- Email: [email protected]
RNAi-mediated silencing of leptin gene expression increases cell death in C6 glioblastoma cells
Molecular Brain Research 139 (2005) 357 – 360 www.elsevier.com/locate/molbrainres
Short Communication
RNAi-mediated silencing of leptin gene expression increases cell death in C6 glioblastoma cells Russell Browna, Barbara Morashb, Ehud Urb,c, Michael Wilkinsona,b,c,* a
Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3K 6R8 Division of Endocrinology and Metabolism, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3K 6R8 c Department of Obstetrics and Gynaecology, Faculty of Medicine, Dalhousie University, Nova Scotia, Canada B3K 6R8
b
Accepted 12 May 2005 Available online 16 June 2005
Abstract We previously demonstrated that the brain, pituitary, and C6 glioblastoma cells express leptin. To determine the physiological role of brain-derived leptin, we sought to selectively silence its expression using RNA interference (RNAi) in vitro. One of four potential targets, siRNA L7, reduced leptin mRNA by 50% (P < 0.05) and protein by 55% (P < 0.0001) in C6 cells. RNAi also induced a twofold increase in cell death as seen by ethidium homodimer-1 (P < 0.015) and TUNEL (P < 0.005) staining. These data suggest that endogenous leptin may be a critical factor promoting cell survival in the brain. D 2005 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic:Neuroendocrine regulation: other Keywords: Leptin; Brain; Reverse transcription polymerase chain reaction; Small interfering RNA; RNA interference; Cell death
Leptin is secreted predominantly from adipocytes and is assumed to enter the brain to regulate metabolism, feeding behavior, and reproduction [22]. We reported that leptin mRNA is also transcribed in the brain and pituitary gland, as well as C6 glioblastoma cells [19,20,28]. These findings were confirmed by others [8,15,24]. Moreover, the human neuroblastoma cell line, SH-SY5Y, expresses leptin and its receptors [21]. In the rat brain, leptin immunoreactivity (ir) was localized to cells, including neurons, in the arcuate nucleus of the hypothalamus (ARC), piriform cortex, hippocampus, supraoptic and paraventricular nuclei [27], brain regions which possess leptin receptors (OBR) [10,11]. This co-localization of leptin mRNA, leptin-ir, and OBR in multiple brain regions is consistent with reports that leptin * Corresponding author. Department of Obstetrics and Gynaecology, Faculty of Medicine, Dalhousie University, IWK Health Centre, 5980 University Avenue, P.O. Box 9070, Halifax, Nova Scotia, Canada B3K 6R8. Fax: +1 902 470 7192. E-mail address: [email protected] (M. Wilkinson). 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2005.05.009
regulates brain development, neuronal projections, and the expression of neuronal and glial proteins [2,3,26], and possesses both antiapoptotic [21] and neuroprotective properties [6]. To definitively establish the role(s) of endogenous brainderived leptin, a specific disruption of central leptin gene expression is required. We hypothesized that RNA interference (RNAi), a powerful post-transcriptional silencing technique [7,12,18], could selectively silence brain leptin expression and provide insight into its potential physiological roles. Experiments carried out in a model system, C6 glioblastoma cells, demonstrated successful silencing of the leptin gene. Rat C6 glioblastoma cells (ATCC # CCL 107; Manassas, Virginia) were maintained in DMEM (Gibco; Burlington, Ont.) supplemented with 2– 10% fetal bovine serum (FBS), depending on the experiment. Cells were plated at a density of 30,000 cells per well in 12-well plates (NUNC) and grown at 37 -C in 5% CO2/95% air for 24 h. Cells were transfected in OptiMEM (500 AL Gibco; Burlington, Ont.)
