猪转录因子SOX2下游调控蛋白筛选及调控网络建立
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摘要
SOX2是早期胚胎发育与干细胞多能性维持相关的重要转录因子。从猪孤雌囊胚中克隆获得猪SOX2基因并构建对应过表达体系。通过猪pEF细胞系与PK15细胞系中过表达SOX2基因及qPCR技术,检测89个下游调控候选基因表达情况,建立猪SOX2下游调控网络。结果发现,GATA4、MSC、GSC、FGF4、STELLA、ACACA、FN1、KLF4以及MEK基因在pEF细胞系与PK15细胞系间呈差异性调控表达。BMP4、FGFR2、ACAA2、ACSL3、β-CATENIN、c-MYC、MYST3、GSK3β、ALDH3A2、STAT3、SMAD2、KDM6A、ACADL、PI3K与WDR3基因在两种细胞系中呈同趋势性调控表达;经JASPAR数据库预测发现,WDR3、β-CATENIN、ACAA2、KDM6A、STAT3、ACADL、GSK3β与ALDH3A2基因启动子区域存在高相关性的SOX2蛋白结合位点,推测这些基因受SOX2蛋白直接调控;同时构建针对猪SOX2基因的高效敲除体系。研究结果为建立物种特异性的猪SOX2调控网络提供数据基础。
SOX2 is an important transcription factor in early embryo development and the pluripotency maintenance of stem cell. The porcine SOX2 gene was cloned from parthenogenesis blastocyst and the overexpression vector was constructed. The SOX2 gene was overexpressed in pEF and PK15 cell lines and the expression of 89 downstream regulatory candidate genes were detected by qPCR to establish the regulation network of porcine SOX2. The results showed that GATA4, MSC,GSC, FGF4, STELLA, ACACA, FN1, KLF4 and MEK were differentially regulated between pEF and PK15 cell lines. BMP4, FGFR2, ACAA2, ACSL3, β-CATENIN, c-MYC, MYST3, GSK3β, ALDH3 A2,STAT3, SMAD2, KDM6 A, ACADL, PI3 K and WDR3 were expressed in the same trend in both cell lines.And there existed high relative motifs for SOX2 binding in WDR3, β-CATENIN, ACAA2, KDM6 A,STAT3, ACADL, GSK3β and ALDH3 A2 gene promoter regions through the JASPAR analysis. These genes were predicted to be directly regulated by SOX2 protein. The gene knockout system designed for the porcine SOX2 could efficiently knockout the SOX2 gene. These results provided a data base for establishing a porcine species-specific SOX2 regulatory network.
SOX2 is an important transcription factor in early embryo development and the pluripotency maintenance of stem cell. The porcine SOX2 gene was cloned from parthenogenesis blastocyst and the overexpression vector was constructed. The SOX2 gene was overexpressed in pEF and PK15 cell lines and the expression of 89 downstream regulatory candidate genes were detected by qPCR to establish the regulation network of porcine SOX2. The results showed that GATA4, MSC,GSC, FGF4, STELLA, ACACA, FN1, KLF4 and MEK were differentially regulated between pEF and PK15 cell lines. BMP4, FGFR2, ACAA2, ACSL3, β-CATENIN, c-MYC, MYST3, GSK3β, ALDH3 A2,STAT3, SMAD2, KDM6 A, ACADL, PI3 K and WDR3 were expressed in the same trend in both cell lines.And there existed high relative motifs for SOX2 binding in WDR3, β-CATENIN, ACAA2, KDM6 A,STAT3, ACADL, GSK3β and ALDH3 A2 gene promoter regions through the JASPAR analysis. These genes were predicted to be directly regulated by SOX2 protein. The gene knockout system designed for the porcine SOX2 could efficiently knockout the SOX2 gene. These results provided a data base for establishing a porcine species-specific SOX2 regulatory network.
引文
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[15] Liu S, Bou G, Sun R, et al. SOX2 is the faithful marker for pluripotency in pig:Evidence from embryonic studies[J]. Developmental Dynamics, 2015, 244(4):619-627.
[16] Narayan S, Bryant G, Shah S, et al. OCT4 and SOX2 work as transcriptional activators in reprogramming human fibroblasts[J]. Cell Reports, 2017, 20(7):1585-1596.
[17] Rizzino A, Wuebben E L. SOX2/OCT4:A delicately balanced partnership in pluripotent stem cells and embryogenesis[J]. Biochimica et Biophysica Acta, 2016, 1859(6):780-791.
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[19] Khan A, Fornes O, Stigliani A, et al. JASPAR 2018:Update of the open-access database of transcription factor binding profiles and its web framework[J]. Nucleic Acids Res, 2018, 46(1):260-266.
[20] Cong L, Ran F A, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121):819-823.
[21] Mali P, Yang L, Esvelt K M, et al. RNA-guided human genome engineering via Cas9[J]. Science, 2013, 339(6121):823-826.
[22] Tang L, Wang D, Gu D. Knockdown of SOX2 inhibits OS cells invasion and migration via modulating Wnt/beta-catenin signaling pathway[J]. Pathology Oncology Research, 2018, 24(4):907-913.
[23] Mansukhani A, Ambrosetti D, Holmes G, et al. Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation[J]. The Journal of Cell Biology, 2005,168(7):1065-1076.
