毛壳素对绒山羊ADSCs组蛋白甲基化修饰的影响研究

doi:10.11843/j.issn.0366-6964.2022.07.034

摘要/Abstract

摘要： 为了探究毛壳素对绒山羊脂肪间充质干细胞(gADSCs)组蛋白甲基化修饰差异的影响。本研究采用不同浓度的毛壳素对gADSCs进行不同时间的处理，以期筛选出对细胞活性影响最低且能够最大程度抑制gADSCs组蛋白甲基化的药物处理浓度及时间。以最适药物浓度和时间处理组为试验组，无药物处理组为对照组，通过实时荧光定量PCR检测H3K9 me2和H3K9 me3甲基化相关酶和胚胎发育多潜能基因mRNA表达水平的改变，并检测毛壳素对组蛋白H3K9 me2和H3K9 me3蛋白表达水平的影响。结果显示，20 nmol·L^-1的毛壳素处理gADSCs 48 h时对细胞活性影响较小，组蛋白甲基化转移酶G9A的表达显著降低(P<0.05)，为最适处理浓度和时间。实时PCR结果显示，试验组H3K9 me2和H3K9 me3甲基化转移酶EHMT1、EHMT2、SUV39H1、SUV39H2表达显著降低(P<0.05)，H3K9 me2和H3K9 me3去甲基化酶KDM3A、KDM3B、KDM4B、KDM4D表达显著增高(P<0.05)，多潜能性相关基因SOX2、OCT4、NANOG表达量增高。免疫荧光和WB结果显示，处理组H3K9 me2蛋白水平无明显变化，而H3K9 me3蛋白水平显著降低(P<0.05)。20 nmol·L^-1的毛壳素处理gADSCs 48 h后可以显著降低组蛋白H3K9 me2和H3K9 me3的甲基化修饰作用，提高多潜能性相关基因的表达。

关键词: 毛壳素, 脂肪间充质干细胞, 表观遗传修饰, H3K9 me2, H3K9 me3

Abstract: The study aimed to explore the effects of chaetocin on histone methylation modification in adipose-derived stem cells (gADSCs) of cashmere goat. In this study, gADSCs were treated with different concentrations of chaetocin for different durations, and the concentration and duration of chaetocin treatment that had the least effects on cell viability and the greatest inhibition on histone methylation in gADSCs were screened. The optimal concentration and duration of chaetocin treatment was used as the experimental group, and the chaetocin-free treatment was used as the control group. The expression of H3K9 me2 and H3K9 me3 methylation-related enzymes and embryonic development pluripotency genes were detected by RT-qPCR, and the effects of chaetocin on histone H3K9 me2 and H3K9 me3 proteins expression were observed as well. The results showed that there was little effect on cell viability when gADSCs were treated with 20 nmol·L^-1 chaetin for 48 h, and the expression of histone methyltransferase G9A significantly reduced (P<0.05), the optimal chaetin treatment concentration and duration was confirmed. RT-qPCR results showed that the expression of H3K9 me2 and H3K9 me3 methyltransferase EHMT1, EHMT2, SUV39H1 and SUV39H2 significantly reduced(P<0.05), and the expression of H3K9 me2 and H3K9 me3 demethylase KDM3A, KDM3B, KDM4B and KDM 4Dsignificantly increased(P<0.05),the expression of multipotency-related genes SOX2, OCT4, NANOG increased in experimental group. The results of immunofluorescence and WB experiments showed that the H3K9 me2 protein level did not significantly change, while the H3K9 me3 protein level significantly reduced(P<0.05) in the experimental group. The modification of histone H3K9 me2 and H3K9 me3 in gADSCs treated with 20 nmol·L^-1 chaetocin for 48 h could be reduced significantly, and the expression of multipotency-related genes could be increased.

Key words: chaetocin, adipose-derived stem cells, epigenetic modification, H3K9 me2, H3K9 me3

中图分类号:

S827.2

孙浩, 者小书, 张文奇, 郝斐, 李文楠, 刘洁, 刘东军. 毛壳素对绒山羊ADSCs组蛋白甲基化修饰的影响研究[J]. 畜牧兽医学报, 2022, 53(7): 2380-2389.

SUN Hao, ZHE Xiaoshu, ZHANG Wenqi, HAO Fei, LI Wennan, LIU Jie, LIU Dongjun. Effects of Chaetocin on Histone Methylation Modification of Cashmere Goats ADSCs[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2380-2389.