358
R. Brown et al. / Molecular Brain Research 139 (2005) 357 – 360
using conditions that were optimized for Oligofectamine (6.4 AL:1 Ag siRNA; Invitrogen; Burlington, Ont.) with a fluorescein-labeled (FAM) siRNA (Ambion; Austin, TX). Multiple targets were designed against leptin mRNA [9] and specificity was confirmed using the BLAST tool on the NCBI website. A non-specific scramble control sequence was also generated. The silencer siRNA construction kit (Ambion; Austin TX) was used to synthesize the multiple siRNAs, including the control. Cells were treated with one of the 4 targets (100 nM), or the scramble control. Subsequently, the most effective target, siRNA L7, and the scramble control, were synthesized in both the unmodified and the stability-enhanced forms (siSTABLE) (Dharmacon, Lafayette, CO) for further experiments. Cells were transfected for 4 h, and propagated for a further 20 h prior to RNA isolation using the RNeasy mini kit following the DNase protocol (Qiagen; Mississauga, Ont.). Leptin gene expression was analyzed using semi-quantitative RT-PCR analysis for leptin [19], and normalized to cyclophilin [17]. Data were expressed as a percentage of the control T SEM, and were analyzed by ANOVA using the Newman – Keuls post hoc test and considered significant if P < 0.05. To characterize the silencing effect, C6 cells were transfected with various doses of siRNA L7 (25, 50, 100, 200 nM), and siRNA Scramble, and RNA was isolated 27 h later for RT-PCR analysis. In addition, we transfected cells, grown for 18 h in serum-free medium (OptiMEM; Gibco; Burlington, Ont.), with either siRNA L7 (100 nM), or the scramble control to establish if it could block dbcAMPmediated (Sigma; St. Louis, MO) stimulation of leptin gene expression, as reported [20]. Finally, the temporal effects of RNAi-mediated suppression of leptin mRNA were investigated by treating cells with 100 nM siSTABLE L7, and propagating them for 24, 48, 72, and 96 h prior to RNA isolation. Control cells were transfected with the siSTABLE scramble and cultured for 96 h. Western blot analysis was performed as follows: cells (70,000/well) were propagated in 6-well plates and transfected at 24 h with 100 nM siSTABLE L7, or the siSTABLE control, as described above. Subsequently, cells were propagated in DMEM containing 2% FBS for a further 72 h. Total cell lysates were subjected to SDS-PAGE and Western blotting using a leptin-specific antibody (1:1000; Y-20;SC-843; Santa Cruz Biotechnology; Santa Cruz, CA). Bands were detected by chemiluminescence and quantified using NIH Image (v1.60). Data were expressed as a percentage of control (TSEM), and were analyzed using the Student’s t test, with P < 0.05 being considered significant. To determine if leptin knockdown affected cell death, cells were cultured on glass cover slips in 12-well plates. Cells were transfected at 24 h using either 100 nM siSTABLE L7, or siSTABLE scramble, and propagated for a further 72 h in DMEM containing 2% FBS. Cells were stained using either Ethidium homodimer-1 (EthD-1, Molecular Probes; Eugene, OR), which accesses the nuclear region of dead cells, as described by the manufacturer. A further group of cells was
Fig. 1. siRNA L7 reduces leptin gene expression. (A) siRNA L7 was engineered against leptin following the criteria of Elbashir et al. (2002) [9]. (B) RT-PCR analysis of leptin gene expression, normalized to cyclophilin, reveals that only siRNA L7 (100 nM) reduced leptin mRNA by 50% (*P < 0.05, n = 4). (C) Dose – response for siRNA L7 inhibition of leptin expression. (*P < 0.05; **P < 0.005 vs. CTL, n = 3). (D) Blockade of the dbcAMP stimulation of leptin gene expression. Transfecting cells with siRNA L7 (100 nM) blocked the subsequent dbcAMP (1 mM) stimulation of leptin gene expression (**P < 0.005 vs. dbcAMP alone, n = 6) (***P < 0.001 vs. unstimulated, n = 6). All values are means T SEM. Representative agarose gels stained with ethidium bromide are shown for leptin and cyclophilin for each experiment.
