Downregulation of Long Noncoding RNA TUG1 Attenuate MTDH /NF-κB/IL-1β mediated inflammatory damage via Targeting miR-29b-1-5p After Spinal cord ischemia reperfusion in rats CURRENT STATUS: POSTED

Background: Spinal cord ischemia reperfusion (IR) is associated with an inflammatory response. The long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) and microRNA-29b (miR-29b) family are frequently dysregulated in neuro-ischemic diseases. However, their potential roles in spinal cord IR injury (IR) are unknown. Methods: A spinal cord IR model was established in rats by14-minute occlusion of aortic arch. The aberrant miRNAs were identified by microarray analysis, and qRT-PCR was used to validate the lncRNA and microRNA levels. The motor function of the differentially-treated animals was assessed by Tarlov scores, and the leakage of Blood-spinal cord barrier (BSCB) was measured in terms of the extravasation of Evans blue (EB) dye. The expression levels of different proteins were analyzed by Western blotting and immunofluorescence. The interaction between TUG1 and miR-29b-1-5p, TRIL and miR-29b-1-5p, and MTDH and miR-29b-1-5p were determined using bioinformatics programs and the dual-luciferase reporter assay. Results: MiR-29b-1-5p was significantly downregulated and TUG1 was upregulated in the spinal cord of rats after IR. In addition, TRIL and MTDH protein levels were also significantly increased after IR. MTDH was predicted as a target of miR-29b-1-5p and its knockdown downregulated NF-κB and IL-1β levels. In addition, a direct interaction was observed between TUG1 and miR-29b-1-5p, and knocking down TUG1 upregulated the miRNA. Furthermore, overexpression of miR-29b-1-5p or TUG1 knockdown alleviated BSCB leakage and improved hind-limb motor function, and downregulated MTDH and its downstream pro-inflammatory cytokines. Suppression of miR-29b-1-5p reversed the neuroprotective effect of TUG1 knockdown, restored the levels of MTDH/ NF-κB/IL-1β and activated astrocytes. Conclusion: Downregulation of TUG1 alleviated MTDH/NF-κB/IL-1β pathway-mediated inflammatory damage after IR by targeting miR-29b-1-5p.

3 aneurysm surgery [1], and can progress to paralysis [2]. However, the underlying molecular mechanisms are poorly understood. Therefore, it is essential to identify the factors involved in regulating spinal cord IR in order to improve patient prognosis. MicroRNAs (miRNAs) are ~ 22 nucleotides long small non-coding RNAs [3] that repress gene expression by binding to the 3'untranslated region (UTR) of the target mRNAs. Several miRNAs have been identified as potential therapeutic targets in tumors [5], infections [6], immune disorders [7], cardiovascular diseases [8], as well as neurodegenerative diseases [9] like spinal cord IR [10]. MiR-29 is involved in the pathogenesis of various neurological diseases [11,12] and nerve injury.
The NF-κB mediated inflammatory signaling pathway also plays a vital role in aggravating spinal cord IR [13]. In addition, the TLR4 interactor with leucine-rich repeats (TRIL), a ligand of TLR4, is highly expressed in the spinal cord after IR [14]. Likewise, metadherin (MTDH) or astrocyte elevated gene 1 is upregulated in various tumors [15] and neurodegeneration diseases [16], and triggers the NF-κB pathway [17], although its potential role in spinal cord IR is unknown. Long non-coding RNAs (lncRNAs) are a class of non-coding transcripts longer than 200 bases [18] that regulate gene expression by targeting the miRNAs as endogenous RNAs (ceRNAs) [19]. Taurine-upregulated gene 1 (TUG1) promotes the normal development of photoreceptors of retina [20], and is aberrantly expressed during neurodegeneration [21,22] and inflammation [23]. In our previous study, we found that knocking down TUG1 inhibited NF-κB pathway-induced inflammatory damage in spinal cord IR [14].

Establishment of rat spinal cord IR model
The IR model was induced as previously reported [14] in 8-weeks old male Sprague-Dawley rats.
Briefly, the descending aorta was cross-clamped distal to the left subclavian artery following left thoracotomy in order to obstruct arterial flow to the spinal cord. The occlusion was removed 14 4 minutes later to restore perfusion. The sham-operated rats underwent thoracotomy without arterial occlusion. All animal experiments were approved by the China Medical University Animal Care and Use Committee (2017105).
Hierarchical clustering was performed to detect the aberrantly expressed miRNAs using MEV software (version 4.6; TIGR, Microarray Software Suite4, Boston, United States).

