Sains Malaysiana 44(10)(2015): 1481–1488

U6 snRNA is a Suitable Endogenous Control for microRNA-124 and -134 in Cultured Rat Hippocampal Neurons

(U6 snRNA adalah kawalan Endogen yang sesuai untuk microRNA-124 dan -134 dalam neuron hipokampus tikus berkultur)

AI SZE CHING & AZLINA AHMAD-ANNUAR*

Department of Biomedical Science, Faculty of Medicine, University of Malaya,

50603 Kuala Lumpur, Malaysia

Diserahkan: 21 Julai 2014/Diterima: 23 Jun 2015

ABSTRACT

As researchers seek to determine the cellular mechanisms underlying biological processes, they have turned to analyze the functional role of microRNAs to understand this process in details. Here, we investigated the expression pattern of two microRNAs, miR-124 and -134 in maturing neurons and found that the choice of endogenous controls influenced the observed expression levels of these microRNAs. We have cultured rat hippocampal neurons and performed quantitative PCR on the microRNAs using Taqman gene expression assays. The expression of miRNAs was normalised with selected endogenous controls. Using BestKeeper and NormFinder software, we found that 18S rRNA and 5S rRNA to be unsuitable as endogenous controls in this system, while normalising to U6 snRNA produced more consistent results. Our study would like to highlight the importance of empirically testing proposed endogenous controls in any model system before data interpretation is carried out.

Keywords: Endogenous controls; hippocampal neuron culture; microRNAs; miR-124; miR-134

ABSTRAK

Dalam usaha untuk menentukan mekanisme sel dalam proses biologi, para penyelidik telah beralih untuk menganalisis peranan fungsi mikroRNA untuk memahami proses ini dengan lebih mendalam. Kami telah menentukan tahap ekspresi untuk dua jenis mikroRNA, miR-124 dan -134 dalam sel-sel neuron yang semakin matang dan mendapati bahawa pilihan untuk gen endogen boleh mempengaruhi tahap ekspresi yang diperhatikan untuk mikroRNA tersebut. Kami telah mengkultur sel neuron daripada hipokampus tikus dan menggunakan teknik PCR kuantitatif dengan asai ekspresi gen Taqman. Ekspresi miRNA gen telah dipenormal dengan gen endogen. Dengan menggunakan perisian BestKeeper dan NormFinder, kami mendapati bahawa rRNA 18S dan rRNA 5S merupakan gen endogen yang tidak sesuai dalam sistem ini, manakala keputusan lebih konsisten apabila snRNA U6 digunakan. Hasil penyelidikan kami menunjukkan kepentingan untuk menggunakan pendekatan secara empirik semasa menimbangkan pilihan gen endogen yang ingin digunakan.

Kata kunci: Kawalan endogen; kultur neuron hipokampus; mikroRNAs; miR-124; miR-134

RUJUKAN

Allison, L.A., North, M.T., Murdoch, K.J., Romaniuk, P.J., Deschamps, S. & le Maire, M. 1993. Structural requirements of 5S rRNA for nuclear transport, 7S ribonucleoprotein particle assembly, and 60S ribosomal subunit assembly in Xenopus oocytes. Mol. Cell. Biol. 13(11): 6819-6831.

Andersen, C.L., Jensen, J.L. & Orntoft, T.F. 2004. Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64(15): 5245-5250.

Arvanitis, D.N., Jungas, T., Behar, A. & Davy, A. 2010. Ephrin-B1 reverse signaling controls a posttranscriptional feedback mechanism via miR-124. Mol. Cell Biol. 30(10): 2508-2517.

Bak, M., Silahtaroglu, A., Moller, M., Christensen, M., Rath, M.F., Skryabin, B., Tommerup, N. & Kauppinen, S. 2008. MicroRNA expression in the adult mouse central nervous system. RNA 14(3): 432-444.

Brett, J.O., Renault, V.M., Rafalski, V.A., Webb, A.E. & Brunet, A. 2011. The microRNA cluster miR-106b~25 regulates adult neural stem/progenitor cell proliferation and neuronal differentiation. Aging (Albany NY) 3(2): 108-124.

Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J. & Wittwer, C.T. 2009. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55(4): 611-622.

Caputo, V., Sinibaldi, L., Fiorentino, A., Parisi, C., Catalanotto, C., Pasini, A., Cogoni, C. & Pizzuti, A. 2011. Brain derived neurotrophic factor (BDNF) expression is regulated by microRNAs miR-26a and miR-26b allele-specific binding. PLoS One 6(12): e28656.

Cheng, L.C., Pastrana, E., Tavazoie, M. & Doetsch, F. 2009. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat. Neurosci. 12(4): 399-408.

Dotti, C.G., Sullivan, C.A. & Banker, G.A. 1988. The establishment of polarity by hippocampal neurons in culture. J. Neurosci. 8(4): 1454-1468.

