Abstract
Purpose:
To investigate the contribution and mechanism of miRNAs and autophagy in diabetic peripheral neuropathy.
Methods:
In this study, we used streptozotocin (STZ)-induced type I diabetes C57 mice as animal models, and we detected the expression of miR-34c and autophagic intensity in trigeminal ganglion (TG) tissue. The bioinformatics software was used to predict and analyze the potential targets of miR-34c. Primary trigeminal ganglion neurons were cultured in vitro to investigate the effect of miR-34c on axon growth and survival of TG cells. A corneal epithelial damage-healing model was established on the diabetic mice, then miR-34c antagomir was injected subconjunctivally. The condition of corneal epithelial healing was observed through sodium fluorescein staining, and the peripheral nerve degeneration of the cornea was evaluated by β-tublin corneal nerve staining.
Results:
The expression of miR-34c was significantly increased in TG tissue of type I diabetic mice by real-time PCR. Western blot showed that autophagy-related proteins Atg4B and LC3-II were significantly down-regulated in diabetes TG compared with normal control. Trigeminal neuron immunofluorescence showed that the length of the trigeminal ganglion cell synapses was significantly increased after miR-34c antagomir treatment compared with normal cultures. Subconjunctival injection of miR-34c antagomir can significantly promote corneal epithelium healing of diabetic mice and appreciably promote the regeneration of corneal nerve. At the same time, it can significantly increase the expression of autophagy in TG tissue of type I diabetic mice.
Conclusions:
In this study , miR-34c was found to affect the growth of trigeminal sensory neurons and the repair of diabetic corneal nerve endings by acting directly on Atg4B.
Diabetes mellitus (DM) is a major disease worldwide, and people with diabetes develop various types of corneal lesions that can cause irreversible visual impairment.
1,2 The cornea is rich in nerve fibers, and corneal fibers are sensitive to cold, heat, pain, and touch pressure. Corneal innervation is derived from branching of the trigeminal ganglia.
3 The corneal nerve supports corneal epithelial nerve function
4 and plays an important role in corneal epithelial wound healing through interactions with TG sensory neurons.
5 Corneal nerve injury can cause corneal hypoesthesia, leading to neurotrophic corneal epithelial lesions.
6 A previous study
7 has shown that diabetes is a risk factor for corneal neuropathy, which can lead to corneal sensory, nutritional, and metabolic dysfunction, causing keratitis and delayed healing of corneal ulcers.
Autophagy is an important cellular pathway in which lysosomes degrade large quantities of protein and organelles, such as mitochondria.
8 Autophagy maintains intracellular balance by removing protein aggregates and damaged or excess organelles.
9 At present, studies on autophagy in the field of diabetes are increasing. Fang et al.
10 reported that high glucose can inhibit basal autophagy in cultured podocytes by reducing the expression of Beclin1, Atg5-12, and LC3, and rapamycin can recover high-glucose-induced autophagy defects, thereby protecting cultured podocytes from high-glucose-induced injury. It is currently believed that diabetic peripheral neuropathy is associated with ROS-mediated mitochondrial autophagy defects. Mitochondria are important capacity organelles in cells and are also the main source of reactive oxygen species (ROS). In a diabetic state, neuronal glucose metabolism is impaired and oxidative stress is enhanced, leading to mitochondrial function disorder. Impaired mitochondria cannot be discharged in time due to defects in mitochondrial autophagy, causing neuronal apoptosis or even death. Axonal retrograde transport disorders interfere with neuronal protein synthesis, causing axonal degeneration, atrophy, trigger diabetic peripheral neuropathy (DPN).
11 The long-term neurological tissue of diabetic patients is in the state of ischemia, hypoxia, and nutrient and neurotrophic deficiency. In addition, persistent oxidative stress damage constitutes the premise and basis of abnormal autophagy. Autophagy in spinal dorsal root neurons of STZ-induced diabetic rats significantly increases, and autophagy could also occur in human neuroblastoma cells (SH-SY5Y) cultured in vitro by patients with diabetic peripheral neuropathy. Increase in autophagy is closely related to the increase in IgG and IgM in the serum of these patients. The FADD-caspase-8 death signaling pathway is involved in activation of autophagy, and when autophagy is mediated by autoimmune globulin, it is significantly reduced by prior treatment with Fas receptor blockers. Autophagy has the potential to protect against apoptosis through activation of the FAS autoantibodies.
