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ciRS-7 targeting miR-135a-5p promotes neuropathic pain in CCI rats via inflammation and autophagy
Journal Pre-proofs Research paper ciRS-7 targeting miR-135a-5p promotes neuropathic pain in CCI rats via inflammation and autophagy Wei Cai, Yang Zhang, Zhen Su PII: DOI: Reference:
S0378-1119(20)30055-X https://doi.org/10.1016/j.gene.2020.144386 GENE 144386
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
2 November 2019 10 January 2020 17 January 2020
Please cite this article as: W. Cai, Y. Zhang, Z. Su, ciRS-7 targeting miR-135a-5p promotes neuropathic pain in CCI rats via inflammation and autophagy, Gene Gene (2020), doi: https://doi.org/10.1016/j.gene.2020.144386
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ciRS-7 targeting miR-135a-5p promotes neuropathic pain in CCI rats via inflammation and autophagy Wei Cai1*, Yang Zhang2*, Zhen Su2 1.Department of Orthopedics, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University, Huai’an Jiangsu, China. 2.Department of Anesthesiology, The Affiliated Huai’an NO.1 Correspondence to Zhen Su; Department of Anesthesiology, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University, Huai’an Jiangsu, China. Email: [email protected]
ciRS-7 targeting miR-135a-5p promotes neuropathic pain in CCI rats via inflammation and autophagy Wei Cai1*, Yang Zhang2*, Zhen Su2 1.Department of Orthopedics, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University, Huai’an Jiangsu, China. 2.Department of Anesthesiology, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University, Huai’an Jiangsu, China. Correspondence to Zhen Su; Department of Anesthesiology, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University, Huai’an Jiangsu, China. Email: [email protected] Abstract Neuropathic pain, caused by damage to the nerve system, is one of the most challenging diseases in the world. Moreover, the etiology remains unclear. Circular RNAs (circRNAs) have been revealed to participate in various biological progress, including neuropathic pain. However, the way circRNAs participate in the progress of
neuropathic pain still needs further study. In this research, we established CCI rat models and measured the expression level of ciRS-7 in the spinal dorsal horn in the postoperative rats. The level of ciRS-7 was positively associated with the progress of neuropathic pain. Next, we test the expression of autophagy and inflammation in the CCI rats, and the results indicate that ciRS-7 associates with the progress of neuropathic pain partly by upregulated the expression level of autophagy and inflammation in the CCI rats. Furthermore, we found ciRS-7 regulates neuropathic pain progress by sponging to miR-135a-5p. In CCI rats, inhibiting miR-135a-5p decreases the level of autophagy and inflammation and alleviates neuropathic pain. We present this research that might provide a new insight for neuropathic pain study. Keywords Circular RNA; Neuropathic pain; ciRS-7; miR-135a-5p; Autophagy; Inflammation 1 Introduction Neuropathic pain, there are about to 30% high prevalence in the general population(Bouhassira et al., 2008), caused by the surgery, trauma, toxicity or other disease affecting the somatosensory nervous system(Blom et al., 2014; Colvin and Dougherty, 2015). Furthermore, it has brought poor life experience to the patients like depression and/or anxiety and a significant health burden to the world(McCarberg and Billington, 2006). The development and progress of neuropathic pain involve many signal cascades and molecules such as autophagy and neuroinflammation makes it one of the most difficult pain to manage(Bennett et al., 2006; Shi et al., 2013; Marinelli et al., 2014; Kiguchi et al., 2017; Sommer et al., 2018). Moreover, the outcomes of neuropathic pain are currently unsatisfied. Though after many efforts have been made to reveal the etiology of neuropathic pain, the molecular mechanisms for the development of neuropathic pain remain unclear and need further elucidation.
Circular RNA (circRNA) is one kind of non-coding RNAs which abundantly expressed in mammals(Jeck and Sharpless, 2014). CircRNA has higher stability due to the single circRNA’s molecular ends that could be covalently linked(Memczak et
al., 2013; Li et al., 2015) in comparison with linear types of RNA. In the past two decades, lots of research have been made to identify the mechanisms of circRNA. With the blossom development of RNA bioinformatics analyses and technology of deep sequencing, circRNA regulates gene expression at the level of transcriptional or after transcriptional and inhibiting their function by binding to microRNA or other ways like circRNA-microRNA-mRNA axis has been well demonstrated(Salzman et al., 2012; Salzman et al., 2013; Zhang et al., 2013). Recently it has been reported that neuropathic pain, which induced by the sciatic chronic constriction injury (CCI), changes the level of circRNA expression in the spinal dorsal horn of rats, indicating that circRNA might associates with the progress of neuropathic pain(Cao et al., 2017; Zhou et al., 2017). Recently, ciRS-7, circular RNA sponge for miR-7, as termed as CDR1as, has been deeply investigated and is highly expressed circular RNA(Hansen et al., 2011; Hentze and Preiss, 2013). As a well-known sponge for miR-7, ciRS-7 consisted of over seventy binding sites. ciRS-7 acts as a regulator of miR-7 by binding it(Hansen et al., 2013b), and participates in various diseases progression(Hsiao et al., 2015; Nagano et al., 2016; Wang et al., 2016; Yue et al., 2016). However, the function of ciRS-7 in the progress of neuropathic pain remains.
