miRNA Therapy in Laboratory Models of Acute Spinal Cord Injury in Rodents: A Meta-analysis

miRNA therapy is popularly investigated in treating acute spinal cord injury (SCI) and offers a significant prospect for the treatment of acute SCI. We aimed to provide pre-clinical validations of miRNA in the treatment of SCI. A systematic search of EMBASE, PubMed, Web of Science, the Cochrane Library, and Scopus databases was performed. Rats, which were the most used animals (70%, n = 46 articles), receiving miRNA therapy got prominent recovery in SCI models [BBB score, SMD 3.90, 95% CI 3.08–4.73, p < 0.01]. Locomotor function of fore and hind limbs in SCI mice receiving miRNA therapy (30%, n = 19 articles) [grip strength, SMD 3.22, 95% CI 2.14–4.26; p < 0.01; BBB score, SMD 3.47, 95% CI 2.38–4.56, p < 0.01; BMS, SMD 2.27, 95% CI 1.34–3.20, p < 0.01] also recovered better than mice in control group. Then, we conducted the subgroup analysis and did find that high-quality articles trended to report non-therapeutic effect of miRNA. Furtherly, we analyzed 46 miRNAs, including 9 miRNA families (miR-21-5p/34a-3p/124-3p/126-3p/223-3p/543-3p/30-3p/136-3p/15-5p), among which miR-30-3p/136-3p/15-5p family were not effective in recovering locomotor function of rats. Conclusively, miRNAs are curative drugs for SCI, however, appropriate miRNA carrier and which miRNA is the most efficacious for SCI should be furtherly investigated.


Introduction
Spinal cord injury is a sophisticated pathological process, which often leads to loss of sensation and motor function below the injured segment (Eldahan and Rabchevsky 2018;Park et al. 2017). Mechanically damaged spinal cord tissue and subsequent inflammatory cascades seriously challenge the therapeutics of SCI (Hede 2013). The main treatments are classified into drug and surgical therapy aiming to protect the central neurons and reduce the inflammatory level (Koda et al. 2007;Park et al. 2017). However, drug development stagnates and no ideal agent has been in clinical utilization (Huang et al. 2017).
miRNAs are a class of highly conserved non-coding RNAs associated with multiple biological processes in vivo (Nieto-Diaz et al. 2014;Sun et al. 2018). Moreover, miRNAs participate in the whole process of SCI from acute phase to chronic phrase Xie et al. 2018). Recent researches have shown that the deterioration of SCI can be intercepted and the spinal cord plasticity is greatly improved through blockage and activation of some miRNAs in animal models in vivo (such as miR-21-5p/34a-3p/124-3p/126-3p/223-3p/543-3p/30-3p/136-3p/15-5p) Li et al. 2019aLi et al. , b, 2020Theis et al. 2017;Wu et al. 2019;Xu et al. 2019;Yue et al. 2019). These small RNA molecules provide alternatives for drug research and development. Yang Wang and Hanxiao Yi have contributed equally to this manuscript.
1 3 miRNAs are known to be a series of non-coding RNAs that suppress specific gene expressions by modulation of the transcriptional and post-transcriptional process (Shi et al. 2017;Zhou et al. 2020). It has been widely demonstrated that miRNAs participate in the pathophysiological process of SCI (Rodrigues et al. 2018). Recently, miRNAs are discovered to play a therapeutic role in SCI treatment through the promotion of neuron repair and regeneration (Hu et al. 2019), the promotion of angiogenesis (Liu et al. 2015), the suppression of secondary inflammation (Li et al. 2019a, b), the prevention of neuronal apoptosis (Tao and Shi 2016) for decades. In our research, miR-21/34a/124-3p/126-3p/223/543 family were found therapeutic and further investigation hints that the JAK-STAT pathway (Ning et al. 2019), PI3K/AKT (Jiang et al. 2020) and NF-κB pathway ) are the main pathways entangled with the treatment of SCI. Regretfully, we failed to compare the therapeutic effectiveness among these miRNA families.
Up to date, no article systematically reviewed the protective effectiveness of miRNAs in SCI treatment. We collected all articles reporting miRNAs involving in the promotion of the locomotor function in laboratory models to assess the efficacy of miRNAs in SCI treatment. Owing to the emerging role of exosomes as a naturally biological carrier, these extracellular vesicles can be the ideal delivery system for manually synthesized miRNAs (Kumar et al. 2020;Xu et al. 2020). This review does not discuss the delivery system for miRNAs but aims to comprehensively dissect whether miR-NAs are efficacious in treating SCI in rodents and which miRNAs can potentially be translated into drugs.

Limits
The search interfaces of all databases were limited to the following restrictions: • Other species. • From onset to 21st, March 2021.

