Cell Biochem Biophys DOI 10.1007/s12013-014-0029-y

ORIGINAL PAPER

MiR-29b Protects Dorsal Root Ganglia Neurons from Diabetic Rat Xiaona Zhang • Xu Gong • Shuhai Han Yang Zhang



Ó Springer Science+Business Media New York 2014

Abstract Accumulated evidences implicated that microRNAs may be involved in diabetic neuropathy. Here, we investigated miR-29’s roles in primary isolated dorsal root ganglion (DRG) neurons from STZ-induced diabetic rats. First, miR-29b was found down-regulated after STZinjection. Inhibitions were increased with time course. Down-regulation of miR-29b was associated with higher apoptosis rate and more serious axonal swelling. Meanwhile, axonogeneration genes were inhibited, whereas neurodegenerative genes were stimulated. Restoration of miR-29b by mimic experiment could reverse the above neuropathy. Furthermore, western blot analysis disclosed that miR-29b could abolish Smad3 activation. In conclusion, the present study identifies that miR-29b could protect DRG from diabetic rats. This protective effects suggested potential therapeutic application of miR-29b in diabetic neuropathy. Keywords Smad3

miR-29b  Diabetic  Neuropathy  Apoptosis 

X. Zhang  S. Han Department of Anesthesiology, The First Affiliated Hospital of Jilin University, 71 Xinmin Avenue, Changchun, Jilin, China X. Gong Department of Hand and Podiatric Surgery, The First Affiliated Hospital of Jilin, University, 71 Xinmin Avenue, Changchun, Jilin, China Y. Zhang (&) Department of Neurosurgery, The First Affiliated Hospital of Jilin University, 71 Xinmin Avenue, Changchun, Jilin, China e-mail: [email protected]

Introduction Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes. The prevalence of DPN is estimated to affect 30–50 % of individuals with diabetes [1]. DPN is characterized by symmetrical distal degeneration of peripheral nerves, leading to symptoms of pain and sensory loss. As the disease progresses, symptoms can improve, predisposing the patients to diabetic ulceration and nontraumatic amputation [2]. So far, there are no effective treatment options to prevent, slow or reverse the progression of diabetic neuropathy other than control of blood glucose levels. The underlying pathology in the distal symmetric sensory polyneuropathy (DSSP) has been shown to consist of a distal axonal degeneration of dying back type 2 with relative preservation of dorsal root ganglion (DRG) cells [3–5]. Alteration of gene expression and signals in DRG neurons may contribute to distal axonal damage in DPN [6–8]. MicroRNAs (miRNAs) have been identified to play an important role in the neuropathy [9–13], miRNAs are short noncoding RNA molecules and regulate gene expression by base pairing with the 30 UTR of target mRNAs to inhibit both translation and decrease mRNA stability [14–22]. A lot of evidences highlight the important role of miRNAs in the regulation of neurodegeneration [23]. However, few data are available for their role in diabetic neuropathy. Interestingly, recent studies identified several miRNAs among diabetic patients, which included miR-15a, miR-20, miR-21, miR-24, miR-29b, miR-126, miR-144, miR-150, miR-197, miR-223, miR-191, miR-320a, miR-486-5p, and miR-28-3p. Among them, miR-29b was also documented its role in neuropathy [24, 25]. Therefore, current study investigated miR-29b expression and its function in the diabetic neuropathy.

123

Cell Biochem Biophys

Materials and Methods

Table 1 List of forward and reverse primers for quantitative realtime PCR

STZ-Induced Diabetic Model

Primers names

Primers sequence (50 ? 30 )

Seven-week-old male Wista rats were intraperitoneally injected with STZ (75 mg/kg; Sigma) and maintained in parallel with age-matched control animals for different time points. 25 animals were randomly grouped for each time point. All rats were housed individually in wire mesh cages. The animals were fed on a standard rat diet, and water was ad libitum. All experimental procedures were carried out in accordance with guide-lines of Animal Care and Use Committee. Tail blood glucose was assayed 2 week later after injection to confirm diabetes. Glucose was determined using Randox kit. HbA1C was determined using Stanbio kits. Only animals with blood glucose values higher than 20 mM were considered diabetic.

