Functional Screening and Biosafety Identify of the Potential Application of Let-7a in Injured Peripheral Nerve Regeneration

Proper supporting factor can possess the ability to enhance neuron regeneration, for instance, neurotrophic effects especially nerve growth factor (NGF). However, the in vivo applications of NGF are largely limited by its intrinsic disadvantages. Considering that let-7 targets and regulates NGF, and let-7 is also the core and harbor regulators in peripheral nerve repair and regeneration, we evaluated the potential application in clinical. We rstly screened the let-7a as the most ideal let-7 family molecular by gene expression analysis and functional approach. We further evaluated the in vivo safety, the cell permeability of 3 main cells in regeneration micro-environment, and the morphological and functional indicators. Our study provides an essential basis for in vivo application of let-7 and pictured a vision for the clinical translation of miRNA as a prospective alternative for regenerative medicine. weeks after surgery, the anterior tibial muscles and gastrocnemius muscles of rats in each group were collected to determine muscle wet weight ratio. Muscles on both the injured site and the contralateral uninjured site were weighed. The wet weight ratio was calculated by dividing the wet weight of muscle on the injured site to muscle on the contralateral uninjured site. belly of anterior tibial muscle was collected, embedded, and stained with Masson trichrome. bers stained


Introduction
Peripheral nerve injury is a common clinical problem that affects about 13 to 23 per 100,000 persons per year in the developed countries [1,2]. Currently, the gold standard treatment for peripheral nerve injury is autologous nerve grafting. The application of autologous nerve graft, however, is largely limited by its intrinsic disadvantages such as limited donor nerve source, functional loss of donor nerve, and size differences of donor nerve and recipient nerve [3,4]. Therefore, it has an urgent demand to develop and construct tissue engineered nerve grafts with better clinical effects to repair injured peripheral nerve.
Tissue engineered nerve grafts have been designed as a prospective alternative for regenerative medicine [5,6]. Tissue engineered nerve grafts generally contain scaffolds and embedded supporting cells and/or biological cues. Morphological and cellular molecular studies demonstrated that immediately axonal regeneration involves the precise coordination of numerous cells in the environment to provide an optimal regeneration microenvironment. Macrophages, Fibroblasts, and Schwann cells gather to the injury site to perform different functions and help to establish an allowable regenerative pathway, as to achieve impaired peripheral nerve function recovery [7][8][9]. Many growth factors with neurotrophic effects, especially NGF bene t myelin sheath formation, possess the ability to enhance axon regeneration and have been used in neural tissue engineering [3,10]. However, the in vivo applications of these growth factors are largely restricted by their low stability, short half-life periods, and high costs [11,12]. Emerging studies showed that a variety of microRNAs (miRNAs) were dysregulated after peripheral nerve injury [3,[13][14][15][16][17][18]. Therefore, it is feasible to incorporate miRNAs into biomaterial scaffolds and to reshape the microenvironment so as to achieve injured nerve regeneration [10,[19][20][21][22]. Let-7, as the rst identi ed human miRNA, is involved in many important biological processes [23][24][25][26].
The Let-7 family controls developmental timing and differentiation and strong evidence of let-7 has been shown to act as key regulators in various disease in ammation and cancer [27,28]. Previous study showed that decreased let-7 elevated the secretion of NGF from Schwann cells, increased the proliferation and migration of Schwann cells in vitro, and promoted Schwann cell migration and axon outgrowth in vivo [29,30]. The further study showed that let-7 is a core and harbor regulators affecting nerve repair and regeneration [31]. Based on these hypotheses and preliminary study, we screened the ideal target member selection of the let-7 family, the cell permeability of 3 main cells in regeneration micro-environment, evaluated in vivo safety and indicated the morphological and functional to evaluate the repairing effect.