[24] Raz R, Lee C K, Cannizzaro L A, et al. Essential role of STAT3for embryonic stem cell pluripotency[J]. Proc Natl Acad Sci USA,1999, 96(6):2846-2851.
[25] Humphrey R K, Beattie G M, Lopez A D, et al. Maintenance of pluripotency in human embryonic stem cells is STAT3 independent[J]. Stem Cells, 2004, 22(4):522-530.
[26] Chen H, Aksoy I, Gonnot F, et al. Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency[J]. Nature Communications, 2015, 6:7095.
[27] Kuo H Y, Hsu H T, Chen Y C, et al. Galectin-3 modulates the EGFR signalling-mediated regulation of SOX2 expression via cMyc in lung cancer[J]. Glycobiology, 2016, 26(2):155-165.
[2] Campolo F, Gori M, Favaro R, et al. Essential role of sox2 for the establishment and maintenance of the germ cell line[J]. Stem Cells, 2013, 31(7):1408-1421.
[3] Sarlak G, Vincent B. The roles of the stem cell-controlling SOX2transcription factor:from neuroectoderm development to alzheimer's disease?[J]. Molecular Neurobiology, 2016, 53(3):1679-1698.
[4] Boyer L A, Lee T I, Cole M F, et al. Core transcriptional regulatory circuitry in human embryonic stem cells[J]. Cell, 2005, 122(6):947-956.
[5] Wang Z, Oron E, Nelson B, et al. Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells[J]. Cell stem cell, 2012, 10(4):440-454.
[6] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell, 2006, 126(4):663-676.
[7] Rudin C M, Durinck S, Stawiski E W, et al. Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene insmall-celllungcancer[J].NatureGenetics, 2012,44(10):1111-1116.
[8] Belotte J, Fletcher N M, Alexis M, et al. SOX2 gene amplification significantly impacts overall survival in serous epithelial ovarian cancer[J]. Reproductive Sciences, 2015, 22(1):38-46.
[9] Long K B, Hornick J L. SOX2 is highly expressed in squamous cell carcinomas of the gastrointestinal tract[J]. Human Pathology,2009, 40(12):1768-1773.
[10] Swindle M M, Makin A, Herron A J, et al. Swine as models in biomedical research and toxicology testing[J]. Veterinary Pathology,2012, 49(2):344-356.
[11] Hunter P. Xeno's paradox:Why pig cells are better for tissue transplants than human cells[J]. EMBO Reports, 2009, 10(6):554-557.
[12] Hou D R, Jin Y, Nie X W, et al. Derivation of porcine embryonic stem-like cells from in vitro-produced blastocyst-stage embryos[J]. Sci Rep, 2016, 6:25838.
[13] Xue B, Li Y, He Y, et al. Porcine pluripotent stem cells derived from IVF embryos contribute to chimeric development in vivo[J].PLoS One, 2016, 11(3):e0151737.
[14] Sun R, Lei L, Liu S, et al. Morphological changes and germ layer formation in the porcine embryos from days 7-13 of development[J]. Zygote, 2015, 23(2):266-276.
[15] Liu S, Bou G, Sun R, et al. SOX2 is the faithful marker for pluripotency in pig:Evidence from embryonic studies[J]. Developmental Dynamics, 2015, 244(4):619-627.
[16] Narayan S, Bryant G, Shah S, et al. OCT4 and SOX2 work as transcriptional activators in reprogramming human fibroblasts[J]. Cell Reports, 2017, 20(7):1585-1596.
[17] Rizzino A, Wuebben E L. SOX2/OCT4:A delicately balanced partnership in pluripotent stem cells and embryogenesis[J]. Biochimica et Biophysica Acta, 2016, 1859(6):780-791.
[18] Kim J B, Greber B, Arauzo-bravo M J, et al. Direct reprogramming of human neural stem cells by OCT4[J]. Nature, 2009, 461(7264):649-653.
[19] Khan A, Fornes O, Stigliani A, et al. JASPAR 2018:Update of the open-access database of transcription factor binding profiles and its web framework[J]. Nucleic Acids Res, 2018, 46(1):260-266.
[20] Cong L, Ran F A, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121):819-823.
[21] Mali P, Yang L, Esvelt K M, et al. RNA-guided human genome engineering via Cas9[J]. Science, 2013, 339(6121):823-826.
[22] Tang L, Wang D, Gu D. Knockdown of SOX2 inhibits OS cells invasion and migration via modulating Wnt/beta-catenin signaling pathway[J]. Pathology Oncology Research, 2018, 24(4):907-913.
[23] Mansukhani A, Ambrosetti D, Holmes G, et al. Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation[J]. The Journal of Cell Biology, 2005,168(7):1065-1076.
[24] Raz R, Lee C K, Cannizzaro L A, et al. Essential role of STAT3for embryonic stem cell pluripotency[J]. Proc Natl Acad Sci USA,1999, 96(6):2846-2851.
[25] Humphrey R K, Beattie G M, Lopez A D, et al. Maintenance of pluripotency in human embryonic stem cells is STAT3 independent[J]. Stem Cells, 2004, 22(4):522-530.
[26] Chen H, Aksoy I, Gonnot F, et al. Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency[J]. Nature Communications, 2015, 6:7095.
[27] Kuo H Y, Hsu H T, Chen Y C, et al. Galectin-3 modulates the EGFR signalling-mediated regulation of SOX2 expression via cMyc in lung cancer[J]. Glycobiology, 2016, 26(2):155-165.
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