MicroRNAs that target RGS5

Document Type : Original Article

Authors

1 Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran

2 Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

3 School of Biology, College of Science, University of Tehran, Tehran, Iran

4 Stem Cell Technology Research Center, Tehran, Iran

5 Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

6 Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran. School of Biology, College of Science, University of Tehran, Tehran, Iran

Abstract

Objective(s):An earlier meta-analysis on gene expression data derived from four microarray, two cDNA library, and one SAGE experiment had identified RGS5 as one of only ten non-housekeeping genes that were reported to be expressed in human trabecular meshwork (TM) cells by all studies. RGS5 encodes regulator of G-protein signaling-5. The TM tissue is the route of aqueous fluid outflow, and is relevant to the pathology of glaucoma. MicroRNAs constitute the most recently identified components of the cellular machinery for gene regulation in eukaryotic cells. Given our long standing interest in glaucoma, we aimed to identify miRNAs that may target RGS5.
Materials and Methods: Eight miRNAs were selected for study using bioinformatics tools and available data on miRNAs expressed in the eye. Their effects were assessed using the dual luciferase assay.  3'-UTR segments of RGS5 mRNA were cloned downstream of a luciferase coding gene in psiCHECK2 vectors, and these were co-transfected with each of the miRNAs into HEK293 cells.
Results: The outcomes evidenced that one or more of the segments are in fact targeted by miR-7, miR-9, miR-96, miR-23a, miR-23b, miR-204, and miR-211. Down regulations by the miRNAs were statistically significant. The effect of miR-204 is considered particularly important as this miRNA is well known to regulate eye development and to affect multiple ocular functions.
Conclusion: Our results justify further studies on regulation of RGS5 expression and RGS5 downstream functions by these miRNAs.