R. Brown et al. / Molecular Brain Research 139 (2005) 357 – 360
stained using the DeadEndi Colorimetric TUNEL System (Promega; Madison WI) according to the manufacturer’s protocol. Dead cells were counted and data were expressed as a percentage of control (TSEM). Initial experiments revealed that 1 of the 4 targets, siRNA L7 (Fig. 1A), reduced leptin expression by 50% (P < 0.05), relative to the non-specific scramble control (Fig. 1B). Fig. 1C illustrates the dose-dependant silencing of leptin gene expression using siRNA L7, with the knockdown of leptin mRNA being significant when doses were 50 nM (P < 0.01). Prior transfection of siRNA L7 significantly attenuated the stimulatory effect of dbcAMP (P < 0.005) (Fig. 1D), whereas the control siRNA was ineffective. Using the unmodified siRNA L7, we achieved a maximal knockdown 30 h post-transfection, with normal gene expression resuming by 48 h (data not shown). In marked contrast, the stability-enhanced siRNA, siSTABLE L7, significantly reduced (¨50%; P < 0.005) leptin expression for up to 48 h (Fig. 2A). When cell growth was slowed by propagating cells in DMEM containing 2% FBS, the silencing effect was prolonged (data not shown). Further, using Western blot analysis, we observed a 55%
Fig. 2. Investigation of a stability-enhanced siRNA L7 (siSTABLE L7). (A) Leptin gene expression was significantly attenuated for up to 48 h, and remained lower for up to 96 h, relative to the control (siSTABLE CTL; 100 nM). (*P < 0.01; **P < 0.005, n = 3). Representative agarose gels stained with ethidium bromide are shown for leptin and cyclophilin. (B) Western blot analysis revealed that leptin protein was reduced by 55% following transfection with siSTABLE L7 (100 nM; 72 h) (*P < 0.05, n = 6). A representative blot is shown for leptin following a knockdown experiment.
359
Fig. 3. Effect of siSTABLE L7 on cell death. (A) Cells stained with ethidium homodimer-1 72 h post-transfection of siSTABLE L7 (100 nM) reveal a 2-fold increase in cell death relative to the control (siSTABLE CTL). (B) TUNEL staining cells revealed a 2.5-fold increase in cell death 72 h post-transfection of siSTABLE L7 relative to the control (siSTABLE CTL) (*P < 0.01, **P < 0.005). All values are means T SEM (n = 7 – 8).
reduction in leptin protein levels in cells treated with siSTABLE L7, relative to the siSTABLE scramble control (Fig. 2B; P < 0.05; 72 h). Our data provide the first evidence that leptin mRNA and protein can be significantly reduced using RNA interference. The silencing effect in C6 cells was target-, time-, and dose-dependant. The only effective target, siRNA L7, was also potent in blocking the cAMP-dependant stimulation of leptin mRNA in C6 cells. Chemical modification of siRNA L7 (siSTABLE L7) prolonged its half life such that leptin gene expression remained significantly attenuated 48 h posttransfection, compared to a complete disappearance of the silencing effect of the unmodified siRNA L7. In addition, chemical modification may enhance target recognition and reduce non-specific effects [4]. This is also supported by our determination of a dose – response relationship showing that the lowest effective dose was 50 nM for siSTABLE L7 [5,23]. Given that three other targets, and the scramble control had no effect on leptin expression or on cell death (results not shown), we conclude that siRNA L7 is specific for the leptin gene. However, investigation of alternative effective targets remains an important goal [14]. A functional consequence of this inhibition was a marked induction of cell death in C6 cells, consistent with the recent demonstration that leptin treatment of the human neuroblastoma cell line, SH-SY5Y, increased cell proliferation by inhibiting apoptosis [21]. The degree of leptin knockdown achieved in C6 glioblastoma was capable of inducing cell death. A reduction in leptin production, using siSTABLE L7, led to a twofold increase in cell death (EthD-1 staining) [16]
360
R. Brown et al. / Molecular Brain Research 139 (2005) 357 – 360
relative to the siSTABLE scramble control (Fig. 3A; P < 0.01) and a 2.5-fold increase assessed by TUNEL staining (Fig. 3B; P < 0.005) [25]. These data suggest, but do not prove, that silencing of leptin expression induces apoptosis in C6 glioblastoma cells. This would be consistent with the report that leptin reduced apoptosis in human neuroblastoma cells [21]. However, studies are required to exclude the possibility of necrosis [13]. The pro-survival influence of leptin in human neuroblastoma cells was dependant on the long form of the leptin receptor (OBRb). We reported that C6 cells express the short form of the leptin receptor, OBRa, but not OBRb [19], which implies that the OBRa is also capable of mediating the pro-survival effects of leptin. This might occur via alternative OBRa-dependant signaling pathways such as MAPK and PI3-K [21]. However, note that the absence of leptin (in ob/ob mice) or OBRb (db/db mice) results in neurodegeneration and smaller brains compared to control mice [2]. Since the db/db mouse has a normal brain complement of OBRa [1], this receptor alone is clearly insufficient for neuronal survival in the presence of leptin. We hypothesize that leptin may be signaling via other isoforms of the leptin receptor (OBRc – OBRf) to prevent C6 cell death. In conclusion, our data suggest that RNAi is an effective tool to explore the physiological roles of leptin in the nervous system, especially in terms of cell survival.