Drd2 forward

CTCCAATCCACTCCACCACT

Isolation of Dorsal Root Ganglia (DRG) Neurons from Diabetic Rat Dissociated cultures were prepared from L4–6 DRGs, and cells were isolated from different time points in diabetic rats using a previously described method [26]. Neurons were cultured in defined Hams F12 media in the presence of modified Bottensteins N2 supplement without insulin (0.1-mg/ml transferrin, 20-nM progesterone, 0.01-mM cytosine arabinoside, 100-lM putrescine, 30-nM sodium selenite, and 1-mg/ml BSA; all additives were from Sigma; culture medium was from Life Technologies). The media was also supplemented with a subsaturating cocktail of neurotrophic factors (0.3-ng/ml NGF, 5-ng/ml GDNF, 1-ng/ml NT-3, and 0.1-nM insulin). Normal neurons were cultured in the presence of 10-mM D-glucose and 1-nM insulin. Diabetic neurons were exposed to 25-mM D-glucose and no insulin.

Drd2 reverse

CCCATCCACAGCCTCTTCTA

Notch1 forward

CCAGGAAAGAGGGCAGCAG

Notch1 reverse

TTGGCTGGGAGCATCTCAAG

Nrcam forward

AAGGACAATGGAGAGCTGCC

Nrcam reverse

CACTGTCCAGCGTTGTGTTG

S100A6 forward S100A6 reverse

TGTCGACGTGTGCTTCTAGC TCACCCTCCTTGCCAGAGTA

S100B forward

GCTTGTCCTCTGTGCAAACG

S100B reverse

GTACTTGGTAACAAACCGCGA

Uch-L1 forward

TGAAGCAGACCATCGGGAAC

Uch-L1 reverse

GAGTCATGGGCTGCCTGAAT

c-Synuclein forward

AATGCCGTGAGTGAAGCTGT

c-Synuclein reverse

GTTCCAAGTCCTCCTTGCGT

SOD1 forward

AGGGCGTCATTCACTTCGAG

SOD1 reverse

CCTCTCTTCATCCGCTGGAC

GAPDH forward

GGCAAGTTCAATGGCACAGT

GAPDH reverse

TGGTGAAGACGCCAGTAGACTC

Real-Time PCR Total RNA of primary isolated cells were extracted by Trizol (invitrogen). 500-ng RNA was used for reverse transcription. TaqMan miRNA assays of miR29b were performed on an Applied Biosystems Instrument (Applied Biosystems). The relative expression levels of miRNAs were normalized to endogenous U6 snRNA expression for each sample. For genes expression assay, Sybr-green-based method was used. Total primers are listed in Table 1. Each assay was done in triplicate for each sample tested. Relative quantities were calculated using the 2-DDCt method.

TUNEL Staining

Western Blot Analysis

Apoptotic cell death in DRG cultures was evaluated with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining using a TMR red in situ cell death detection kit (Roche). Cells were stained after fixation for 30 min with 4 % paraformaldehyde in PBS (pH 7.4) and permeabilization with 0.1 % Triton X-100 in 0.1 % sodium citrate. Cells were then stained with the TUNEL reaction mixture for 1 h in a humidified chamber in the dark at 37 °C. Negative controls were evaluated using all reagents except terminal transferase. Stained DRG cultures were counterstained by DAPI and analyzed using fluorescence microscopy. TUNEL-positive cells containing red fluorescent nuclei were quantitatively assessed by ImageJ (NIH).

Briefly, equal amounts of proteins were loaded on 4–20 % gradient sodium dodecyl sulfate (SDS)–polyacrylamide gel. After electrophoresis, the proteins were transferred to PVDG membranes, and the blots were subsequently probed with the following antibodies: phospho-Smad3 (Cell Signaling Technology), Smad3 (Upstate Biotechnology), and b-actin (Santa Cruz Biotechnology). For detection, horseradish peroxidaseconjugated secondary antibodies were used followed by enhanced chemiluminescence development (Pierce, Rockford).

123

Axonal Swelling Assay Primary DRG neurons were fixed with 2 % paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) for

Cell Biochem Biophys

15 min at room temperature then permeabilized with 0.3 % Triton X-100 in PBS for 5 min. Primary antibodies to (E)4-hydroxy-2-nonenal adducts (1:500) (Alexis Biochemicals) were incubated at 4 °C overnight. The following day, the cells were incubated with FITC-conjugated secondary antibodies for 1 h at room temperature, mounted and imaged using fluorescence microscope. Images were captured using with Q-capture software. Quantification of 4-HNE immunofluorescent staining intensity was assessed using ImageJ software. Five axons/images were traced using the polygon tool, and the average pixel intensity was recorded. To quantify the number of abnormal swellings, the total number of swellings was divided by the total number of axons per image. Mimic miR-29b in DRG from Diabetic Rats Primary isolated cells were seeded onto 60-mm cell culture dishes at a density of 6 9 105 cells, and transfected with 100 pmol of miScript miR-29b mimic (Qiagen) or miRNA negative control (Ambion) using commercial medium (Opti-MEM; Invitrogen). Level of miR-29b was confirmed by qPCR before any mimic experiment.

