MiR-96-5p relieves neuropathic pain by targeting ZEB1

Background MicroRNAs (miRNAs) gradually attracts researchers’ attention in regulating the development of neuropathic pain, and many miRNAs have been reported that were able to alleviate neuropathic pain. Meantime, neuroinammations promote the process of neuropathic pain. MiR ‐ 96 ‐ 5p was associated with many pathological diseases, including many kinds of cancers. However, we knew little about the biological function of miR ‐ 96 ‐ 5p in the regulation of neuropathic pain development. Hence, we focused our study on the biological function of miR ‐ 96 ‐ 5p in neuropathic pain. Results As the result, a decrease of miR ‐ 96 ‐ 5p expression was observed in CCI rats model. Meanwhile, the overexpression of miR-96-5p resulted in inhibition of inammation ‐ correlated biomarkers of IL ‐ 6, IL ‐ 1β and Cox-2. In addition, we predicted the zinc nger E ‐ box binding homeobox 1 (ZEB1) was one target of miR ‐ 96 ‐ 5p, with a conserved interaction site at 3′ ‐ untranslated region of ZEB1. Furthermore, we observed the increased expression of ZEB1 in CCI rats at both of transcriptional and translational level and miR ‐ 96 ‐ 5p could regulate that negatively. And overexpressing ZEB1 would disrupt the miR-96-5p inducing alleviation of neuropathic pain, along with IL ‐ 6, IL ‐ 1β and Cox-2 up-regulated. Conclusions Our study demonstrated that miR ‐ 96 ‐ 5p could relieve neuropathic pain by targeting ZEB1 in vivo.


Introduction
As a result of the somatosensory nervous system affected by lesions or disease, neuropathic pain could caused by trauma, compression, autoimmune disease and diabetes, which had disrupted the quality of many patients' live, with a population prevalence estimate of neuropathic pain ranging from 6.9%-10% (Baron, Binder, & Wasner, 2010;Campbell & Meyer, 2006;van Hecke, Austin, Khan, Smith, & Torrance, 2014). The neuropathic pain development was associated with neuronal pathways, peripheral immune system, satellite cells, Schwann cells, spinal microglia and astrocytes (Scholz & Woolf, 2007;Woolf & Mannion, 1999). However, the mechanism of neuropathic pain is still to be investigated deeply(de Moraes Vieira, Garcia, da Silva, Mualem Araujo, & Jansen, 2012). According to resent reports, there is a strong recommendation for drug proposal in neuropathic pain for pregabalin and gabapentin (Finnerup et al., 2015). But neuropathic pain remains di cult to be cure, because of which the development of effective therapeutic treatments is necessary.
Along with the deepening of investigation to miRNA, it has draw our attention to the linkage between miRNAs and pain.
Here, our recent study was focused on the functions of miR-96-5p in regulating neuropathic pain. First of all, we observed a down-regulation of miR-96-5p in chronic constriction injury (CCI) rats. Meanwhile, the overexpression of miR-96-5p repressed in ammatory cytokines in our rats model. Further, we predicted that ZEB1 was one of targets of miR-96-5p, because it could be negatively regulation by miR-96-5p. As the result of overexpressing ZEB1, the alleviation of neuropathic pain by miR-96-5p was reversed. So we hypothesized miR-96-5p could relieve neuropathic pain by inhibiting ZEB1 level.

Experimental Section
Animal studies In our study, S-D rats (adult, female, weighed 190-210 g) were purchased from Shanghai Animal Laboratory Center. Every cage kept ve rats in a constant environment at 25 °C with 55% ± 5% humidity. All rats were observed to detect any abnormal behaviors every day and randomly assigned after seven days. The intrathecal administration was performed after CCI surgery for 7 days. The spinal cord of rats were collected after rats sacri ced for next experiments. The procedures of our all experiments were under the requirements of Guide for the Care and Use of Laboratory Animals by National Institutes of Health.

CCI rats model
The neuropathic pain rats model was established through CCI method (Bennett & Xie, 1988). Sham control groups were sciatic nerve exposed and isolated and not ligated. At days 0, 3, 7, 14, and 21, the transected and midline-cut dorsal spinal cords were harvested and stored at − 80 °C for further experiments.
qRT-PCR RNAiso Plus (Takara Bio, China) was used to extract total RNA of rats tissues. Then reverse transcription and quantitative PCR were performed through the PrimeScript™ RT Master Mix and SYBR Premix Ex Taq™ II (Takara Bio, China), respectively. The primers were shown in Table 1. All reactions were performed in the QuantStudio6 Flex (Fuji lm, Japan).

Statistical analysis
With at least three independent experiments, all data were exhibited as the form of mean ± standard deviation. Student's t-test and One-Way ANOVA analysis were employed to compare quantitative variables. When the P values were less than 0.05, the differences were signi cant. The SPSS 19.0 (SPSS Inc, USA) was used.

