key: cord-0028770-p1kta11y
authors: Yi, Min; Dai, Xingping; Li, Qiuxia; Xu, Xia; Chen, Yanyi; Wang, Dongsheng
title: Downregulated lncRNA CRNDE contributes to the enhancement of nerve repair after traumatic brain injury in rats
date: 2019-07-29
journal: Cell Cycle
DOI: 10.1080/15384101.2019.1647024
sha: 11f8cba9ab2401da4c44e11038d01a407d148b9c
doc_id: 28770
cord_uid: p1kta11y

Objective: Long non-coding RNAs (lncRNAs) have recently been demonstrated to be involved in craniocerebral disease, but their expression in traumatic brain injury (TBI) is still unearthed. Therefore, we aimed to elucidate the effect of lncRNA CRNDE on TBI. Methods: Firstly, CRNDE expression was determined in serum of TBI patients and healthy controls. The TBI rat model was established based on Feeney’s freefall impact method. The modeled rats were injected with siRNA against CRNDE, and the rats’ neurobehavioral function were measured. Besides, expression of inflammatory factors, size, shape and number of hippocampal neurons, neuron apoptosis, Beclin I, LC3-I, LC3-II, glial fibrillary acidic protein (GFAP), BrdU, nerve growth factor (NGF), nestin, and neuronal nuclei (NeuN) expression were detected through different methods. Results: In TBI, CRNDE was found to be upregulated. Downregulated CRNDE improved neurobehavioral function, repressed expression of neuroinflammatory factors, elevated number of Nissl bodies, as well as restricted neuronal apoptosis and autophagy in TBI rats. Besides, downregulated CRNDE also promoted expression of GFAP, BrdU, NGF, nestin, and NeuN, thus induced the differentiation of neurons and the directional growth and regeneration of nerve fibers. Conclusion: Altogether, we found that silencing of CRNDE might be able to promote the nerve repair after TBI in rats.

Traumatic brain injury (TBI) is a main reason of death and morbidity, especially at both ends of the age range, which brings enormous costs to society [1] . Statistics have shown increased incidence, mortality, and disability of TBI in recent years [2] . As reported, primary mechanical injury and secondary injury are two well-known mechanisms of TBI [3] . As one highly complex disease, patients suffering TBI present different degrees of contusion, hemorrhage, and hypoxia [4] . To be specific, TBI causes secondary biochemical changes which contributes to neurological dysfunction, delayed neuroinflammation, as well as nerve cell death [5] . Nowadays, although many efforts have been made, there is no treatment to alleviate or prevent nerve dysfunction after TBI [6] . Researchers are now turning to biomarkers, objective molecular characteristics of injury, as a platform for finding more sensitive and specific approaches for TBI treatment and diagnosis.

Long non-coding RNAs (lncRNAs) are vital in multiple kinds of pathophysiological processes of brain diseases [7] . Moreover, lncRNAs are proved to be closely related to nervous system development and neurodegenerative diseases [8, 9] . Colorectal neoplasia differentially expressed (CRNDE) transcripts are described as one lncRNA and positioned on chromosome 16, exerting oncogenic functions in diverse cancers [10] . In addition, CRNDE is known to be highly expressed in multiple kinds of cancerous diseases, particularly in cancers of the brain [11] . Furthermore, upregulation of CRNDE was demonstrated to promote glioma cell viability, migration, and invasion [12] . Song H et al. also proposed that highly expressed CRNDE was found in medulloblastoma, knowing as the most frequent malignant brain tumor in children [13] . Thus, we speculate that CRNDE may exert affect in TBI. Herein, we initially detected CRNDE expression in clinical serum sample from patients with TBI and rat serum and hippocampal tissues. Furthermore, the effects of CRNDE on nerve repair were fully investigated using siRNA-mediated silencing.

The study was permitted by the Ethics Committee of Xiangya Hospital, Central South University and an informed consent form were signed with the patient's family. The experimental animal program has been permitted by the Laboratory Animal Ethics Committee. It conformed to the principles of animal protection, animal welfare, and ethics, as well as the relevant regulations of the national ethics of laboratory animal welfare.

