黑色素细胞中过量表达Pax610Neu基因对MITF和TYR的影响

doi:10.3864/j.issn.0578-1752.2016.11.017

摘要/Abstract

摘要： 【目的】 MITF和β-catenin是黑色素生成通路中重要的调节基因，而Pax6通过调控MITF和β-catenin来调控黑色素细胞的黑色素生成，通过对只含有正常Pax6 paird domain 的前102个氨基酸的Pax6^10Neu的研究，来探究Pax6^10Neu是否还有生物学功能，以及Pax6对其下游基因的作用机理。【方法】根据NCBI查找到的Pax6^10Neu基因序列设计PCR引物，克隆Pax6^10Neu，收集所得基因片段送华大基因测序确认。后通过对Pax6^10Neu和慢病毒表达载体的序列分析来筛选合适的酶切位点，通过分析选取Sal I和Xba I作为酶切位点，设计含有Sal I和Xba I酶切位点的PCR引物，大量克隆Pax6^10Neu基因，从而通过胶回收得到含有Sal I和Xba I酶切位点的Pax6^10Neu基因。将含有酶切位点的目的片段与T载体相连，并送华大基因测序。将与T载体连接成功的质粒导入感受态细胞使其大量扩增，提取质粒，双酶切后，胶回收，得到大量含有Sal I和Xba I酶切位点的Pax6^10Neu基因片段，将其与含有小鼠tyrp2特异性启动子和绿色荧光蛋白(GFP)的慢病毒表达载体相连接，送华大基因测序确认。将连接好的慢病毒表达载体导入感受态细胞使其大量扩增，通过质粒中提试剂盒获得大量去内毒素的Pax6^10Neu慢病毒真核表达载体。将慢病毒真核表达载体通过细胞转染导入到培养的绵羊黑色素细胞中，使其过量表达。收集细胞，分别通过观察绿色荧光蛋白和使用RT-PCR来检测转染效率，使用RT-PCR和Western blot来检测MITF和TYR在mRNA和蛋白水平的变化，同时检测黑色素细胞中黑色素生成量的变化。【结果】与正常组相比，在mRNA水平，MITF显著升高3.2倍（P＜0.05），TYR升高1.31倍，由此得出，Pax6^10Neu可以有效促进MITF mRNA 的产生，对TYR mRNA产生的影响不是太显著；在蛋白质水平，MITF显著升高8.24倍(P＜0.001)，TYR显著升高2.09倍(P＜0.001)，由此得出，Pax6^10Neu可以显著提高MITF 、TYR 蛋白的产生；同时黑色素细胞产生黑色素的量显著升高1.22倍(P＜0.05)，由此得出Pax6^10Neu可以有效促进黑色素细胞黑色素的生成。【结论】只含有正常Pax6 paird domain前102个氨基酸的Pax6^10Neu，仍然可以在黑色素细胞中与MITF相互作用，同时间接调控TYR的表达，从而使黑色素细胞黑色素的生成量发生改变。

关键词: Pax6^10Neu, Pax6, MITF, TYR, 黑色素

Abstract: 【Objective】 MITF and β-catenin are important regulatory genes of the melanin system. Pax6 regulates the production of melanin indirectly, through regulates MITF and TYR. In order to explore the function of Pax6^10Neu and the mechanism of Pax6 with its target genes, we explored the function of Pax6^10Neu, which only included 102 N-terminalanimo acids of the paird domain. 【Method】 According to the sequence of Pax6^10Neu in NCBI, the primers of Pax6^10Neu were designed and synthesized by BGI. The coding sequences of Pax6^10Neu were PCR amplified and then confirmed by sequencing. Sal I and Xba I restriction sites were selected by analyzing the sequences of Pax6^10Neu and the mammalian expression vector. The Pax6^10Neu was cloned into the T-Vector, meanwhile, confirmed by sequencing. The fragment was then subcloned into a mammalian expression vector, resulting in a construction that contained a specific mouse tyrp2 promoter driving the expression of the green fluorescent protein (GFP) and the target fragment with Sal I and Xba I restriction sites. The plasmid vector was confirmed by sequencing. The expression vector was amplified by competent cells and obtained without endotoxin by the plasmid midiprep system. Then, the sheep melanocytes were transfected with the vector using Liposome 2000. There were three methods used in the results test which were quantitatively real-time PCR, western blot, and melanin content measurement. 【Result】 The results showed that the MITF mRNA, MITF and TYR protein, melanin content were significantly increased. MITF mRNA was significantly increased to 3.2 times (P＜0.05) and TYR mRNA was increased to 1.31 times, compared with control group, MITF protein was significantly increased to 8.24 times (P＜0.001) and TYR protein was significantly increased to 2.09 times (P＜0.001). Meanwhile, the melanin content was significantly increased to 1.22 times (P＜0.05). 【Conclusion】 We demonstrated that the Pax6^10Neustill interacts with MITF, and then regulated TYR indirectly, meanwhile changing the production of melanin of melanocytes.

