miR-140在内耳毛细胞中的表达及其对突触蛋白Dynamin-1的调控作用
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摘要
目的研究miR-140在正常小鼠内耳毛细胞的表达分布及其对编码突触蛋白Dynamin-1的Dnm1基因的靶向调控作用。方法取正常成年C57BL/6小鼠耳蜗基底膜,免疫荧光染色和原位杂交法观察Dynamin-1与miR-140的表达定位情况。构建荧光素酶报告基因载体转染293T细胞,通过荧光素酶活性变化明确Dnm1是否是miR-140的靶基因。携带miR-140前体及空载的腺相关病毒(adenoassociated virus,AAV)通过圆窗膜注射传递到耳蜗中,14 d后RT-PCR检测miR-140及Dnm1的mRNA表达水平,Western blot检测Dynamin-1蛋白的表达水平。结果 miR-140表达于内外毛细胞细胞质,Dynamin-1主要表达于内毛细胞细胞质;荧光素酶活性检测结果显示,miR-140抑制Dnm1的3'UTR荧光素酶报告基因活性(P<0.05)。RT-PCR及Western blot检测结果显示:转染AAV-miR-140后miR-140的相对表达量明显升高,Dnm1的mRNA及其蛋白表达水平显著降低(P<0.05)。结论Dnm1是miR-140的靶基因,且miR-140可以抑制内耳毛细胞中Dynamin-1的表达,表明miR-140可能通过抑制Dynamin-1蛋白负向调控内毛细胞突触囊泡内吞过程,进而影响听觉信号的传递。
Objective To investigate the expression and distribution of miR-140 in mouse inner ear hair cells and its targeting regulation on Dnm1,the gene encoding synaptophysin Dynamin-1. Methods After the cochlear basement membrane was harvested from healthy adult C57 BL/6 J mice( 8 weeks old),immunofluorescence staining and in situ hybridization were used to observe the expression and location of Dynamin-1 and miR-140. The luciferase reporter gene vector was constructed and then transfected into 293 T cells to determine whether Dnm1 is the target gene of miR-140. Adeno-associated viral vectors carrying miR-140 precursor and blank gene were injected into the cochlea of the mice via circular window membrane.After 14 d,the mRNA expression of miR-140 and Dnm1 was detected by RT-PCR,and Dynamin-1 protein was detected by Western blotting. Results miR-140 was expressed in the cytoplasm of inner and outer hair cells,and Dynamin-1 was mainly expressed in the cytoplasm of inner hair cells. The luciferase activity assay showed that miR-140 could inhibit the enzyme reporter activity of 3' UTR of Dnm1( P < 0. 05). The mRNA expression of miR-140 was significantly increased after transfection of AAV-miR-140,while the mRNA level of Dnm1 and its protein level were decreased significantly( P < 0. 05). Conclusion Dnm1 is the target gene of miR-140,and the latter can inhibit the expression of Dynamin-1 in inner ear hair cells,suggesting that it may negatively regulate the process of synaptic vesicle endocytosis of inner hair cells by inhibiting Dynamin,and then affect the transmission of auditory signals.
Objective To investigate the expression and distribution of miR-140 in mouse inner ear hair cells and its targeting regulation on Dnm1,the gene encoding synaptophysin Dynamin-1. Methods After the cochlear basement membrane was harvested from healthy adult C57 BL/6 J mice( 8 weeks old),immunofluorescence staining and in situ hybridization were used to observe the expression and location of Dynamin-1 and miR-140. The luciferase reporter gene vector was constructed and then transfected into 293 T cells to determine whether Dnm1 is the target gene of miR-140. Adeno-associated viral vectors carrying miR-140 precursor and blank gene were injected into the cochlea of the mice via circular window membrane.After 14 d,the mRNA expression of miR-140 and Dnm1 was detected by RT-PCR,and Dynamin-1 protein was detected by Western blotting. Results miR-140 was expressed in the cytoplasm of inner and outer hair cells,and Dynamin-1 was mainly expressed in the cytoplasm of inner hair cells. The luciferase activity assay showed that miR-140 could inhibit the enzyme reporter activity of 3' UTR of Dnm1( P < 0. 05). The mRNA expression of miR-140 was significantly increased after transfection of AAV-miR-140,while the mRNA level of Dnm1 and its protein level were decreased significantly( P < 0. 05). Conclusion Dnm1 is the target gene of miR-140,and the latter can inhibit the expression of Dynamin-1 in inner ear hair cells,suggesting that it may negatively regulate the process of synaptic vesicle endocytosis of inner hair cells by inhibiting Dynamin,and then affect the transmission of auditory signals.
引文
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[2]RAIMONDI A,FERGUSON S M,LOU X,et al.Overlapping role of dynamin isoforms in synaptic vesicle endocytosis[J].Neuron,2011,70(6):1100-1114.DOI:10.1016/j.neuron.2011.04.031.