A total of 43 patients with TBI (male, n = 25; female, n = 18; mean age of 63.7 ± 4.9 years; American Society of Anesthesiologists grade II or Ⅲ) diagnosed in Xiangya Hospital, Central South University from June 2017 to June 2018 were collected as the case group. All patients had preoperative Glasgow Coma Scale (GCS) score of 6-10 points. Patients were included if they had no obvious history of heart, lung, liver, kidney disease before operation; they had no long-term history of psychotropic drugs and alcoholism; they undergo craniotomy under general anesthesia with drug sedation required during the perioperative period. Patients were excluded if they had dysfunction of blood coagulation; they had GCS score less than four points; they need deep sedation due to pregnancy, coma, and elevated intracranial pressure; they died within 72 h after operation. Besides, 32 healthy physical examiners (male, n = 15; female, n = 17; mean age of 54.7 ± 7.8 years) were randomly selected as the control group. There was no statistical significance in age, gender, and state of an illness between the two groups.

A sum of 55 healthy Sprague-Dawley rats (aged about 6-8 weeks and weighed 180-200 g) obtained from Henan Experimental Animal Center were selected for our study. The feeding conditions shall be carried out according to the standard of the animal at clean level. All rats were assigned into a sham group (n = 10) and a TBI group (n = 45) randomly. The rat model of TBI was established according to Feeney's freefall impact method [14, 15] . Rats were fixed on brain stereotactic instrument with prone position, and the bumper was placed on the exposed right dural. Rats were anesthetized with 1% pentobarbital sodium (40 mg/kg) via an intraperitoneal injection. And then, 40 g weight was dropped from 25 cm height along the peripheral catheter, and the bone window was sealed with bone wax after impact. Rats in the sham group only received cranial window opening which was then sealed with bone wax. After modeling, the rats were placed in the cage according to the label and fed with adequate food and water at room temperature.

A total of 40 rats were randomly selected from 42 rats with successful modeling and assigned into the following groups: TBI group (TBI rats without any other treatment), si-negative control (NC) group (TBI rats injected with si-NC plasmid of CRNDE), si-CRNDE-1 group (TBI rats injected with CRNDE interference plasmid 1) and si-CRNDE-2 group (TBI rats injected with CRNDE interference plasmid 2). The transfection plasmids were provided by GenePharma Co. Ltd., Shanghai, China (Shanghai, China). In the sham group, rats received no treatment.

The spontaneous activity of rats was analyzed by Smart-128 small animal behavior analysis system (Panlab, Barcelona, Spain). After 30 min of the last administration for rats in each group, a single rat was placed in an observation box (42 cm × 42 cm × 30 cm) with a camera placed in at the top. The activities of rats were tracked by video, and the tracks of rats were recorded spontaneously, and the distance of activities was calculated. The total activity distance of rats within a short period of time was used to evaluate the index of spontaneous activity.

On the 5 th day of administration, the rats were placed in the bright room of the IA training instrument with their back facing the isolation door. When the rats were facing the lift door, the door was raised with the darkroom exposed. When all 4 feet of the rat entered the dark room, the lift door will be closed, the rat foot will be given a single click (0.4 m, 2 s), and then the rat will be taken out. Twenty-four hours later, the IA memory of the rats was measured, and the time (latent period) of staying in the bright room before entering the darkroom was recorded.

Morris water maze test [16] was used for assessing the learning and memory capacity and evaluating the degree of memory impairment in rats in the second week of cultivation. The test was conducted in rats of each group at the 2 nd and 4 th week after operation, respectively. Each test began by placing the rats in the water close to and facing the wall of the pool in one of the four entry points. The times across the original platform within 90 s was recorded. After behavioral examination, 2 mL orbital blood was taken and then the rats were euthanized with their brain tissues collected. Afterwards, the tissues were fixed by 4% paraformaldehyde, dewaxed by routine gradient alcohol and embedded by paraffin. The coronal sections were continuously cut at the optic intersection, and the sections were 4 μm thick.

