| Abstract No.: | C-C3115 |
| Country: | Canada |
| | |
| Title: | REDUCING MRNA EXPRESSION OF THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS IN CULTURED RAT DORSAL ROOT GANGLIONS USING RNA INTERFERENCE |
| | |
| Authors/Affiliations: | 1 Dorothy Thomas*; 1 José Martinez; 1 Christine Webber; 1 Cory Toth;
1 University of Calgary, AB, Canada
|
| | |
| Content: | Introduction: Diabetic peripheral neuropathy (DPN) is a frequent and debilitating complication of Type I and II diabetes (4). One potential mechanism of DPN involves the activation of the receptor for advanced glycation end products (RAGE) which has been implicated in diabetic complications in other tissues, such as vasculature (5). Functional RAGE expression has been located in dorsal root ganglion (DRG) neurons and activates signalling pathways leading to oxidative stress and nerve damage (7). In addition, RAGE null mice develop significantly less diabetic neuropathies than wildtype mice with highly expressed RAGE (6). Collectively, this suggests that blocking RAGE activation in DRG neurons may mitigate the development of DPN. Using the novel technique of RNA interference with small interfering RNA (siRNA) (1,2), RAGE signalling pathways have been successfully reported to be downregulated in cultured astrocytes (3).
Objective: The aim of this study was to evaluate the effectiveness of siRNA treatment on RAGE expression in non-diabetic cultures of DRG neurons.
Materials and Methods: Primary DRG cell cultures from non-diabetic Sprague-Dawley rats were established by plating cells at high density on poly-L-lysine/laminin pre-coated glass slides in 24-well plates (~750 cells/well). The following day cells were transfected with 75 ng of either RAGE-specific siRNA (Qiagen, SI00274904), scrambled siRNA (Qiagen, 1027310), or an equivalent volume of ddH2O for 24 hours. All treatments used 3μL/well of transfection reagent (Qiagen, #301705) as a carrier. Immunocytochemistry was completed on selected wells which used fluorescently tagged siRNAs (Alexa-488 tagged RAGE-specific siRNA; fluorescein tagged scrambled siRNA) and were counterstained with NF200:CY3 antibodies. Quantification of mRNA expression of key molecules including RAGE and NF-κB was performed using qRT-PCR analysis.
Results: Successful co-localisation of DRGs and RAGE-specific/scrambled siRNA was observed in immunocytochemistry images. Further, no staining was observed in water controls. In addition, RAGE expression was downregulated in RAGE-specific siRNA treated cultures compared to scrambled and water-treated controls. Concomitantly, the transcription factor downstream to RAGE signalling, NF-κB, was similarly downregulated. Within the two control treatments, no differences were observed in the expression levels of either of these molecules.
Conclusion:
This study confirmed that siRNA can be successfully delivered to DRGs in-vitro and resulted in downregulation of RAGE and the transcription factor for RAGE signalling. Consequently, siRNA treatment is a promising technique for future studies of the RAGE signalling pathway. Future work will evaluate this technique within the context of streptozotocin-induced diabetic animals to evaluate whether this method would prove beneficial to avert neuropathic complications.
References:
1. Fire et al., 1998. Nature 391:806-11.; 2. Medema, 2004. Biochem J. 380:593-603.; 3. Ponath et al., 2007. J Neuroimmunol. 184:214-22.; 4. Pop-Busui et al, 2006. Diabetes Metab Res Rev. 22(4):257-73; 5. Schmidt et al, 1999. Circ Res. 84:489-97.; 6.Toth et al, 2007 Diabetes E-pub.; 7. Vincent et al, 2007. Endocrinology. 148(2):548-58
|
| | |
| Back |
|