Ethics statement
Experimental animals were purchased from the Animal Experimental Center of Nantong University, Jiangsu, China. Experimental procedures were conducted in accordance with Institutional Animal Care guidelines of Nantong University and were ethically approved by Administration Committee of Experimental Animals, Jiangsu, China.
Quantitative RT-PCR Total RNAs were isolated from cultured Schwann cells and reversely transcribed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and stem-loop RT primers (Ribobio).
Quantitative RT-PCR was performed using QuantiNova SYBR Green PCR Kit (Qiagen, Hilden, Germany) on an Applied Biosystems Stepone real-time PCR System (Applied Biosystems, Foster City, CA). Relative expression levels of let-7 were determined by the ΔΔCt method.
EdU cell proliferation assay Schwann cells were resuspended, seeded onto 96-well plates at a volume of 100 µL and a density of 2×10 5 cells/ml, and transfected with let-7 mimic, let-7 inhibitor, or the corresponding controls for 24 hours. 100 µM EdU was added to the cell culture medium and cells were cultured for additional 12 hours.
After the xation with 4% paraformaldehyde, the proliferation rate of Schwann cells was measured with Cell-Light EdU DNA Cell Proliferation Kit (RiboBio). Images were captured with a DMR uorescence microscope (Leica Microsystems, Bensheim, Germany).

Transwell-based cell migration assay
Schwann cells transfected with let-7 mimic, let-7 inhibitor, or the corresponding controls were resuspended in DMEM medium and seeded onto the upper chamber of a 6.5 mm transwell with 8 µm pores (Corning, Tewksbury, MA) at a volume of 100 µL and a density of 3×10 5 cells/ml. The bottom chamber of transwell was lled with 500 µL culture medium. After 24 hours incubation, Schwann cells left in the upper surface of the upper chamber were cleaned with a cotton swab while Schwann cells migrated to the bottom surface were stained with 0.1% crystal violet. Images were captured with a Leica DMI3000 B (Leica Microsystems). Migrated cells were dissolved in crystal violet with 33% acetic acid and measured the intensity of the absorbance of crystal violet staining using a SynergyTM 2 Multi-Mode Microplate Reader (BioTek, Burlington, VT, USA).

Biosafety assessment
Adult, male SD rats was anesthetized and injected with 100 nmol let-7a antagomir (RiboBio) dissolved in 1 ml saline through caudal vein injection. At 5 days after injection, rats were sacri ced and their hearts, livers, spleens, lungs, and kidneys were collected and xed in 4% PFA. Hetamotylin-eosin staining was performed for histopathological examinations. At 5 days and 4 weeks after injection, rats were subjected to blood sample test. Blood cellular and electrolyte parameters, biochemical parameters, and immunological parameters were measured.
Cells were then stained with Alexa Fluor 488 donkey anti-rabbit IgG and subjected to ow cytometry analysis (BD Bioscience, San Jose, CA). The positive ratio of anti-let-7a containing cells was calculated with the following formula: Positive ratio=UR/(UR+LR) ×100%, where UR represented upper right and LR represented left right.
Animal surgery and application of let-7 antagomir 5 nmol let-7a antagomir was dissolved in 20 µL DEPC-treated water and mixed with 10 µL hydrogel (Beaver for Life Sciences, Suzhou, Jiangsu, China). The mixture of let-7a antagomir and hydrogel was injected into chitosan conduit to construct a let-7a antagomir. Adult, male SD rats (Anti-let-7a group) was subjected to 7 mm sciatic nerve transection and their nerve gaps were bridged with let-7a antagomir. Rats in the control group were bridged with chitosan scaffolds containing 5 nmol let-7a antagomir nontargeting control, 20 µL DEPC-treated water, and 10 µL hydrogel.
Compound muscle action potential (CMAP) recording At 8 weeks after surgery, rats in the control group and the Anti-let-7 group were used for CMAP recording by using a Keypoint 2 portable electromyography (Dantec, Denmark). Recording electrodes were inserted into the mid-belly of gastrocnemius and stimulating electrodes were inserted into the proximal and distal sciatic nerve stumps. An electric stimulus of 5 mV was delivered to evoke CMAP responses. CMAP amplitudes both at the proximal nerve stump and the distal nerve stumps were recorded.

