LncRNA-177922靶向MAPK15调节B16-F10黑色素瘤细胞黑色素生成、增殖、迁移和自噬

黑色素瘤是一种预后较差的侵袭性癌症。了解黑色素瘤的分子机制和诊断标志物对黑色素瘤的防治极为重要。LncRNAs在肿瘤的发生发展中发挥重要作用。与正常黑色素细胞相比,LncRNA-177922在B16-F10黑色素瘤中高表达。丝裂源活化蛋白激酶15 (mitogen-activated protein kinase 15, MAPK15) 的缺失影响肿瘤的发生和进展。本研究中,LncRNA-177922在B16-F10细胞中过表达,结果显示,黑色素生成、增殖、迁移相关基因的mRNA和蛋白质水平显著上调(P< 0. 05),自噬相关基因的mRNA水平和蛋白质丰度下调(P< 0. 05),PI3K/AKT/mTOR通路被激活。同时,进一步验证了细胞迁移、增殖和自噬的表型。结果提示,LncRNA-177922靶向MAPK15通过交叉点细胞外调节蛋白激酶 (extracellular regulated protein kinases, ERK)参与调控黑色素生成、增殖、迁移、自噬等B16-F10细胞的生物学特性,可能是一个新的治疗靶点和诊断标志物。     

小鼠正常黑素细胞与黑色素瘤细胞（B16）mRNA表达差异分析

黑色素瘤是一种高侵袭性的恶性皮肤肿瘤,转移率高、预后差。研究黑素瘤细胞生物学特性对黑素瘤的治疗和控制具有重要的意义。本研究以C57BL/6J小鼠的正常黑色素细胞及B16黑色素瘤细胞为研究对象,采用二代测序技术分析两种细胞间的转录组表达差异,筛选差异基因,为后续黑色素瘤的形成机制研究提供理论依据。采用差异倍数及错误率分析测序数据,鉴定出1 436个新的mRNA和4 086个差异表达的已知mRNA。GO数据库和KEGG数据库分析显示,差异表达的mRNAs参与了149个调控途径, 主要集中在疾病调控、细胞周期调节和环境信息调控方面。qRT-PCR及Western印迹检测发现,调节细胞增殖、迁移的Pdgf-B、Integrin-β1和Integrin-β5以及调节黑色素颗粒增加的Mitf、Tyr、Tyrp1和Tyrp2在B16细胞中的表达量显著高于在正常黑色素细胞中的表达。本研究获得的差异基因为后续黑色素瘤的研究提供了新的候选基因。     

小鼠正常黑色素细胞与黑色素瘤细胞(B16)mRNA表达差异分析

黑色素瘤是一种高侵袭性的恶性皮肤肿瘤,转移率高、预后差。研究黑素瘤细胞生物学特性对黑素瘤的治疗和控制具有重要的意义。本研究以C57BL/6J小鼠的正常黑色素细胞及B16黑色素瘤细胞为研究对象,采用二代测序技术分析两种细胞间的转录组表达差异,筛选差异基因,为后续黑色素瘤的形成机制研究提供理论依据。采用差异倍数及错误率分析测序数据,鉴定出1 436个新的mRNA和4 086个差异表达的已知mRNA。GO数据库和KEGG数据库分析显示,差异表达的mRNAs参与了149个调控途径,主要集中在疾病调控、细胞周期调节和环境信息调控方面。qRT-PCR及Western印迹检测发现,调节细胞增殖、迁移的Pdgf-B、Integrinβ1和Integrinβ5以及调节黑色素颗粒增加的Mitf、Tyr、Tyrp1和Tyrp2在B16细胞中的表达量显著高于在正常黑色素细胞中的表达。本研究获得的差异基因为后续黑色素瘤的研究提供了新的候选基因。     

体色差异豹纹鳃棘鲈的色素及酶含量分析

鱼类体色发育在生物进化、生理生态等方面具有重要的意义,豹纹鳃棘鲈是一种体色变异丰富的珊瑚礁鱼类,在不同环境中呈现出显著差异的体色。本文选取了豹纹鳃棘鲈体色差异个体,并对不同部位的皮肤颜色、色素分布和相关色素酶含量进行了检测和分析。结果显示,鱼体表皮分布着大量黑色素细胞和红色素细胞。黑色个体的黑色素细胞密度较大、黑色素含量较高,黑色素酶含量较低;而红色个体的红色素细胞密度更大、胡萝卜素与类胡萝卜素含量更高,黑色素酶的含量也更高。实验结果表明,豹纹鳃棘鲈的体色差异与黑色素颗粒的聚集、分散程度以及黑色素代谢酶的含量相关,体色鲜艳程度与红色素细胞数量以及胡萝卜素和类胡萝卜素含量相关。本文解释了红黑豹纹鳃棘鲈体色在色素和酶含量方面的差异,为进一步研究其体色变异机制提供了理论依据。     