Acknowledgments These studies were funded by the NSHRF, the IWK Health Centre, and the Atlee Endowment (Department of Obstetrics and Gynaecology). RB is the recipient of a NSHRF Graduate Studentship and BM an IWK Research Associateship. We are indebted to Diane Wilkinson and Dr. C. Too for their help.
References [1] R.S. Ahima, J.S. Flier, Leptin, Annu. Rev. Physiol. 62 (2000) 413 – 437. [2] R.S. Ahima, C. Bjorbaek, S. Osei, J.S. Flier, Regulation of neuronal and glial proteins by leptin: implications for brain development, Endocrinology 140 (1999) 2755 – 2762. [3] S.G. Bouret, S.J. Draper, R.B. Simerly, Trophic action of leptin on hypothalamic neurons that regulate feeding, Science 304 (2004) 108 – 110. [4] D.A. Braasch, S. Jensen, Y. Liu, K. Kaur, K. Arar, M.A. White, D.R. Corey, RNA interference in mammalian cells by chemically-modified RNA, Biochemistry 42 (2003) 7967 – 7975. [5] J. Couzin, Molecular biology. RNAi shows cracks in its armor, Science 306 (2004) 1124 – 1125. [6] E. Dicou, S. Attoub, P. Gressens, Neuroprotective effects of leptin in vivo and in vitro, NeuroReport 12 (2001) 3947 – 3951. [7] G. Dorn, S. Patel, G. Wotherspoon, M. Hemmings-Mieszczak, J. Barclay, F.J. Natt, P. Martin, S. Bevan, A. Fox, P. Ganju, W. Wishart, J. Hall, siRNA relieves chronic neuropathic pain, Nucleic Acids Res. 32 (2004) e49.
[8] R.A. Ehrhardt, A.W. Bell, Y.R. Boisclair, Spatial and developmental regulation of leptin in fetal sheep, Am. J. Physiol.: Regul., Integr. Comp. Physiol. 282 (2002) R1628 – R1635. [9] S.M. Elbashir, J. Harborth, K. Weber, T. Tuschl, Analysis of gene function in somatic mammalian cells using small interfering RNAs, Methods 26 (2002) 199 – 213. [10] J.K. Elmquist, C. Bjorbaek, R.S. Ahima, J.S. Flier, C.B. Saper, Distributions of leptin receptor mRNA isoforms in the rat brain, J. Comp. Neurol. 395 (1998) 535 – 547. [11] M.L. Hakansson, H. Brown, N. Ghilardi, R.C. Skoda, B. Meister, Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus, J. Neurosci. 18 (1998) 559 – 572. [12] J.D. Hommel, R.M. Sears, D. Georgescu, D.L. Simmons, R.J. DiLeone, Local gene knockdown in the brain using viral-mediated RNA interference, Nat. Med. 9 (2003) 1539 – 1544. [13] M.J. Ishimaru, C. Ikonomidou, T.I. Tenkova, T.C. Der, K. Dikranian, M.A. Sesma, J.W. Olney, Distinguishing excitotoxic from apoptotic neurodegeneration in the developing rat brain, J. Comp. Neurol. 408 (1999) 461 – 476. [14] A.L. Jackson, P.S. Linsley, Noise amidst the silence: off-target effects of siRNAs? Trends Genet. 