In this research, we tested the level of ciRS-7 in the CCI rats’ spinal dorsal horn and found that ciRS-7 associates with the development of neuropathic pain by sponging miR-135a-5p. Furthermore, ciRS-7/miR-135a-5p regulates the level of autophagy and inflammation in the CCI rats’ spinal dorsal horn, which affects the progress of neuropathic pain. 2 Materials and methods 2.1 Chronic-constrictive injury model Establishing rat model of CCI-induced neuropathic pain was according to the previous report(Bennett and Xie, 1988). Male Sprague Dawley (SD) rats (weighing 200-300g) were purchased from the Experimental Animal Center of Zhengzhou University, China. The Rats were maintained in separate cages (humidity, 60 ± 5%; temperature, 23 ± 4°C, 12-12 h light/dark cycle). Briefly, in the neuropathic pain rats,
near the important branch of the sciatic nerve were placed two loose ligatures, and in sham rat, sciatic nerve was just exposure without ligation. The animal surgeries were performed underlying the guidelines of the National Institutes of Health on animal care. This research was approved and authorized by Department of Orthopedics, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University. 2.2 Determination of mechanical and thermal hyperalgesia Paw withdrawal threshold (PWT) indicates Mechanical allodynia, the stimulation of mechanic of rats tested by pain gauge measurement (von Frey; IITC, CA). Paw withdrawal latencies (PWL) indicate Thermal hyperalgesia, the stimulation of radiant heat of rats by using a pain threshold detector. The experimental procedures were performed in accordance with the former report(Chaplan et al., 1994). 2.3 Quantitative Real-Time PCR RNA was extracted from separated spinal cord tissue by employing TRIzol Reagent (Thermo Fisher) followed by the manufacturer’s methods. The extracted total RNA was measured by nanodrop 2000 (Thermo Fisher Scientific) for its quality. Reverse transcription was performed by employing Maxima First Strand cDNA Synthesis kit (cat no. K1642; Thermo Fisher Scientific, Inc.) followed by the manufacturer’s protocol. In accordance with the standard operating procedures, and qRT‐PCR was performed by employing SYBR FAST qPCR Master Mix (2×) Kit (Kapa Biosystems) on an ABI7500 system (Thermo Fisher Scientific) in triplicate. And the internal reference gene GAPDH was used to normalize the relative expression level of mRNAs and LncRNA, respectively. Relative gene level was calculated using 2ΔΔCt
method. The primers were as shown: ciRS-7, sense 5’-
TCTCTGGTGTACCGGTCGTCTAAC-3; and antisense 5’CTCAATATCTACGTCTTCCAACG-3. miR-135a-5p, sense 5′′CTCCTAGGTATGGCTTTTTATTC-3′; and antisense 5′′TCAACTGGTGTCGTGGAGTC-3′. GAPDH, sense 5′′CCTGCCTCTACTGGCGCTGC-3′; and antisense 5′′GCAGTGGGGACACGGAAGGC-3′.
2.4 Cells and transfection The 293T cells and Rat microglial cells were pursued from the American Type Culture Collection (Manassas, VA, USA). DMEM medium was used to culture cells with 10% HyClone (Logan, UT), Cells were grown at 37°C in a water-saturated atmosphere of 5% CO2 in the air. In according to the previous description(Hansen et al., 2013a), pcDNA3.1 expression vector was used to harbor human ciRS-7 cDNA. And Lipofectamine 2000 (Invitrogen, Waltham, MA) was used to transfect the cells with Vector‐ciRS-7, siRNA‐ciRS-7, miR‐135a-5p mimics or negative control were synthesized by Sangon Biotech (Shanghai,China).
2.5 Intrathecal injection procedure A PE-10 polyethylene catheter was planted into the cisterna magna to perform the intrathecal implantation. By employing a microinjection syringe, recombinant siRNA, plasmids, inhibitor or their control (10 μL, Sangon) was injected into the intrathecal catheter. For each intrathecal administration. The L4–L5 lumbar spinal cords were harvested for biological test After the experiments.
2.6 WB analysis The protein from spinal cord tissues (L4-5) was harvested by employing Radio immunoprecipitation assay (RIPA) lysis buffer (Boster, Wuhan, China). Protein qualified by using BCA protein assay kit (Sigma Aldrich). To perform the western blot analysis, 10% SDS–PAGE gel was used to separate protein, then electrophoretically transferred onto PVDF membrane (Millipore, IPVH85R). At room temperature, membrane was blocked with 5% milk in TBST, 0.1% Tris buffered saline Tween 20, for 1 hour, TBST was used to wash the membrane three times and incubated with primary antibody overnight at 4°C, then incubated with secondary antibody. Antibodies were purchased from Abcam. And results were detected with a Bio-Rad imaging system (Hercules, CA, United States).
2.7 Luciferase Reporter Assay PmirGLO dual-luciferase reporter plasmids (Genechem, China) were harboring the fragments of ciRS-7 3′-UTRs which consisting the wild-type (WT) seed-matched or mutant (MT) binding sites. And about 5 × 104 293T cells were placed into a 12‐well plate overnight. Then psiCHECK2‐ciRS‐7 plasmid and WT type, miR-135a-5p mimics were transfected into 293T cells. After 2 days, cells were lysed, and the luciferase activity was measured according to the protocol of luciferase reporter gene system (Promega).
2.8 RNA‐binding protein immunoprecipitation (RIP) miR-135a-5p mimics and Myc-AGO2 were transfected into cells. And after 48 hours, AGO2 protein immunoprecipitation assay was performed. RNA immunoprecipitation (RIP) buffer was used to lyse treated cells, which is consisted of 5mM DTT, 5mM EDTA, 0.5% Triton X-100, 150mM KCL, 25mM pH 7.4 Tris-HCL with proteinase inhibitor and RNase inhibitor. Rabbit anti‐Myc tag antibody and IgG negative control antibody (Abcam) which consists sepharose beads (Sigma, USA) was cultivated at 4 °C, and with cycle rotation. Subsequently, in a designated buffer. And then, by adding 1 mL of Trizol, RNA was isolated from the IP's and input. Detectting the binding targets of miR‐135a-5p, the eluted RNA was carried out by synthesizing cDNA for qRT-PCR. 2.9 Statistical analysis Statistical analyses were carried out by employing GraphPad Prism 8 and SPSS17.0 statistical software. The assays were repeated in triplicate. The results are represented as means ± SD. Student's t-test was employed to estimate the differences between. Spearman's correlation test was used to estimate the correlation between miR-135a-5p and ciRS-7. with significant levels set at *P < 0.05 and **P < 0.01. 3 Results 3.1 ciRS-7 was increased in CCI rats and promotes CCI-induced neuropathic pain.