F ratio gives P \ 0.05, comparisons between individual groups were been made by Bonferroni’s post hoc. Significant difference was determined when F ratio gives P \ 0.05 Results Down-Regulation of miR-29b in Diabetic DRG To validate diabetic model in rat, we monitored weight, blood glucose, and HbA1c. Diabetic rats exhibit lower body weight (data not shown), and elevated blood glucose (Fig. 1a), and HbA1c levels (Fig. 1b). Peak level of glucose and HbA1c, both appeared in 5 month after STZinjection. Next, we conducted real-time PCR to detect miR-29b level in primary DRG cells. The results indicated down-regulation of miR-29b with the time course. The miR-29b levels were significantly lower than age-matched control. Maximum of down-regulation was found 5 month later after STZ-injection. According to our data, also considering diabetic neuropathy is the complication, which happened in late phase of diabetic, we choose 5 month DRG cells for the following function experiment.

Statistics

Down-Regulation of miR-29b Associated with Diabetic DRG Neuropathy

Data were subjected to one-way analysis of variance (ANOVA) using the Statistical Package SPSS/PC? (SPSS, Chicago).

Next, we first analyzed diabetic neuropathy at 5 month of animal model. Simultaneously with the down-regulation of

Fig. 1 Down-regulation of miR-29b in Diabetic DRG. a Blood glucose level in different time points after STZinjection. b HbA1c level in different time points after STZinjection. c Real-time PCR for miR-29b expression in diabetic DRG (***P \ 0.001, compared with control, ###P \ 0.001; compared with the rest of groups)

123

Cell Biochem Biophys

miR-29b, diabetic DRG neurons were characterized by higher percentage of TUNNEL positive staining and higher percentage of 4-HNE positive staining, which indicated more apoptosis and more serious axonal swelling, respectively (P \ 0.001) (Fig. 2a, b). For associated genes analysis, dopamine receptor D2 (Drd2), notch 1 (Notch1), neuronal cell adhesion molecule (Nrcam), S100 calcium binding protein A6 (S100a6), and S100 calcium binding protein B (S100b) were selected for axonogeneration, meanwhile, ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) (Uchl1), synuclein, gamma (breast cancer-specific protein 1) (Sncg), and superoxide dismutase 1 (Sod1) were selected for neurodegeneration. With no surprise, all selected axonogeneration genes were dramatically inhibited, and all neurodegenerative genes were increased (Fig. 2c, d). We examined whether miR-29b was involved in neuropathy processes to the level in MM cells using a miR-29b lentivirus (Fig. 1b). The restoration of miR-29b in the DRG reduced apoptosis, axon swelling, and degeneration whereas increased axon regeneration in gene

Fig. 2 Down-regulation of miR-29b associated with diabetic DRG neuropathy. a Representative microscopic images showed TUNELpositive DRG neurons in both control and diabetic (Db) groups. The percentage of TUNEL-positive cells was showed by the graph, Db [ control; b 4-HNE staining (green) was used for axon swelling

123

expression. The effect of miR-29b restoration was further assessed by determining downstream effector Smad3. Mimic miR-29b Protected Diabetic DRG Neuropathy In order to evaluate miR-29b’s roles in diabetic neuropathy, we conducted mimic experiment to restore miR-29b expression. Data showed mimic miR-29b reduced TUNNEL positive staining and 4-HNE staining (P \ 0.001) (Fig. 3a, b). At the same time, restoration of miR-29b promoted axonogeneration and inhibited neurodegeneration (Fig. 3c, d). These findings implicated miR-29b could protect DRG neuron from diabetic rats. MiR-29b Abolished Smad3 Activation Finally, we explored whether miR-29b’s function in diabetic neuropathy shared same pathway with diabetic nephropathy. Phospho-Smad3 was detected by Western Blot for TGF-b/Smad3 pathway activation (Fig. 4).

analysis. Image J was used to quantify the average pixel intensity per unit area of axons for 4-HNE staining. c Real-time PCR for neurodegeneration-related genes assay. d Real-time PCR for axonogenesis-related genes assay. ***P \ 0.001 (Color figure online)