20 (2004) 521 – 524. [15] L. Jin, S. Zhang, B.G. Burguera, M.E. Couce, R.Y. Osamura, E. Kulig, R.V. Lloyd, Leptin and leptin receptor expression in rat and mouse pituitary cells, Endocrinology 141 (2000) 333 – 339. [16] A. Levesque, A. Paquet, M. Page, Improved fluorescent bioassay for the detection of tumor necrosis factor activity, J. Immunol. Methods 178 (1995) 71 – 76. [17] Y.J. Ma, D.F. Hill, M.P. Junier, M.E. Costa, S.E. Felder, S.R. Ojeda, Expression of epidermal growth factor receptor changes in the hypothalamus during the onset of female puberty, Mol. Cell. Neurosci. 5 (1994) 246 – 262. [18] H. Makimura, T.M. Mizuno, J.W. Mastaitis, R. Agami, C.V. Mobbs, Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake, BMC Neurosci. 3 (2002) 18. [19] B. Morash, A. Li, P.R. Murphy, M. Wilkinson, E. Ur, Leptin gene expression in the brain and pituitary gland, Endocrinology 140 (1999) 5995 – 5998. [20] B. Morash, J. Johnstone, C. Leopold, A. Li, P. Murphy, E. Ur, M. Wilkinson, The regulation of leptin gene expression in the C6 glioblastoma cell line, Mol. Cell. Endocrinol. 165 (2000) 97 – 105. [21] V.C. Russo, S. Metaxas, K. Kobayashi, M. Harris, G.A. Werther, Antiapoptotic effects of leptin in human neuroblastoma cells, Endocrinology 145 (2004) 4103 – 4112. [22] A. Sahu, Minireview: a hypothalamic role in energy balance with special emphasis on leptin, Endocrinology 145 (2004) 2613 – 2620. [23] D. Semizarov, L. Frost, A. Sarthy, P. Kroeger, D.N. Halbert, S.W. Fesik, Specificity of short interfering RNA determined through gene expression signatures, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 6347 – 6352. [24] N. Smolinska, J. Przala, T. Kaminski, G. Siawrys, A. Gajewska, K. Kochman, S. Okrasa, Leptin gene expression in the hypothalamus and pituitary of pregnant pigs, Neuro-Endocrinol. Lett. 25 (2004) 191 – 195. [25] A. Spanova, H. Kovaru, V. Lisa, E. Lukasova, B. Rittich, Estimation of apoptosis in C6 glioma cells treated with antidepressants, Physiol. Res. 46 (1997) 161 – 164. [26] C.M. Steppan, A.G. Swick, A role for leptin in brain development, Biochem. Biophys. Res. Commun. 256 (1999) 600 – 602. [27] E. Ur, D.A. Wilkinson, B.A. Morash, M. Wilkinson, Leptin immunoreactivity is localized to neurons in rat brain, Neuroendocrinology 75 (2002) 264 – 272. [28] M. Wilkinson, B. Morash, E. Ur, The brain is a source of leptin, Front. Horm. Res. 26 (2000) 106 – 125.