Intrathecal Injection
The animals were intrathecally injected with the synthetic miRs or siRNAs in the L4-L6 segments 3 days before IR induction as previously reported [14]. The hind-limb motor function of the rats was observed after intrathecal injection, and only those with normal movement were selected for the subsequent experiments. According to the manufacturer's instructions, 30μl of the synthetic miRs, siRNAs or corresponding controls (GenePharma, Shanghai, China) was infused intrathecally along with Total RNA of L4 to L6 spinal cord segments was isolated by Trizol reagent (Takara, Otsu, Japan) as previously reported [25]. TaqMan MicroRNA Assay Kit (Applied Biosystems) was used for quantifying miR-29b-1-5p with the following primer: forward 5′-CGCGCGTTTCATATGGTGGTTTAGATTT-3′. U6 was used as the internal control (forward 5′-CTCGCTTCGGCAGCACA-3′). For TUG1 expression analysis, Prime-Script RT reagent Kit with gDNA Eraser (Takara) was used for reversed transcription, and GAPDH was used as the internal control. The primer sequences (Sangon Biotech, Shanghai, China) were as follows: TUG1: forward 5'-TGCCACCAGCACTGTCACT-3' and reverse 5'-ACGGTCCAGGTGAATGAACA -3'; GADPH: forward 5'-GGGGCTCTCTGCTCCTCCCTG-3' and reverse 5'-AGGCGTCCGATACGGCCAAA-3'. Relative expression levels were measured using the 2 -△△ Ct method.

Western Blotting
Western blotting was performed as previously described [26] using primary antibodies against TRIL

Neurological Assessment
Hind limb motor function was assessed 12h after IR by two observers blinded to the experimental grouping using the Tarlov scale [10]. The locomotor function was scored as follows: 0 -lower limb function deficiency, 1 -visible lower limb movement but weak against gravity, 2 -occasional lower limb movement and against gravity but inability to stand, 3 -abnormal standing posture and walk, and 4 -completely normal movement.

6
Evans blue (EB) fluorescence was used to assess blood-spinal cord barrier (BSCB) leakage as previously described [14]. Briefly, EB was injected into the rats 1h before sacrifice via their tail vein.
The L4-L6 spinal cord segments were later dissected and fixed in paraformaldehyde (Beyotime Biotechnology), cut into 10μm-thick sections, and viewed under a fluorescence microscope (Olympus, Melville, NY) using the green filter.

Statistical Analysis
All data were expressed as mean ± standard error (SEM) and SPSS software (version 22.0, SPSS, Inc., USA) was used for statistical analysis. Student t-test or two-way analysis of variance (ANOVA) were used to compare two or multiple groups respectively. The correlation between TUG1 and miR-29b-1-5p expression levels was assessed by Spearman correlation test. Kruskal-Wallis test with Bonferroni correction was used to assess Tarlov scores, and p<0.008, p<0.005 and p<0.003 were considered statistically significant for 4, 5 and 6 groups. For other analyses, P<0.05 was regarded as statistically 7 significant.

MicroRNA expression profiles in rats spinal cord after IR
A total of 141 miRNAs were identified in the microarray, of which one was upregulated (up to >2 fold) and 19 were downregulated (<0.5 fold) after IR ( Figure 1a, Table 1). Compared to the sham group, miR-29b-1-5p was seriously downregulated after IR in a time-dependent manner and reached the minimum levels after 12h (Figure 1b). Therefore, subsequent experiments were conducted at the 12h time point.

MiR-29b-1-5p mimic alleviated the neurological deficit after IR
In order to elucidate the neurological function of miR-29b-1-5p, the spinal cord IR-modeled rats were injected with the mimic. As shown in Figure 2a, mimic-29b-1-5p significantly increased the miRNA levels after IR compared to the untreated and NC-29b-1-5p-treated groups. Furthermore, mimic-29b-1-5p also restored the Tarlov scores of locomotor function that were decreased significantly due to IR, whereas the NC-29b-1-5p had no discernible effect (Figure 2b). Consistent with this, ectopic level of miR-29b-1-5p also significantly attenuated the IR-induced BSCB leakage and EB extravasation ( Figure   2c, d). These results point to a protective role of miR-29b-1-5p in spinal cord IR.