[3]BOUMIL R M,LETTS V A,ROBERTS M C,et al.A missense mutation in a highly conserved alternate exon of dynamin-1 causes epilepsy in fitful mice[J].PLo S Genet,2010,6(8):e1001046.DOI:10.1371/journal.pgen.1001046.
[4]蔡婷,陈知己,顾翔,等.Dynamin在不同年龄段小鼠耳蜗中的表达[J].中华耳鼻咽喉头颈外科杂志,2015,50(7):583-586.DOI:10.3760/cma.j.issn.1673-0860.2015.07.012.CAI T,CHEN Z J,GU X,et al.Expression of dynamin in mouse cochlea of different ages[J].Chin J Otorhinolaryngol Head Neck Surg,2015,50(7):583-586.DOI:10.3760/cma.j.issn.1673-0860.2015.07.012.
[5]USHAKOV K,RUDNICKI A,AVRAHAM K B.MicroRNAs in sensorineural diseases of the ear[J].Front Mol Neurosci,2013,6:52.DOI:10.3389/fnmol.2013.00052.
[6]HUANG W,LI M D.Nicotine modulates expression of miR-140*,which targets the 3'-untranslated region of dynamin 1gene(Dnm1)[J].Int J Neuropsychopharmacol,2009,12(4):537-546.DOI:10.1017/S1461145708009528.
[7]PATEL M,HU B H.MicroRNAs in inner ear biology and pathogenesis[J].Hear Res,2012,287(1/2):6-14.DOI:10.1016/j.heares.2012.03.008.
[8]AKIL O,ROUSE S L,CHAN D K,et al.Surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane[J].J Vis Exp,2015(97):52187.DOI:10.3791/52187.
[9]徐延军,胡吟燕,翟所强,等.耳后入路圆窗膜显微注射小鼠耳蜗基因转染新途径的研究[J].听力学及言语疾病杂志,2009,17(3):279-282.DOI:10.3969/j.issn.1006-7299.2009.03.023.XU Y J,HU Y Y,ZHAI S Q,et al.The study of a new approach to postauricular microinjection via the round window membrane for cochlear gene transfection in mouse[J].J Audiol Speech Pathol,2009,17(3):279-282.DOI:10.3969/j.issn.1006-7299.2009.03.023.
[10]蔡婷,袁伟.突触内吞关键蛋白Dynamin在小鼠耳蜗内毛细胞中的表达研究[J].中华耳科学杂志,2014,12(4):643-646.DOI:10.3969/j.issn.1672-2922.2014.04.033.CAI T,YUAN W.Study on the expression of synaptic endocytosis key protein dynamin in mice cochlear inner hair cells[J].Chin J Otology,2014,12(4):643-646.DOI:10.3969/j.issn.1672-2922.2014.04.033.
[11]ZHANG R,MA J,YAO J.Molecular mechanisms of the cartilage-specific microRNA-140 in osteoarthritis[J].Inflamm Res,2013,62(10):871-877.DOI:10.1007/s00011-013-0654-8.
[12]YU L,LU Y,HAN X,et al.microRNA-140-5p inhibits colorectal cancer invasion and metastasis by targeting ADAMTS5 and IGFBP5[J].Stem Cell Res Ther,2016,7(1):180.DOI:10.1186/s13287-016-0438-5.
[13]YUAN Y,SHEN Y,XUE L,et al.miR-140 suppresses tumor growth and metastasis of non-small cell lung cancer by targeting insulin-like growth factor 1 receptor[J].PLo S ONE,2013,8(9):e73604.DOI:10.1371/journal.pone.0073604.
[14]GU R,SUN Y F,WU M F,et al.Biological roles of microRNA-140 in tumor growth,migration,and metastasis of osteosarcoma in vivo and in vitro[J].Tumour Biol,2016,37(1):353-360.DOI:10.1007/s13277-015-3801-8.
[15]WANG X R,ZHANG X M,ZHEN J,et al.MicroRNA expression in the embryonic mouse inner ear[J].Neuroreport,2010,21(9):611-617.DOI:10.1097/WNR.0b013e328338864b.
[16]PANG J,XIONG H,LIN P,et al.Activation of miR-34a impairs autophagic flux and promotes cochlear cell death via repressing ATG9A:implications for age-related hearing loss[J].Cell Death Dis,2017,8(10):e3079.DOI:10.1038/cddis.2017.462.
[17]LI Y,LI A,WU J,et al.MiR-182-5p protects inner ear hair cells from cisplatin-induced apoptosis by inhibiting FOXO3a[J].Cell Death Dis,2016,7(9):e2362.DOI:10.1038/cddis.2016.246.
[18]LI Y,PENG A,GE S,et al.miR-204 suppresses cochlear spiral ganglion neuron survival in vitro by targeting TMPRSS3[J].Hear Res,2014,314:60-64.DOI:10.1016/j.heares.2014.05.002.