Short Communication
RNAi-mediated silencing of leptin gene expression increases cell death in C6 glioblastoma cells Russell Browna, Barbara Morashb, Ehud Urb,c, Michael Wilkinsona,b,c,* a
Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3K 6R8 Division of Endocrinology and Metabolism, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3K 6R8 c Department of Obstetrics and Gynaecology, Faculty of Medicine, Dalhousie University, Nova Scotia, Canada B3K 6R8
b
Accepted 12 May 2005 Available online 16 June 2005
Abstract We previously demonstrated that the brain, pituitary, and C6 glioblastoma cells express leptin. To determine the physiological role of brain-derived leptin, we sought to selectively silence its expression using RNA interference (RNAi) in vitro. One of four potential targets, siRNA L7, reduced leptin mRNA by 50% (P < 0.05) and protein by 55% (P < 0.0001) in C6 cells. RNAi also induced a twofold increase in cell death as seen by ethidium homodimer-1 (P < 0.015) and TUNEL (P < 0.005) staining. These data suggest that endogenous leptin may be a critical factor promoting cell survival in the brain. D 2005 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic:Neuroendocrine regulation: other Keywords: Leptin; Brain; Reverse transcription polymerase chain reaction; Small interfering RNA; RNA interference; Cell death
Leptin is secreted predominantly from adipocytes and is assumed to enter the brain to regulate metabolism, feeding behavior, and reproduction [22]. We reported that leptin mRNA is also transcribed in the brain and pituitary gland, as well as C6 glioblastoma cells [19,20,28]. These findings were confirmed by others [8,15,24]. Moreover, the human neuroblastoma cell line, SH-SY5Y, expresses leptin and its receptors [21]. In the rat brain, leptin immunoreactivity (ir) was localized to cells, including neurons, in the arcuate nucleus of the hypothalamus (ARC), piriform cortex, hippocampus, supraoptic and paraventricular nuclei [27], brain regions which possess leptin receptors (OBR) [10,11]. This co-localization of leptin mRNA, leptin-ir, and OBR in multiple brain regions is consistent with reports that leptin * Corresponding author. Department of Obstetrics and Gynaecology, Faculty of Medicine, Dalhousie University, IWK Health Centre, 5980 University Avenue, P.O. Box 9070, Halifax, Nova Scotia, Canada B3K 6R8. Fax: +1 902 470 7192. E-mail address: [email protected] (M. Wilkinson). 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2005.05.009
regulates brain development, neuronal projections, and the expression of neuronal and glial proteins [2,3,26], and possesses both antiapoptotic [21] and neuroprotective properties [6]. To definitively establish the role(s) of endogenous brainderived leptin, a specific disruption of central leptin gene expression is required. We hypothesized that RNA interference (RNAi), a powerful post-transcriptional silencing technique [7,12,18], could selectively silence brain leptin expression and provide insight into its potential physiological roles. Experiments carried out in a model system, C6 glioblastoma cells, demonstrated successful silencing of the leptin gene. Rat C6 glioblastoma cells (ATCC # CCL 107; Manassas, Virginia) were maintained in DMEM (Gibco; Burlington, Ont.) supplemented with 2– 10% fetal bovine serum (FBS), depending on the experiment. Cells were plated at a density of 30,000 cells per well in 12-well plates (NUNC) and grown at 37 -C in 5% CO2/95% air for 24 h. Cells were transfected in OptiMEM (500 AL Gibco; Burlington, Ont.)
358
R. Brown et al. / Molecular Brain Research 139 (2005) 357 – 360
using conditions that were optimized for Oligofectamine (6.4 AL:1 Ag siRNA; Invitrogen; Burlington, Ont.) with a fluorescein-labeled (FAM) siRNA (Ambion; Austin, TX). Multiple targets were designed against leptin mRNA [9] and specificity was confirmed using the BLAST tool on the NCBI website. A non-specific scramble control sequence was also generated. The silencer siRNA construction kit (Ambion; Austin TX) was used to synthesize the multiple siRNAs, including the control. Cells were treated with one of the 4 targets (100 nM), or the scramble control. Subsequently, the most effective target, siRNA L7, and the scramble control, were synthesized in both the unmodified and the stability-enhanced forms (siSTABLE) (Dharmacon, Lafayette, CO) for further experiments. Cells were transfected for 4 h, and propagated for a further 20 h prior to RNA isolation using the RNeasy mini kit following the DNase protocol (Qiagen; Mississauga, Ont.). Leptin gene expression was analyzed using semi-quantitative RT-PCR analysis for leptin [19], and normalized to cyclophilin [17]. Data