Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models.
Elbashir, S.M.; Harborth, J.; Lendeckel, W.; Yalch, A.; Weber, K.; Tuschl, T. Duplex of 21-nucleotide RNAs mediates RNA interference in cultured mammalian cells. Nature 2001, 411, 494–498.
[4]
Wianny, F.; Zrnicka-Goetz, M. Specific interference with gene function by double-stranded RNA in early mouse development. Nature Cell Biol. 2000, 2, 70–75, doi:10.1038/35000016.
[5]
Sharma, S.; Rao, A. RNAi screening: Tips and techniques. Nat. Immunol. 2009, 10, 799–804, doi:10.1038/ni0809-799.
[6]
Seyhan, A.A.; Ryan, T.E. RNAi screening for the discovery of novel modulators of human diseases. Curr. Pharmaceut. Biotechnol. 2010, 11, 735–756, doi:10.2174/138920110792927766.
[7]
Mohr, S.; Bakal, C.; Perrimon, N. Genomic screening with RNAi: Results and challenges. Ann. Rev. Biochem. 2010, 79, 37–64, doi:10.1146/annurev-biochem-060408-092949.
[8]
Sood, P.; Krek, A.; Zavolan, M.; Macino, G.; Rajewsky, N. Cell-type-specific signatures of microRNAs on target mRNA expression. Proc. Natl. Acad. Sci. USA 2006, 103, 2746–2751.
[9]
Baek, D.; Villen, J.; Shin, C.; Camargo, F.D.; Gygi, S.P.; Bartel, D.P. The impact of microRNAs on protein output. Nature 2008, 455, 64–71.
Schena, M.; Shalon, D.; Davis, R.W.; Brown, P.O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995, 270, 467–470.
[20]
Cheung, V.G.; Morley, M.; Aguilar, F.; Massimi, A.; Kucherlapati, R.; Childs, G. Making and Reading Microarrays. Available online: http://www.rose-hulman.edu/~ahmed/making%20and%20reading%20cdna%20microarrays.pdf (accessed on 8 February 2013).
[21]
Barbulovic-Nad, I.; Lucente, M.; Sun, Y.; Zhang, M.; Wheeler, A.R.; Bussmann, M. Bio-microarray fabrication techniques—A review. Crit. Rev. Biotechnol. 2006, 26, 237–259, doi:10.1080/07388550600978358.
[22]
Ziauddin, J.; Sabatini, D.M. Micro-array of cells expressing defined cDNAs. Nature 2001, 411, 107–110, doi:10.1038/35075114.
Karin, M.; Greten, F.R. NF-κB: Linking inflammation and immunity to cancer development and progression. Nat. Rev. Immunol. 2005, 5, 739–759.
[26]
Karin, M. Nuclear factor-κB in cancer development and progression. Nature , 2006 441, 431–436.
[27]
Hoffmann, A.; Baltimore, D. Circuitry of nuclear factor κB signaling. Immunol. Rev. 2006, 210, 171–186, doi:10.1111/j.0105-2896.2006.00375.x.
[28]
Oeckinghaus, A.; Ghosh, S. The NF-κB family of transcription factors and its regulation. Cold Spring Harb. Perspct. Biol. 2009, doi:10.1101/cshprespect.a000034.
He, L.; Thomson, J.M.; Hemann, M.T.; Hernando-Monge, E.; Mu, D.; Goodson, S.; Powers, S.; Cordon-Cardo, C.; Lowe, S.W.; Hannon, G.J.; Hammond, S.M. A microRNA polycistron as a potential human oncogene. Nature 2005, 435, 828–833, doi:10.1038/nature03552.
[31]
Mraz, M.; Pospisilova, S.; Malinova, K.; Slapak, I.; Mayer, J. MicroRNAs in chronic lymphocytic leukemia pathogenesis and disease subtypes. Leuk. Lymphoma 2009, 50, 506–509, doi:10.1080/10428190902763517.