To find out whether the expression of ciRS-7 was associated with the progress of neuropathic pain. Rat models of chronic constriction injury (CCI) were generated. And then, as showed in Fig.1A, we assessed the expression level of ciRS-7 in CCI rats at postoperative days 0, 3, 7, 11, 15, 20. The results shown that, comparing with the control group, the level of ciRS-7 was significantly increased in a time-dependent manner and keep stable since day15, indicating that a close association between ciRS7 and neuropathic pain. Subsequently, we observed the expression level of ciRS-7 in CCI rats, which infected the si-ciRS-7 and si-NC of post-operation day 7. The results showed in the Fig.1B; the ciRS-7 level was remarkably decreased after si-ciRS-7 infection. Next, after intrathecal administration of ciRS-7, we tested the function of ciRS-7 on neuropathic pain by evaluating the mechanical allodynia and thermal hyperalgesia. Paw withdrawal latency (PWL) to radiant heat, which manifests thermal hyperalgesia, showed that comparing with the si-ciRS-7 group, PWL was reduced in the si-NC group, indicating that ciRS-7 could reinforce the thermal hyperalgesia in CCI rats. Subsequently, Paw withdrawal threshold (PWT) tested at days 0, 3, and 7, 15 and 20, postoperative, showed that ciRS-7 could also enhance the mechanical allodynia in CCI models. And results manifest that ciRS-7 significantly promotes neuropathic pain progression.
3.2 Increased ciRS-7 induced neuroinflammation and autophagy in CCI rat models To elucidate the effect of ciRS-7 in the progress of neuropathic pain. We assessed the neuroinflammation and autophagy biomarkers in the dorsal spinal cord of rats at postoperative days 3, 7, 15, and 20, respectively. The results, as shown in Fig 2A. The level of inflammation biomarkers IL-6, IL-12, TNF-α, and Iba1 was increased and keep stable at postoperative day 15. Next, we examined the level of autophagy biomarkers Beclin-1, LC3-I, lC3-II, and p62 at postoperative days 3, 7, 11, 15, and 20. In a time-dependent manner, Beclin-1, LC3-I, LC3-II was increased, P62 was decreased and kept stable at postoperative day 15, as showed in Fig.2C. Then we assessed the expression level of neuroinflammation and autophagy biomarkers level
in the rats affected si-ciRS-7 and negative control group after three days. The results showed in Fig.2B, D. IL-6, IL-12, TNF-α, and Iba1 were decreased. Moreover, Beclin-1, LC3-I, LC3-II was decreased, P62 was increased. Indicating that ciRS-7 promotes the development of neuropathic pain partly depends on neuroinflammation and autophagy in CCI rats. 3.3 miR-135a-5p was directly target of ciRS-7 To elucidate the underlying molecular mechanisms of ciRS-7 in the progress of neuropathic pain. We favorably identified miR-135a-5p as a downstream target gene of ciRS-7 by performing bioinformatics analysis using TargetScan, ENCORI. Subsequently, the WT and Mut miR-135a-5p was manifested, as shown in Fig.3A. Next, we measured the expression level of miR-135a-5p in the si-NC and si-ciRS-7 infected rat microglial cells. As shown in Fig.3B, with the level of ciRS-7 decreased, the level of miR-135a-5p also decreased, indicating that the level of miR-135a-5p is related to ciRS-7 in the microglial cells. Then the Dual-luciferase reporter analysis was performed. The luciferase vector harboring WT‐miR-135a-5p co-transfected with ciRS-7, and the relative luciferase activity was decreased. However, co-transfection with negative control upregulated the relative luciferase activity. This result indicates that miR-135a-5p was a down-stream target gene of ciRS-7(Fig.3C). RNA-induced silencing complexes (RISCs) are generated by miRNA ribonucleoprotein complexes(miRNPs), presenting in anti-Ago2 immunoprecipitants. As a result, miRNAs and their relative RNA components were contained in anti-Ago2 immunoprecipitants(Zhang et al., 2016; King and Borchert, 2017). RIP was carried out by using anti-Ago2 in rat microglial cells isolation. Comparing the anti-IgG immunoprecipitates, ciRS-7 and miR-135a-5p were gathered in miRNPs which containing Ago2 immunoprecipitates (Fig.3D). Furthermore, the results of RNA pulldown assay elucidate that ciRS-7 is significantly gathered in the WT-miR-135a-5p compared with that in the Mut-miR-135a-5p (Fig 3E). Those results revealed that miR-135a-5p is a downstream target gene of ciRS-7 and directly binding to it. 3.4 Inhibiting miR-135a-5p abrogated the effect of ciRS-7.
To validate whether ciRS-7 promoting neuropathic pain through miR-135a-5p. We investigated the biological function of miR-135a-5p in the progress of neuropathic pain. miR-135a-5p inhibitor and control were infected in microglial cell and CCI rats, then the expression of miR-135a-5p was tested by RT-qPCR (Fig.4A). Subsequently, we examined the PWT and PWL on the miR-135a-5p treated CCI rats. The results are shown in Fig B and C. Decreasing the expression of miR-135a-5p inhibits neuropathic pain in CCI rats. Next, we tested the expression of neuroinflammation and autophagy biomarkers in ciRS-7 and miR-135a-5p inhibitor infected microglial cells and CCI rats. As shown in Fig D and F, in microglial cells, the level of neuroinflammation and autophagy in ciRS-7 with miR-135a-5p inhibitor group are decreased comparing with ciRS-7 with the negative control group. And in CCI rats, the level of neuroinflammation and autophagy in CCI with miR-135a-5p inhibitor group are significantly decreased compared with CCI with the NC group. Those results were indicating that ciRS-7 promoting neuropathic pain in CCI rats partly through mediating miR-135a-5p. Discussion Due to the difficulty management, worse outcome, and ineffective therapy of neuropathic pain. It has become a global health burden, and lots of efforts have been made to understand the molecular mechanisms underlying the development of neuropathic pain, but the mystery of neuropathic pain still unclear currently. In the past two decades, the function of circRNA in the progress of various diseases has been deeply investigated. The function of circRNA in neuropathic pain progress has been rarely studied.
