LncRNA UCA1 alleviates aberrant hippocampal neurogenesis through regulating miR-375/SFRP1-mediated WNT/β-catenin pathway in kainic acid-induced epilepsy

1Department of Neurology, The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning, Guangxi, 530023, China; 2Department of Numerical Control Technology, Guangxi Technological College of Machinery and Electricity, Nanning, Guangxi, 530007, China; 3Department of Graduate School, Guangxi University of Chinese Medicine, Nanning, Guangxi, 530023, China; 4Department of Laboratory, The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning, Guangxi, 530023, China


INTRODUCTION
Temporal lobe epilepsy (TLE) is one of the most common chronic diseases of the nervous system, and its clinical treatment is extremely scarce worldwide (Bartolomei et al., 2008). It is well known that TLE is a very common locally related epilepsy, usually drug-resistant (Asadi-Pooya et al., 2017), and known as hippocampal sclerosis, which is characterized by the loss of a large number of neurons and severe glial hyperplasia (Tai et al., 2018). Abnormal neurogenesis and reorganization of neural circuits in the hippocampus cause spontaneous or recurrent seizures of epileptic foci and participate in the process of temporal lobe epilepsy . Although recent data shows that abnormal neurogenesis is caused by acute attacks or sudden injuries, the mechanism of abnormal neurogenesis in temporal lobe epilepsy is still elusive.
Studies have shown that long non-coding RNAs (long non-coding RNAs, lncRNAs) are also key regulators of the occurrence and development of diseases including epilepsy (Qiao et al., 2018). Urothcarcinoma associated 1 (UCA1) is a lncRNA originally found in bladder transitional cell carcinoma (Fan et al., 2014). Down-regulation of UCA1 plays a role in promoting apoptosis in primary cardiomyocytes by promoting p27 expression (Liu et al., 2015). Recently, UCA1 has been shown to promote the proliferation and differentiation of neural stem cells, suggesting that UCA1 may play an important role in the nervous system (Zheng et al., 2017). Recent studies have found that UCA1 has a certain role in epilepsy. For example, UCA1 inhibits epilepsy and epilepsy-induced brain injury by regulating miR-495/Nrf2-ARE signaling pathway (Zheng et al., 2017). UCA1 inhibits temporal lobe epilepsy star by regulating JAK/STAT signaling pathway Activation of shaped cells (Geng et al., 2018). However, the role and mechanism of UCA1 in abnormal neurogenesis caused by epilepsy has not been investigated.
Recently, the role of small non coding RNAs, especially microRNAs (miRNAs), in epilepsy and neurodegenerative diseases (Alzheimer's disease and Huntington's disease) has attracted great attention (Hébert et al., 2008;Johnson et al., 2008;Wu et al., 2019). MiR-375 has been reported to be involved in neuromodulation. For example, miR-375 inhibits neuronal differentiation by reducing HUD levels (Abdelmohsen et al., 2010), and miR-375 inhibits neuronal apoptosis and promotes growth in cerebral ischemia-reperfusion (Ou et al., 2017). It is reported that miR-375 is up-regulated in the KA- induced SD epilepsy rat model (Henshall 2013). However, whether the abnormal expression of miR-375 in the hippocampus of patients with temporal lobe epilepsy is related to neuronal hyperplasia has not been reported. The current study explored the effect of UCA1 on hippocampal neurogenesis in KA-induced epilepsy mice and its potential molecular mechanism. It was found that UCA1 regulates the expression of Secreted Frizzled Related Protein 1 (Sfrp1) by targeting miR-375, thereby regulating the WNT/β-catenin signaling pathway and participating in abnormal neurogenesis caused by epilepsy. Our findings provide a theoretical basis for the clinical application of UCA1.

Animal modeling and grouping
All animal experiments were approved by the Guangxi University of Chinese Medicine Institutional Review Board. All procedures were conducted according to the Guide for the Use of Laboratory Animals (National Academy Press). 24 adult male C57Bl/6 mice (10 weeks, weighing 20-25 g) were obtained from Hunan Slake Jingda Experimental Animal Co. LTD (SCXK(HU)2016-0002). The mice were housed in a constant environment (temperature 25°C) with a 12-12 h light-dark cycle and free accesses of food and water. The mice were divided into two groups (n=12): Control group and kainic acid (KA) group. The modeling is carried out according to the method of Beamer et al., (Beamer et al., 2018). In short, the mice were deeply anesthetized with 5% isoflurane. Then, a guide sleeve was installed on the dura mater of mice and fixed with gutta percha for KA injection. KA was injected into the amygdala under a unilateral stereotaxic microscope (0.3 μg KA in 0.2 μl solvent, pH 7.4). Non epileptic control mice were injected with 2 μl of solvent in the same way. Three days later, the mice were exposed to Ad-vector, Ad-Uca1 or Ad-shSfrp1, respectively. Finally, the mice were euthanized by cervical dislocation, and PBS and 4% paraformaldehyde (PFA) were perfused into the brain for analysis.

Isolation of primary mouse hippocampal neurons
After the hippocampal tissue was cut into pieces, Dhank's solution and 0.25% trypsin were added, gently blown and centrifuged, and all supernatant was collected. Sieve with 200 mesh and centrifuge for 5 min at 1500 r/ min. Discard supernatant, add medium to resuspend cells. Cells (50 000 cells/cm 2 ) were cultured in Neurobasal Media including 5% FBS, B27, 2 mM GlutaMAX-I, penicillin/streptomycin and 15 mM glucose at 37°C with 5% CO 2 in a humidified incubator, and transfected using Lipofectamine2000 (Invitrogen).