The rats were euthanized to collect serum with brain tissue separated and preserved. According to the instructions of ELISA kits, the serum levels of tumor necrosis factor (TNF)-α (SBJ-R0040), interleukin (IL)-1β (SBJ-R0024), IL-6 (SBJ-R0755) and IL-10 (SBJ-R0786) (SenBeiJia Biological Technology Co., Ltd. Nanjing, China) were detected. The optical density (OD) values measured at 450 nm by a microplate reader (BS-1101, Detie Experimental equipment Co., Ltd., Nanjing, China).

The paraffin-embedded sections were treated with xylene, absolute ethyl alcohol, alcohol (95%, 80%, and 70%), and rinsed by distilled water. Afterwards, the sections were stained with 1% tar violet or 1% thionine for 10 to 60 min, dehydrated with alcohol, absolute ethyl alcohol, and cleared with xylene. After that, the sections were sealed with 1, 3-diethyl-8-phenylxanthine (DPX) and observed under a microscope. The Nissl body was purple, and the nucleus was lilac.

After 24-h transfection, the cells were fixed with 2%formaldehyde and permeated with 0.1% Triton X-100 (Solarbio Science & Technology Co., Ltd., Beijing, China). According to the instructions, the cells were incubated with 50 μl TUNEL reaction mixture (Roche, Basel, Switzerland) in the dark. The results were observed under an inverted fluorescence microscope (Leica, Microsystems AG, Wetzlar, Germany). Apoptosis rate = the apoptotic positive cells number/total cell count × 100%.

The tissue sections were fixed by 4% paraformaldehyde, treated with 0.2% TritonX-100, sealed by 3% BSA at 4°C, and next, incubated with fluorescence primary antibody GFAP (ab33922, 1:500, Abcam, Shanghai, China) and BrdU (ab6326, 1:200, Abcam, Shanghai, China). After that, the sections were incubated with fluorescence secondary antibody (1:500) in the dark for 2 h, and reacted with DAPI (ab104139, 1:100, Abcam, Shanghai, China) in darkness for 10 min. The sections were sealed and observed under an inverted fluorescence microscope.

The fresh hippocampal tissue was fixed in 2.5% glutaraldehyde solution, fixed with 1% osmic acid (Rongbai biological technology Co., Ltd., Shanghai, China), and washed with 0.1 M buffer solution for 5 min. After that, the tissues were dehydrated by gradient ethanol, soaked by 90% ethanol acetone, 90% acetone and 100% acetone (each for 5 min), by a mixture of acetone and epoxy resin (1:1) for 2 h, embedded, polymerized for 10 h and made into ultrathin sections (70 nm). Subsequently, the sections were stained by uranyl acetate and acetate leaching, respectively, for 10 min with the ultrastructure of hippocampal CA1 region observed by a transmission electron microscopy (HTT700, Hitachi, Tokyo, Japan).

The extraction of total RNA was carried out using the Trizol kit (16,096,020, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA), and 5 µg of total protein was reversely transcribed into complementary DNA (cDNA) based on the RT-qPCR kit (ABI Company, Oyster Bay, NY). The primers (Invitrogen, Shanghai, China) used are listed in Table 1 . Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the internal parameter with the 2 −ΔΔCt [17] used for data analysis.

Brain tissues were extracted, lysed and centrifuged at 12,000 r/min to remove tissue fragment or cell debris. After that, a bicinchoninic acid kit (Beyotime Biotechnology, Shanghai, China) was utilized for protein concentration determination. Next, 50 µg protein was collected and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane. Afterwards, the membrane was sealed with 5% skimmed milk and incubated with the diluted primary antibodies: Beclin I (ab62557, 1:500), LC3-I (ab48394, 1:1000), LC3-II (ab51520, 1:3000), NGF (ab6199, 1:1000), Nestin (ab6142, 1:1000), NeuN (ab236870, 1:1000) (all from Abcam, Shanghai, China), and GAPDH (1:500, ab8245, Abcam, Cambridge, UK). On the following day, the membrane was further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. After that, the protein bands were viewed with an enhanced chemiluminescence reagent (BB-3501, Amersham Company, Piscataway, NJ, USA), which were imaged on Bio-Rad image analysis system (Bio-Rad Laboratories, Hercules, CA, USA). Image analysis was followed using Quantity One v4.6.2 software. The target proteins were quantified as relative gray values against the internal reference GAPDH.