In this study, we revealed that circRNA ciRS-7 was positively related to the progress of CCI-induced neuropathic pain, and with the upregulation of ciRS-7, the neuropathic pain developed. We also found that ciRS-7 promotes neuropathic pain partly through upregulating the level of autophagy and inflammation. In the CCI rats, with the level of ciRS-7 increased, the biomarkers of autophagy Beclin-1, LC3-I, LC3-II were increased, and P62 was decreased. Moreover, the biomarkers of
inflammation IL-6, IL-12, Iba1, and TNF-α were increased. We further demonstrated that ciRS-7 regulates autophagy and inflammation in CCI rats by directly targeting miR-135a-5p. Inhibiting miR-135a-5p in CCI rats or microglial cells decreased the level of autophagy and inflammation and alleviated neuropathic pain in CCI rats.
Our investigation presents the evidence that ciRS-7 can promote neuropathic pain by targeting miR-135a-5p through increases in the level of autophagy and inflammation. Furthermore, reducing the expression level of ciRS-7 or using miR-135a-5p inhibitor can hold back this phenomenon. We propose that this could be the potential treatment for neuropathic pain.
Acknowledgement N.A Data availability statement The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Declaration of competing interest There is no conflict of interests to declare in the current study. Funding N.A
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Figure 1 ciRS-7 was increased in CCI rats and promotes neuropathic pain. A, ciRS-7 expression level in the rats’ L4-6 dorsal spinal cord. At postoperative days 0, 3, 7, 15 and 20, qRT-PCR was performed. B, Expression level of ciRS-7 in CCI rats which injected with si-ciRS-7 or si-NC day 7 after sugery. C, the function of ciRS-7 on
mechanical allodynia was estimated by PWT. D, the function of ciRS-7 on thermal hyperalgesia was tested by PWL. Figure 2 Increased ciRS-7 induced neuroinflammation and autophagy in CCI rats. A and C. The protein level of IL-6, IL-12, TNF-α, Iba1 and Beclin-1, P62, LC3-I, LC3-II at postoperative days 3, 7, 11, 15,20 were tested by western blot. B and C. The protein level of IL-6, IL-12, TNF-α, Iba1 and Beclin-1, P62, LC3-I, LC3-II at postoperative days 3 after si-NC and si-ciRS-7 infection were tested by western blot. Figure 3 ciRS-7 was directly targeting miR-135a-5p. A; The binding sites of ciRS-7 with miR135a-5p and the Mut type of miR-135a-5p. B; The expression of ciRS-7 and miR135a-5p in the si-NC and si-ciRS-7 infected rat microglial cells. C; Luciferase reporter gene assays were used to estimate the luciferase activity in the rat microglial cells. **P<0.01.D; The enrichment of ciRS-7 and miR-135a-5p immunoprecipiated complex were delivered to Anti-Argonaute 2 RNA immunoprecipitation (RIP) assays and anti-immunoglobulin (IgG) was used as the control. **P<0.01. E; The rat microglial cells were infected with WT miR-135a-5p and Mut miR-135a-5p. Realtime quantitative polymerse chain reaction (RT-qPCR) were used to measure the RNA levels of ciRS-7 and GAPDH. **P<0.01. Figure 4 Inhibiting miR-135a-5p abrogated the function of ciRS-7. A. the expression of miR135a-5p in the NC and inhibitor treated microglial cells and CCI rats. B. the function of miR-135a-5p inhibitor and negative control on mechanical allodynia was assessed by PWT. C, the effect of miR-135a-5p inhibitor and negative control on thermal hyperalgesia was evaluated by PWL. CCI, chronic constrictive injury. D,E the protein level of IL-6, IL-12, TNF-α, and GAPDH in the ciRS-7 and miR-135a-5p inhibitor treated microglial cells and CCI rats were tested by western blot. F,G the protein level of Beclin-1, P62, LC3-I, LC3-II and GAPDH in the ciRS-7 and miR-135a-5p inhibitor treated microglial cells and CCI rats were tested by western blot. *P<0.05.
Highlights • CircRNA ciRS-7 was upregulated and involved in the neuropathic pain development. • CircRNA ciRS-7 promotes neuroinflammation and autophagy level in CCI rat models • CircRNA ciRS-7 promotes neuroinflammation and autophagy by sponging to miR-13a-5p
Declaration of Interest Statement The authors certify that they have participated sufficiently in the work to take public responsibility for the appropriateness of the experimental design, method, collection, analysis, and interpretation of the data. This manuscript has not been published in whole or in part nor is it being considered for publication elsewhere. The authors declare that there are no financial or other relationships that might lead to a conflict of interest of the present article. All authors have reviewed the final version of the manuscript and approved it for publication.