[32]
Cordes, K.; Srivastava, D. MicroRNA regulation of cardiovascular development. Circ. Res. 2009, doi:10.1161/circresaha.108.192872.
[33]
Ma, X.; Becker Buscaglia, L.E; Barker, J.R.; Li, Y. MicroRNAs in NF-κB signaling. J. Mol. Cell Biol. 2011, doi:10.1093/jmcb/mjr007.
[34]
Liu, P.; Wilson, M.J. MiR-520c and miR373 upregulate MMP9 expression by targeting mTOR and SIRT1, and activate the Ras/Raf/MEK/Erk signaling pathway and NF-κB factor in Human fibrosarcoma cells. J. Cell. Physiol. 2012, 277, 867–876.
[35]
Wang, L.; Kang, F.; Shan, B.; Liu, L.; Sang, M. Targeting NF-κB p65 with an artificial microRNA suppress growth of MDA-MB-231 human triple-negative breast cancer cell line. Gene Ther. Mol. Biol. 2012, 14, 30–41.
[36]
Huang, S.; Robinson, J.B.; Deguzman, A.; Bucana, C.D.; Fidler, I.J. Blockade of nuclear factor-κB signaling inhibits angiogenesis and tumorigenecity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8. Cancer Res. 2000, 60, 5334–5339.
[37]
Huang, Q.; Gumireddy, K.; Schrier, M.; le Sage, C.; Nagel, R.; Nair, S.; Egan, D.A.; Li, A.; Huang, G.; Pure, E.; Agami, R. The microRNAs miR-373 and miR-520c promote tumor invasion and metastasis. Nat. Cell Biol. 2008, 10, 202–210, doi:10.1038/ncb1681.
[38]
Erfle, H.; Neumann, B.; Liebel, U.; Rogers, P.; Held, M.; Walter, T.; Ellenberg, J.; Pepperkok, R. Reverse transfection on cell arreys for high content screening microscopy. Nat. Protocol. 2007, 2, 392–399.
[39]
Erfle, H.; Neumann, B.; Rogers, P.; Bulkescher, J.; Ellenberg, J.; Pepperkok, R. Work flow for multiplexing siRNA assays by solid-pahse reverse trasnfection in multiwell plates. J. Biomol. Screen. 2008, 13, 575–580, doi:10.1177/1087057108320133.
[40]
Genovesio, A.; Giardini, M.A.; Kwon, Y.J.; Dossin, F.D.M.; Choi, S.Y.; Kim, N.Y.; Kim, H.C.; Jung, S.Y.; Schenkman, S.; Almeida, I.C.; Emans, N.; Freitas-Junior, L.H.F. Visual genome-wide RNAi screening to identify human hostfactors requered for Trypanosoma cruzi infection. PLoS One 2011, 6, e19733, doi:10.1371/journal.pone.0019733.
[41]
Genovesio, A.; Kwon, Y.J.; Windisch, M.P.; Kim, N.Y.; Choi, S.Y.; Kim, H.C.; Jung, S.; Mammano, F.; Perrin, V.; Boese, A.S.; Casartelli, N.; Swartz, O.; Nehrbass, U.; Emans, N. Automated genome-wide visual profiling of cellular proteins involved in HIV infection. J. Biomol. Screen. 2011, 16, 945–958, doi:10.1177/1087057111415521.
Tavazoie, S.F.; Alarcon, C.; Oskarsson, T.; Padua, D.; Wang, Q.; Bos, P.D.; Gerald, W.L.; Massague, J. Endogenous human microRNAs that suppress breast cancer metastasis. Nature 2008, 451, 147–152.
[46]
Dai, R.; Zhang, Y.; Khan, D.; Heid, B.; Caudell, D.; Crasta, O.; Ahmed, S.A. Identification of a common lupus disease-associated microRNA expression pattern in three different murine models of Lupus. PLoS One 2010, 5, e14302, doi:10.1371/journal.pone.0014302.