Expression of recombinant plasmid of homo heterogeneous nuclear ribonucleoprotein E1 in SH-SY5Y cells

doi:10.3969/j.issn.2095-4344.2014.42.009

Abstract

Abstract:

BACKGROUND: The functions of homo heterogeneous ribonucleoprotein E1 are very wide. It can participate in the expression of skeleton proteins in the nervous system.
OBJECTIVE: To construct the recombinant plasmid of homo heterogeneous ribonucleoprotein E1 and observe its expression in nerve cells for further studying the functions of it in neurocytes.
METHODS: Using pcDNATM4/His C, the homo heterogeneous ribonucleoprotein E1 was subcloned into recombinant plasmid E1-pcDNATM4/His C, followed by enzyming and sequencing. After SH-SY5Y cells were transfected with the recombinant plasmid, western blot analysis and real time RT-PCR were used to detect the expression of homo heterogeneous ribonucleoprotein E1 in SH-SY5Y cells. And the growth of SH-SY5Y cells was observed.
RESULTS AND CONCLUSION: We successfully constructed the eukaryotic expressed vector of homo heterogeneous ribonucleoprotein E1. The recombinant plasmids were verified to express in SH-SY5Y cells correctly at mRNA and protein levels. And SH-SY5Y cells generated quickly after homo heterogeneous ribonucleoprotein E1 was over-expressed. The homo heterogeneous ribonucleoprotein E1 is an important protein in neural development. And this vector offers the premise for further studying its function in nervous system.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: ribonucleoprotein, heterogeneous, neurons, transfection

CLC Number:

R318

Huo Li-rong, Wang Lan-ying, Zou Jun-hua, Zhong Nan . Expression of recombinant plasmid of homo heterogeneous nuclear ribonucleoprotein E1 in SH-SY5Y cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(42): 6769-6774.

