小鼠正常黑色素细胞与黑色素瘤细胞(B16)mRNA表达差异分析
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摘要
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。
Melanoma is a type of strongly invasive malignant skin tumor with high rate of metastasis and poor prognosis. Analysis of the biological characteristics of melanoma cells is important for finding potential treatment for melanoma. In this study, second generation sequencing was used to analyze the difference of transcriptome expression between normal melanocytes and B16 melanoma cells of C57BL/6 J mice, which may provide further clues in understanding of the mechanism of melanoma tumorigenesis. Totally 1 436 novel mRNAs and 4 086 known mRNAs which were differentially expressed were identified. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis revealed that the differentially expressed mRNAs were involved in 149 regulatory pathways, and the significantly enriched pathways in B16 were involved in human diseases, cellular processes, and environmental information processing. Among the differentially expressed genes, Pdgf-B, Integrin β1 and Integrin β5 that regulate cell migration and Mitf, Tyr, Tyrp1 and Tyrp2 that regulate melanogenesis were upregulated significantly in B16 melanoma cells. The results may screen potential candidates as therapeutic targets of melanoma.
Melanoma is a type of strongly invasive malignant skin tumor with high rate of metastasis and poor prognosis. Analysis of the biological characteristics of melanoma cells is important for finding potential treatment for melanoma. In this study, second generation sequencing was used to analyze the difference of transcriptome expression between normal melanocytes and B16 melanoma cells of C57BL/6 J mice, which may provide further clues in understanding of the mechanism of melanoma tumorigenesis. Totally 1 436 novel mRNAs and 4 086 known mRNAs which were differentially expressed were identified. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis revealed that the differentially expressed mRNAs were involved in 149 regulatory pathways, and the significantly enriched pathways in B16 were involved in human diseases, cellular processes, and environmental information processing. Among the differentially expressed genes, Pdgf-B, Integrin β1 and Integrin β5 that regulate cell migration and Mitf, Tyr, Tyrp1 and Tyrp2 that regulate melanogenesis were upregulated significantly in B16 melanoma cells. The results may screen potential candidates as therapeutic targets of melanoma.
引文
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[18] Scholl FA, Kamarashev J, Murmann OV, et al. PAX3 is expressed in human melanomas and contributes to tumor cell survival [J]. Cancer Res, 2001, 61(3): 823-826
[19] Bittner M, Meltzer P, Chen Y, et al. Molecular classification of cutaneous malignant melanoma by gene expression profiling [J]. Nature, 2000, 406(6795): 536-540
[20] Weeraratna AT, Jiang Y, Hostetter G, et al. Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma [J]. Cancer Cell, 2002, 1(3): 279-288
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[25] Gray-Schopfer VC, da Rocha Dias S, Marais R. The role of B-RAF in melanoma [J]. Cancer Metastasis Rev, 2005, 24(1): 165-183
[26] Tanaka Y, Gavrielides MV, Mitsuuchi Y, et al. Protein Kinase C Promotes Apoptosis in LNCaP Prostate Cancer Cells through Activation of p38 MAPK and Inhibition of the Akt Survival Pathway [J]. J Biol Chem, 2003, 278(36): 33753-33762
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[28] Bhowmick NA, Zent R, Ghiassi M, et al. Integrin β1 Signaling is necessary for transforming growth factor-β activation of p38MAPK and epithelial plasticity [J]. J Biol Chem, 2001, 276(50): 46707-46713
[29] Sharma A, Trivedi NR, Zimmerman MA, et al. Mutant V599EB-Raf regulates growth and vascular development of malignant melanoma tumors [J]. Cancer Res, 2005, 65(6): 2412-2421
[30] Costa PM, Cardoso AL, Pereira de Almeida LF, et al. PDGF-B-mediated downregulation of miR-21: new insights into PDGF signaling in glioblastoma [J]. Hum Mol Genet, 2012, 21(23): 5118-5130
[31] Ji K,Zhang P, Zhang J, et al. MicroRNA 143-5p regulates alpaca melanocyte migration, proliferation, and melanogenesis [J]. Exp Dermatol, 2018, 27(2): 166-171
[32] Hearing VJ. Biochemical control of melanogenesis and melanosomal organization [J].J Investig Dermatol Symp Proc, 1999, 4(1): 24-28
[33] Levy C, Khaled M, Fisher DE. MITF: master regulator of melanocyte development and melanoma oncogene [J]. Trends Mol Med, 2006, 12 (9): 406-414
[2] Hale LP. Zinc alpha-2-glycoprotein regulates melanin production by normal and malignant melanocytes [J]. J Invest Dermatol, 2002, 119(2): 464-470
[3] Amer MH. Gene therapy for cancer: present status and future perspective [J]. Mol Cell Ther, 2014, 2: 27
