Altered microRNA expression following sciatic nerve resection in dorsal root ganglia of rats

Acta Biochim Biophys Sin 2011, 43: 909 – 915 | ª The Author 2011. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmr083. Advance Access Publication 10 September 2011 Short Communication Altered microRNA expression following sciatic nerve resection in dorsal root ganglia of rats Bin Yu, Songlin Zhou, Tianmei Qian, Yongjun Wang, Fei Ding, and Xiaosong Gu* Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, China *Correspondence address. Tel: þ86-513-85051801; Fax: þ86-513-85511585; E-mail: Keywords microRNA; regeneration Received: June 3, 2011 dorsal root ganglia; nerve Accepted: August 2, 2011 Introduction The peripheral nervous system (PNS) differs from the central nervous system (CNS) in that it is capable of extraordinary regeneration even after severe injury. After an injury, both PNS and CNS axons distal to the lesion degenerate, but only PNS axons re-grow and re-connect to their targets [1,2]. In order to achieve successful nerve repair, neuronal loss has to be prevented, axons have to re-grow and arrive at their correct target cells, and myelin sheaths have to be re-synthesized. This series of events is at least a partial re-capitulation of molecular and cellular mechanisms occurring during development. However, the widely elucidatory molecular mechanisms that are responsible for PNS injury and the subsequent restoration of nerve remain largely unclear. MicroRNAs (miRNAs) are small, non-coding RNAs that are emerging as important post-transcriptional regulators and have been implicated in developmental and disease processes [3–5]. miRNAs largely act as repressors of gene expression either by guiding the cleavage of their target mRNAs or by inhibiting their translation [3,6]. Their ability to potentially regulate large numbers of target genes simultaneously suggests that they may be important sculptors of transcriptional networks. As such, they are attractive candidates for regulating repair and restoration of PNS. Given the importance of miRNAs, we were interested in the expression profile of miRNAs at different time points involved in sciatic nerve regeneration. We addressed this issue using a microarray-based screening approach in the dorsal root ganglia (DRG) involved in resection of the sciatic nerve in a rat model. Materials and Methods Animal surgery and tissue preparation Adult, male Sprague–Dawley rats weighing 180–220 g (supplied by the Experimental Animal Center of Nantong University) were used in this study. Forty-two rats were randomly divided into seven groups of six animals each. The rats were anesthetized by an intraperitoneal injection of complex narcotics (85 mg/kg trichloroacetaldehyde monohydrate, 42 mg/kg magnesium sulfate, 17 mg/kg sodium pentobarbital), and the sciatic nerve was exposed and lifted through an incision on the lateral aspect of the mid-thigh of left hind limb. A 1-cm-long segment of sciatic nerve was then resected at the site just proximal to its division to tibial and common peroneal nerves, and the incision sites were then closed [7]. Lumbar 4–6 DRGs Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 11 | Page 909 MicroRNAs (miRNAs) are a class of small, non-coding RNAs (∼22 nucleotides) that negatively regulate gene expression post-transcriptionally, either through translational inhibition or degradation of target mRNAs. We uncovered a previously unknown alteration in the expression of miRNAs in the dorsal root ganglia (DRG) at 1, 4, 7, and 14 days after resection of the sciatic nerve in rats using microarray analysis. Thirty-two significantly upregulated and 18 downregulated miRNAs were identified in the DRG at four time points following sciatic nerve injury. The expression of four consecutively deregulated miRNAs, analyzed by real-time Taqman polymerase chain reaction, was in agreement with the microarray data (upregulated: miR-21, miR-221; downregulated: miR-500, miR-551b). The potential targets for these miRNAs, altered after sciatic nerve resection, are involved mainly in nervous system development, multi-cellular organismal development, and the regulation of cellular processes. This study demonstrated a different involvement of miRNAs in the DRG after resection of the sciatic nerve in a rat model, and it may also contribute in illustrating the molecular mechanisms responsible for nerve regeneration. miRNA alteration after nerve injury were collected at 0, 1, 4, 7, 14, 21, and 28 days after injury, respectively. All the experimental procedures involving animals were conducted in accordance with Institutional Animal Care guidelines and approved ethically by the Administration Committee of Experimental Animals, Jiangsu Province, China. MicroRNA microarray A miRNA microarray (Agilent Technology), containing probes for the complete Sanger miRBase 10.0, was used to screen RNA from rat DRG of 0-, 1-, 4-, 7-, and 14-day groups. The experiment was repeated three times. The labeling and hybridization were performed at the Shanghai Biochip Company, according to the protocols of the Agilent miRNA microarray system. Agilent Scan Control software was used for scanning the microarray slides, and Agilent Feature Extraction (FE) software version 9.5.3 was used for image analysis. Microarray data were analyzed using GeneSpring GX v11.0 software (Agilent Technology). Raw data were normalized by Quantile algorithm and analyzed statistically using the two-sample independent group t-test, and differences were considered statistically significant by using a cut-off of 1.4-fold change at P , 0.05. Real-time Taqman polymerase chain reaction For mature miRNAs, we used the TaqManw MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA) to synthesize cDNA from 20 ng of RNA. TaqMan microRNA assays for miR-21, miR-221, miR-500, and miR-551b (Applied Biosystems) that include specific RT primers (TM000397, TM000524, TM002606, TM002760), and TaqMan probes (RT000397, RT000524, RT002606, RT002760) were used to quantify the expression of mature miRNAs. Quantitative real-time polymerase chain reaction was performed with the 7300 realtime PCR system (Applied Biosystems). The relative expression of each miRNA was calculated using the comparative 22DCt method and was normalized using RNU6B mature miRNA. All data were expressed as mean + SD. Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 11 | Page 910 Results We examined the expression of 350 Rattus norvegicus-miRNAs based on Version 10.0 of the Sanger miRBase (Sanger Institute, Cambridge, UK; http://microrna.sanger.ac.uk/sequences) in the DRG at 0, 1, 4, 7, and 14 days after sciatic nerve amputation. Using the miRNA-based array screening, we identified differentially expressed miRNAs in the 1-, 4-, 7-, and 14-day groups compared with the 0-day group. Fou (...truncated)