Cell Biochem Biophys

Fig. 3 Mimic miR-29b protect diabetic DRG neuropathy. a Representative microscopic images showed TUNEL-positive DRG neurons in both diabetic (Db) and diabetic DRG transfected with miR-29b groups (miR-29b). The percentage of TUNEL-positive cells was showed by the graph, Db [ miR-29b; b 4-HNE staining (green) was

used for axon swelling analysis. Image J was used to quantify the average pixel intensity per unit area of axons for 4-HNE staining. c Real-time PCR for neurodegeneration-related genes assay. d Realtime PCR for axonogenesis-related genes assay. ***P \ 0.001 (Color figure online)

According to western blot, under diabetic condition, phosphor-Smad3 was significantly increased, which indicated TGF-b/Smad3 activation, whereas, restoration of miR-29b could abolish this activation.

death [27]. Kole et al. reported that miR-29b functions as an anti-apoptotic factor protecting neural cells against apoptosis induced by neurotrophic withdrawal, DNA damage, and ER-stress. Their data further demonstrated that the level of miR-29b is developmentally regulated in the mouse sympathetic neurons. It was low before postnatal day 13 (PD 13) and was markedly increased thereafter. More importantly, the increase of miR-29b protected mature neurons against apoptosis induced by diverse insults [29]. Excitingly, we also found down-regulation of miR-29b may be involved in diabetic neuropathy. Our data indicated that down-regulation of miR-29b was associated with increased apoptosis, axonal swelling, and abnormal genes expression profiles in diabetic DRG (Fig. 2a–d). However, mimic miR-29b expression in the diabetic DRG protected neurons against apoptosis and reduced axonal swelling. Meanwhile, mimic miR-29b stimulated axon regenerationrelated genes expression and inhibited neuron degeneration-related genes expression (Fig. 3a–d). So far, pathogenic mechanisms of diabetic neuropathy included, nonenzymatic glycation of proteins involved in

Discussion In this study, we found down-regulation of miR-29b in diabetic DRG neuron. These abnormal changes became more serious with longer high glucose exposure (Fig. 1c). MiRNA-29 (miR-29) family includes miR-29a, -29b1, -29b2, and -29c. Human miR-29a and -29b1 are processed from an intron located on chromosome 7, and miR-29b2 and -29c are from chromosome 1. They are highly conserved in human, mouse, and rat [27]. Since miR-29b1 and -29b2 have identical sequences, they are collectively referred to as miR-29b. MiR-29b has been implicated in neuronal survival. Decrease of miR-29b has been observed in the brain of patients with Alzheimer’s disease (AD) and causes Ab generation in the neurons [28]. Loss of miR-29b following acute ischemic stroke contributes to neural cell

123

Cell Biochem Biophys

Fig. 4 Mimic miR-29b abolished Smad3 activation. Western blot analysis for phosphor-Smad3 protein level probe. Total Smad3 was used for normalization. Actin is used for loading control

neural function, changes in neural polyol metabolism, prevention of production of angiogenic and neurotrophic growth factors following hyperglycemia, production of ROS, and micro-vascular disease with impaired blood circulation in diabetic nerves as described [30–32]. However, underlying series molecular events remain unclear. It was reported that miR-29b may be involved in several important pathway like Smad, ERK, p38 MAPK, and Wnt [33–38]. Among those pathways, we checked TGF-b/ Smad3 pathway, since it was reported that miR-29b inhibits diabetic nephropathy through this pathway [37]. It could target the coding sequence of TGF-b1 exon 3 to inhibit diabetic nephropathy by suppressing TGF-b1 expression, thereby inactivating Smad3 signaling and Smad3-dependent pathology. We observed the same Smad3 inactivation after miR-29b restoration in diabetic neuropathy. A lot of evidence supported that TGF-b/ Smad3 was the link between ROS and insulin resistance [38–41]. ROS levels were dramatically increased in diabetic neuropathy. That may partially explain miR-29b protective effects against diabetic neuropathy. In conclusion, the present study identifies that miR-29b could protect DRG from diabetic rats. These protective effects strongly suggested potential therapeutic application of miR-29b in diabetic neuropathy.

References 1. Deshpande, A. D., Harris-Hayes, M., & Schootman, M. (2008). Epidemiology of diabetes and diabetes-related complications. Physical Therapy, 88, 1254–1264. 2. Boulton, A. J., Vinik, A. I., Arezzo, J. C., Bril, V., Feldman, E. L., & American Diabetes Association. (2005). Diabetic neuropathies A statement by the American Diabetes Association. Diabetes Care, 28, 956–962. 3. Said, G., Slama, G., & Selva, J. (1983). Progressive centripetal degeneration of axons in small fibre diabetic neuropathy. Brain, 106, 791–807.