参考文献 40

[1]	YOU H J,HAN S K. Cell therapy for wound healing[J].J Korean Med Sci,2014,29(3):311-319.
[2]	BAKSH D,SONG L,TUAN R S.Adult mesenchymal stem cells:characterization,differentiation,and application in cell and gene therapy[J].J Cell Mol Med,2004,8(3):301-316.
[3]	刘贤俊,刘海亮,景波,等.人不同组织间充质干细胞免疫调控能力的比较[J].同济大学学报:医学版,2017,38(6):12-17.LIU X J,LIU H L,JING B,et al.Comparison of immune regulatory properties of human mesenchymal stem cells derived from different tissues[J].Journal of Tongji University:Medical Science,2017,38(6):12-17.(in Chinese)
[4]	祁旻龙,朱良,CIERVO Y,等.过表达趋化因子受体2促进脂肪间充质干细胞对皮肤损伤的修复[J].同济大学学报:医学版,2018,39(5):23-27,33.QI M L,ZHU L,CIERVO Y,et al.Overexpression of chemokine receptor type 2 in adipose mesenchymal stem cells promotes cutaneous wound healing[J].Journal of Tongji University:Medical Science,2018,39(5):23-27,33.(in Chinese)
[5]	杨旭琼,吴珍芳,李紫聪.哺乳动物体细胞核移植表观遗传重编程研究进展[J].遗传,2019,41(12):1099-1109.YANG X Q,WU Z F,LI Z C.Advances in epigenetic reprogramming of somatic cells nuclear transfer in mammals[J].Hereditas (Beijing),2019,41(12):1099-1109.(in Chinese)
[6]	OGURA A,INOUE K,WAKAYAMA T.Recent advancements in cloning by somatic cell nuclear transfer[J].Philos Trans Roy Soc B Biol Sci,2013,368(1609):20110329.
[7]	GOUVEIA C,HUYSER C,EGLI D,et al.Lessons learned from somatic cell nuclear transfer[J].Int J Mol Sci,2020,21(7):2314.
[8]	SRIRATTANA K,KANEDA M,PARNPAI R.Strategies to improve the efficiency of somatic cell nuclear transfer[J].Int J Mol Sci,2022,23(4):1969.
[9]	LOI P,IUSO D,CZERNIK M,et al.A new,dynamic era for somatic cell nuclear transfer?[J].Trends Biotechnol,2016,34(10):791-797.
[10]	PEAT J R,REIK W.Incomplete methylation reprogramming in SCNT embryos[J].Nat Genet,2012,44(9):965-966.
[11]	ATLASI Y,STUNNENBERG H G.The interplay of epigenetic marks during stem cell differentiation and development[J].Nat Rev Genet,2017,18(11):643-658.
[12]	DIXON J R,SELVARAJ S,YUE F.Topological domains in mammalian genomes identified by analysis of chromatin interactions[J]. Nature,2012,485(7398):376-380.
[13]	WANG J L,ZHANG M,ZHANG Y,et al.The histone demethylase JMJD2C is stage-specifically expressed in preimplantation mouse embryos and is required for embryonic development[J].Biol Reprod,2010,82(1):105-111.
[14]	VARGHES B,DEL GAUDIO N,COBELLIS G,et al.KDM4 involvement in breast cancer and possible therapeutic approaches[J].Front Oncol,2021,11:750315.
[15]	ROSS P J,SAMPAIO R V.Epigenetic remodeling in preimplantation embryos:cows are not big mice[J].Anim Reprod,2018, 15(3):204-214.
[16]	MATOBA S,LIU Y T,LU F L,et al.Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation[J].Cell,2014,159(4):884-895.
[17]	ZHANG Y M,GAO E E,WANG Q Q,et al.Effects of histone methyltransferase inhibitor chaetocin on histone H3K9 methylation of cultured ovine somatic cells and development of preimplantation cloned embryos[J].Reprod Toxicol,2018,79:124-131.
[18]	JAFARPOUR F,GHAZVINI ZADEGAN F,OSTADHOSSEINI S,et al.siRNA inhibition and not chemical inhibition of Suv39 h1/2 enhances pre-implantation embryonic development of bovine somatic cell nuclear transfer embryos[J].PLoS One,2020, 15(6):e0233880.
[19]	GREINER D,BONALDI T,ESKELAND R,et al.Identification of a specific inhibitor of the histone methyltransferase SU (VAR)3-9[J].Nat Chem Biol,2005,1(3):143-145.
[20]	SOUFI A,DONAHUE G,ZARET K S.Facilitators and impediments of the pluripotency reprogramming factors'initial engagement with the genome[J].Cell,2012,151(5):994-1004.
