异补骨脂甲素调控miR-124对小鼠胚胎干细胞向神经细胞分化的影响
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摘要
目的探讨异补骨脂甲素(isobavachin,IBA)调控微小RNA-124(miR-124)对小鼠J1胚胎干细胞向神经细胞分化的影响。方法采用qRT-PCR和免疫荧光染色分别检测IBA干预或抑制miR-124表达的J1细胞中miR-124、八聚体转录因子4(octamer transcription factor-4,OCT4)和神经微丝H(neurofilament-H, NFH)的表达。使用Targetscan在线预测、双荧光素酶报告基因和Western blot实验验证miR-124和锌指蛋白18(zinc finger and BTB domain containing 18, ZBTB18)的靶向关系。在J1细胞中转染pcDNA-ZBTB18或miR-124 mimics、ZBTB18 shRNA,免疫荧光染色检测细胞中OCT4和NFH的表达。结果 IBA可显著抑制J1细胞中OCT4蛋白表达,并上调miR-124和NFH表达;而抑制miR-124表达后所得结果相反。miR-124可靶向调控ZBTB18蛋白表达。在J1细胞中过表达ZBTB18,OCT4阳性表达率升高而NFH阳性表达率降低;敲减ZBTB18可在一定程度上减弱miR-124对IBA干预J1细胞OCT4和NFH表达的促进或抑制作用。结论 IBA能够促进J1细胞中miR-124表达,而miR-124可靶向调控ZBTB18,从而调节OCT4和NFH蛋白表达,这可能是IBA促进小鼠胚胎干细胞分化为神经细胞的作用机制。
Objective To investigate the effect of isobavachin(IBA) on neurogenesis of mouse J1 embryonic stem cells and explore the mechanism in light of microRNA-124(miR-124) regulation. Methods The expression of miR-124, octamer transcription factor-4(OCT4) and neurofilament-H(NFH) were detected by qRT-PCR in J1 cells after treatment with IBA or transfection with miR-124 inhibitor. Targetscan online prediction, dual-luciferase assay and Western blotting were used for verifying the targeting of miR-124 to Zinc finger and BTB domain containing 18(ZBTB18). The expression levels of OCT4 and NFH were detected with immunofluorescence assay in J1 cells transfected with pcDNA-ZBTB18 or with miR-124 mimics along with ZBTB18 shRNA. Results IBA significantly inhibited the expression of OCT4 and promoted the expression of miR-124 and NFH, and inhibition of miR-124 obviously enhanced OCT4 expression in J1 cells. The rsults of Targetscan online prediction, dual-luciferase assay and Western blotting indicated that miR-124 regulated the expression of ZBTB18 protein. Overexpression of ZBTB18 significantly increased the expression of OCT4 and decreased the expression of NFH in IBA-treated J1 cells; ZBTB18 knockdown in the cells attenuated the regulatory effects of miR-124 on the expression of OCT4 and NFH in IBA-treated J1 cells. Conclusion IBA promotes the expression of miR-124 in J1 cells, and the latter regulates the expression of OCT4 and NFH proteins by targeting ZBTB18, which can be a possible mechanism by which IBA promotes neurogenesis of mouse embryonic stem cells in vitro.
Objective To investigate the effect of isobavachin(IBA) on neurogenesis of mouse J1 embryonic stem cells and explore the mechanism in light of microRNA-124(miR-124) regulation. Methods The expression of miR-124, octamer transcription factor-4(OCT4) and neurofilament-H(NFH) were detected by qRT-PCR in J1 cells after treatment with IBA or transfection with miR-124 inhibitor. Targetscan online prediction, dual-luciferase assay and Western blotting were used for verifying the targeting of miR-124 to Zinc finger and BTB domain containing 18(ZBTB18). The expression levels of OCT4 and NFH were detected with immunofluorescence assay in J1 cells transfected with pcDNA-ZBTB18 or with miR-124 mimics along with ZBTB18 shRNA. Results IBA significantly inhibited the expression of OCT4 and promoted the expression of miR-124 and NFH, and inhibition of miR-124 obviously enhanced OCT4 expression in J1 cells. The rsults of Targetscan online prediction, dual-luciferase assay and Western blotting indicated that miR-124 regulated the expression of ZBTB18 protein. Overexpression of ZBTB18 significantly increased the expression of OCT4 and decreased the expression of NFH in IBA-treated J1 cells; ZBTB18 knockdown in the cells attenuated the regulatory effects of miR-124 on the expression of OCT4 and NFH in IBA-treated J1 cells. Conclusion IBA promotes the expression of miR-124 in J1 cells, and the latter regulates the expression of OCT4 and NFH proteins by targeting ZBTB18, which can be a possible mechanism by which IBA promotes neurogenesis of mouse embryonic stem cells in vitro.
引文
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[2] HU J,YANG Z Y,WANG J B,et al.Zinc chloride transiently maintains mouse embryonic stem cell pluripotency by activating stat3 signaling[J].PLoS ONE,2016,11(2):e0148994.DOI:10.1371/journal.pone.0148994.