Key words: Pax6^10Neu, Pax6, MITF, TYR, melanin

聂瑞强，杨玉静，谢建山，范瑞文，高文俊，董常生. 黑色素细胞中过量表达Pax6^10Neu基因对MITF和TYR的影响[J]. 中国农业科学, 2016, 49(11): 2214-2221.

NIE Rui-qiang, YANG Yu-jing, XIE Jian-shan, FAN Rui-wen, GAO Wen-jun, DONG Chang-sheng. The Influences of Over-Expressing Pax6^10Neu on MITF and TYR in Melanocytes[J]. Scientia Agricultura Sinica, 2016, 49(11): 2214-2221.

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参考文献

[1] PAN L, MA X, WEN B, SU Z, ZHENG X, LIU Y, LI H, CHEN Y, WANG J, LU F, QU J, HOU L. Microphthalmia-associated transcription factor/T-box factor-2 axis acts through Cyclin D1 to regulate melanocyte proliferation. Cell Proliferation, 2015, 48: 631-642.

[2] KURITA K, NISHITO M, SHIMOGAKI H, TAKADA K, YAMAZAKI H, KUNISADA T. Suppression of progressive loss of coat color in microphthalmia-vitiligo mutant mice. The Journal of Investigative Dermatology, 2005, 125(3):538-544.

[3] YUSNIZAR Y, WILBE M, HERLINO A O, SUMANTRI C, NOOR R R, BOEDIONO A, ANDERSSON L, ANDERSSON G. Microphthalmia- associated transcription factor mutations are associated with white- spotted coat color in swamp buffalo. Animal Genetics, 2015, 46: 676-682.

[4] DONG C, WANG H, XUE L, DONG Y, YANG L, FAN R, YU X, TIAN X, MA S, SMITH G W. Coat color determination by miR-137 mediated down-regulation of microphthalmia-associated transcription factor in a mouse model. RNA, 2012, 18(9):1679-1686.

[5] RAVIV S, BHARTI K, RENCUS-LAZAR S, COHEN-TAYAR Y, SCHYR R, EVANTAL N, MESHORER E, ZILBERBERG A, IDELSON M, REUBINOFF B. PAX6 regulates melanogenesis in the retinal pigmented epithelium through feed-forward regulatory interactions with MITF. PLoS Genetics, 2014, 10(5):e1004360.

[6] BHARTI K, GASPER M, OU J, BRUCATO M, CLORE- GRONENBORN K, PICKEL J, ARNHEITER H. A regulatory loop involving PAX6, MITF, and WNT signaling controls retinal pigment epithelium development. PLoS Genetics, 2012, 8(7):e1002757.

[7] BAUMER N, MARQUARDT T, STOYKOVA A, SPIELER D, TREICHEL D, ASHERY-PADAN R, GRUSS P. Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6. Development (Cambridge, England), 2003, 130(13): 2903-2915.

[8] 朱芷葳, 贺俊平, 于秀菊, 李鹏飞, 程志学, 董常生. PAX3转录因子在羊驼皮肤组织中的表达和定位分析. 畜牧兽医学报, 2012(5): 729-734.

ZHU Z W, HE J P, YU X J, LEI P F, CHENG Z X, DONG C S. Expression and location analysis of pax3 transcription factor in Alpaca skin. Acta Veterinaria et Zootechnica Sinica, 2012, 43(5): 729-734. (in Chinese)

[9] FUJIMURA N, KLIMOVA L, ANTOSOVA B, SMOLIKOVA J, MACHON O, KOZMIK Z. Genetic interaction between Pax6 and β-catenin in the developing retinal pigment epithelium. Development Genes and Evolution, 2015, 225(2):121-128.