All experimental data were processed with SPSS 21.0 statistical software (IBM Corp., Armonk, NY, USA). All data are consistent with normality distribution and variance homogeneous test. Measurement data are reported as mean ± standard deviation. Differences between two groups were compared using independent t-test, while among multiple groups, using one-way analysis of variance, followed by Tukey's post hoc test. A P value <0.05 was described as statistically significant.

In order to elucidate the relationship between CRNDE and TBI, the expression of CRNDE in serum of the patients with TBI was determined. The results revealed ( Figure 1A ) that expression of CRNDE elevated in the serum of patients with TBI. Besides, the CRNDE expression in serum and hippocampal tissues of TBI rats was also detected, and the results also revealed highly expressed CRNDE (P < 0.05) ( Figure 1B and C) . All the above results suggested that CRNDE was highly expressed in TBI. 

Subsequently, we evaluated the interference efficiency of CRNDE after interfering through detecting the CRNDE expression in cells by RT-qPCR. Following that, two of them which have good interference efficiency were selected for subsequent experiments (Figure 2A) . Besides, behavioral changes of rats were detected, and the results showed that the total distance of spontaneous activity prolonged ( Figure 2B ), the 24h memory latency ( Figure 2C ) and the number of crossing the platform ( Figure 2D ) decreased in rats with TBI. In contrast to the si-NC group, the total distance of spontaneous activity shortened, and the 24-h memory latency and the number of crossing the platform increased in the si-CRNDE-1 and the si-CRNDE-2 groups (all P < 0.05).

After TBI, the blood-brain barrier was destroyed and the pro-inflammatory factors in the vascular system were expressed with the inflammatory cells aggregated, which released a large number of inflammatory factors, thus aggravating the secondary TBI. Therefore, we would like to explore whether CRNDE could inhibit pro-inflammatory factors with the help of ELISA. The results suggested that expression of pro-inflammatory factor TNF-α, IL-1β, and IL-6 in serum of TBI rats increased, while the expression of IL-10 decreased in rats with TBI. TNF-α, IL-1β, and IL-6 expression declined in serum of rats in the si-CRNDE-1 and the si-CRNDE-2 groups, while the IL-10 expression increased relative to the si-NC group (P< 0.05) ( Table 2 ). These results implied the neuroprotective effect of silencing of CRNDE on rats with TBI may be related to the inhibition of neuroinflammation.

According to Nissl's staining, the morphological changes of nerve cells in the injured area were observed under a common optical microscope, and the quantitative analysis of the number of neurons under a high magnification microscope was carried out with computer software. The results revealed that in the sham group, the cells in the cortex were arranged orderly and the shape was regular; the Nissl body was blue and clearly visible with the cell numbers of (79.78 ± 5.63). In the TBI and si-NC groups, the cells in the injured area were disordered and sparse with decreased Nissl body, the cell structure was not clear, and the number of nerve cells was less than that in the sham group. In the si-CRNDE-1 group and the si-CRNDE-2 group, the cell arrangement was disordered, the cell volume was slightly smaller, the shape was irregular, the Nissl body was decreased, Notes: A, The expression of CRNDE in serum of TBI patients was detected by RT-qPCR; B, The expression of CRNDE in serum of TBI rats was detected by RT-qPCR; C, The expression of CRNDE in hippocampal tissues of TBI rats was detected by RT-qPCR; All data are measurement data and presented as mean ± standard deviation. Differences between two groups were compared using independent sample t-test; * compared with the control or sham groups, P < 0.05; In panel A, control group, n = 32; case group, n = 43; In panel B and C, sham group, n = 10; TBI group, n = 10.