[21]	KREIMER A,ASHUACH T,INOUE F,et al.Massively parallel reporter perturbation assays uncover temporal regulatory architecture during neural differentiation[J].Nat Commun,2022,13(1):1504.
[22]	STILLMAN B.Histone modifications:insights into their influence on gene expression[J].Cell,2018,175(1):6-9.
[23]	PAN G J,THOMSON J A.Nanog and transcriptional networks in embryonic stem cell pluripotency[J].Cell Res,2007,17(1):42-49.
[24]	AVILION A A,NICOLIS S K,PEVNY L H,et al.Multipotent cell lineages in early mouse development depend on SOX2 function[J].Genes Dev,2003,17(1):126-140.
[25]	CHEN J,CHEN X L,LI M,et al.Hierarchical Oct4 binding in concert with primed epigenetic rearrangements during somatic cell reprogramming[J].Cell Rep,2016,14(6):1540-1554.
[26]	LOH Y H,WU Q,CHEW J L,et al.The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells[J].Nat Genet,2006,38(4):431-440.
[27]	李海艳.毛壳素对德保黑猪核移植胚胎体外发育潜能影响的初步研究[D].南宁:广西大学,2017.LI H Y.Effects of chaetocin on the in vitro development of Debao black porcine somatic cell nuclear transfer embryos[D].Nanning:Guangxi University,2017.(in Chinese)
[28]	王婷,李海艳,刘晨,等.毛壳素对猪核移植胚胎体外发育潜能的影响研究[J].中国兽医科学,2021,51(6):792-797.WANG T,LI H Y,LIU C,et al.Effects of chaetocin treatment on in vitro developmental potential of nuclear transfer embryos of pig[J].Chinese Veterinary Science,2021,51(6):792-797.(in Chinese)
[29]	JEONG P S,SIM B W,PARK S H,et al.Chaetocin improves pig cloning efficiency by enhancing epigenetic reprogramming and autophagic activity[J].Int J Mol Sci,2020,21(14):4836.
[30]	EILERTSEN K J,POWER R A,HARKINS L L,et al.Targeting cellular memory to reprogram the epigenome,restore potential,and improve somatic cell nuclear transfer[J].Anim Reprod Sci,2007,98(1-2):129-146.
[31]	CHEN P R,REDEL B K,KERNS K C,et al.Challenges and considerations during in vitro production of porcine embryos[J]. Cells,2021,10(10):2770.
[32]	JEONG P S,YANG H J,PARK S H,et al.Combined chaetocin/trichostatin a treatment improves the epigenetic modification and developmental competence of porcine somatic cell nuclear transfer embryos[J].Front Cell Dev Biol,2021,9:709574.
[33]	CHUNG Y G,MATOBA S,LIU Y T,et al.Histone demethylase expression enhances human somatic cell nuclear transfer efficiency and promotes derivation of pluripotent stem cells[J].Cell Stem Cell,2015,17(6):758-766.
[34]	WENG X G,CAI M M,ZHAN Y T,et al.Improvement in the in vitro development of cloned pig embryos after kdm4a overexpression and an H3K9 me3 methyltransferase inhibitor treatment[J].Theriogenology,2020,146:162-170.
[35]	SAMPAIO R V,SANGALLI J R,DE BEM T H C,et al.Catalytic inhibition of H3K9 me2 writers disturbs epigenetic marks during bovine nuclear reprogramming[J].Sci Rep,2020,10(1):11493.
[36]	RUAN Z Y,ZHAO X,LI Z D,et al.Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos[J].Reprod Domest Anim,2019,54(1):11-22.
[37]	YU J Y,VODYANIK M A,SMUGA-OTTO K,et al.Induced pluripotent stem cell lines derived from human somatic cells[J]. Science, 2007,318(5858):1917-1920.
[38]	LEWITZKY M,YAMANAKA S.Reprogramming somatic cells towards pluripotency by defined factors[J].Curr Opin Biotechnol, 2007, 18(5):467-473.
[39]	HUSSENET T,DALI S,EXINGER J,et al.SOX2 is an oncogene activated by recurrent 3q26.3 amplifications in human lung squamous cell carcinomas[J].PLoS One,2010,5(1):e8960.
[40]	WILBERTZ T,WAGNER P,PETERSEN K,et al.SOX2 gene amplification and protein overexpression are associated with better outcome in squamous cell lung cancer[J].Mod Pathol,2011,24(7):944-953.