[4] Brunet JF, Denizot F, Luciani MF, et al. A new member of the immunoglobulin superfamily--CTLA-4 [J]. Nature, 1987, 328(6127): 267-270
[5] Thompson CB, Allison JP. The emerging role of CTLA-4 as an immune attenuator [J]. Immunity, 1997, 7(4): 445-450
[6] Lipson EJ, Drake CG. Ipilimumab: an anti-CTLA-4 antibody for metastatic melanoma [J]. Clin Cancer Res, 2011, 17(22): 6958-6962
[7] Haass NK, Smalley KS, Herlyn M. The role of altered cell-cell communication in melanoma progression [J]. J Mol Histol, 2004, 35(3): 309-318
[8] Bissell MJ, Radisky D. Putting tumours in context [J]. Nat Rev Cancer, 2001, 1(1): 46-54
[9] Haass NK, Smalley KS, Li L, et al. Adhesion, migration and communication in melanocytes and melanoma [J]. Pigment Cell Res, 2005, 18 (3): 150-159
[10] Shi Z, Ji K, Yang SS, et al. Biological characteristics of mouse skin melanocytes [J]. Tissue Cell, 2016, 48: 114-120
[11] Shao B, Wang H, Li Y. Trinity: a distributed graph engine on a memory cloud [J]. SIGMOD Conference, 2013: 505-516,(ACM)
[12] Ferguson GD, Vician L, Herschman HR. Synaptotagmin IV:biochemistry,behavior,and possible links to human psychiatris disease [J]. Mol Neurobiol, 2001, 23(2-3): 173-185
[13] Zhang Y, Akintola OS, Liu KJA, et al. Membrane gene ontology bias in sequencing and microarray obtained by housekeeping-gene analysis [J]. Gene, 2016, 575(2 Pt 2): 559-566
[14] Kotera M, Moriya Y, Tokimatsu T, et al. KEGG and GenomeNet, New Developments, Metagenomic Analysis [J]. Encyclopedia Metagenomics, 2013: 329-339.doi:10.1007/978-1-4899-7478-5-694
[15] Lee HO, Levorse JM, Shin MK. The endothelin receptor-B is required for the migration of neural crest-derived melanocyte and enteric neuron precursors [J]. Dev Biol, 2003, 259(1): 162-175
[16] Tan SH, Pal M, Tan MJ, et al. Regulation of cell proliferation and migration by TAK1 via transcriptional control of von Hippel-Lindau tumor suppressor [J]. J Biol Chem, 2009, 284(27): 18047-18058
[17] Hoek K, Rimm DL, Williams KR, et al. Expression profiling reveals novel pathways in the transformation of melanocytes to melanomas [J]. Cancer Res, 2004, 64(15): 5270-5282
[18] Scholl FA, Kamarashev J, Murmann OV, et al. PAX3 is expressed in human melanomas and contributes to tumor cell survival [J]. Cancer Res, 2001, 61(3): 823-826
[19] Bittner M, Meltzer P, Chen Y, et al. Molecular classification of cutaneous malignant melanoma by gene expression profiling [J]. Nature, 2000, 406(6795): 536-540
[20] Weeraratna AT, Jiang Y, Hostetter G, et al. Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma [J]. Cancer Cell, 2002, 1(3): 279-288
[21] Wen D, Suggs SV, Karunagaran D, et al. Structural and functional aspects of the multiplicity of Neu differentiation factors [J]. Mol Cell Biol, 1994, 14(3): 1909-1919
[22] Carraway KL 3rd, Cantley LC. A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signaling [J]. Cell, 1994, 78(1): 5-8
[23] Citri A, Skaria KB, Yarden Y. The deaf and the dumb: The biology of ErbB-2 and ErbB-3 [J]. Exp Cell Res, 2003, 284(1): 54-65
[24] Gray-Schopfer V, Wellbrock C, Marais R. Melanoma biology and new targeted therapy [J]. Nature, 2007, 445(7130): 851-857
[25] Gray-Schopfer VC, da Rocha Dias S, Marais R. The role of B-RAF in melanoma [J]. Cancer Metastasis Rev, 2005, 24(1): 165-183
[26] Tanaka Y, Gavrielides MV, Mitsuuchi Y, et al. Protein Kinase C Promotes Apoptosis in LNCaP Prostate Cancer Cells through Activation of p38 MAPK and Inhibition of the Akt Survival Pathway [J]. J Biol Chem, 2003, 278(36): 33753-33762
[27] Galliher AJ, Schiemann WP. Src phosphorylates Tyr284 in TGF-beta type II receptor and regulates TGF-beta stimulation of p38 MAPK during breast cancer cell proliferation and invasion [J]. Cancer Res, 2007, 67(8): 3752-3758
[28] Bhowmick NA, Zent R, Ghiassi M, et al. Integrin β1 Signaling is necessary for transforming growth factor-β activation of p38MAPK and epithelial plasticity [J]. J Biol Chem, 2001, 276(50): 46707-46713
[29] Sharma A, Trivedi NR, Zimmerman MA, et al. Mutant V599EB-Raf regulates growth and vascular development of malignant melanoma tumors [J]. Cancer Res, 2005, 65(6): 2412-2421
[30] Costa PM, Cardoso AL, Pereira de Almeida LF, et al. PDGF-B-mediated downregulation of miR-21: new insights into PDGF signaling in glioblastoma [J]. Hum Mol Genet, 2012, 21(23): 5118-5130
[31] Ji K,Zhang P, Zhang J, et al. MicroRNA 143-5p regulates alpaca melanocyte migration, proliferation, and melanogenesis [J]. Exp Dermatol, 2018, 27(2): 166-171
[32] Hearing VJ. Biochemical control of melanogenesis and melanosomal organization [J].J Investig Dermatol Symp Proc, 1999, 4(1): 24-28
[33] Levy C, Khaled M, Fisher DE. MITF: master regulator of melanocyte development and melanoma oncogene [J]. Trends Mol Med, 2006, 12 (9): 406-414