but the number of cells was more than that in the si-NC group (all P< 0.05) ( Figure 3A and B). At the same time, TUNEL assay was used to detect neuronal apoptosis in each group. The results showed that the neuronal apoptosis was higher in rats with TBI; the neuronal apoptosis weakened in the si-CRNDE-1 and the si-CRNDE-2 groups versus the si-NC group (P < 0 05) ( Figure 3C and D) . These results revealed that silencing of CRNDE may increase the number of Nissl bodies and inhibit neuronal apoptosis in rats.

Evidence has shown that GFAP, as the intermediate filament protein of astrocyte cytoskeleton, is a new marker of astrocytes which are involved in the repair and regeneration of nerve injury [18] . The increased expression of BrdU positive cells contributes to the induction of the differentiation process of endogenous neural stem cells and promote neural repair after TBI in rats [19, 20] . So, immunofluorescence assay was applied for GFAP and BrdU protein expression evaluation in brain tissues. The results revealed that GFAP and BrdU protein expression elevated in rats with TBI; GFAP and BrdU protein expression enhanced in the si-CRNDE-1 and the si-CRNDE-2 groups relative to the si-NC group (P < 0 05) ( Figure  4A and B) . The results showed that GFAP and BrdU expression could be promoted after TBI, and downregulated CRNDE could further decrease GFAP and BrdU protein expression in brain tissues, which might promote the nerve repair of rats after TBI.

The neuronal cells were observed under a transmission electron microscopy ( Figure 5A ), in the sham group, neuronal cells had normal morphology and structure, complete mitochondrial structure and abundant endoplasmic reticulum; In the TBI group, the ultrastructure of the cells was disordered, mitochondria were seriously damaged with obvious swelling, rough endoplasmic reticulum structure was not clear, and a large number of vacuolar degeneration could be seen in the matrix. After silencing of CRNDE, in contrast to the TBI group, the ultrastructural damage of neuronal cells was alleviated, a few vacuolar degeneration in the matrix and slight damage of mitochondria were observed. At the same time, the expres- sion of autophagy-related factors Beclin-1, and LC3-II /I was detected by western blot analysis ( Figure 5B ). Expression of Beclin-1 and LC3-II/I increased in rats with TBI. In comparison to the si-NC group, Beclin-1 and LC3-II/I protein expression declined in the si-CRNDE-1 and the si-CRNDE-2 groups (P < 0.05).

The above results clarified that downregulated CRNDE reduces the autophagy of neuronal cells in TBI rats.

Silencing of CRNDE elevates the expression of NGF, nestin, and NeuN in brain tissues in TBI rats TBI can trigger the expression of many kinds of nutritive factors in the injured tissues. Therefore, expression of NGF, nestin, and NeuN in the brain tissues of each group was determined. The results indicated that expression of NGF, nestin and NeuN protein increased in rats with TBI; and the expression of NGF, nestin and NeuN protein increased in the si-CRNDE-1 group and the si-CRNDE-2 groups relative to those in the si-NC group (P < 0 05) ( Figure 6A and B). These results presented that silencing of CRNDE may enhance the expression of many kinds of nutrient factors, thus promote the differentiation of neurons and induce the directional growth and regeneration of nerve fibers.

TBI, characterized by a vast array of evolving neuropathologies, is now regarded as a major health issue and a well-known pathogenic factor for the later development of neurodegenerative disorders [21] . Therefore, effective therapies are needed to reduce TBI-related disabilities. It is reported that lncRNAs have been revealed to present strong expression in adult brains and are proved to be implicated in not only neural differentiation but also synaptic plasticity Figure 4 . Silencing of CRNDE increases the expression of GFAP and BrdU protein in brain tissues in TBI rats. Note: A, Detection of GFAP protein expression in brain tissues by immunofluorescence assay (× 200); B, Detection of BrdU protein expression in brain tissues by immunofluorescence assay (× 200); All data are measurement data and presented as mean ± standard deviation. * compared with the sham group, P < 0.05; # compared with the si-NC group, P< 0.05; n = 10. [22] [23] [24] . In our study, we tried to figure out the regulatory role of CRNDE in nerve repair after TBI to find a new treatment target for TBI. The present study made conclusion that silencing of CRNDE could promote the nerve repair after TBI in rats.

