microRNA-222 Targeting PTEN Promotes Neurite Outgrowth from Adult Dorsal Root Ganglion Neurons following Sciatic Nerve Transection

et al. (2012) microRNA-222 Targeting PTEN Promotes Neurite Outgrowth from Adult Dorsal Root Ganglion Neurons following Sciatic Nerve Transection. PLoS ONE 7(9): e44768. doi:10.1371/journal.pone.0044768 microRNA-222 Targeting PTEN Promotes Neurite Outgrowth from Adult Dorsal Root Ganglion Neurons following Sciatic Nerve Transection Songlin Zhou 0 Dingding Shen 0 Yongjun Wang 0 Leilei Gong 0 Xiaoyan Tang 0 Bin Yu 0 Xiaosong Gu 0 Fei Ding 0 Kin-Sang Cho, Schepens Eye Research Institute, Harvard Medical School, United States of America 0 1 Jiangsu Key Laboratory of Neuroregeneration, Nantong University , Nantong , China , 2 Key Lab of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai , China Dorsal root ganglia (DRG) neurons spontaneously undergo neurite growth after nerve injury. MicroRNAs (miRNAs), as small, non-coding RNAs, negatively regulate gene expression in a variety of biological processes. The roles of miRNAs in the regulation of responses of DRG neurons to injury stimuli, however, are not fully understood. Here, microarray analysis was performed to profile the miRNAs in L4-L6 DRGs following rat sciatic nerve transection. The 26 known miRNAs were differentially expressed at 0, 1, 4, 7, 14 d post injury, and the potential targets of the miRNAs were involved in nerve regeneration, as analyzed by bioinformatics. Among the 26 miRNAs, microRNA-222 (miR-222) was our research focus because its increased expression promoted neurite outgrowth while it silencing by miR-222 inhibitor reduced neurite outgrowth. Knockdown experiments confirmed that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major inhibitor of nerve regeneration, was a direct target of miR-222 in DRG neurons. In addition, we found that miR-222 might regulate the phosphorylation of cAMP response element binding protein (CREB) through PTEN, and c-Jun activation might enhance the miR-222 expression. Collectively, our data suggest that miR-222 could regulate neurite outgrowth from DRG neurons by targeting PTEN. - Funding: This study was supported by National Natural Science Foundation of China (Grant No. 81130080, 81171180, and 30870811), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Upon injury to peripheral nerves, the proximal nerve stump will spontaneously regenerate due to activation of the intrinsic growth capacity of neurons. The sciatic nerve, comprising a mixed population of motor and sensory axons, is a commonly used model in nerve regeneration studies. The sensory neurons extending into the sciatic nerve are located in the L4-L6 dorsal root ganglia (DRGs). Once primary sensory neurons are primed by peripheral axonal injury, they grow more rapidly in response to a subsequent lesion, which has been known as the conditioning effect [1,2]. Peripheral axonal injury triggers the intrinsic growth of DRG neurons, conditions the neurons to grow extensively in vitro [3], and facilitates peripheral nerve regeneration in vivo [4]. The conditioning injuries also permit DRG neuronal growth on normally nonpermissive central myelin substrates, such as myelinassociated glycoprotein (MAG) or myelin [5], and enable the central axons of DRG neurons to regenerate into and beyond the injury site in the inhibitory environment of the spinal cord [6]. Strikingly, regeneration is enhanced in axons re-injured 12 weeks after a conditioning lesion [7]. In other words, peripheral conditioning lesion indeed switches DRG neurons from a transmitter to a regenerative state, indicating that the robust response of peripheral axons to injury is not merely a default state, but results from activation of injury signals, which travel retrogradely from the peripheral lesion site to the cell body and enhance the intrinsic growth capacity of the neurons [8]. It is generally known that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), an endogenous inhibitor of the phosphoinositide 3-kinase (PI3K) pathway, is important for central axon growth [9]. PTEN inhibition facilitates the intrinsic regenerative outgrowth of adult peripheral axons, and PTEN might act as an intrinsic brake on the regenerative outgrowth [10]. Now, the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling and the c-Jun transcription factor have been known to activate the intrinsic growth capacity mainly at the transcriptional level after peripheral nerve injury [11,12]. The detailed signaling pathways responsible for the intrinsic regeneration, however, remain to be further investigated. microRNAs (miRNAs), a class of approximately 22 nucleotide non-coding RNA molecules, negatively regulate the expression of a wide variety of genes mainly through a direct interaction with the 39-untranslated regions (39-UTRs) of their corresponding mRNA targets [13]. It has been estimated that miRNAs regulate up to 60% of the total human genes at the post-transcriptional level [14], indicating that miRNAs play pivotal roles in physiological and pathological processes. The importance of miRNA in neural development and neurodegeneration is starting to be recognized [15-17], and miRNA expression profiles have been found to be significantly altered in the spinal cord injury model of adult rats [18,19]. We also performed microarray and deep sequencing to show that abnormal expressions of miRNA in DRGs might be involved in molecular mechanisms of nerve regeneration after sciatic nerve transection [20,21]. These results are consistent with the previous findings that Dicer-mediated miRNAs pathway is required for effective and timely regeneration of peripheral nerves in vivo or for regenerative axon growth in vitro [22,23]. In particular, the role of miRNA in neurite outgrowth from DRG neurons has recently been shown by two independent studies [24,25]. While the post-injury time points chosen for analyzing miRNA expression profiles are different among studies, each study shows that axotomy-induced miRNAs could regulate neurite growth. For example, miR-21 promotes neurite outgrowth by directly down-regulating Sprouty2 expression; while miRNA145 inhibites neurite outgrowth by inhibiting Robo2 expression. To date, however, no reports are available on profiling miRNAs following peripheral nerve injury to elucidate, in a systematic way, vital roles of miRNAs in controlling the phenotypic changes of DRG neurons throughout the time period of nerve regeneration. In this study we aimed to investigate the role of miRNAs in regulating gene expression and functions of DRG neurons during peripheral nerve regeneration. Our results indicated that miR-222 d (...truncated)