MiR-381下调Hes1表达调控神经干细胞增殖和向神经元的分化
|
摘要
目的:探讨miRNA-381在神经干细胞(neural stem cell,NSCs)的体外增殖、分化中的作用及相关机制。方法:采用光镜观察法、免疫组化法对原代获取及诱导分化后的NSCs予以鉴定;QPCR及Western blot法检测miRNA-381、nestin、β-tubulin-Ⅲ和Hes1的mRNA及蛋白表达;CCK-8法检测不同时间点NSCs的增殖情况;荧光素酶报告基因法验证miR-381对Hes1野生型及突变型3'非编码结合区的靶向作用。结果:原代培养细胞呈明显的神经球形态,且高表达nestin蛋白,经b FGF诱导后则高表达β-tubulin-Ⅲ;miR-381转染后,NSCs中miR-381、nestin、β-tubulin-Ⅲ的mRNA和蛋白水平均明显升高(p<0.001),免疫荧光法也进一步验证miR-381转染后的NSCs其β-tubulin-Ⅲ的荧光强度显著增强;此外,miR-381转染24 h、48 h及72 h后,NSCs的增殖能力均高于阴性对照组(p24h<0.01,p48h<0.001,p72h<0.001);荧光素酶法结果显示miR-381能显著降低野生型Hes1载体的3'非编码区的荧光素酶活性,而Hes1基因载体的突变型3'UTR的荧光素酶活性不受到miR-381的影响(p<0.001);另外,miR-381过表达能明显降低Hes1蛋白的表达;而二者共转染的NSCs增殖能力在转染后24 h、48 h及72 h,均明显低于单纯miR-381转染组(p<0.01或p<0.001);同时,共转染后β-tubulin Ⅲ的mRNA及蛋白水平均明显低于单纯miR-381转染组(p<0.001)。结论:miR-381通过下调Hes1表达促进NSCs的增殖和向神经元的分化。
Objective: To explore the role of miRNA-381 in the proliferation and differentiation of neural stem cells(NSCs), as well as the relative mechanisms. Methods: Observation under light microscope and immunohistochemistry were used to identify NSCs.QPCR and Western blot were selected to detect both mRNA levels and protein levels of miRNA-381, nestin, β-tubulin-Ⅲ and Hes1 expression. The proliferation of NSCs was detected by CCK-8 assay, and Luciferase reporter gene assay was processed to measure effects of miR-381 on wild type/mutant 3' UTR of Hes1 vector. Results: Primary cultured neuronal cells showed typical morphology of neurosphere and highly expressed nestin protein, while cells presented high expression of β-tubulin-Ⅲ after b FGF induction. The mRNA and protein levels of miR-381, nestin, β-tubulin-Ⅲ in NSCs after miR-381 transfection were significantly higher than that of negative control group(p < 0.001), besides, the fluorescence intensity of β-tubulin-Ⅲ was also obviously enhanced. What's more, the proliferation of NSCs after 24 h, 48 h and 72 h transfection by miR-381 was statistical higher than control group(p24h < 0.01, p48 h <0.001, p72 h <0.001); luciferase enzyme results showed that the luciferase activity of 3'UTR of wild-type Hes1 vector was significantly reduced by miR-381 over expression, however, the 3'UTR of mutant Hes1 vector was not influenced by miR-381(p < 0.001); additionally, miR-381 over expression obviously reduced HES1 protein expression, and the proliferative ability of NSCs, as well as the mRNA and protein expression of β-tubulin Ⅲ, were down-regulated by miR-381 and Hes1 co-transfection compared with that of miR-381 single transfection group(p < 0.01, p < 0.001). Conclusions: MiR-381 promotes the proliferation of NSCs and the differentiation into neurons via down-regulating Hes1 expression.