Fiore, R., Khudayberdiev, S., Christensen, M., Siegel, G., Flavell, S.W., Kim, T.K., Greenberg, M.E. & Schratt, G. 2009. Mef2- mediated transcription of the miR379-410 cluster regulates activity-dependent dendritogenesis by fine-tuning Pumilio2 protein levels. EMBO J. 28(6): 697-710.

Genovesi, L.A., Anderson, D., Carter, K.W., Giles, K.M. & Dallas, P.B. 2012. Identification of suitable endogenous control genes for microRNA expression profiling of childhood medulloblastoma and human neural stem cells. BMC Res. Notes 5: 507.

Godlewski, J., Nowicki, M.O., Bronisz, A., Williams, S., Otsuki, A., Nuovo, G., Raychaudhury, A., Newton, H.B., Chiocca, E.A. & Lawler, S. 2008. Targeting of the Bmi-1 oncogene/ stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal. Cancer Res. 68(22): 9125- 9130.

Kang, H. & Schuman, E.M. 1996. A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity. Science 273(5280): 1402-1406.

Kim, J., Krichevsky, A., Grad, Y., Hayes, G.D., Kosik, K.S., Church, G.M. & Ruvkun, G. 2004. Identification of many microRNAs that copurify with polyribosomes in mammalian neurons. Proc. Natl. Acad Sci. USA 101(1): 360-365.

Krichevsky, A.M., King, K.S., Donahue, C.P., Khrapko, K. & Kosik, K.S. 2003. A microRNA array reveals extensive regulation of microRNAs during brain development. RNA 9(10): 1274-1281.

Lim, Q.E., Zhou, L., Ho, Y.K., Wan, G. & Too, H.P. 2011. snoU6 and 5S RNAs are not reliable miRNA reference genes in neuronal differentiation. Neuroscience 199: 32-43.

Livak, K.J. & Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4): 402-408.

Madhani, H.D., Bordonne, R. & Guthrie, C. 1990. Multiple roles for U6 snRNA in the splicing pathway. Genes Dev. 4(12B): 2264-2277.

Makeyev, E.V., Zhang, J., Carrasco, M.A. & Maniatis, T. 2007. The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol. Cell 27(3): 435-448.

Mishima, T., Yamamoto, K., Sugimoto, T., Sakakibara, K., Uehara, A. & Yoshii, S. 2010. Severe aortic regurgitation resulting from a downward displacement of anterior aortic annulus and fibrous strands in the bicuspid aortic valve. Ann. Thorac. Cardiovasc. Surg. 16(1): 57-59.

Paschou, M. & Doxakis, E. 2012. Neurofibromin 1 is a miRNA target in neurons. PLoS One 7(10): e46773.

Pfaffl, M.W., Tichopad, A., Prgomet, C. & Neuvians, T.P. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-- Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26(6): 509-515.

Ponomarev, E.D., Veremeyko, T., Barteneva, N., Krichevsky, A.M. & Weiner, H.L. 2011. MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-alpha-PU.1 pathway. Nat. Med. 17(1): 64-70.

Rabl, J., Leibundgut, M., Ataide, S.F., Haag, A. & Ban, N. 2011. Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1. Science 331(6018): 730-736.

Rajasethupathy, P., Fiumara, F., Sheridan, R., Betel, D., Puthanveettil, S.V., Russo, J.J., Sander, C., Tuschl, T. & Kandel, E. 2009. Characterization of small RNAs in Aplysia reveals a role for miR-124 in constraining synaptic plasticity through CREB. Neuron 63(6): 803-817.

Schratt, G.M., Nigh, E.A., Chen, W.G., Hu, L. & Greenberg, M.E. 2004. BDNF regulates the translation of a select group of mRNAs by a mammalian target of rapamycin-phosphatidylinositol 3-kinase-dependent pathway during neuronal development. J. Neurosci. 24(33): 7366-7377.

Schratt, G.M., Tuebing, F., Nigh, E.A., Kane, C.G., Sabatini, M.E., Kiebler, M. & Greenberg, M.E. 2006. A brain-specific microRNA regulates dendritic spine development. Nature 439(7074): 283-289.

Smirnova, L., Grafe, A., Seiler, A., Schumacher, S., Nitsch, R. & Wulczyn, F.G. 2005. Regulation of miRNA expression during neural cell specification. Eur. J. Neurosci. 21(6): 1469-1477.

Wang, W.X., Wilfred, B.R., Baldwin, D.A., Isett, R.B., Ren, N., Stromberg, A. & Nelson, P.T. 2008. Focus on RNA isolation: Obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue. Biochim. Biophys. Acta 1779(11): 749-757.

*Pengarang untuk surat-menyurat; email: azlina_aa@um.edu.my