Abbreviations List CCI, chronic constriction injury; circRNA, circular RNA; qRT-PCR, Quantitative reverse transcription PCR; IP, immunoprecipitation; RIP, RNA‐binding protein immunoprecipitation; 3′-UTR,3′-Untranslated Region; PWT, Paw withdrawal threshold; PWL, Paw withdrawal latencies
Credit Author Statement
Author contributions Wei Cai: Conceptualization, Methodology, Software, Investigation, Writing - Original Draft. Yang Zhang: Validation, Formal analysis, Visualization, Software. Zhen Su: Resources, Writing - Review & Editing, Supervision, Data Curation. Wei Cai: Writing: Review & Editing. Yang Zhang: Writing: Review & Editing. Zhen Su: Writing: Review & Editing
S0378-1119(20)30055-X https://doi.org/10.1016/j.gene.2020.144386 GENE 144386
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
2 November 2019 10 January 2020 17 January 2020
Please cite this article as: W. Cai, Y. Zhang, Z. Su, ciRS-7 targeting miR-135a-5p promotes neuropathic pain in CCI rats via inflammation and autophagy, Gene Gene (2020), doi: https://doi.org/10.1016/j.gene.2020.144386
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ciRS-7 targeting miR-135a-5p promotes neuropathic pain in CCI rats via inflammation and autophagy Wei Cai1*, Yang Zhang2*, Zhen Su2 1.Department of Orthopedics, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University, Huai’an Jiangsu, China. 2.Department of Anesthesiology, The Affiliated Huai’an NO.1 Correspondence to Zhen Su; Department of Anesthesiology, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University, Huai’an Jiangsu, China. Email: [email protected]
ciRS-7 targeting miR-135a-5p promotes neuropathic pain in CCI rats via inflammation and autophagy Wei Cai1*, Yang Zhang2*, Zhen Su2 1.Department of Orthopedics, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University, Huai’an Jiangsu, China. 2.Department of Anesthesiology, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University, Huai’an Jiangsu, China. Correspondence to Zhen Su; Department of Anesthesiology, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University, Huai’an Jiangsu, China. Email: [email protected] Abstract Neuropathic pain, caused by damage to the nerve system, is one of the most challenging diseases in the world. Moreover, the etiology remains unclear. Circular RNAs (circRNAs) have been revealed to participate in various biological progress, including neuropathic pain. However, the way circRNAs participate in the progress of
neuropathic pain still needs further study. In this research, we established CCI rat models and measured the expression level of ciRS-7 in the spinal dorsal horn in the postoperative rats. The level of ciRS-7 was positively associated with the progress of neuropathic pain. Next, we test the expression of autophagy and inflammation in the CCI rats, and the results indicate that ciRS-7 associates with the progress of neuropathic pain partly by upregulated the expression level of autophagy and inflammation in the CCI rats. Furthermore, we found ciRS-7 regulates neuropathic pain progress by sponging to miR-135a-5p. In CCI rats, inhibiting miR-135a-5p decreases the level of autophagy and inflammation and alleviates neuropathic pain. We present this research that might provide a new insight for neuropathic pain study. Keywords Circular RNA; Neuropathic pain; ciRS-7; miR-135a-5p; Autophagy; Inflammation 1 Introduction Neuropathic pain, there are about to 30% high prevalence in the general population(Bouhassira et al., 2008), caused by the surgery, trauma, toxicity or other disease affecting the somatosensory nervous system(Blom et al., 2014; Colvin and Dougherty, 2015). Furthermore, it has brought poor life experience to the patients like depression and/or anxiety and a significant health burden to the world(McCarberg and Billington, 2006). The development and progress of neuropathic pain involve many signal cascades and molecules such as autophagy and neuroinflammation makes it one of the most difficult pain to manage(Bennett et al., 2006; Shi et al., 2013; Marinelli et al., 2014; Kiguchi et al., 2017; Sommer et al., 2018). Moreover, the outcomes of neuropathic pain are currently unsatisfied. Though after many efforts have been made to reveal the etiology of neuropathic pain, the molecular mechanisms for the development of neuropathic pain remain unclear and need further elucidation.
Circular RNA (circRNA) is one kind of non-coding RNAs which abundantly expressed in mammals(Jeck and Sharpless, 2014). CircRNA has higher stability due to the single circRNA’s molecular ends that could be covalently linked(Memczak et
al., 2013; Li et al., 2015) in comparison with linear types of RNA. In the past two decades, lots of research have been made to identify the mechanisms of circRNA. With the blossom development of RNA bioinformatics analyses and technology of deep sequencing, circRNA regulates gene expression at the level of transcriptional or after transcriptional and inhibiting their function by binding to microRNA or other ways like circRNA-microRNA-mRNA axis has been well demonstrated(Salzman et al., 2012; Salzman et al., 2013; Zhang et al., 2013). Recently it has been reported that neuropathic pain, which induced by the sciatic chronic constriction injury (CCI), changes the level of circRNA expression in the spinal dorsal horn of rats, indicating that circRNA might associates with the progress of neuropathic pain(Cao et al., 2017; Zhou et al., 2017). Recently, ciRS-7, circular RNA sponge for miR-7, as termed as CDR1as, has been deeply investigated and is highly expressed circular RNA(Hansen et al., 2011; Hentze and Preiss, 2013). As a well-known sponge for miR-7, ciRS-7 consisted of over seventy binding sites. ciRS-7 acts as a regulator of miR-7 by binding it(Hansen et al., 2013b), and participates in various diseases progression(Hsiao et al., 2015; Nagano et al., 2016; Wang et al., 2016; Yue et al., 2016). However, the function of ciRS-7 in the progress of neuropathic pain remains.