Objective: To explore the role of miRNA-381 in the proliferation and differentiation of neural stem cells(NSCs), as well as the relative mechanisms. Methods: Observation under light microscope and immunohistochemistry were used to identify NSCs.QPCR and Western blot were selected to detect both mRNA levels and protein levels of miRNA-381, nestin, β-tubulin-Ⅲ and Hes1 expression. The proliferation of NSCs was detected by CCK-8 assay, and Luciferase reporter gene assay was processed to measure effects of miR-381 on wild type/mutant 3' UTR of Hes1 vector. Results: Primary cultured neuronal cells showed typical morphology of neurosphere and highly expressed nestin protein, while cells presented high expression of β-tubulin-Ⅲ after b FGF induction. The mRNA and protein levels of miR-381, nestin, β-tubulin-Ⅲ in NSCs after miR-381 transfection were significantly higher than that of negative control group(p < 0.001), besides, the fluorescence intensity of β-tubulin-Ⅲ was also obviously enhanced. What's more, the proliferation of NSCs after 24 h, 48 h and 72 h transfection by miR-381 was statistical higher than control group(p24h < 0.01, p48 h <0.001, p72 h <0.001); luciferase enzyme results showed that the luciferase activity of 3'UTR of wild-type Hes1 vector was significantly reduced by miR-381 over expression, however, the 3'UTR of mutant Hes1 vector was not influenced by miR-381(p < 0.001); additionally, miR-381 over expression obviously reduced HES1 protein expression, and the proliferative ability of NSCs, as well as the mRNA and protein expression of β-tubulin Ⅲ, were down-regulated by miR-381 and Hes1 co-transfection compared with that of miR-381 single transfection group(p < 0.01, p < 0.001). Conclusions: MiR-381 promotes the proliferation of NSCs and the differentiation into neurons via down-regulating Hes1 expression.
引文
[1]Gonzalez C,Bonilla S,Flores AI,et al.An Update on Human Stem Cell-Based Therapy in Parkinson's Disease[J].Curr Stem Cell Res Ther,2016,11(7):561-568
[2]Shalaby T,Fiaschetti G,Baumgartner M,et al.Micro RNA signatures as biomarkers and therapeutic target for CNS embryonal tumors:the pros and the cons[J].Int J Mol Sci,2014,15(11):21554-21586
[3]Singh DK,Bose S,Kumar S.Role of microRNA in regulating cell signaling pathways,cell cycle,and apoptosis in non-small cell lung cancer[J].Curr Mol Med,2016[Epub ahead of print]
[4]Li S,Zhao W,Xu Q,et al.Micro RNA-765 regulates neural stem cell proliferation and differentiation by modulating Hes1 expression[J].Am J Transl Res,2016,8(7):3115-3123
[5]K atz S,Cussigh D,Urban N,et al.A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States[J].Cell Rep,2016,17(5):1383-1398
[6]De Filippis L,Binda E.Concise review:self-renewal in the central nervous system:neural stem cells from embryo to adult[J].Stem Cells Transl Med,2012,1(4):298-308
[7]Drago D,Basso V,Gaude E,et al.Metabolic determinants of the immune modulatory function of neural stem cells[J].J Neuro inflammation,2016,13(1):232
[8]Morgado AL,Rodrigues CM,Sola S.Micro RNA-145 Regulates Neural Stem Cell Differentiation Through the Sox2-Lin28/let-7 Signaling Pathway[J].Stem Cells,2016,34(5):1386-1395
[9]Wienholds E,Kloosterman WP,Miska E,et al.Micro RNA expression in zebrafish embryonic development[J].Science,2005,309(5732):310-311
[10]Krichevsky AM,King KS,Donahue CP,et al.A microRNA array reveals extensive regulation of microRNAs during brain development[J].Rna,2003,9(10):1274-1281
[11]Tang H,Liu X,Wang Z,et al.Interaction of hsa-miR-381 and glioma suppressor LRRC4 is involved in glioma growth[J].Brain Res,2011,1390:21-32
[12]Kuzontkoski PM,Mulligan-Kehoe MJ,Harris BT,et al.Inhibitor of DNA binding-4 promotes angiogenesis and growth of glioblastoma multiforme by elevating matrix GLA levels[J].Oncogene,2010,29(26):3793-3802
[13]Garg N,Po A,Miele E,et al.microRNA-17-92 cluster is a direct Nanog target and controls neural stem cell through Trp53inp1[J].Embo J,2013,32(21):2819-2832
[14]Li X,Feng R,Huang C,et al.Micro RNA-351 regulates TMEM 59(DCF1)expression and mediates neural stem cell morphogenesis[J].RNA Biol,2012,9(3):292-301
[15]Dailey DD,Anfinsen KP,Pfaff LE,et al.HES1,a target of Notch signaling,is elevated in canine osteosarcoma,but reduced in the most aggressive tumors[J].BMC Vet Res,2014,9:130