12,13
Atg4B is a cysteine protease that cleaves the C-terminal amino acid of pro-Atg8, thereby exposing a C-terminal glycine. Atg4B is a key member of autophagy core proteins and affects autophagic membrane extension, closure, and maturation. Studies have found that Atg4B is the strongest activator of LC3, an autophagosome marker. During autophagy, LC3 is cleaved by Atg4B to the 5 amino acid residues at the C-terminus, forming an activated form of LC3-I. LC3-I is modified and processed by a ubiquitin-like system, including Atg7 and Atg3, to produce LC3-II and then localize to autophagosomes. Thus, LC3-II was used as autophagy markers for autophagy, and the amount of LC3-II is directly proportional to the degree of autophagy.
MicroRNAs (miRNAs or miRs) are a class of small, endogenous, non-coding single-stranded RNAs that are widely expressed in vivo. miRNAs regulate the expression of their target genes at the post-transcriptional level by specifically binding to the 3′ untranslated region (3′UTR) of the target mRNA.
14,15 It is well known that miRNAs play an important role in multiple biological processes, including growth, cell death, central nervous system function, and tumor formation.
16–19 A number of studies using microRNA microarray analyses in mouse and model cells have shown that expression of the miR-34 family increases with age.
20 Recent studies have shown that miR-34 overexpression accelerates the aging process of human diploid cells
21 and endothelial progenitor cells.
22 Moreover, the latest research has shown that some miR-34 target genes (such as SIRT1 and Bcl-2) are also important regulators of autophagic degradation.
23,24 Previously, Yang et al.
25 found that miR-34 represses autophagy by directly inhibiting the expression of autophagy-related protein Atg9 in mammalian cells. All these studies support the hypothesis that miR-34 may have a role in regulating autophagy.
Taken together, these previous findings suggest a possible regulatory association among miR-34c, autophagy, and diabetes. Therefore, we sought to determine whether miR-34c is involved in the pathology of diabetic corneal neuropathy and whether miR-34c contributes to diabetic corneal neuropathy by regulating autophagy. Herein, we have investigated this hypothesis using in vivo and in vitro models.
C57BL/6 mice (6–8 weeks old, male) were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All animal care and procedures were carried out according to the Principles of Laboratory Animal Care. All animals were treated in accordance with the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research during the study. We developed mice with type 1 diabetes through intraperitoneal injections of low-dose streptozotocin (STZ, 50 mg/kg; Sigma-Aldrich Corp., St. Louis, MO, USA) in ice-cold citrate-citric acid buffer (pH 4.5) for 5 consecutive days. Twelve weeks after the streptozotocin injections, animals with HbA1c values higher than 6.5% and blood glucose levels higher than 16.7 mmol/L were considered to have DM and were used for experiments.
All miRNA antagomir and agomir sequences are fully complementary to the miRNA and are single-stranded. The non-targeting negative controls were from Guangzhou RiboBio, Co., Ltd. (Guangzhou, China). Trigeminal nerve cells were seeded on 6-well plates in completed KSFM medium to 80% confluence. The cells were then shifted to KSFM medium with no EGF and BPE overnight for starvation. Next, the cells were transiently transfected with a miRNA antagomir or non-targeting negative control using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) for 6 hours according to the manufacturer's protocol. The cells were then transferred to KSFM medium and were collected for further analysis 48 hours after transfection. The expression of miR-34c was detected by qRT-PCR and the expression of autophagy protein were detected by Western blot.
The total protein from mouse TG or cultured neurons was extracted with RIPA lysate. The homogenates, which contained 20 μg of protein, were run on 12% SDS-PAGE gels and then transferred to a PVDF membrane (Millipore Sigma). The PVDF membrane was blocked with 5% non-fat dry milk for 1 hour at room temperature on a shaker and incubated overnight at 4°C with primary antibodies, including LC3 (1:300; Abcam), Atg4b (1:1000; GTX), and GAPDH (1:3000; Abcam).
Downregulation of miR-34c Promotes Corneal Epithelial Wound Healing in Diabetes Via Autophagy In Vivo
Inhibition of miR-34c Promotes Innervations of the Corneal Nerve in Diabetic Mice
Supported by the National Natural Science Foundation of China, Beijing, China (Grant 81670822), the Science and Technology Innovation Joint Fund Project of Fujian Province, Fuzhou, China (Grant 2016Y9013), and Natural Science Foundation of Fujian Province, Fuzhou, China (Grant 2017J01280), the Open Program of Shandong Provincial Key Laboratory of Ophthalmology (Grant 2018-02). The authors alone are responsible for the content and writing of the paper.
Disclosure: J. Hu, None; X.Y. Hu, None; T. Kan, None