were expressed as a percentage of the control T SEM, and were analyzed by ANOVA using the Newman – Keuls post hoc test and considered significant if P < 0.05. To characterize the silencing effect, C6 cells were transfected with various doses of siRNA L7 (25, 50, 100, 200 nM), and siRNA Scramble, and RNA was isolated 27 h later for RT-PCR analysis. In addition, we transfected cells, grown for 18 h in serum-free medium (OptiMEM; Gibco; Burlington, Ont.), with either siRNA L7 (100 nM), or the scramble control to establish if it could block dbcAMPmediated (Sigma; St. Louis, MO) stimulation of leptin gene expression, as reported [20]. Finally, the temporal effects of RNAi-mediated suppression of leptin mRNA were investigated by treating cells with 100 nM siSTABLE L7, and propagating them for 24, 48, 72, and 96 h prior to RNA isolation. Control cells were transfected with the siSTABLE scramble and cultured for 96 h. Western blot analysis was performed as follows: cells (70,000/well) were propagated in 6-well plates and transfected at 24 h with 100 nM siSTABLE L7, or the siSTABLE control, as described above. Subsequently, cells were propagated in DMEM containing 2% FBS for a further 72 h. Total cell lysates were subjected to SDS-PAGE and Western blotting using a leptin-specific antibody (1:1000; Y-20;SC-843; Santa Cruz Biotechnology; Santa Cruz, CA). Bands were detected by chemiluminescence and quantified using NIH Image (v1.60). Data were expressed as a percentage of control (TSEM), and were analyzed using the Student’s t test, with P < 0.05 being considered significant. To determine if leptin knockdown affected cell death, cells were cultured on glass cover slips in 12-well plates. Cells were transfected at 24 h using either 100 nM siSTABLE L7, or siSTABLE scramble, and propagated for a further 72 h in DMEM containing 2% FBS. Cells were stained using either Ethidium homodimer-1 (EthD-1, Molecular Probes; Eugene, OR), which accesses the nuclear region of dead cells, as described by the manufacturer. A further group of cells was
Fig. 1. siRNA L7 reduces leptin gene expression. (A) siRNA L7 was engineered against leptin following the criteria of Elbashir et al. (2002) [9]. (B) RT-PCR analysis of leptin gene expression, normalized to cyclophilin, reveals that only siRNA L7 (100 nM) reduced leptin mRNA by 50% (*P < 0.05, n = 4). (C) Dose – response for siRNA L7 inhibition of leptin expression. (*P < 0.05; **P < 0.005 vs. CTL, n = 3). (D) Blockade of the dbcAMP stimulation of leptin gene expression. Transfecting cells with siRNA L7 (100 nM) blocked the subsequent dbcAMP (1 mM) stimulation of leptin gene expression (**P < 0.005 vs. dbcAMP alone, n = 6) (***P < 0.001 vs. unstimulated, n = 6). All values are means T SEM. Representative agarose gels stained with ethidium bromide are shown for leptin and cyclophilin for each experiment.
R. Brown et al. / Molecular Brain Research 139 (2005) 357 – 360
stained using the DeadEndi Colorimetric TUNEL System (Promega; Madison WI) according to the manufacturer’s protocol. Dead cells were counted and data were expressed as a percentage of control (TSEM). Initial experiments revealed that 1 of the 4 targets, siRNA L7 (Fig. 1A), reduced leptin expression by 50% (P < 0.05), relative to the non-specific scramble control (Fig. 1B). Fig. 1C illustrates the dose-dependant silencing of leptin gene expression using siRNA L7, with the knockdown of leptin mRNA being significant when doses were 50 nM (P < 0.01). Prior transfection of siRNA L7 significantly attenuated the stimulatory effect of dbcAMP (P < 0.005) (Fig. 1D), whereas the control siRNA was ineffective. Using the unmodified siRNA L7, we achieved a maximal knockdown 30 h post-transfection, with normal gene expression resuming by 48 h (data not shown). In marked contrast, the stability-enhanced siRNA, siSTABLE L7, significantly reduced (¨50%; P < 0.005) leptin expression for up to 48 h (Fig. 2A). When cell growth was slowed by propagating cells in DMEM containing 2% FBS, the silencing effect was prolonged (data not shown). Further, using Western blot analysis, we observed a 55%
Fig. 2. Investigation of a stability-enhanced siRNA L7 (siSTABLE L7). (A) Leptin gene expression was significantly attenuated for up to 48 h, and remained lower for up to 96 h, relative to the control (siSTABLE CTL; 100 nM). (*P < 0.01; **P < 0.005, n = 3). Representative agarose gels stained with ethidium bromide are shown for leptin and cyclophilin. (B) Western blot analysis revealed that leptin protein was reduced by 55% following transfection with siSTABLE L7 (100 nM; 72 h) (*P < 0.05, n = 6). A representative blot is shown for leptin following a knockdown experiment.