[16]Lillycrop KA,Costello PM,Teh AL,et al.Association between perinatal methylation of the neuronal differentiation regulator HES1and later childhood neurocognitive function and behaviour[J].Int J Epidemiol,2015,44(4):1263-1276
[17]Wang W,Wang P,Li S,et al.Methylprednisolone inhibits the proliferation and affects the differentiation of rat spinal cord-derived neural progenitor cells cultured in low oxygen conditions by inhibiting HIF-1alpha and Hes1 in vitro[J].Int J Mol Med,2014,34(3):788-795
[18]Kobayashi T,Kageyama R.Expression dynamics and functions of Hes factors in development and diseases[J].Curr Top Dev Biol,2014,110:263-283
[19]Wang C,Bian W,Xia C,et al.Visualization of b HLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET[J].Cell Res,2006,16(6):585-598
[20]Imayoshi I,Ishidate F,Kageyama R.Real-time imaging of b HLH transcription factors reveals their dynamic control in the multipotency and fate choice of neural stem cells[J].Front Cell Neurosci,2015,9:288
[2]Shalaby T,Fiaschetti G,Baumgartner M,et al.Micro RNA signatures as biomarkers and therapeutic target for CNS embryonal tumors:the pros and the cons[J].Int J Mol Sci,2014,15(11):21554-21586
[3]Singh DK,Bose S,Kumar S.Role of microRNA in regulating cell signaling pathways,cell cycle,and apoptosis in non-small cell lung cancer[J].Curr Mol Med,2016[Epub ahead of print]
[4]Li S,Zhao W,Xu Q,et al.Micro RNA-765 regulates neural stem cell proliferation and differentiation by modulating Hes1 expression[J].Am J Transl Res,2016,8(7):3115-3123
[5]K atz S,Cussigh D,Urban N,et al.A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States[J].Cell Rep,2016,17(5):1383-1398
[6]De Filippis L,Binda E.Concise review:self-renewal in the central nervous system:neural stem cells from embryo to adult[J].Stem Cells Transl Med,2012,1(4):298-308
[7]Drago D,Basso V,Gaude E,et al.Metabolic determinants of the immune modulatory function of neural stem cells[J].J Neuro inflammation,2016,13(1):232
[8]Morgado AL,Rodrigues CM,Sola S.Micro RNA-145 Regulates Neural Stem Cell Differentiation Through the Sox2-Lin28/let-7 Signaling Pathway[J].Stem Cells,2016,34(5):1386-1395
[9]Wienholds E,Kloosterman WP,Miska E,et al.Micro RNA expression in zebrafish embryonic development[J].Science,2005,309(5732):310-311
[10]Krichevsky AM,King KS,Donahue CP,et al.A microRNA array reveals extensive regulation of microRNAs during brain development[J].Rna,2003,9(10):1274-1281
[11]Tang H,Liu X,Wang Z,et al.Interaction of hsa-miR-381 and glioma suppressor LRRC4 is involved in glioma growth[J].Brain Res,2011,1390:21-32
[12]Kuzontkoski PM,Mulligan-Kehoe MJ,Harris BT,et al.Inhibitor of DNA binding-4 promotes angiogenesis and growth of glioblastoma multiforme by elevating matrix GLA levels[J].Oncogene,2010,29(26):3793-3802
[13]Garg N,Po A,Miele E,et al.microRNA-17-92 cluster is a direct Nanog target and controls neural stem cell through Trp53inp1[J].Embo J,2013,32(21):2819-2832
[14]Li X,Feng R,Huang C,et al.Micro RNA-351 regulates TMEM 59(DCF1)expression and mediates neural stem cell morphogenesis[J].RNA Biol,2012,9(3):292-301
[15]Dailey DD,Anfinsen KP,Pfaff LE,et al.HES1,a target of Notch signaling,is elevated in canine osteosarcoma,but reduced in the most aggressive tumors[J].BMC Vet Res,2014,9:130
[16]Lillycrop KA,Costello PM,Teh AL,et al.Association between perinatal methylation of the neuronal differentiation regulator HES1and later childhood neurocognitive function and behaviour[J].Int J Epidemiol,2015,44(4):1263-1276
[17]Wang W,Wang P,Li S,et al.Methylprednisolone inhibits the proliferation and affects the differentiation of rat spinal cord-derived neural progenitor cells cultured in low oxygen conditions by inhibiting HIF-1alpha and Hes1 in vitro[J].Int J Mol Med,2014,34(3):788-795
[18]Kobayashi T,Kageyama R.Expression dynamics and functions of Hes factors in development and diseases[J].Curr Top Dev Biol,2014,110:263-283
[19]Wang C,Bian W,Xia C,et al.Visualization of b HLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET[J].Cell Res,2006,16(6):585-598
[20]Imayoshi I,Ishidate F,Kageyama R.Real-time imaging of b HLH transcription factors reveals their dynamic control in the multipotency and fate choice of neural stem cells[J].Front Cell Neurosci,2015,9:288