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References

[1] Akindahunsi AA, Bandiera A, Manzini G.Vertebrate 2xRBD hnRNP proteins:a comparative analysis of genome, mRNA and protein sequences. Comput Biol Chem.2005;29(1): 13-23.
[2] Choi HS, Hwang CK, Song KY, et al. Poly(C)-binding proteins as transcriptional regulators of gene expression. Biochem Bioph Res Co.2009; 380(3): 431-436.
[3] Cho SJ, Jung YS, Chen X. Poly (C)-binding protein 1 regulates p63 expression through mRNA stability. PLoS One.2013;8 (8): e71724.
[4] Kim JH, Hahm B, Kim YK, et al. Protein–protein interaction among hnRNPs shuttling between nucleus and cytoplasm. J Mol Biol.2000;298(3): 395-405.
[5] Sola I, Mateos-Gomez PA, Almazan F,et al. RNA-RNA and RNA-protein interactions in coronavirus replication and transcription. RNA Biol.2011;8(2): 237-248.
[6] Xiao XL, Tang YS, Janet MY, et al. Isolation and characterization of a folate receptor mRNA-binding trans-factor from human placenta.J Biol Chem.2001;276(44): 41510-41517.
[7] Chkheidze AN, Liebhaber SA.A Novel Set of Nuclear Localization Signals Determine Distributions of the αCP RNA-Binding Proteins. Mol Cell Biol.2003;23(23): 8405-8415.
[8] Gamarnik A, Andino R. Two functional complexes formed by KH domain containing proteins with the 5’noncoding region of poliovirus RNA. RNA.1997;3(8): 882-892.
[9] Grüllich C,Duvoisin RM, Wiedmann M. Inhibition of 15-lipoxygenase leads to delayed organelle degradation in the reticulocyte. FEBS Lett.2001;489 (1): 51-54.
[10] Ostareck DH, Ostareck-Lederer A, Wilm M, et al. mRNAsilencing in erythroid differentiation: hnRNPK and hnRNP E1 regulate 15-lipoxygenase translation from the 3’end. Cell. 1997;89(4):597-606.
[11] Ghanem LR, Chatterji P, Liebhaber SA. Specific enrichment of the RNA-binding proteins PCBP1 and PCBP2 in chief cells of the murine gastric mucosa. Gene Expr Patterns. 2014;14(2): 78-87.
[12] Leffers H, Dejgaard K, Celis J. Characterisation of two major cellular poly (rC)-binding human proteins, each containing three Khomologous (KH) domains. Eur J Biochem. 1995;230(2): 447-453.
[13] Kuhn H, Heydeck D, Brinckman R, et al. Regulation of cellular 15-lipoxygenase activity on pretranslational, translational, and posttranslational levels. Lipids. 1999; 34(Suppl): S273-S279.
[14] Reimann I, Huth A, Thiele H, et al. Suppression of 15-lipoxygenase synthesis by hnRNP E1 is dependent on repetitive nature of LOX mRNA 30-UTR control element DICE. J Mol Biol.2002;315(5): 965-974.
[15] Walter BL,Parsley TB, Ehrenfeld E, et al. Distinct poly(rC) binding protein KH domain determinants for poliovirus translation initiation and viral rna replication. J Virol.2002; 76(23):12008-12022.
[16] Yeap BB, Voon DC, Vivian JP,et al. Novel binding of HuR and poly(C)-binding protein to a conserved UC-rich motif within the 30-untranslated region of the androgen receptor messenger RNA. J Biol Chem.2002; 277(30): 27183-27192.
[17] Mili S., Shu H.J.,Zhao Y. and Pinol-Roma S. Distinct RNP complexes of shuttling hnRNP proteins with pre-mRNA and mRNA: candidate intermediates in formation and export of mRNA. Mol Cell Biol. 2001;21(21):7307-7319.
[18] Shi HF, Bencze KZ, Stemmler TL, et al. A cytosolic iron chaperone that delivers iron to ferritin. Science.2008;320 (5880):1207-1210.
[19] Leidgens S, Bullough KZ, Shi H, et al. Each member of the poly-r(C)-binding protein 1(PCBP)family exhibits iron chaperone activity toward ferritin. J Biol Chem. 2013; 288(24): 17791- 17802.
[20] Yanatori I, Yasui Y, Tabuchi M,et al.Chaperone protein involved in transmembrane transport of iron. Biochem J. 2014 May 23. [Epub ahead of print].
[21] Thyagarajan A, Szaro BG. Dynamic endogenous association of neurofilament mRNAs with K-homology domain ribonucleoproteins in developing cerebral cortex. Brain Res. 2008;1189:33-42.
[22] Song Q, Sheng W, Zhang X,et al.ILEI drives epithelial to mesenchymal transition and metastatic progression in the lung cancer cell line A549. Tumour Biol. 2014;35(2): 1377-1382.
[23] Zhang HY, Dou KF. PCBP1 is an important mediator of TGF-β-induced epithelial to mesenchymal transition in gall bladder cancer cell line GBC-SD. Mol Biol Rep. 2014 Jun 3. [Epub ahead of print]
[24] 霍丽蓉,邹俊华,钟南.酵母双杂交系统3筛选人源核不均一核糖核蛋白E1的功能伙伴[J].中国组织工程研究与临床康复, 2010, 14(20):3714-3717.
[25] Makeyev AV, Liebhaber SA. The poly(C)-binding proteins: A multiplicity of functions and a search for mechanisms. RNA. 2002;8: 265-278.
[26] Thyagarajan A, Szaro BG. Phylogenetically conserved binding of specific K homology domain proteins to the 3’-untranslated region of the vertebrate middle neurofilament mRNA. J Biol Chem.2004;279 (48):49680-49688.
[27] Kosturko LD,Maggipinto MJ,Korza G,et al.Heterogeneous nuclear ribonucleoprotein (hnRNP) E1 binds to hnRNP A2 and inhibits translation of A2 response element mRNAs. Mol Biol Cell.2006;17 (8): 3521-3533.