Article Selection
First, duplicates of all articles were eliminated. Two participants initially screened all abstracts to pre-include all articles which were closely related to our research objectives. Then, full manuscripts that potentially met the eligibility criteria were obtained and carefully reviewed to identify whether major indicators were reported. A third referee would judge the outcome if any discrepancies occurred.

Eligibility Criteria
Articles reporting the following contents were considered eligible:

Data Extraction
We retrieved baselines, including author, year, miRNA, change of miRNA (regulation), species, gender, agent, anaesthesia, injured segment, SCI model, behavior assessment, administration method, the timing of injection, and dose, from all included articles (YW and HXY). All BBB and BMS values were extracted by Engauge Digitizer software and stored in the excel. All miRNA sequences are supplied in Additional file 2.

Quality Assessment
We assessed the quality in each study using the SYRCLE's tool. This scale mainly comprises five entries, that is selection bias, detection bias, reporting bias, attrition bias and other biases. Each item was marked as low risk of bias, high risk of bias or unclear risk of bias.

Subgroup Analysis
Subgroup analysis concerning the species, model of SCI, period, gender, and miRNA family, miRNA carrier and administration method was conducted. First, the pooled

Statistical Analysis
All data were calculated and exported using the R software 3.6.3 version (University of Auckland, New Zealand) and meta-package. A random-effect model was performed to synthesize pooled-effect if p ≤ 0.05, or I 2 > 50%; otherwise, a fixed-effect model was utilized. Then, the calculated data of BBB score, BMS score and limb strength (left, right limb and pair of forelimbs) by R software presented in this metaanalysis were re-exhibited as standard mean differences SMD with 95% confidence intervals (CI) for outcomes instead of forest plot. Trial sequential analysis (TSA) was conducted using TSA software. Publication bias was tested by Egger's test with R software 3.6.3 version and exhibited as a funnel plot.

Results
The items of this meta-analysis were reported according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) (Additional file 3).

Article Selection
Approximately, 2784 articles were found after the initial search. 2674 irrelevant studies were excluded via browsing the abstract and 45 articles were precluded because of unexpected outcomes and interventions. For example, some of the excluded studies used the cavity of the injured spinal cord, the function of bladder, motor evoked potential (MEP) and (SEP) sensory evoked potential as the endpoints. Unfortunately, these nonfunctional measures are not primary goals. Models used in the rest of the excluded studies are constructed by heat, ultrasound, electricity and magnetism and did not meet the preset criteria (damage caused by ischemia and force).
Finally, 65 experimental trials were taken into analysis, including 46 articles investigating rats and 19 articles investigating mice (Additional file 4).

Characteristics of Included Studies
The primary objectives utilized in SCI models were rats and mice. In rats, the most injured segment ranged from T8-T11 (Liu et al. 2015;Qi et al. 2019), however, the injured segment of mice was ranging from C5 to T12 (Wan et al. 2020;Wang et al. 2019;Zhou et al. 2020). Four SCI models, including ischemia, strike, compression, and transection model, were performed in these studies. The agents were manually synthesized oligonucleotides or other vectors carrying these oligonucleotides. Agents were delivered by either tail vein injection or intrathecal injection. The drug was always immediately injected or continuously injected for 3 days ( Table 1).

Assessment of Locomotor Function in Rats
We analyzed the SMD value of BBB score between SCI and miRNA treatment group at the time when the first and the last assessment of hindlimbs locomotor function were performed. It was demonstrated that significant locomotor function had been discovered at the first assessment (SMD 0.67, 95% CI 0.43-0.90, p < 0.01) in the treatment group. At the last assessment, a greater SMD value of BBB score was observed (SMD 3.90, 95% CI 3.08-4.73, p < 0.01) ( Fig. 2A). Then, the subgroup analysis demonstrated that none of these factors, including injection manner, vectors, and gender, significantly impacted the outcome from the final assessment (Fig. 2B).
Subsequently, we calculated the SMD value of BBB score from the 1st to 35th day post-injury. Pooled analysis showed that miRNAs can effectively boost the locomotor function of rats and the SMD value of BBB score between two groups gradually increased over time (Fig. 1C). Furtherly, we identified that miRNAs were effective in transection model, and strike model but not in the ischemia model (Fig. 1D). As for the ischemia model, significant recovery of locomotor function was identified 7 days post-injury but this significance vanished at the 14th, 21st, and 28th day post-injury.

Analysis of Up and DownRegulated miRNAs in Rats
miRNA therapy aims to upregulate or downregulate the specific miRNA level in vivo. Hence, these articles were categorized based on the miRNA alteration. The outcome showed that miRNA therapy can remarkably promote the locomotor function of rats from the 1st to the 35th day postinjury ( Fig. 2).