In this research, we tested the level of ciRS-7 in the CCI rats’ spinal dorsal horn and found that ciRS-7 associates with the development of neuropathic pain by sponging miR-135a-5p. Furthermore, ciRS-7/miR-135a-5p regulates the level of autophagy and inflammation in the CCI rats’ spinal dorsal horn, which affects the progress of neuropathic pain. 2 Materials and methods 2.1 Chronic-constrictive injury model Establishing rat model of CCI-induced neuropathic pain was according to the previous report(Bennett and Xie, 1988). Male Sprague Dawley (SD) rats (weighing 200-300g) were purchased from the Experimental Animal Center of Zhengzhou University, China. The Rats were maintained in separate cages (humidity, 60 ± 5%; temperature, 23 ± 4°C, 12-12 h light/dark cycle). Briefly, in the neuropathic pain rats,
near the important branch of the sciatic nerve were placed two loose ligatures, and in sham rat, sciatic nerve was just exposure without ligation. The animal surgeries were performed underlying the guidelines of the National Institutes of Health on animal care. This research was approved and authorized by Department of Orthopedics, The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University. 2.2 Determination of mechanical and thermal hyperalgesia Paw withdrawal threshold (PWT) indicates Mechanical allodynia, the stimulation of mechanic of rats tested by pain gauge measurement (von Frey; IITC, CA). Paw withdrawal latencies (PWL) indicate Thermal hyperalgesia, the stimulation of radiant heat of rats by using a pain threshold detector. The experimental procedures were performed in accordance with the former report(Chaplan et al., 1994). 2.3 Quantitative Real-Time PCR RNA was extracted from separated spinal cord tissue by employing TRIzol Reagent (Thermo Fisher) followed by the manufacturer’s methods. The extracted total RNA was measured by nanodrop 2000 (Thermo Fisher Scientific) for its quality. Reverse transcription was performed by employing Maxima First Strand cDNA Synthesis kit (cat no. K1642; Thermo Fisher Scientific, Inc.) followed by the manufacturer’s protocol. In accordance with the standard operating procedures, and qRT‐PCR was performed by employing SYBR FAST qPCR Master Mix (2×) Kit (Kapa Biosystems) on an ABI7500 system (Thermo Fisher Scientific) in triplicate. And the internal reference gene GAPDH was used to normalize the relative expression level of mRNAs and LncRNA, respectively. Relative gene level was calculated using 2ΔΔCt
method. The primers were as shown: ciRS-7, sense 5’-
TCTCTGGTGTACCGGTCGTCTAAC-3; and antisense 5’CTCAATATCTACGTCTTCCAACG-3. miR-135a-5p, sense 5′′CTCCTAGGTATGGCTTTTTATTC-3′; and antisense 5′′TCAACTGGTGTCGTGGAGTC-3′. GAPDH, sense 5′′CCTGCCTCTACTGGCGCTGC-3′; and antisense 5′′GCAGTGGGGACACGGAAGGC-3′.
2.4 Cells and transfection The 293T cells and Rat microglial cells were pursued from the American Type Culture Collection (Manassas, VA, USA). DMEM medium was used to culture cells with 10% HyClone (Logan, UT), Cells were grown at 37°C in a water-saturated atmosphere of 5% CO2 in the air. In according to the previous description(Hansen et al., 2013a), pcDNA3.1 expression vector was used to harbor human ciRS-7 cDNA. And Lipofectamine 2000 (Invitrogen, Waltham, MA) was used to transfect the cells with Vector‐ciRS-7, siRNA‐ciRS-7, miR‐135a-5p mimics or negative control were synthesized by Sangon Biotech (Shanghai,China).
2.5 Intrathecal injection procedure A PE-10 polyethylene catheter was planted into the cisterna magna to perform the intrathecal implantation. By employing a microinjection syringe, recombinant siRNA, plasmids, inhibitor or their control (10 μL, Sangon) was injected into the intrathecal catheter. For each intrathecal administration. The L4–L5 lumbar spinal cords were harvested for biological test After the experiments.
2.6 WB analysis The protein from spinal cord tissues (L4-5) was harvested by employing Radio immunoprecipitation assay (RIPA) lysis buffer (Boster, Wuhan, China). Protein qualified by using BCA protein assay kit (Sigma Aldrich). To perform the western blot analysis, 10% SDS–PAGE gel was used to separate protein, then electrophoretically transferred onto PVDF membrane (Millipore, IPVH85R). At room temperature, membrane was blocked with 5% milk in TBST, 0.1% Tris buffered saline Tween 20, for 1 hour, TBST was used to wash the membrane three times and incubated with primary antibody overnight at 4°C, then incubated with secondary antibody. Antibodies were purchased from Abcam. And results were detected with a Bio-Rad imaging system (Hercules, CA, United States).
2.7 Luciferase Reporter Assay PmirGLO dual-luciferase reporter plasmids (Genechem, China) were harboring the fragments of ciRS-7 3′-UTRs which consisting the wild-type (WT) seed-matched or mutant (MT) binding sites. And about 5 × 104 293T cells were placed into a 12‐well plate overnight. Then psiCHECK2‐ciRS‐7 plasmid and WT type, miR-135a-5p mimics were transfected into 293T cells. After 2 days, cells were lysed, and the luciferase activity was measured according to the protocol of luciferase reporter gene system (Promega).
2.8 RNA‐binding protein immunoprecipitation (RIP) miR-135a-5p mimics and Myc-AGO2 were transfected into cells. And after 48 hours, AGO2 protein immunoprecipitation assay was performed. RNA immunoprecipitation (RIP) buffer was used to lyse treated cells, which is consisted of 5mM DTT, 5mM EDTA, 0.5% Triton X-100, 150mM KCL, 25mM pH 7.4 Tris-HCL with proteinase inhibitor and RNase inhibitor. Rabbit anti‐Myc tag antibody and IgG negative control antibody (Abcam) which consists sepharose beads (Sigma, USA) was cultivated at 4 °C, and with cycle rotation. Subsequently, in a designated buffer. And then, by adding 1 mL of Trizol, RNA was isolated from the IP's and input. Detectting the binding targets of miR‐135a-5p, the eluted RNA was carried out by synthesizing cDNA for qRT-PCR. 2.9 Statistical analysis Statistical analyses were carried out by employing GraphPad Prism 8 and SPSS17.0 statistical software. The assays were repeated in triplicate. The results are represented as means ± SD. Student's t-test was employed to estimate the differences between. Spearman's correlation test was used to estimate the correlation between miR-135a-5p and ciRS-7. with significant levels set at *P < 0.05 and **P < 0.01. 3 Results 3.1 ciRS-7 was increased in CCI rats and promotes CCI-induced neuropathic pain.