Keywords


1. Sierra DA, Gilbert DJ, Householder D, Grishin NV, Yu K, Ukidwe P, et al. Evolution of the regulators of G-protein signaling multigene family in mouse and human. Genomics 2002; 79:177-185.
2. Xie GX, Palmer PP. How regulators of G protein signaling achieve selective regulation. J Mol Biol 2007; 366:349-365.
3. Benjamin LE, Golijanin D, Itin A, Pode D, Keshet E. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest 1999; 103:159-165.
4. Zhou J, Moroi K, Nishiyama M, Usui H, Seki N, Ishida J, et al. Characterization of RGS5 in regulation of G protein-coupled receptor signaling. Life Sci 2001; 68:1457-1469.
5. Berman DM, Gilman AG. Mammalian RGS proteins: barbarians at the gate. J Biol Chem 1998; 273:1269-1272.
6. Anger T, Klintworth N, Stumpf C, Daniel WG, Mende U, Garlichs CD. RGS protein specificity towards Gq- and Gi/o-mediated ERK 1/2 and Akt activation, in vitro. J Biochem Mol Biol 2007; 40:899-910.
7. Anger T, Grebe N, Osinski D, Stelzer N, Carson W, Daniel WG, et al. Role of endogenous RGS proteins on endothelial ERK 1/2 activation. Exp Mol Pathol 2008; 85:165-173.
8. Wang Q, Liu M, Mullah B, Siderovski DP, Neubig RR. Receptor-selective effects of endogenous RGS3 and RGS5 to regulate mitogen-activated protein kinase activation in rat vascular smooth muscle cells. J Biol Chem 2002; 277:24949-24958.
9. Sato M, Moroi K, Nishiyama M, Zhou J, Usui H, Kasuya Y, et al. Characterization of a novel C. elegans RGS protein with a C2 domain: evidence for direct association between C2 domain and Galphaq subunit. Life Sci 2003; 73:917-932.
10. Sjogren B, Neubig RR. Thinking outside of the "RGS box": new approaches to therapeutic targeting of regulators of G protein signaling. Mol Pharmacol 2010; 78:550-557.
11. Mitchell TS, Bradley J, Robinson GS, Shima DT, Ng YS. RGS5 expression is a quantitative measure of pericyte coverage of blood vessels. Angiogenesis 2008; 11:141-151.
12. Li H, He C, Feng J, Zhang Y, Tang Q, Bian Z, et al. Regulator of G protein signaling 5 protects against cardiac hypertrophy and fibrosis during biomechanical stress of pressure overload. Proc Natl Acad Sci USA 2010; 107:13818-13823.
13. Holobotovskyy V, Manzur M, Tare M, Burchell J, Bolitho E, Viola H, et al. Regulator of G-protein signaling 5 controls blood pressure homeostasis and vessel wall remodeling. Circ Res 2013; 112:781-791.
14. Jin Y, An X, Ye Z, Cully B, Wu J, Li J. RGS5, a hypoxia-inducible apoptotic stimulator in endothelial cells. J Biol Chem 2009; 284:23436-23443.
15. Silini A, Ghilardi C, Figini S, Sangalli F, Fruscio R, Dahse R, et al. Regulator of G-protein signaling 5 (RGS5) protein: a novel marker of cancer vasculature elicited and sustained by the tumor's proangiogenic microenvironment. Cell Mol Life Sci 2012; 69:1167-1178.
16. Huang G, Song H, Wang R, Han X, Chen L. The relationship between RGS5 expression and cancer differentiation and metastasis in non-small cell lung cancer. J Surg Oncol 2012; 105:420-424.
17. Boss CN, Grunebach F, Brauer K, Hantschel M, Mirakaj V, Weinschenk T, et al. Identification and characterization of T-cell epitopes deduced from RGS5, a novel broadly expressed tumor antigen. Clin Cancer Res 2007; 13:3347-3355.
18. Yao M, Huang Y, Shioi K, Hattori K, Murakami T, Sano F, et al. A three-gene expression signature model to predict clinical outcome of clear cell renal carcinoma. Int J Cancer 2008; 123:1126-1132.
19. Wang JH, Huang WS, Hu CR, Guan XX, Zhou HB, Chen LB. Relationship between RGS5 expression and differentiation and angiogenesis of gastric carcinoma. World J Gastroenterol 2010; 16:5642-5646.
20. Liang  Y, Li C, Burke J, Protzman C, Krauss A-H, Nieves A, et al. Upregulation of regulator of G protein signaling 5 (RGS5) in the iris-ciliary body of chronic ocular hypertensive primates. Invest Ophthalmol Vis Sci 2002; 43:3387.
21. Liang Y, Li C, Guzman VM, Chang WW, Evinger AJ, 3rd, Sao D, et al. Identification of a novel alternative splicing variant of RGS5 mRNA in human ocular tissues. FEBS J 2005; 272:791-799.
22. Gasiorowski JZ, Russell P. Biological properties of trabecular meshwork cells. Exp Eye Res 2009; 88:671-675.
23. Sommer A. Intraocular pressure and glaucoma. Am J Ophthalmol 1989; 107:186-188.
24. Paylakhi SH, Yazdani S, April C, Fan JB, Moazzeni H, Ronaghi M, et al. Non-housekeeping genes expressed in human trabecular meshwork cell cultures. Mol Vis 2012; 18:241-254.
25. Zhao X, Pearson KE, Stephan DA, Russell P. Effects of prostaglandin analogues on human ciliary muscle and trabecular meshwork cells. Invest Ophthalmol Vis Sci 2003; 44:1945-1952.