[3] ANTONOV S A,MANUILOVA E S,DOLOTOV O V,et al.Effect of nerve growth factor on neural differentiation of mouse embryonic stem cells[J].Bull Exp Biol Med,2017,162(5):679-683.DOI:10.1007/s10517-017-3686-1.
[4] GUIDOLIN D,TORTORELLA C,MARCOLI M,et al.Neuroglobin,a factor playing for nerve cell survival[J].Int J Mol Sci,2016,17(11):E1817.DOI:10.3390/ijms17111817.
[5] LIU Z J,JIANG Y Y,LI X Y,et al.Embryonic stem cell-derived peripheral auditory neurons form neural connections with mouse central auditory neurons in vitro via the α2δ1 receptor[J].Stem Cell Rep,2018,11(1):157-170.DOI:10.1016/j.stemcr.2018.05.006.
[6] HUANG M L,MICHALAK A L,FISHER C J,et al.Small molecule antagonist of cell surface glycosaminoglycans restricts mouse embryonic stem cells in a pluripotent state[J].Stem Cells,2018,36(1):45-54.DOI:10.1002/stem.2714.
[7] WANG D Y,HU Y Z,KONG S S,et al.Promoting effects of isobavachin on neurogenesis of mouse embryonic stem cells were associated with protein prenylation[J].Acta Pharmacol Sin,2011,32(4):425-432.DOI:10.1038/aps.2011.5.
[8] WANG X,ZHAO Y N,XIAO Z F,et al.Alternative translation of OCT4 by an internal ribosome entry site and its novel function in stress response[J].Stem Cells,2009,27(6):1265-1275.DOI:10.1002/stem.58.
[9] CHIN T K,HARDING S E,EAGLES P A.Characterization of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH[J].Biochem J,1989,264(1):53-60.
[10] 李建宁,任维,刘一辉.外周神经系统中神经微丝蛋白研究现状[J].山东医药,2012,52(27):88-90.DOI:10.3969/j.issn.1002-266X.2012.27.035.LI J N,REN W,LIU Y H.Current status of research on neurofilament proteins in peripheral nervous system [J].Shandong Med J,2012,52(27):88-90.DOI:10.3969/j.issn.1002-266X.2012.27.035.
[11] CARRO M S,LIM W K,ALVAREZ M J,et al.The transcriptional network for mesenchymal transformation of brain tumours[J].Nature,2010,463(7279):318-325.DOI:10.1038/nature08712.
[12] TATARD V M,XIANG C M,BIEGEL J A,et al.ZNF238 is expressed in postmitotic brain cells and inhibits brain tumor growth[J].Cancer Res,2010,70(3):1236-1246.DOI:10.1158/0008-5472.CAN-09-2249.
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[22] LI D W,SHEN D C,TAI H F,et al.Neurofilaments in CSF as diagnostic biomarkers in motor neuron disease:a meta-analysis[J].Front Aging Neurosci,2016,8:290.DOI:10.3389/fnagi.2016.00290.
[23] 王伟英,李海明,戴艺民,等.植物锌指蛋白的功能研究进展[J].中国园艺文摘,2016,32(7):3-5,12.DOI:10.3969/j.issn.1672-0873.2016.07.002.WANG W Y,LI H M,DAI Y M,et al.Advances on the function of plant zinc finger protein[J].Chin Hort Abstr,2016,32(7):3-5,12.DOI:10.3969/j.issn.1672-0873.2016.07.002.
[24] ZHU C,CHEN G,ZHAO Y,et al.Regulation of the development and function of B cells by ZBTB transcription factors[J].Front Immunol,2018,9:580.DOI:10.3389/fimmu.2018.00580.
[25] MATHEW R,SEILER M P,SCANLON S T,et al.BTB-ZF factors recruit the E3 ligase cullin 3 to regulate lymphoid effector programs[J].Nature,2012,491(7425):618-621.DOI:10.1038/nature11548.
[26] MAEDA T.Regulation of hematopoietic development by ZBTB transcription factors[J].Int J Hematol,2016,104(3):310-323.DOI:10.1007/s12185-016-2035-x.
[27] 赵倩倩,班博,邵倩.锌指蛋白ZBTB38调控生长发育相关研究进展[J].中国儿童保健杂志,2018,26(1):44-47.ZHAO Q Q,BAN B,SHAO Q.Research advances on zinc finger protein ZBTB38 regulating growth and development[J].Chin J Child Health Care,2018,26(1):44-47.
[28] 刘淦,陈李斌佶,谢志芳,等.锌指蛋白ZBTB20生物学功能的研究进展[J].第二军医大学学报,2015,36(5):525-529.DOI:10.3724/SP.J.1008.2015.00525.LIU G,CHEN L,XIE Z F,et al.Biological functions of zinc finger protein ZBTB20:recent advance[J].Acad J Second Mil Med Univ,2015,36(5):525-529.DOI:10.3724/SP.J.1008.2015.00525.
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[30] VAN DER SCHOOT V,DE MUNNIK S,VENSELAAR H,et al.Toward clinical and molecular understanding of pathogenic variants in the ZBTB18 gene[J].Mol Genet Genomic Med,2018,6(3):393-400.DOI:10.1002/mgg3.387.