CatWalk gait analysis
At 8 weeks after surgery, rats in the control group and the Anti-let-7a group were used for CatWalk gait analysis. The CatWalk XT system (Noldus Information Technology, the Netherlands) with a high-speed camera that detects digital images at a high-speed rate was used to determine the intensity of rat paws as previously described [32]. Sciatic function index (SFI) was calculated with the following formula: Muscle weight measurement and Masson trichrome staining At 4 and 8 weeks after surgery, the anterior tibial muscles and gastrocnemius muscles of rats in each group were collected to determine muscle wet weight ratio. Muscles on both the injured site and the contralateral uninjured site were weighed. The wet weight ratio was calculated by dividing the wet weight of muscle on the injured site to muscle on the contralateral uninjured site. The belly of anterior tibial muscle was collected, para n embedded, and stained with Masson trichrome. Muscle bers were stained in red while collagen bers were stained in blue.

Statistical analysis
Student's t-test and ANOVA by Dunnett's post hoc test were used to compare the statistical differences among groups. Statistical analysis and histograms were conducted with GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA). A p-value < 0.05 was considered signi cant.
Let-7a, let-7c, and let-7d induced changes of gene expressions of other family members of let-7 Notably, members of the let-7 family and their negative regulator LIN28 possess a double-negative feedback loop. Changes of one member of the let-7 family may further affect other members of the let-7 family via the regulatory effect of LIN28. Considering the importance of Schwann cells in peripheral nerve repair and regeneration, Schwann cells were transfected with the mimics of top 3 highest expressed let-7 miRNAs, let-7a, let-7c, and let-7d. Besides the elevation of the abundance of let-7a, transfection with let-7a mimic elevated the expressions of all other members of let-7d, let-7e, and let-7f (Fig. 2a). Analogously, transfection with let-7c mimic and let-7d mimic increased the expressions of let-7e and let-7f (Fig. 2b). Transfection with let-7d, however, did not signi cantly affect the expressions of other family members of let-7 (Fig. 2c). Schwann cells were further transfected with inhibitors of let-7a, let-7c, or let-7d. The inhibitors of let-7a, let-7c, and let-7d, by contrast with the mimic, reduced the expressions of some members of the let-7 family (Fig. 2d, 2e & 2f). Consistently, the same results were observed in broblasts (Fig. 2g& 2i) and macrophages (Fig. 2h & 2j) transfected with let-7a mimic and let-7a inhibitor. These studies indicated that let-7a is the most ideal molecule for studying the let-7 family.
Our previous study demonstrated that let-7d could strongly inhibit Schwann cell proliferation and migration [29]. Here, the functional effects of let-7a and let-7c were also examined. EdU cell proliferation assay showed that similar as let-7d mimic, Schwann cells transfected with let-7a and let-7c mimic exhibited signi cantly reduced cell proliferation rate (Fig. 3a). On the contrast, cells transfected with let-7a inhibitor, let-7c inhibitor, or let-7d inhibitor exhibited elevated cell proliferation rates (Fig. 3b). Transwellbased cell migration assay showed that let-7a mimic, let-7c mimic and let-7d mimic induced an inhibitory effect on Schwann cell migration while let-7a inhibitor, let-7c inhibitor, and let-7d inhibitor induced a promoting effect on Schwann cell migration (Fig. 3c&3d). These observations suggested that let-7a was the most ideal molecule for studying the let-7 family and let-7a could strongly suppress the proliferation and migration of Schwann cells.
Let-7a did not induce morphological, biochemical, or immunological changes The biosafety of let-7a antagomir was examined by directly introducing high dose (100 nmol) of let-7a antagomir into rats by caudal vein injection. Morphological characteristics of rat heart, liver, spleen, lung, and kidney were determined by hetamotylin-eosin staining at 5 days after let-7a antagomir injection. The external appearances and weights of these organs in rats injected with let-7a antagomir were similar with rats injected with saline only (Fig. 4a). Images from hetamotylin-eosin staining further demonstrated that the histopathological properties of these organs were not affected by let-7a antagomir injection (Fig. 4b).
The examination of rat blood samples collected at 5 days and 4 weeks after let-7a antagomir injection showed that blood cellular and electrolyte parameters, biochemical parameters, and immunological parameters of rats treated with let-7a antagomir were not signi cantly different from saline-treated control rats (Table 1). Table 1 Blood sample examinations of rats injected with let-7a antagomir. Blood cellular and electrolyte parameters, biochemical parameters, and immunological parameters of rats at 5 days and 4 weeks after let-7a antagomir injection were determined. let-7a antagomir realized sustained promoting effect on peripheral nerve regeneration Rat sciatic nerve crush injury was performed and rat sciatic nerve stumps were subjected to ow cytometry analysis to determine whether let-7a antagomir could enter into cells. At 1 day post injury, let-7a antagomir positive ratios in Schwann cells, broblast, and macrophages were about 33.7%, 61.5%, and 81.1%, respectively. At 4 days post injury, let-7a antagomir positive ratios in Schwann cells, broblast, and macrophages were about 54.6%, 31.7%, and 78.3%, respectively (Fig. 5a). Immuno uorescence outcomes directly showed that the antagomir of let-7a could enter into Schwann cells, broblasts, and macrophages (Fig. 5b). The uorescent signal of Cy3 could be detected at 4 weeks after the nerve grafting, suggesting that the controlled release of let-7 antagomir was achieved (Fig. 5c).
The effect of let-7a antagomir on peripheral nerve regeneration was examined by NF-200 staining. Quantitative analysis showed that the uorescence of NF-200 in the distal nerve stump in the Anti-let-7 group was much less than in the normal group but signi cantly higher than the control group (Fig. 6a).
The morphology of motor endplates in gastronomies muscles observed at 8 weeks showed that the motor endplates in the Anti-let-7 group were also obviously larger than those in the control group ( Fig. 6b).
Let-7a antagomir bene ted the functional recovery of injured peripheral nerves CMAP recording showed that at 8 weeks after injury, in normal rat nerves, the peak amplitudes at both the proximal site and the distal site were about 15 mV. In the control group, the detected peak amplitudes were obviously lower. Peak amplitudes in the Anti-let-7a group were much higher than those in the control group, reached about 8 mV (Fig. 7a). Automatic CatWalk track analysis detected the functional of injured peripheral nerves rats was increasing recovered. The measurement of the intensities of rat hindpaw showed that in Anti-let-7a-injected rats, the force and touch of the injured hindpaw were more closed to that of the uninjured hindpaw. At 8 weeks after injury, rats in the control group had a SFI of around -87 while rats in the Anti-let-7 group had a signi cantly higher SFI value of around -70, suggesting that the functional recovery of injured sciatic nerves was much better in the Anti-let-7 group (Fig. 7b).
The weight and morphology of the target muscles were also measured. At 4 weeks after surgery, there existed no difference in-between the wet weight ratios of anterior tibial muscle and gastrocnemius muscle in the control group and the Anti-let-7 group. However, at 8 weeks after surgery, the wet weight ratios of anterior tibial muscle and gastrocnemius muscle in the Anti-let-7 group were signi cantly higher than those in the control group (Fig. 8a). Observations from Masson trichrome staining showed that compared with muscles in the control group, muscle bers were much larger and collagen bers were relatively less in the Anti-let-7 group at 4 and 8 weeks after surgery (Fig. 8b).