26. Zhao X, Ramsey KE, Stephan DA, Russell P. Gene and protein expression changes in human trabecular meshwork cells treated with transforming growth factor-beta. Invest Ophthalmol Vis Sci 2004; 45:4023-4034.
27. Paylakhi SH, Fan JB, Mehrabian M, Sadeghizadeh M, Yazdani S, Katanforoush A, et al. Effect of PITX2 knockdown on transcriptome of primary human trabecular meshwork cell cultures. Mol Vis 2011; 17:1209-1221.
28. Gonzalez P, Epstein DL, Borras T. Characterization of gene expression in human trabecular meshwork using single-pass sequencing of 1060 clones. Invest Ophthalmol Vis Sci 2000; 41:3678-3693.
29. Tomarev SI, Wistow G, Raymond V, Dubois S, Malyukova I. Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis. Invest Ophthalmol Vis Sci 2003; 44:2588-2596.
30. Liu Y, Munro D, Layfield D, Dellinger A, Walter J, Peterson K, et al. Serial analysis of gene expression (SAGE) in normal human trabecular meshwork. Mol Vis 2011; 17:885-893.
31. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116:281-297.
32. Ambros V. The functions of animal microRNAs. Nature 2004; 431:350-355.
33. Ouellet DL, Perron MP, Gobeil LA, Plante P, Provost P. MicroRNAs in gene regulation: when the smallest governs it all. J Biomed Biotechnol 2006; 2006:69616.
34. Hughes AE, Bradley DT, Campbell M, Lechner J, Dash DP, Simpson DA, et al. Mutation altering the miR-184 seed region causes familial keratoconus with cataract. Am J Hum Genet 2011; 89:628-633.
35. Liu N, Landreh M, Cao K, Abe M, Hendriks GJ, Kennerdell JR, et al. The microRNA miR-34 modulates ageing and neurodegeneration in Drosophila. Nature 2012; 482:519-523.
36. Zhang N, Li X, Wu CW, Dong Y, Cai M, Mok MT,             et al. microRNA-7 is a novel inhibitor of YY1 contributing to colorectal tumorigenesis. Oncogene 2013; 32:5078-5088.
37. Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature 2008; 455:64-71.
38. Nilsen TW. Mechanisms of microRNA-mediated gene regulation in animal cells. Trends Genet 2007; 23:243-249.
39. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature 2008; 455:58-63.
40. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 2009; 19:92-105.
41. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120:15-20.
42. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell 2003; 115:787-798.
43. Ritchie W, Rasko JE, Flamant S. MicroRNA target prediction and validation. Adv Exp Med Biol 2013; 774:39-53.
44. Witkos TM, Koscianska E, Krzyzosiak WJ. Practical Aspects of microRNA Target Prediction. Curr Mol Med 2011; 11:93-109.
45. Karali M, Peluso I, Marigo V, Banfi S. Identification and characterization of microRNAs expressed in the mouse eye. Invest Ophthalmol Vis Sci 2007; 48:509-515.
46. Conte I, Carrella S, Avellino R, Karali M, Marco-Ferreres R, Bovolenta P, et al. miR-204 is required for lens and retinal development via Meis2 targeting. Proc Natl Acad Sci USA 2010; 107:15491-15496.
47. Dunmire JJ, Lagouros E, Bouhenni RA, Jones M, Edward DP. MicroRNA in aqueous humor from patients with cataract. Exp Eye Res 2013; 108:68-71.
48. Hoffmann A, Huang Y, Suetsugu-Maki R, Ringelberg CS, Tomlinson CR, Del Rio-Tsonis K, et al. Implication of the miR-184 and miR-204 competitive RNA network in control of mouse secondary cataract. Mol Med 2012; 18:528-538.
49. Li G, Luna C, Qiu J, Epstein DL, Gonzalez P. Role of miR-204 in the regulation of apoptosis, endoplasmic reticulum stress response, and inflammation in human trabecular meshwork cells. Invest Ophthalmol Vis Sci 2011; 52:2999-3007.
50. Paylakhi SH, Moazzeni H, Yazdani S, Rassouli P, Arefian E, Jaberi E, et al. FOXC1 in human trabecular meshwork cells is involved in regulatory pathway that includes miR-204, MEIS2, and ITGbeta1. Exp Eye Res 2013; 111:112-121.
51. Dweep H, Sticht C, Pandey P, Gretz N. miRWalk--database: prediction of possible miRNA binding sites by "walking" the genes of three genomes. J Biomed Inform 2011; 44:839-847.
52. Li G, Luna C, Qiu J, Epstein DL, Gonzalez P. Modulation of inflammatory markers by miR-146a during replicative senescence in trabecular meshwork cells. Invest Ophthalmol Vis Sci 2010; 51:2976-2985.
53. Luna C, Li G, Qiu J, Epstein DL, Gonzalez P. MicroRNA-24 regulates the processing of latent TGFbeta1 during cyclic mechanical stress in human trabecular meshwork cells through direct targeting of FURIN. J Cell Physiol 2011; 226:1407-1414.
54. Gessert S, Bugner V, Tecza A, Pinker M, Kuhl M. FMR1/FXR1 and the miRNA pathway are required for eye and neural crest development. Dev Biol 2010; 341:222-235.
55. Xu S, Witmer PD, Lumayag S, Kovacs B, Valle D.
MicroRNA (miRNA) transcriptome of mouse retina and identification of a sensory organ-specific miRNA cluster. J Biol Chem 2007; 282:25053-25066.