To find out whether the expression of ciRS-7 was associated with the progress of neuropathic pain. Rat models of chronic constriction injury (CCI) were generated. And then, as showed in Fig.1A, we assessed the expression level of ciRS-7 in CCI rats at postoperative days 0, 3, 7, 11, 15, 20. The results shown that, comparing with the control group, the level of ciRS-7 was significantly increased in a time-dependent manner and keep stable since day15, indicating that a close association between ciRS7 and neuropathic pain. Subsequently, we observed the expression level of ciRS-7 in CCI rats, which infected the si-ciRS-7 and si-NC of post-operation day 7. The results showed in the Fig.1B; the ciRS-7 level was remarkably decreased after si-ciRS-7 infection. Next, after intrathecal administration of ciRS-7, we tested the function of ciRS-7 on neuropathic pain by evaluating the mechanical allodynia and thermal hyperalgesia. Paw withdrawal latency (PWL) to radiant heat, which manifests thermal hyperalgesia, showed that comparing with the si-ciRS-7 group, PWL was reduced in the si-NC group, indicating that ciRS-7 could reinforce the thermal hyperalgesia in CCI rats. Subsequently, Paw withdrawal threshold (PWT) tested at days 0, 3, and 7, 15 and 20, postoperative, showed that ciRS-7 could also enhance the mechanical allodynia in CCI models. And results manifest that ciRS-7 significantly promotes neuropathic pain progression.
3.2 Increased ciRS-7 induced neuroinflammation and autophagy in CCI rat models To elucidate the effect of ciRS-7 in the progress of neuropathic pain. We assessed the neuroinflammation and autophagy biomarkers in the dorsal spinal cord of rats at postoperative days 3, 7, 15, and 20, respectively. The results, as shown in Fig 2A. The level of inflammation biomarkers IL-6, IL-12, TNF-α, and Iba1 was increased and keep stable at postoperative day 15. Next, we examined the level of autophagy biomarkers Beclin-1, LC3-I, lC3-II, and p62 at postoperative days 3, 7, 11, 15, and 20. In a time-dependent manner, Beclin-1, LC3-I, LC3-II was increased, P62 was decreased and kept stable at postoperative day 15, as showed in Fig.2C. Then we assessed the expression level of neuroinflammation and autophagy biomarkers level
in the rats affected si-ciRS-7 and negative control group after three days. The results showed in Fig.2B, D. IL-6, IL-12, TNF-α, and Iba1 were decreased. Moreover, Beclin-1, LC3-I, LC3-II was decreased, P62 was increased. Indicating that ciRS-7 promotes the development of neuropathic pain partly depends on neuroinflammation and autophagy in CCI rats. 3.3 miR-135a-5p was directly target of ciRS-7 To elucidate the underlying molecular mechanisms of ciRS-7 in the progress of neuropathic pain. We favorably identified miR-135a-5p as a downstream target gene of ciRS-7 by performing bioinformatics analysis using TargetScan, ENCORI. Subsequently, the WT and Mut miR-135a-5p was manifested, as shown in Fig.3A. Next, we measured the expression level of miR-135a-5p in the si-NC and si-ciRS-7 infected rat microglial cells. As shown in Fig.3B, with the level of ciRS-7 decreased, the level of miR-135a-5p also decreased, indicating that the level of miR-135a-5p is related to ciRS-7 in the microglial cells. Then the Dual-luciferase reporter analysis was performed. The luciferase vector harboring WT‐miR-135a-5p co-transfected with ciRS-7, and the relative luciferase activity was decreased. However, co-transfection with negative control upregulated the relative luciferase activity. This result indicates that miR-135a-5p was a down-stream target gene of ciRS-7(Fig.3C). RNA-induced silencing complexes (RISCs) are generated by miRNA ribonucleoprotein complexes(miRNPs), presenting in anti-Ago2 immunoprecipitants. As a result, miRNAs and their relative RNA components were contained in anti-Ago2 immunoprecipitants(Zhang et al., 2016; King and Borchert, 2017). RIP was carried out by using anti-Ago2 in rat microglial cells isolation. Comparing the anti-IgG immunoprecipitates, ciRS-7 and miR-135a-5p were gathered in miRNPs which containing Ago2 immunoprecipitates (Fig.3D). Furthermore, the results of RNA pulldown assay elucidate that ciRS-7 is significantly gathered in the WT-miR-135a-5p compared with that in the Mut-miR-135a-5p (Fig 3E). Those results revealed that miR-135a-5p is a downstream target gene of ciRS-7 and directly binding to it. 3.4 Inhibiting miR-135a-5p abrogated the effect of ciRS-7.
To validate whether ciRS-7 promoting neuropathic pain through miR-135a-5p. We investigated the biological function of miR-135a-5p in the progress of neuropathic pain. miR-135a-5p inhibitor and control were infected in microglial cell and CCI rats, then the expression of miR-135a-5p was tested by RT-qPCR (Fig.4A). Subsequently, we examined the PWT and PWL on the miR-135a-5p treated CCI rats. The results are shown in Fig B and C. Decreasing the expression of miR-135a-5p inhibits neuropathic pain in CCI rats. Next, we tested the expression of neuroinflammation and autophagy biomarkers in ciRS-7 and miR-135a-5p inhibitor infected microglial cells and CCI rats. As shown in Fig D and F, in microglial cells, the level of neuroinflammation and autophagy in ciRS-7 with miR-135a-5p inhibitor group are decreased comparing with ciRS-7 with the negative control group. And in CCI rats, the level of neuroinflammation and autophagy in CCI with miR-135a-5p inhibitor group are significantly decreased compared with CCI with the NC group. Those results were indicating that ciRS-7 promoting neuropathic pain in CCI rats partly through mediating miR-135a-5p. Discussion Due to the difficulty management, worse outcome, and ineffective therapy of neuropathic pain. It has become a global health burden, and lots of efforts have been made to understand the molecular mechanisms underlying the development of neuropathic pain, but the mystery of neuropathic pain still unclear currently. In the past two decades, the function of circRNA in the progress of various diseases has been deeply investigated. The function of circRNA in neuropathic pain progress has been rarely studied.