Discussion
Therapeutics targeting miRNAs have been proved to be potential treatments for genetic disorders and regenerative medicine [33][34][35]. Since miRNAs regulate cell fate of neurons and glial cells, the potential therapeutic applications of miRNAs in neural tissue engineering have engrossed much attention [36].
Here, we constructed a let-7 antagomir-incorporated biomaterials to bridge peripheral nerve gaps in rats and largely contributes to the translational use of miRNAs.
Quantitative analysis of these let-7 family members in Schwann cells, the main cell type in sciatic nerve stumps, showed that let-7a, let-7c, and let-7d were expressed in relatively high levels while let-7a was the most abundant. Schwann cells were transfected with these highly expressed miRNAs to examine their regulatory effects on the expressions of other members of the let-7 family. Transfection with let-7a mimic, let-7a inhibitor, let-7c mimic and let-7c inhibitor signi cantly affected the abundances of other let-7 family members, indicating that let-7a and let-7c would elicit synergetic effects to enlarge their effectiveness.
Moreover, let-7a mimic and inhibitor showed the most robust effects on Schwann cell proliferation and migration. Therefore, let-7a antagomir was used for generating neural tissue engineered graft.
The cellular uptake and in vivo stability of miRNAs are main barriers of the effective delivery of miRNAs [36]. We examined the presence of let-7a antagomir by immunohistochemistry staining and found that let-7a antagomir could enter into Schwann cells, broblasts, and macrophages. To achieve a stable and controlled release of let-7a antagomir, we innovatively dissolved let-7a antagomir in DEPC-treated water and mixed let-7a antagomir with hydrogel. By using hydrogel as the stabilizer and repository of let-7a antagomir, we found that let-7a antagomir could exist in the injured site for at least 4 weeks after nerve injury and repair. The mixture of let-7a antagomir and hydrogel was then injected into the chitosan conduit to effectively deliver let-7a antagomir into the injured site. Chitosan possesses favorable biocompatibility, biodegradability, and permeability. In our laboratory, chitosan-based arti cial nerve grafts have been applied for peripheral nerve regeneration for a long period of time [37][38][39]. Therefore, we took the advantages of the properties of chitosan, used the chitosan tube as a nerve guidance conduit, and achieved the local delivery of let-7a antagomir. The joint use of hydrogel and chitosan conduit provided a helpful method for the long storage and controlled local release of miRNA agomir and/or antagomir.
The biological effect of let-7a antagomir was fully investigated by histological, morphological, and electrophysiological examinations. Immunohistochemistry staining, CMAP recording, CatWalk gait analysis, muscle weight measurement, and Masson trichrome staining demonstrated that let-7a antagomir could signi cantly promote axon elongation, increase electrophysiological response and sciatic function index, prevent muscle atrophy, and improve nerve innervation. Histopathological, biochemical, and immunological examinations also indicated that there existed no obvious adverse effects of let-7a antagomir. Our study incorporated let-7a into neural arti cial nerve graft and successfully achieved controlled long-time release of let-7a antagomir and signi cantly promoted the growth of axons and the reinnervation of target muscles. The in vivo application of let-7a antagomir offers the possibility to bypass the disadvantages of the direct use of NGF and provides an essential basis for the clinical translation of miRNA-incorporated therapy in neural regeneration. Figure 1 The expression levels of let-7 family members in (a) Schwann cells, (b) broblasts, and (c) macrophages. Histogram showed the relative expression levels of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, and let-7i.