[10] ZHU P Y, YIN W H, WANG M R, DANG Y Y, YE X Y. Andrographolide suppresses melanin synthesis through Akt/GSK3β/β- catenin signal pathway. Journal of Dermatological Science, 2015, 79(1):74-83.

[11] PARK H J. CARI ONE induces anagen phase of telogenic hair follicles through regulation of β-catenin, stimulation of dermal papilla cell proliferation, and melanogenesis. Journal of Dietary Supplements, 2014, 11(4):320-333.

[12] LANG D, POWELL S K, PLUMMER R S, YOUNG K P, RUGGERI B A. PAX genes: roles in development, pathophysiology, and cancer. Biochemical Pharmacology, 2007, 73(1):1-14.

[13] PAIXAO-CORTES V R, SALZANO F M, BORTOLINI M C. Origins and evolvability of the PAX family. Seminars in Cell & Developmental Biology, 2015, 44: 64-74.

[14] BLAKE J A, ZIMAN M R. Pax genes: regulators of lineage specification and progenitor cell maintenance. Development (Cambridge, England), 2014, 141(4): 737-751.

[15] MONSORO-BURQ A H. PAX transcription factors in neural crest development. Seminars in Cell & Developmental Biology, 2015, 44:87-96.

[16] 王秀, 王蔚, 王义权. Pax基因功能及其选择性剪接的研究进展. 生命科学, 2008, 20(1):125-130.

WANG X, WANG W, WANG Y Q. Progress in the study of Pax gene family and its alternative splicing. Chinese Bulletin of Life Sciences, 2008, 20(1):125-130. (in Chinese)

[17] EBERHARD D, JIMENEZ G, HEAVEY B, BUSSLINGER M. Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family. The EMBO Journal, 2000, 19(10):2292-2303.

[18] FAVOR J, PETERS H, HERMANN T, SCHMAHL W, CHATTERJEE B, NEUHAUSER-KLAUS A, SANDULACHE R. Molecular characterization of Pax6(2Neu) through Pax6(10Neu): an extension of the Pax6 allelic series and the identification of two possible hypomorph alleles in the mouse Mus musculus. Genetics, 2001, 159(4): 1689-1700.

[19] DONG Y, WANG H, CAO J, REN J, FAN R, HE X, SMITH GW, DONG C. Nitric oxide enhances melanogenesis of alpaca skin melanocytes in vitro by activating the MITF phosphorylation. Molecular and Cellular Biochemistry, 2011, 352(1/2):255-260.

[20] MAZUR M A, WINKLER M, GANIC E, COLBERG J K, JOHANSSON J K, BENNET H, FEX M, NUBER U A, ARTNER I. Microphthalmia transcription factor regulates pancreatic β-cell function. Diabetes, 2013, 62(8):2834-2842.

[21] HODGKINSON C A, MOORE K J, NAKAYAMA A, STEINGRIMSSON E, COPELAND N G, JENKINS N A, ARNHEITER H. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell, 1993, 74(2):395-404.

[22] SHIBAHARA S, YASUMOTO K, AMAE S, UDONO T, WATANABE K, SAITO H, TAKEDA K. Regulation of pigment cell-specific gene expression by MITF//Pigment cell research sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society, 2000, 13(8):98-102.

[23] SPENCE J R, MADHAVAN M, AYCINENA J C, DEL RIO-TSONIS K. Retina regeneration in the chick embryo is not induced by spontaneous Mitf downregulation but requires FGF/FGFR/MEK/Erk dependent upregulation of Pax6. Molecular Vision, 2007, 13:57-65.

[24] ZHAO Y, WANG P, MENG J, JI Y, XU D, CHEN T, FAN R, YU X, YAO J, DONG C. MicroRNA-27a-3p Inhibits Melanogenesis in Mouse Skin Melanocytes by Targeting Wnt3a. International Journal of Molecular Sciences, 2015, 16(5):10921-10933.

[25] YUN C Y, YOU S T, KIM J H, CHUNG J H, HAN S B, SHIN E Y, KIM E G. p21-activated kinase 4 critically regulates melanogenesis via activation of the CREB/MITF and β-catenin/MITF pathways. The Journal of Investigative Dermatology, 2015, 135(5):1385-1394.