冬虫夏草提取物的美白作用

以冬虫夏草提取物(Chinese cordyceps extract)为研究对象,通过蘑菇酪氨酸酶活性抑制试验和小鼠皮肤黑色素瘤细胞(B16-F10)黑素合成抑制试验考察冬虫夏草提取物的美白活性。结果显示冬虫夏草提取物(质量浓度40～200 mg/m L)呈剂量依赖性抑制蘑菇酪氨酸酶的活性,且在安全剂量(质量浓度0.1～0.5 mg/m L)下显著抑制小鼠皮肤黑色素瘤细胞(B16-F10)内的黑色素合成(P0.05)。说明冬虫夏草提取物能通过抑制酪氨酸酶活性有效阻滞黑色素的合成,从而实现美白作用。     

OCA2基因的研究进展

OCA2基因为哺乳动物重要的色素基因,参与黑色素合成反应,同时也是人眼睛颜色的主要调控基因。其编码一个110 kD大小的跨膜蛋白,该蛋白质定位于黑素体膜。研究表明, OCA2主要通过影响黑素体成熟、调节黑素体pH值以及参与黑色素合成途径第一步这3个方面影响黑色素合成。相邻基因HERC2的rs12913832增强子位点通过影响OCA2表达水平,实现对人眼睛颜色的调控。同时, OCA2基因是Ⅱ型眼皮肤白化病(oculocutaneousalbinism typeⅡ, OCA2)的致病基因,与普拉德-威利综合征(Prader-Willi syndrome, PWS)、天使综合征(Angelman syndrome, AS)和黑色素瘤之间也存在间接关联,这些患者通常表现出皮肤或头发低色素沉着。本文从分子生物学、细胞生物学和遗传学的角度对OCA2基因进行概述,以期为进一步探究OCA2的分子机制以及相关疾病的诊疗提供思路和见解。     

慢性应激通过减少毛囊黑色素细胞和酪氨酸酶活性导致雌性C57小鼠毛发颜色变浅

越来越多的证据表明压力可能会导致头发颜色发生变化,但其潜在机制尚不完全清楚。本研究采用雌性C57BL/6小鼠脚底电刺激结合束缚来建立慢性应激小鼠模型,并用比色法检测小鼠皮肤和B16F10黑色素瘤细胞中黑色素含量和酪氨酸酶活性;通过酶联免疫吸附实验(ELISA)测定小鼠皮肤中肿瘤坏死因子α(tumor necrosis factorα, TNF-α)、白细胞介素1β(interleukin-1β, IL-1β)和白细胞介素6 (interleukin-6, IL-6)含量;通过免疫荧光染色评估小鼠皮肤中核因子κB (nuclear factorκB, NFκB)/p65亚基的含量。结果显示:C57BL/6小鼠在慢性应激下由于皮肤中的毛囊黑色素细胞和酪氨酸酶活性降低,其毛皮颜色从暗色变为棕色。同时,慢性应激小鼠皮肤炎症反应增加,表现为皮肤中NFκB活性和TNF-α表达增加。在体外,TNF-α以剂量依赖性方式降低B16F10黑色素瘤细胞中黑色素生成和酪氨酸酶活性。以上结果表明,慢性应激通过降低雌性C57BL/6小鼠的毛囊黑色素细胞和酪氨酸酶活性来诱导皮毛颜色改变,而TNF-α可能在应激诱导的毛色改变中起重要作用。     

小鼠皮肤黑色素瘤细胞在三种不同基质上的趋电性比较

微小直流电场具有指导细胞进行定向迁移的作用。各种细胞外基质的物理、化学性质会影响细胞的迁移。该研究以小鼠皮肤黑色素瘤细胞（B16-F10）为模型,比较微直流电场（250mV／mm）指导下细胞在平滑基底与两种不同市售基质Matrigel及FNC上的趋电性。结果显示,黑色素瘤细胞在三种基底上均有明显的向电场阴极迁移的趋电运动,但在不同基质上细胞趋电的方向性无显著差异,但细胞迁移速度及在细胞沿电场进行定向迁移的持续性有显著差异。     