Assessment of Locomotor Function in Mice
The recovery of locomotor function in mice was measured by the evaluation of the strength, BBB score and BMS. First, we analyzed the effect of miRNAs in recovering the strength of limbs in mice (Fig. 4A). Interestingly, we found these miRNAs were effective in treating mice in SCI models at the 28th day post-injury; however, non-effectiveness was identified in the pair (SMD 4.88, 95% CI -0.33-10.10, p = 0.07), left (SMD 3.56, 95% CI -0.68-7.80, p = 0.10), and right (SMD 3.82, 95% CI -0.33-7.98, p = 0.07) forelimb (s) at the 14th day post-injury. Then, the analysis of BBB score and BMS showed similar results (Fig. 4B). No significant promotion in BBB score and BMS of mice was identified at the 3rd and 7th day post-injury, respectively. Significant promotion of locomotor function of mice was observed until the 21st day post-injury at the earliest.

Analysis of Quality Score for Rats
The 10 items of SYRCLE's tool symbols 10 scores. If one item is marked as " + ", this study will get 1 score, otherwise, this study gets 0 (Table 2). To evaluate whether article quality impacts the final results, we divided all articles assessing the locomotor function of rats into poor quality (1-4 scores), middle (5-7 scores) and high-quality (8-10 scores) article manually. Finally, 2 articles are marker as high quality, 16 articles are marker as low quality and 28 articles are marked as middle quality. The pooled results from both low quality and middle-quality articles yet demonstrated that miRNAs were potent in recovering hindlimbs locomotor function of rats whenever the BBB scale was performed at the 3rd, 7th, 14th, 21st, 28th, 35th day post-injury (Fig. 5). On the first-day post-injury, only low-quality studies showed that rats receiving miRNA therapy had higher BBB score than SCI rats. As for high-quality studies, no hindlimbs locomotor function was discovered from the first day to the end.

Trial Sequential Analysis
TSAs were performed for rats at the end of the follow-up day in a random-effects model meta-analysis with an overall significance level (α) of 0.05 and a type II error risk (β) of 0.1 (i.e., power 90%) preset (Additional file 5). The cumulative Z-curve for rats crossed the upper monitoring boundary for benefit and the adjusted required information size was calculated as 423 accrued rats, confirming a beneficial effect  Furtherly, we set the value as the least difference of BBB score and Z-curve crossed the upper monitoring boundary for benefit before reaching the adjusted information size 2117 (Additional file 5B).

Publication Bias and Quality Evaluation of Included Articles
Publication biases for BBB scores of rats assessed at first and last measurement of hindlimbs locomotor function (Additional files 6 and 7), and strength (at the 35th day postinjury), BBB score (at the 28th day post-injury), BMS score of mice (at the 35th day post-injury) (Additional files 8-10) were exhibited as funnel plots. We evaluated the article quality using SYRCLE's tool. The results showed that the randomness and blindness were nicely performed by most studies, while the rest merely reported either the randomness or the blindness. Other bias indexes were low risks. Overall, most of the included articles were middle and low quality, and only four articles were considered as high quality. The outcome of the quality assessment is provided in Table 2.

Discussion
miRNA therapy in the treatment of spinal cord injury is an interesting topic because no miRNA drug is now being clinically used or undergoing clinical trials despite the powerful therapeutic effect reported by so many researchers. Possibly, the development of precise delivery and resolution of the off-target effect of miRNA in the future will accelerate translational progress. Herein, we summarized all miRNAs associated with the recovery of motor function and dissected the effect of miRNA in acute traumatic spinal cord injury rodents.