In this study, we revealed that circRNA ciRS-7 was positively related to the progress of CCI-induced neuropathic pain, and with the upregulation of ciRS-7, the neuropathic pain developed. We also found that ciRS-7 promotes neuropathic pain partly through upregulating the level of autophagy and inflammation. In the CCI rats, with the level of ciRS-7 increased, the biomarkers of autophagy Beclin-1, LC3-I, LC3-II were increased, and P62 was decreased. Moreover, the biomarkers of
inflammation IL-6, IL-12, Iba1, and TNF-α were increased. We further demonstrated that ciRS-7 regulates autophagy and inflammation in CCI rats by directly targeting miR-135a-5p. Inhibiting miR-135a-5p in CCI rats or microglial cells decreased the level of autophagy and inflammation and alleviated neuropathic pain in CCI rats.
Our investigation presents the evidence that ciRS-7 can promote neuropathic pain by targeting miR-135a-5p through increases in the level of autophagy and inflammation. Furthermore, reducing the expression level of ciRS-7 or using miR-135a-5p inhibitor can hold back this phenomenon. We propose that this could be the potential treatment for neuropathic pain.
Acknowledgement N.A Data availability statement The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Declaration of competing interest There is no conflict of interests to declare in the current study. Funding N.A
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Figure 1 ciRS-7 was increased in CCI rats and promotes neuropathic pain. A, ciRS-7 expression level in the rats’ L4-6 dorsal spinal cord. At postoperative days 0, 3, 7, 15 and 20, qRT-PCR was performed. B, Expression level of ciRS-7 in CCI rats which injected with si-ciRS-7 or si-NC day 7 after sugery. C, the function of ciRS-7 on
mechanical allodynia was estimated by PWT. D, the function of ciRS-7 on thermal hyperalgesia was tested by PWL. Figure 2 Increased ciRS-7 induced neuroinflammation and autophagy in CCI rats. A and C. The protein level of IL-6, IL-12, TNF-α, Iba1 and Beclin-1, P62, LC3-I, LC3-II at postoperative days 3, 7, 11, 15,20 were tested by western blot. B and C. The protein level of IL-6, IL-12, TNF-α, Iba1 and Beclin-1, P62, LC3-I, LC3-II at postoperative days 3 after si-NC and si-ciRS-7 infection were tested by western blot. Figure 3 ciRS-7 was directly targeting miR-135a-5p. A; The binding sites of ciRS-7 with miR135a-5p and the Mut type of miR-135a-5p. B; The expression of ciRS-7 and miR135a-5p in the si-NC and si-ciRS-7 infected rat microglial cells. C; Luciferase reporter gene assays were used to estimate the luciferase activity in the rat microglial cells. **P<0.01.D; The enrichment of ciRS-7 and miR-135a-5p immunoprecipiated complex were delivered to Anti-Argonaute 2 RNA immunoprecipitation (RIP) assays and anti-immunoglobulin (IgG) was used as the control. **P<0.01. E; The rat microglial cells were infected with WT miR-135a-5p and Mut miR-135a-5p. Realtime quantitative polymerse chain reaction (RT-qPCR) were used to measure the RNA levels of ciRS-7 and GAPDH. **P<0.01. Figure 4 Inhibiting miR-135a-5p abrogated the function of ciRS-7. A. the expression of miR135a-5p in the NC and inhibitor treated microglial cells and CCI rats. B. the function of miR-135a-5p inhibitor and negative control on mechanical allodynia was assessed by PWT. C, the effect of miR-135a-5p inhibitor and negative control on thermal hyperalgesia was evaluated by PWL. CCI, chronic constrictive injury. D,E the protein level of IL-6, IL-12, TNF-α, and GAPDH in the ciRS-7 and miR-135a-5p inhibitor treated microglial cells and CCI rats were tested by western blot. F,G the protein level of Beclin-1, P62, LC3-I, LC3-II and GAPDH in the ciRS-7 and miR-135a-5p inhibitor treated microglial cells and CCI rats were tested by western blot. *P<0.05.
Highlights • CircRNA ciRS-7 was upregulated and involved in the neuropathic pain development. • CircRNA ciRS-7 promotes neuroinflammation and autophagy level in CCI rat models • CircRNA ciRS-7 promotes neuroinflammation and autophagy by sponging to miR-13a-5p
Declaration of Interest Statement The authors certify that they have participated sufficiently in the work to take public responsibility for the appropriateness of the experimental design, method, collection, analysis, and interpretation of the data. This manuscript has not been published in whole or in part nor is it being considered for publication elsewhere. The authors declare that there are no financial or other relationships that might lead to a conflict of interest of the present article. All authors have reviewed the final version of the manuscript and approved it for publication.
Abbreviations List CCI, chronic constriction injury; circRNA, circular RNA; qRT-PCR, Quantitative reverse transcription PCR; IP, immunoprecipitation; RIP, RNA‐binding protein immunoprecipitation; 3′-UTR,3′-Untranslated Region; PWT, Paw withdrawal threshold; PWL, Paw withdrawal latencies
Credit Author Statement
Author contributions Wei Cai: Conceptualization, Methodology, Software, Investigation, Writing - Original Draft. Yang Zhang: Validation, Formal analysis, Visualization, Software. Zhen Su: Resources, Writing - Review & Editing, Supervision, Data Curation. Wei Cai: Writing: Review & Editing. Yang Zhang: Writing: Review & Editing. Zhen Su: Writing: Review & Editing