359
Fig. 3. Effect of siSTABLE L7 on cell death. (A) Cells stained with ethidium homodimer-1 72 h post-transfection of siSTABLE L7 (100 nM) reveal a 2-fold increase in cell death relative to the control (siSTABLE CTL). (B) TUNEL staining cells revealed a 2.5-fold increase in cell death 72 h post-transfection of siSTABLE L7 relative to the control (siSTABLE CTL) (*P < 0.01, **P < 0.005). All values are means T SEM (n = 7 – 8).
reduction in leptin protein levels in cells treated with siSTABLE L7, relative to the siSTABLE scramble control (Fig. 2B; P < 0.05; 72 h). Our data provide the first evidence that leptin mRNA and protein can be significantly reduced using RNA interference. The silencing effect in C6 cells was target-, time-, and dose-dependant. The only effective target, siRNA L7, was also potent in blocking the cAMP-dependant stimulation of leptin mRNA in C6 cells. Chemical modification of siRNA L7 (siSTABLE L7) prolonged its half life such that leptin gene expression remained significantly attenuated 48 h posttransfection, compared to a complete disappearance of the silencing effect of the unmodified siRNA L7. In addition, chemical modification may enhance target recognition and reduce non-specific effects [4]. This is also supported by our determination of a dose – response relationship showing that the lowest effective dose was 50 nM for siSTABLE L7 [5,23]. Given that three other targets, and the scramble control had no effect on leptin expression or on cell death (results not shown), we conclude that siRNA L7 is specific for the leptin gene. However, investigation of alternative effective targets remains an important goal [14]. A functional consequence of this inhibition was a marked induction of cell death in C6 cells, consistent with the recent demonstration that leptin treatment of the human neuroblastoma cell line, SH-SY5Y, increased cell proliferation by inhibiting apoptosis [21]. The degree of leptin knockdown achieved in C6 glioblastoma was capable of inducing cell death. A reduction in leptin production, using siSTABLE L7, led to a twofold increase in cell death (EthD-1 staining) [16]
360
R. Brown et al. / Molecular Brain Research 139 (2005) 357 – 360
relative to the siSTABLE scramble control (Fig. 3A; P < 0.01) and a 2.5-fold increase assessed by TUNEL staining (Fig. 3B; P < 0.005) [25]. These data suggest, but do not prove, that silencing of leptin expression induces apoptosis in C6 glioblastoma cells. This would be consistent with the report that leptin reduced apoptosis in human neuroblastoma cells [21]. However, studies are required to exclude the possibility of necrosis [13]. The pro-survival influence of leptin in human neuroblastoma cells was dependant on the long form of the leptin receptor (OBRb). We reported that C6 cells express the short form of the leptin receptor, OBRa, but not OBRb [19], which implies that the OBRa is also capable of mediating the pro-survival effects of leptin. This might occur via alternative OBRa-dependant signaling pathways such as MAPK and PI3-K [21]. However, note that the absence of leptin (in ob/ob mice) or OBRb (db/db mice) results in neurodegeneration and smaller brains compared to control mice [2]. Since the db/db mouse has a normal brain complement of OBRa [1], this receptor alone is clearly insufficient for neuronal survival in the presence of leptin. We hypothesize that leptin may be signaling via other isoforms of the leptin receptor (OBRc – OBRf) to prevent C6 cell death. In conclusion, our data suggest that RNAi is an effective tool to explore the physiological roles of leptin in the nervous system, especially in terms of cell survival.
Acknowledgments These studies were funded by the NSHRF, the IWK Health Centre, and the Atlee Endowment (Department of Obstetrics and Gynaecology). RB is the recipient of a NSHRF Graduate Studentship and BM an IWK Research Associateship. We are indebted to Diane Wilkinson and Dr. C. Too for their help.
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