123

4. Dolman, C. L. (1963). The morbid anatomy of diabetic neuropathy. Neurology, 13, 135–144. 5. Watkins, P. J., Gayle, C., Alsanjari, N., et al. (1995). Severe sensory-autonomic neuropathy and endocrinopathy in insulindependent diabetes. Quarterly Journal of Medicine, 88, 795–804. 6. Kishi, M., Tanabe, J., Schmelzer, J. D., & Low, P. A. (2002). Morphometry of dorsal root ganglion in chronic experimental diabetic neuropathy. Diabetes, 51, 819–824. 7. Schmeichel, A. M., Schmelzer, J. D., & Low, P. A. (2003). Oxidative injury and apoptosis of dorsal root ganglion neurons in chronic experimental diabetic neuropathy. Diabetes, 52, 165–171. 8. Pickup, J. C. (2004). Inflammation and activated innate immunity in the pathogenesis of type 2 diabetes. Diabetes Care, 27, 813–823. 9. Jeyaseelan, K., Lim, K. Y., & Armugam, A. (2008). MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke, 39, 959–966. 10. Dharap, A., Bowen, K., Place, R., Li, L. C., & Vemuganti, R. (2009). Transient focal ischemia induces extensive temporal changes in rat cerebral microRNAome. Journal of Cerebral Blood Flow and Metabolism, 29, 675–687. 11. Tan, K. S., Armugam, A., Sepramaniam, S., Lim, K. Y., Setyowati, K. D., et al. (2009). Expression profile of microRNAs in young stroke patients. PLoS ONE, 4, e7689. 12. Yin, K. J., Deng, Z., Huang, H., Hamblin, M., & Xie, C. (2010). miR-497 regulates neuronal death in mouse brain after transient focal cerebral ischemia. Neurobiology of Diseases, 38, 17–26. 13. Siegel, C., Li, J., Liu, F., Benashski, S. E., & McCullough, L. D. (2011). miR-23a regulation of X-linked inhibitor of apoptosis (XIAP) contributes to sex differences in the response to cerebral ischemia. Proceedings of National Academy of Sciences, 108, 11662–11667. 14. Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., & Bartel, D. P. (2002). MicroRNAs in plants. Genes & Development, 16, 1616–1626. 15. Bartel, D. P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell, 116, 281–297. 16. Kloosterman, W. P., & Plasterk, R. H. (2006). The diverse functions of microRNAs in animal development and disease. Developmental Cell, 11, 441–450. 17. Bushati, N., & Cohen, S. M. (2007). microRNA functions. Annual Review of Cell and Developmental Biology, 23, 175–205. 18. Rana, T. M. (2007). Illuminating the silence: Understanding the structure and function of small RNAs. Nature Reviews Molecular Cell Biology, 8, 23–36. 19. Eulalio, A., Huntzinger, E., & Izaurralde, E. (2008). Getting to the root of miRNA mediated gene silencing. Cell, 132, 9–14. 20. Filipowicz, W., Bhattacharyya, S. N., & Sonenberg, N. (2008). Mechanisms of posttranscriptional regulation by microRNAs: Are the answers in sight? Nature Reviews Genetics, 9, 102–114. 21. Winter, J., Jung, S., Keller, S., Gregory, R. I., & Diederichs, S. (2009). Many roads to maturity: MicroRNA biogenesis pathways and their regulation. Nature Cell Biology, 11, 228–234. 22. Meijer, H. A., Kong, Y. W., Lu, W. T., Wilczynska, A., Spriggs, R. V., et al. (2013). Translational repression and eIF4A2 activity are critical for microRNA mediated gene regulation. Science, 340, 82–85. 23. Maciotta, S., Meregalli, M., & Torrente, Y. (2013). The involvement of microRNAs in neurodegenerative diseases. Frontiers in Cellular Neuroscience, 7, 265. 24. Packer, A. N., Xing, Y., Harper, S. Q., Jones, L., & Davidson, B. L. (2008). The bifunctional microRNAmiR-9, miR-9* regulates REST and CoREST and is down-regulated in Huntington’s disease. Journal of Neuroscience, 28, 14341–14346.