Summary of Evidence
The motor function of spinal cord injury is usually measured by BBB scale and the BMS scale (He et al. 2016;Jee et al. 2012;Wan et al. 2020). Despite BMS scale from modified BBB scale, we separately collected the data from rats and mice. For mice of cervical spinal cord injury, griping strength meter (GSM) method was used to assess the two forelimbs grip force (Dai et al. 2018). First, we discovered significantly preserved motor function in rats receiving miRNA therapy and this may indicate the anti-apoptotic effects of miRNA for neurons. Then, rats in miRNA group recover much greater motor function at the last measurement than at the first measurement. Through subgroup-analysis, gender (female, male), administration method (tail vein injection, intrathecal injection) and forms of miRNA (viral vector, nucleotide and other vectors) are not potential impactor factors of miRNA therapy. Subsequently, we observed the increasing difference of BBB score of rats in control and miRNA group along with the passing of time. Finally, we analyzed different models of spinal cord injury in rats. miRNA appears to be therapeutic for transection model and strike model rather than ischemia model and compression model. Of rats in transection model and strike model, rats suffering from contusion trends to have higher difference of BBB score than rats subjected to spinal cord transection. But this point lacks solid and direct validation.
Usually, miRNA administration is to systemically and locally upregulate and knock down the specific targets of miRNAs (Song et al. 2017;Tao and Shi 2016). Further subgroup-analysis identified that both miRNA mimic and antisense oligonucleotide are powerful in recovering motor function of SCI rats regardless of the action mode of the miRNA (to upregulate or downregulate the target). Interestingly, SCI rats receiving miRNA mimic trend to have higher difference of BBB score than rats receiving miRNA inhibitor and this may prefer to remind us of the therapeutic power of the upregulation of miRNA rather than the downregulation of miRNA in vivo. Subsequently, we dissected which miRNA families are efficacious in treating SCI and which are not. Totally, nine miRNA families were included into analysis and the results reminds us of the therapeutic of miR-21-5p/34a-3p/124-3p/126-3p/223-3p/543-3p family rather than the rest families.
All studies involved 46 different microRNAs, 18 of which are located in the 1, 9, 13, 14, X chromosome and none of which is located in 4, 10, 12, 15, 20 and Y chromosome (Additional file 11). The rest microRNAs are scatteredly located in 2, 3, 5, 8, 11, 16, 17, 18 and     21chromosome. Further analysis demonstrated that most target genes of these microRNAs are associated with giloma and proteoglycans in cancer pathway, the top 3 significantly impacted pathways are fatty acid metabolism, prion diseases, and fatty acid biosynthesis pathway (Additional file 12). We also discovered that miR-204 and 494 can significantly affect metabolism of xenobiotics by cytochrome P450 pathway, miR-153 significantly affects the prion diseases pathway, miR-15a and 195 significantly affect the metabolism of xenobiotics by cytochrome P450, fatty acid biosynthesis, and fatty acid metabolism pathway (Additional file 13). After absorption by target cells (astrocytes, endothelial cells, microglial cells), the certain miRNA will exert a synthetic function because of the way of miRNA functions. The miRNA functions mainly through binding to the 3'-untranslational region of mRNA following the base paring principles (Bushati and Cohen 2007;Lu and Rothenberg 2018;Mohr and Mott 2015), which suggests that abundant mRNAs can be the target candidates of a single miRNA and unanticipated biological behaviors of target cells would occur followed by non-therapeutic effects on patients. Thus, the specific target cell and major targets of a miRNA should be elucidated in the future if we want to put a miRNA into clinical use.
Additionally, an appropriate carrier shall greatly improve the work efficacy of a miRNA. Currently, a wide range of carriers such as lentivirus (Song et al. 2017), adenovirus (Zhang et al. 2020), microvesicles ) and stem cells (Song et al. 2017) are experimentally applied. Considering the safety and the presence of the blood-brain barrier, the microvesicles derived from stem cells could be a potential candidate for miRNA carrier because our previous research discovered a high affinity of stem cell-derived microvesicles to injured neurons (Wang et al. 2021).

Strength and Weakness
We spotted that almost all researchers used a subjective scale, such as BBB and BMS scale, to assess the motor function of animals and an objective criterion is of absence (Dai et al. 2018;Yu et al. 2019;Zhang et al. 2020;Zheng et al. 2020). A combination of a subjective scale and the motor evoked potential (MEP) shall be suggested. Despite the demonstrated therapeutic effect of miR-21-5p/34a-3p/124-3p/126-3p/223-3p/543-3p family, which miRNA shows the best therapeutic effect and if mixed miRNAs exhibited a more powerful therapeutic effect than a single miRNA remains investigated. Moreover, the preferred target cells on which the miRNA exerts its function should be found out because of at least four types of cells in the damaged spinal cord.
The therapeutic effect of miRNA casts light on the translation from laboratory research to clinical research, however, many issues, such as drug administration method, drug delivery, drug carrier and the off-target effect (the safety issue), set huge obstacles between the laboratory research and the clinical application.
Our research has limitations. First, safety always comes the first priority.
We failed to evaluate the safety of different carriers because of the lack of biochemical and physiological indexes of blood. We cannot speculate if the virus will damage the DNA structure of cells or other components of microvesicles and cells will have an unexpected function when these carriers are clinically utilized. Second, tremendous risk bias exists among articles due to methodology, especially different SCI models, applied in the studies. Universally admitted SCI models, such as the acute SCI model, sub-acute SCI model or chronic SCI model, should be used to promote the compatibility among published data. Third, this is a review summarizing experimental research and its conclusion cannot provide clinical recommendations.

Conclusion
miRNA holds the perspective of being interpreted into the clinical application after considering the gap of the efficacy of miRNA between human beings and experimental animals. Meanwhile, the safety of miRNA should be rigorously tested before clinical trials. Analysis of quality score of articles for rat at the 1st, 3rd, 7th, 14th, 21st, 28th, 35th day post-injury. *microRNA group vs SCI group, p < 0.05, #microRNA group vs SCI group, p > 0.05