Figure 6
Immunohistochemistry examinations of rat sciatic nerves. (a) NF-200 staining of axons in the transverse sections of normal distal nerve stumps (Normal) and distal nerve stumps bridged with let-7a antagomir (Anti-let-7a) or chitosan-hydrogel scaffold containing non-targeting antagomir control (Con) for 8 weeks.

Figure 7
Functional examinations of rat sciatic nerves. (a) CMAP recordings of normal rat sciatic nerves (Normal) and injured nerves bridged with let-7a antagomir (Anti-let-7a) or chitosan-hydrogel scaffold containing non-targeting antagomir control (Con) for 8 weeks. Histogram showed peak CMAP amplitudes at the proximal and the distal nerve. (b) Representative rat paw print images in CatWalk gait analysis and calculated SCI from CatWalk recordings of the paws of injured rats at 8 weeks after surgery. *p-value < 0.05.

Figure 8
Immunohistochemistry examinations of muscles. (a) Wet weight ratios of gastrocnemius and anterior tibialis muscles of rats bridged with let-7a antagomir (Anti-let-7a) or chitosan-hydrogel scaffold containing non-targeting antagomir control (Con) for 4 and 8 weeks. (b) Masson trichrome staining of gastrocnemius muscle of normal rats (Normal) and nerve injured rats at 4 and 8 weeks after surgery. Red indicated muscle ber staining and blue indicated collagen ber staining. *p-value < 0.05.