TUG1 aggravates spinal cord IR by targeting miR-29b-1-5p and activating the MTDH/NF-κB/IL-1β pathway
To further establish the biological relevance of TUG1 in neurological injury post spinal cord IR, we treated the modeled rats with si-TUG1. As shown in Figure 7a, si-TUG1 significantly increased the Tarlov scores after IR along with attenuating BSCB extravasation (Figure 7b), and significantly downregulated MTDH, NF-κB and IL-1β after IR (Figure 8a-d). In line with our findings so far, silencing TUG1 upregulated miR-29b-1-5p during IR, which was neutralized by the specific miRNA inhibitor (P< 0.05; Figure 9a). Furthermore, suppression of miR-29b-1-5p reversed the ameliorative effects of TUG1 9 knockdown on Tarlov scores (Figure 9b) and BSCB leakage (Figure 9c,d), thereby highlighting the regulatory role of the miR-29b-1-5p/TUG1 axis in spinal cord IR. Mechanistically, inhibition of miR-29b-1-5p restored the levels of MTDH, NF-κB p65 and IL-1β after IR in the presence of TUG1 knockdown (Figure 10a-f). Finally, IR in the spinal cord significantly increased the expression of the astrocyte marker GFAP, which was decreased upon TUG1 knockdown, and restored by the inhibition of miR-29b-1-5p (Figure 11a, b). We can conclude therefore that TUG1 plays a pathological role in spinal cord IR by targeting miR-29b-1-5p, which activates the pro-inflammatory NF-κB pathway.
Several miRNAs are aberrantly expressed during spinal cord IR, and may act as protective or pathological factors. For example, increased levels of miR-27a attenuated IR-related damage in the spinal cord by targeting the TLR4 inflammatory pathway [24], while miR-199a-5p also exerted a protective effect by blocking ECE1-mediated apoptosis signaling [28]. In contrast, inhibiting miRNA-124 protected rats against spinal cord IR by inducing mitophagy [29]. Previous reports on spinal cord IR microarrays [30,31] have also identified miR-29, which is involved in neurological diseases such as stroke [11], Huntington's disease [33] Parkinson's disease [32] and Alzheimer's disease [12], and was significantly downregulated in the hippocampal neurons of a rat model of brain hypoxia [34]. We have firstly shown miR-29b-1-5p level was markedly decreased after spinal cord IR, and may therefore play a protective role. Indeed, intrathecal administration of the miR-29b-1-5p mimic improved motor function and decreased blood-spinal barrier (BSCB) leakage in the rats after IR.
BSCB integrity is vital to maintaining homeostasis between capillaries and the spinal cord [35], and is disrupted during an inflammatory response. The inflammatory cytokines disintegrate the tight junction proteins, which increases vascular permeability and exposes the spinal cord to circulating pathogens, thereby increasing the risk of neuronal damage [35]. IR-induced inflammation upregulates the matrix metalloproteinase-9 (MMP-9) and downregulates the xx protein [36]. In addition, intrathecal injection of TLR4 inhibitors attenuated BSCB leakage and inflammatory responses [37].
Therefore, we hypothesized that miR-29b-1-5p exerted a protective function by inhibiting the inflammatory responses.
MiRNAs inhibit gene level by binding to the 3'-UTR of target mRNAs [4]. We identified the inflammation-related proteins TRIL and MTDH as the putative targets of miR-29b-1-5p. In a previous research, we suggested that TRIL was upregulated in the spinal cord after IR and its knockdown reduced the levels of NF-κB-dependent cytokines. Similar results were seen in the present study as well, although we could not establish a direct interaction between the the 3' UTR of TRIL and miR-29b-1-5p. MTDH is overexpressed in multiple tumors and correlates with increased tumor progression, invasion and metastasis, as well as poor prognosis [15]. In addition, MTDH is also involved in central nervous system diseases such as Huntington's disease, HIV-related dementia and migraine [38].
MTDH silencing inhibited proliferation and migration of Schwann cells in distal sciatic nerve injury [39]. It is known to trigger the NF-κB pathway [17], and high levels of MTDH in glioma promotes the release of pro-inflammatory cytokines [40]. We found that IR significantly upregulated MTDH, and its knockdown decreased NF-κB and IL-1β expressions, indicating that the NF-κB pathway lies downstream of MTDH in IR. Furthermore, the dual-luciferase reporter assay established a direct interaction between miR-29b-1-3p and MTDH, and forced expression of the former blocked the MTDH/NF-κB/IL-1β pathway. Thus, miR-29b-1-5p assuages IR by inhibiting the inflammatory pathway via targeting MTDH.
Astrogliosis is a characteristic feature of central nervous system diseases, and accompanied by increased expression of the glial fibrillary acidic protein (GFAP) [50]. IR also activates the astrocytes, which can be attenuated by anti-inflammatory drugs [13]. In the present study, the astrogliosis induced by spinal cord IR was decreased by TUG1 knockdown and restored by the miR-29b-1-5p inhibitor. Previous reports have shown that MTDH colocalizes with the GFAP + reactive astrocytes in the injured area [51], and its knockdown suppresses astrocytes migration and proliferation [16].
MTDH-overexpressing astrocytes correlate significantly with excitotoxic neuronal damage [38]. Our data indicate that the TUG1/ miR-29b-1-5p/MTDH pathway mediates the inflammatory response of astrocytes during spinal cord IR. Furthermore, since an lncRNA can bind to multiple miRNAs [47], and miRNAs also have multiple target [52], it is feasible that TUG1 regulates TRIL directly or via other miRNAs, which is another therapeutic possibility that can be explored in future.

Conclusion
To summarize, the interaction between miR-29b-1-5p and TUG1 regulates the MTDH/NF-κB/IL-1β pathway in the ischemic spinal cord, and is therefore a potential therapeutic target for spinal cord ischemia injury.

Availability of data and materials
The datasets obtained of this article are available upon request.

Authors' contributions
HJ, BF and HM designed the stdy, HJ, ZL and YJZ constructed the IR model, HJ, ZL and YJZ performed the experiments, HJ and YC analyzed the data, and HJ and HM wrote and revised the manuscript. All authors have reviewed and approved the manuscript.

Ethics approval
All animal experiments were approved by the Ethics Committee of China Medical University.