Cell Biochem Biophys 25. Geekiyanage, H., & Chan, C. (2011). MicroRNA-137, 181c regulates serine palmitoyltransferase and inturnamyloid beta, novel targets in sporadic Alzheimer’s disease. Journal of Neuroscience, 31, 14820–14830. 26. Huang, T. J., Verkhratsky, A., & Fernyhough, P. (2005). Insulin enhances mitochondrial inner membrane potential and increases ATP levels through phosphoinositide 3-kinase in adult sensory neurons. Molecular and Cellular Neuroscience, 28, 42–54. 27. Khanna, S., Rink, C., Ghoorkhanian, R., Gnyawali, S., Heigel, M., Wijesinghe, D. S., et al. (2013). Loss of miR-29b following acute ischemic stroke contributes to neural cell death and infarct size. Journal of Cerebral Blood Flow and Metabolism, 33, 1197–1206. 28. Hebert, S. S., Horre, K., Nicolai, L., Papadopoulou, A. S., Mandemakers, W., Silahtaroglu, A. N., et al. (2008). Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer’s disease correlates with increased BACE1/beta-secretase expression. Proceedings of the National Academy of Sciences of the United States of America, 105, 6415–6420. 29. Kole, A. J., Swahari, V., Hammond, S. M., & Deshmukh, M. (2011). miR-29b is activated during neuronal maturation and targets BH3-only genes to restrict apoptosis. Genes & Development, 25, 125–130. 30. Kim, H., Park, J. S., Choi, Y. J., Kim, M. O., Huh, Y. H., Kim, S. W., et al. (2009). Bone marrow mononuclear cells have neurovascular tropism and improve diabetic neuropathy. Stem Cells, 27, 1686–1696. 31. Barber, A. J., Lieth, E., Khim, S. A., Antonetti, D. A., Buchanan, A. G., & Gardner, T. W. (1998). Neural apoptosis in the retina during experimental and human diabetes: early onset and effect of insulin. Journal of Clinical Investigation, 102, 783–791. 32. Russell, J. W., Sullivan, K. A., Windebank, A. J., Herrmann, D. N., & Feldman, E. L. (1999). Neurons undergo apoptosis in animal and cell culture models of diabetes. Neurobiology of Diseases, 6, 347–363.

33. Jeon, E. J., Lee, K. Y., Choi, N. S., Lee, M. H., Kim, H. N., Jin, Y. H., et al. (2006). Bone morphogenetic protein-2 stimulates Runx2 acetylation. Journal of Biological Chemistry, 281, 16502–16511. 34. Maeda, S., Hayashi, M., Komiya, S., Imamura, T., & Miyazono, K. (2004). Endogenous TGF-b signaling suppresses maturation of osteoblastic mesenchymal cells. EMBO Journal, 23, 552–563. 35. Ikenoue, T., Jingushi, S., Urabe, K., Okazaki, K., & Iwamoto, Y. (1999). Inhibitory effects of activin-a on osteoblast differentiation during cultures of fetal rat calvarial cells. Journal of Cellular Biochemistry, 75, 206–214. 36. Eijken, M., Swagemakers, S., Koedam, M., Steenbergen, C., Derkx, P., Uitterlinden, A. G., et al. (2007). The activin A-follistatin system: Potent regulator of human extracellular matrix mineralization. FASEB Journal, 21, 2949–2960. 37. Lynch, M. P., Stein, J. L., Stein, G. S., & Lian, J. B. (1995). The influence of type I collagen on the development and maintenance of the osteoblast phenotype in primary and passaged rat calvarial osteoblasts: Modification of expression of genes supporting cell growth, adhesion, and extracellular matrix mineralization. Experimental Cell Research, 216, 35–45. 38. Houstis, N., Rosen, E. D., & Lander, E. S. (2006). Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature, 440, 944–948. 39. Hirosumi, J., Tuncman, G., Chang, L., Go¨rgu¨n, C. Z., Uysal, K. T., Maeda, K., et al. (2002). A central role for JNK in obesity and insulin resistance. Nature, 420, 333–336. 40. Kamata, H., Honda, S., Maeda, S., Chang, L., Hirata, H., & Karin, M. (2005). Reactive oxygen species promote TNFalphainduced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell, 120, 649–661. 41. Lee, Y. H., Giraud, J., Davis, R. J., & White, M. F. (2003). c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade. Journal of Biological Chemistry, 278, 2896–2902.

123

MiR-29b protects dorsal root ganglia neurons from diabetic rat.

Accumulated evidences implicated that microRNAs may be involved in diabetic neuropathy. Here, we investigated miR-29's roles in primary isolated dorsa...
1MB Sizes 2 Downloads 6 Views