For investigation, we firstly tested CRNDE expression in serum of TBI patients and healthy controls as along with serum and hippocampal tissues of TBI rats. The results in our study found that CRNDE was highly expressed in TBI. In recent years, evidence has revealed that abnormal regulation of lncRNA is common in heterogeneous tumors, which can effectively affect the malignant cells by promoting growth, thereby resulting in unrestrained tumor growth [25] . It is worth noting that the increased expression of CRNDE usually occurs in cancers derived from normal expressed cell types or organs, strongly suggesting that CRNDE is of great significance in the carcinogenesis of human tumors [11] .

Although CRNDE was initially found in colorectal cancer, it was later found that CRNDE was upregulated in other tumors, especially in brain tumors [13] , which was partly in line with our result. After model establishment of TBI via Feeney's freefall impact method, the underlying mechanisms of CRNDE in nerve repair in TBI rats were further explored. Our study pointed out that silencing of CRNDE could inhibit expression of neuroinflammatory factors, neuronal apoptosis, autophagy of neuronal cells as well as increased expression of GFAP and BrdU and expression of nutrition factors. Interestingly, neuroinflammation could result in neurological disorders and tissue damage that the TBI involves, and evidence corroborated that the initiation of neuroinflammation after TBI is essential in the loss of neurological function [26, 27] . In addition, the elevation of proinflammatory cytokines, immune cell proteases, as well as oxidative stress can lead to excess tissue damage, and thus inducing neuronal cell death [28, 29] . The increasing reactive astrocytes as presented by an increased GFAP is recognized as a emblematical sign of injury to the central nervous system [30] . It is reported that neurotropic factors (BDNF and GDNF), play a positive role in development and regeneration of neurons, in fact, these factors can promote survival of neurons following a mechanic damage as in the TBI [31, 32] . Previous evidence has been clarified that CRNDE is highly expressed in multiple cancers associated with blood and brain, and CRNDE is obviously increased in gliomas, which is related to a neurodegenerative process [11] . Remarkably, Lin et al. also pointed out that CRNDE was expressed in the process of neural differentiation from human-induced pluripotent stem cells [33] . Specifically, overexpressed CRNDE might induce inflammation to modulate tumorigenesis in glioma [34] . Furthermore, up-regulation of CRNDE triggered WI-38 cell injuries with obviously suppressed cell viability, enhanced cell apoptosis as well as elevated inflammatory cytokines levels [35] , which was partly consistent with our study.

Finally, we recognized that CRNDE, a lncRNA, was overexpressed in TBI, and silencing of CRNDE could inhibit expression of neuroinflammatory factors, neuronal apoptosis and autophagy of neuronal cells using a series Figure 6 . Silencing of CRNDE elevates the expression of NGF, Nestin, and NeuN in brain tissues in TBI rats. Note: A, grey value analysis of NGF, Nestin, and NeuN in brain tissues; B, protein expression of NGF, Nestin and NeuN was detected by western blot analysis; All data are measurement data and presented as mean ± standard deviation. * compared with the sham group, P < 0.05; # compared with the si-NC group, P< 0.05; n = 10.

of assays, thus promoting nerve repair. The underlying mechanisms contributing to the oncogenic phenotype of CRNDE in TBI remain to be revealed. We provided evidence that CRNDE might serve as a potent therapeutic target for TBI treatment.

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We would like to acknowledge the reviewers for their helpful comments on this paper.

The authors declare that they have no conflicts of interest.

This study was supported by Provincial science and technology innovation platform and talent plan(420010087).

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