慢性应激通过减少毛囊黑色素细胞和酪氨酸酶活性导致雌性C57小鼠毛发颜色变浅（英文）

越来越多的证据表明压力可能会导致头发颜色发生变化,但其潜在机制尚不完全清楚。本研究采用雌性C57BL/6小鼠脚底电刺激结合束缚来建立慢性应激小鼠模型,并用比色法检测小鼠皮肤和B16F10黑色素瘤细胞中黑色素含量和酪氨酸酶活性;通过酶联免疫吸附实验(ELISA)测定小鼠皮肤中肿瘤坏死因子α(tumor necrosis factorα, TNF-α)、白细胞介素1β(interleukin-1β, IL-1β)和白细胞介素6 (interleukin-6, IL-6)含量;通过免疫荧光染色评估小鼠皮肤中核因子κB (nuclear factorκB, NFκB)/p65亚基的含量。结果显示:C57BL/6小鼠在慢性应激下由于皮肤中的毛囊黑色素细胞和酪氨酸酶活性降低,其毛皮颜色从暗色变为棕色。同时,慢性应激小鼠皮肤炎症反应增加,表现为皮肤中NFκB活性和TNF-α表达增加。在体外,TNF-α以剂量依赖性方式降低B16F10黑色素瘤细胞中黑色素生成和酪氨酸酶活性。以上结果表明,慢性应激通过降低雌性C57BL/6小鼠的毛囊黑色素细胞和酪氨酸酶活性来诱导皮毛颜色改变,而TNF-α可能在应激诱导的毛色改变中起重要作用。     

Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte-macrophage colony-stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte-derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor-mediated signaling pathways.     

A melanocyte–melanoma precursor niche in sweat glands of volar skin

Determination of the niche for early‐stage cancer remains a challenging issue. Melanoma is an aggressive cancer of the melanocyte lineage. Early melanoma cells are often found in the epidermis around sweat ducts of human volar skin, and the skin pigmentation pattern is an early diagnostic sign of acral melanoma. However, the niche for melanoma precursors has not been determined yet. Here, we report that the secretory portion (SP) of eccrine sweat glands provide an anatomical niche for melanocyte–melanoma precursor cells. Using lineage‐tagged H2B‐GFP reporter mice, we found that melanoblasts that colonize sweat glands during development are maintained in an immature, slow‐cycling state but renew themselves in response to genomic stress and provide their differentiating progeny to the epidermis. FISH analysis of human acral melanoma expanding in the epidermis revealed that unpigmented melanoblasts with significant cyclin D1 gene amplification reside deep in the SP of particular sweat gland(s). These findings indicate that sweat glands maintain melanocyte–melanoma precursors in an immature state in the niche and explain the preferential distribution of early melanoma cells around sweat glands in human volar skin.     

Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha‐melanocyte‐stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte‐macrophage colony‐stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte‐derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor‐mediated signaling pathways.     

How are proliferation and differentiation of melanocytes regulated?

Coat colors are determined by melanin (eumelanin and pheomelanin). Melanin is synthesized in melanocytes and accumulates in special organelles, melanosomes, which upon maturation are transferred to keratinocytes. Melanocytes differentiate from undifferentiated precursors, called melanoblasts, which are derived from neural crest cells. Melanoblast/melanocyte proliferation and differentiation are regulated by the tissue environment, especially by keratinocytes, which synthesize endothelins, steel factor, hepatocyte growth factor, leukemia inhibitory factor and granulocyte-macrophage colony-stimulating factor. Melanocyte differentiation is also stimulated by alpha-melanocyte stimulating hormone; in the mouse, however, this hormone is likely carried through the bloodstream and not produced locally in the skin. Melanoblast migration, proliferation and differentiation are also regulated by many coat color genes otherwise known for their ability to regulate melanosome formation and maturation, pigment type switching and melanosome distribution and transfer. Thus, melanocyte proliferation and differentiation are not only regulated by genes encoding typical growth factors and their receptors but also by genes classically known for their role in pigment formation.     

MicroRNA‐622 is a novel mediator of tumorigenicity in melanoma by targeting Kirsten rat sarcoma

The network of molecular players is similar when comparing neural crest‐derived, actively migrating melanoblasts to melanoma cells. However, melanoblasts are sensitive to differentiation‐initiating signals at their target site (epidermis), while melanoma cells maintain migratory and undifferentiated features. We aimed at identifying downregulated genes in melanoma that are particularly upregulated in melanoblasts. Loss of such genes could contribute to stabilization of a dedifferentiated, malignant phenotype in melanoma. We determined that microRNA‐622 (miR‐622) expression was strongly downregulated in melanoma cells and tissues compared to melanocytes and melanoblast‐related cells. miR‐622 expression correlated with survival of patients with melanoma. miR‐622 re‐expression inhibited clonogenicity, proliferation, and migration in melanoma. Inhibition of miR‐622 in melanocytes induced enhanced migration. Kirsten rat sarcoma (KRAS) was identified as a major functional target of miR‐622 in melanoma. We conclude that miR‐622 is a novel tumor suppressor in melanoma and identify the miR‐622‐KRAS axis as potential therapeutic target.     

