous system. We previously reported that CDK5 showed differential expression in the transcriptome profiles of the skin of alpacas with different hair colors. To understand the functional role of CDK5 in hair color determination, we constructed CDK5-knockdown mice and identified the effect on the mitogen-activated protein kinase (MAPK) pathway in the mouse skin. Quantitative real-time polymerase chain reaction, co-immunoprecipitation, and western blotting were performed to analyze the effects of CDK5-knockdown on the MAPK pathway in mice. The results showed that MAP3K6 was inhibited by phosphorylated CDK5 through its activator CDK7. The decrease in MAP3K6 levels caused down-regulation of MEK1 and ERK expression, leading to the up-regulation of miR-143-3p, which targets MAP3K6 via Dicer. Taken together, our findings indicate that CDK5 functions in regulating the MAPK pathway. Given that MAP3K6 was inhibited in two directions, this mechanism can provide insight into the contributions of the MAPK/ERK pathway to the inhibition of melanin production.

Long non-coding RNA expression profile in Cdk5-knockdown mouse skin

To elucidate the Cdk5 regulatory molecular mechanism in skin, we generated Cdk5-knockdown mice and subjected their skins to lncRNA sequencing. The results showed that there were 4533 novel lncRNAs from 142 lncRNA families. In total, 693 lncRNAs were significantly differentially expressed. Alignment analysis of the lncRNAs in miRBase identified 45 pre-mRNAs. By KEGG PATHWAY Database analysis, we found that lncRNAs (lnc-NONMMUT064276.2, lnc-NONMMUT075728.1, and lnc-NONMMUT039653.2) may regulate pigmentation by regulating target genes. To reveal potential antisense lncRNA-mRNA interactions, we searched all lncRNA-mRNA duplexes using RNAplex, and found 97 lncRNAs interacted with mRNAs. The luciferase assay confirmed that TCONS_00049140 binded to Krt80 by the co-transfection of pVAX1-TCONS_00049140 and pGL0-Krt80 expression plasmids in 293T cell, based on the bioinformatics analysis. Overexpression of TCONS_00049140 in mouse melanocytes down-regulated Krt80 and resulted in the phenotype of increased cell proliferation and increased melanin production. The results suggested that TCONS_00049140 contributed to skin thickening through Krt80. Our findings provide a direction for research of the molecular mechanism of Cdk5 function.

Differential expression of lncRNAs and predicted target genes in normal mouse melanocytes and B16 cells

Melanoma is a highly invasive and metastatic malignant skin tumor with poor prognosis. Although several widely studied pure melanoma cell lines are available, the precise mechanism underlying transformation of melanocyte to melanoma remains unclear. Long non‐coding RNAs (lncRNAs) represent a vast category of non‐coding RNA molecules, and increasing evidence suggests that lncRNAs are crucial for various biological processes, including those in the skin. Herein, lncRNA sequencing was performed on an Illumina HiSeq platform to identify lncRNAs expressed differently in murine B16 melanoma cells compared to normal mouse melanocytes. Using four computational approaches, 2319 lncRNAs were expressed in both normal melanocytes and B16 cells, with 373 being differentially expressed at a significant level. Of these, 136 lncRNAs were upregulated and 237 were downregulated. KEGG analyses revealed that 467 genes were target genes in the Wnt signalling pathway, TGF‐beta signalling pathway, MAPK signalling pathway, NF‐kappa B signalling pathway, melanoma and several other cancer‐related regulatory pathways. From among the differentially expressed lncRNAs, lnc‐13317.1 was found to play a role in the cell cycle in melanoma by targeting BRCA1. Thus, lnc‐13317.1 might have therapeutic potential in melanoma treatment. The lncRNA profile described here highlights the importance of elucidating the exact function of these lncRNAs in the transformation of melanoma. Lnc‐13317.1 might have therapeutic potential in melanoma treatment by targeting BRCA1.