MiR-96-5p was decreased in CCI rats
To investigate whether miR-96-5p possesses biological functions which could regulate neuropathic pain, we rstly examined the levels of miR-96-5p in the spinal cord of CCI rats through qRT-PCR. The levels of miR-96-5p in CCI rats was signi cantly decreased compared to the expression in sham-operated rats, after CCI surgery for 0, 3,7,14 and 21 days (Fig. 1A). Then, we made miR-96-5p stably overexpress in CCI rats through intrathecal injection of LV-miR-96-5p and the up-regulating expression was observed in LV-miR-96-5p infecting CCI rats (Fig. 1B). Next, the relationship between miR-96-5p and neuropathic pain was measured by evaluating mechanical allodynia and thermal hyperalgesia. Both mechanical allodynia (Fig. 1C) and thermal hyperalgesia (Fig. 1D) were restrained in CCI rats after overexpressing miR-96-5p, which implied that miR-96-5p acted as a repressive role in regulation neuropathic pain.

ZEB1 was a direct binding target of miR-96-5p
Further, we employed Starbase, TargetScan, miRDB, and miRanda to predict potential binding targets of miR-96-5p. According to the results of bioinformatic prediction, it was found that a binding region of miR-96-5p was at ZEB1 3′UTR (Fig. 3A). Then we employed dual-luciferase report system to detect the direct binding of miR-96-5p and ZEB1. The wild type (WT) or mutant (MUT) ZEB1 3′UTR was constructed into a dual-luciferase vector and then co-transfected into HEK-293T cells with miR-96-5p mimics. The reduction of luciferase activity indicated the binding of miR-96-5p and WT-ZEB1 mRNA (Fig. 3B). Meanwhile, we examined the expression of ZEB1 in vivo. A signi cant decrease of ZEB1 was observed after overexpressing miR-96-5p in CCI rats through qRT-PCR (Fig. 3C). In addition, the protein level of ZEB1 was also detected and the down-regulation of ZEB1 protein was observed after miR-96-5p overexpressed (Fig. 3D,E). All of these indicated that miR-96-5p could bind to ZEB1 mRNA and repress the expression of ZEB1 directly.
Many miRNAs have been reported could regulate neuropathic pains through different pathways. We investigated that miR-96-5p could alleviate neuropathic pains by targeting ZEB1, along with the down-regulation of in ammation-associated cytokines.
Through establishing CCI rats model, we observed the decrease of miR-96-5p expression and down-regulation of neuroin ammation-associated cytokines in CCI rats. Then, through employing bioinformatic predictions, ZEB1 was found possessed a binding site of miR-96-5p. Overexpressing miR-96-5p in CCI rats could inhibit ZEB1 by targeting its 3'-UTR conserved binding region. The direct interaction of miR-96-5p and ZEB1 3'-UTR was veri ed, and miR-96-5p mimics could signi cant repress ZEB1. Moreover, we observed an up-regulation of ZEB1 in CCI rats and it would be neutralized by miR-96-5p overexpressing. Also, anaplerotic expression of ZEB1 in miR-96-5p overexpressing CCI rats would reverse the alleviating effect miR-96-5p. Taken together, miR-96-5p regulated neuropathic pain through suppressing ZEB1 in vivo. In addition, with more and more reports, lncRNA may play a role in regulating miRNAs such as molecular sponges (Gao et al., 2018), which points a direction for our further study of miR-96-5p.
It has been reported that ZEB1 was associated with many kinds of cancers including breast cancer, renal clear cell carcinoma, and prostate carcinoma (Graham et al., 2008;Guaita et al., 2002;Krishnamachary et al., 2006;Ma et al., 2016). Meantime, there were some reports that miRNAs was able to be regulators in some diseases by targeting ZEB1, such as miR-409-3p, miR-204, miR-708, miR-873, miR-28-5p and miR-429 (Bao et al., 2018;Jiao, Guo, & Fu, 2019;Luo et al., 2019;Ma et al., 2016;Sun et al., 2019; G. C. Wu et al., 2019). In this study, it was observed that the ZEB1 was up-regulated at both transcriptional and translational level in CCI rats. In addition, ZEB1 was one of binding targets of miR-96-5p in regulating neuropathic pain. MiR-96-5p could inhibit ZEB1, and overexpression of ZEB1 could reverse the alleviating effect of miR-96-5p. In conclusion, miR-96-5p alleviate neuropathic pain through down-regulating ZEB1 in vivo, and depress the neuroin ammations at the same time, which indicated miR-96-5p possesses the potential for clinical therapeutic of neuropathic pain. LW revised the manuscript. All authors approved the nal manuscript.