Novel method for isolating human melanoblasts from keratinocyte culture

The characterization of melanoblasts is important for understanding their in vivo development, melanoma formation, and pigment‐related disorders. However, no methods have been reported for the isolation of melanoblasts from human skin. Using a ‘calcium‐pulse’ technique, involving the differentiation of human keratinocytes with high calcium and the subsequent spontaneous separation of the epidermal sheets, we effectively isolated human melanoblasts (keratinocyte‐adapted melanoblasts, KaMBs) from keratinocyte culture. The KaMBs expressed early melanogenesis‐related genes, including BRN2, which is a known melanoblast marker. Moreover, the KaMBs displayed much higher proliferative and growth capacities than the primary melanocytes. Considering that keratinocytes might provide an in vivo‐like environment for KaMBs during isolation and in vitro culture, the ‘calcium‐pulse’ technique offers an unprecedented, easy, and efficient method for the isolation of human melanoblasts, retaining the original characteristics of these cells.     

Keratinocyte cadherin desmoglein 1 controls melanocyte behavior through paracrine signaling

The epidermis is the first line of defense against ultraviolet (UV) light from the sun. Keratinocytes and melanocytes respond to UV exposure by eliciting a tanning response dependent in part on paracrine signaling, but how keratinocyte:melanocyte communication is regulated during this response remains understudied. Here, we uncover a surprising new function for the keratinocyte‐specific cell–cell adhesion molecule desmoglein 1 (Dsg1) in regulating keratinocyte:melanocyte paracrine signaling to promote the tanning response in the absence of UV exposure. Melanocytes within Dsg1‐silenced human skin equivalents exhibited increased pigmentation and altered dendrite morphology, phenotypes which were confirmed in 2D culture using conditioned media from Dsg1‐silenced keratinocytes. Dsg1‐silenced keratinocytes increased melanocyte‐stimulating hormone precursor (Pomc) and cytokine mRNA. Melanocytes cultured in media conditioned by Dsg1‐silenced keratinocytes increased Mitf and Tyrp1 mRNA, TYRP1 protein, and melanin production and secretion. Melanocytes in Dsg1‐silenced skin equivalents mislocalized suprabasally, reminiscent of early melanoma pagetoid behavior. Together with our previous report that UV reduces Dsg1 expression, these data support a role for Dsg1 in controlling keratinocyte:melanocyte paracrine communication and raise the possibility that a Dsg1‐deficient niche contributes to pagetoid behavior, such as occurs in early melanoma development.     

The patchwork mouse phenotype: implication for melanocyte replacement in the hair follicle.

Mice homozygous for the recessive patchwork (pwk) mutation are characterized by a variegated pigment pattern with a mixture of unpigmented and normally pigmented hairs. The pigmented hair bulbs contain functional melanocytes. By contrast, the unpigmented hair bulbs contain no melanocytes. This lack results from the death of melanoblasts in the hair follicle at the end of embryogenesis. Here, we report that melanoblasts and melanocytes are found in the epidermis of pwk/pwk mice. Furthermore, these epidermal pigment cells are able to colonize new hair follicles after skin wounding. Despite the presence of epidermal pigment cells with a colonization potential, a follicle that had produced an unpigmented hair produces a new unpigmented hair during the successive hair growth cycles. This hair color continuity is also true for the pigmented hair follicles. Thus, in normal conditions, the hair acts as an independent functional unit as regards its pigment cells population.     

Characterization of two novel small molecules targeting melanocyte development in zebrafish embryogenesis

Melanocytes are pigment cells that are closely associated with many skin disorders, such as vitiligo, piebaldism, Waardenburg syndrome, and the deadliest skin cancer, melanoma. Through studies of model organisms, the genetic regulatory network of melanocyte development during embryogenesis has been well established. This network also seems to be shared with adult melanocyte regeneration and melanoma formation. To identify chemical regulators of melanocyte development and homeostasis, we screened a small-molecule library of 6000 compounds using zebrafish embryos and identified five novel compounds that inhibited pigmentation. Here we report characterization of two compounds, 12G9 and 36E9, which disrupted melanocyte development. TUNEL assay indicated that these two compounds induced apoptosis of melanocytes. Furthermore, compound 12G9 specifically inhibited the viability of mammalian melanoma cells in vitro. These two compounds should be useful as chemical biology tools to study melanocytes and could serve as drug candidates against melanocyte-related diseases.