UCA1 overexpression and SFRP1 knockdown
UCA1 gene was integrated into pHBAd-U6 (Wujia, Beijing, China) to construct recombinant vector pHBAd-U6-UCA1. Ad-vector was used as a blank control. The successful construction of the vector was confirmed by sequencing and restriction enzyme digestion. The Sfrp1 shRNA-targeting sequence (sh#1, 5′-CATGGC-CTAACGGACGTAAA-3′) or control shRNA sequence was inserted into pHBAd-U6 for adenovirus production. Mice in the normal group and KA group were injected with 1×10 7 pfu/10μL purified recombinant Ad-Uca1 or Ad-shSfrp1 intraperitoneally to overexpress UCA1 or knock down SFRP1.

RT-qPCR
The expression of Uca1, Sfrp1 and miR-375 in the hippocampus was detected. Total RNA was extracted from hippocampus or cells using Qiazol Lysis Reagent (Qiagen). RNAs were reverse transcribed to cDNA and quantified by qPCR using Quant One Step RT-qPCR Kit (SYBR Green) according to the manufacturer's protocol (Tiangen, Beijing, China). Relative quantities were calculated using 2 ΔΔCT after normalization to Gapdh. Primers are as follows:

Brdu staining
Three days after kainic acid administration and after shRNA administration, the mice were injected intraperitoneally with BrdU at 50 μg/g body weight 4 hours before sacrifice to mark the proliferating cells. BrdUpositive cells were detected using BrdU Labeling and Detection Kit I according to manufacturer's procedures (Roche, Indianapolis, IN)

RNA immunoprecipitation (RIP) assay
RIP assay was conducted using magna RNA binding protein immunoprecipitation kit (Millipore) according to manufacturer's instructions. The cell lysate was incu-bated with human anti-Uca1 antibody (proteintech) or anti-mouse IgG coupled magnetic bead RIP buffer. Coprecipitated RNA was detected by qRT-PCR. To demonstrate that the detected RNA signals specifically bind to Uca1, we examined both total RNA (input control) and IgG controls.

Statistical analysis
Data was exhibited as mean ± standard derivation (mean ± SD) and analyzed using SPSS 21.0 (IBM Corp. Armonk, NY, USA). Differences between two groups were analyzed by t-test, while differences between three groups or more were analyzed by one-way analysis of variance (ANOVA) following post hoc. Pearson was used to analyze the correlation between Uca1 and miR-375, miR-375 and Sfrp1 and Uca1 and Sfrp1 expression. p<0.05 means significant difference.

Correlation of Uca1, miR-375 and Sfrp1 expression in hippocampus of epileptic mice
In this study, the expressions of Uca1, Sfrp1 and miR-375 in hippocampal tissues of KA-induced epileptic mice were firstly investigated. As shown in Fig. 1A-C, the expression of Uca1 and Sfrp1 in hippocampal tissues of epileptic mice were significantly reduced, while the expression of miR-375 was significantly increased compared with the control group. Pearson correlation analysis showed that Uca1 was positively correlated with SFRP1, while miR-375 was negatively correlated with Uca1 and SFRP1 (Fig. 1D). These results indicated that abnormal expressions of Uca1, miR-375 and Sfrp1 were related to epilepsy.

Uca1 overexpression reduced abnormal proliferation of neural progenitors in epileptic mice
To investigate the effect of Uca1 on the proliferation of neural progenitors in epileptic mice, Uca1 was ectopically expressed. As shown in Fig. 2A, the expression of Uca1 in hippocampal nerve tissues of the Ad-Uca1 group was increased sharply in normal mice and epileptic mice compared with the Ad-vector group ( Fig. 2A). Besides, BrdU staining showed that the overexpression of Uca1 in normal mice had no significant effect on the prolif-  Fig. 2B and C). In general, these results showed that up-regulation of Uca1 expression reduced abnormal proliferation of neural progenitors in epileptic mice.

Uca1 was combined with miR-375
The target binding sites of miR-375 and Uca1 was predicted by miRDB. (Fig. 3A). Besides, the targeting relationship between miR-375 and Uca1 was further confirmed by dual luciferase reporter assay and RIP. As shown in Fig. 3B, after co-transfection of Uca1-WT and miR-375 mimic into HEK-293 cells with Lipofectamine 3000, the luciferase activity was decreased significantly, while after co-transfection of Uca1-WT and miR-375 inhibitor, the luciferase activity of HEK-293 cells was increased (Fig. 3B). RIP assay further showed that compared with IgG, UCA1 was significantly enriched in Ago2, indicating that UCA1 directly binds to Ago2 ( Figure 3C). qPCR further showed that Uca1 overexpression significantly reduced the expression of miR-375 in the hippocampal tissues of epileptic mice, but had no significant effect in normal mice (Fig. 3D). Together, these results suggest that Uca1 was combined with miR-375.

Uca1 overexpression promoted Sfrp1 expression through miR-375 to regulate WNT/β-catenin pathway
The targeted binding site of miR-375 to Sfrp1 was predicted by TargetScan (Fig. 4A). Besides, the targeting relationship between miR-375 and Sfrp1 was further confirmed by dual luciferase report assay. As shown in Fig. 4B, after co-transfection of Sfrp1-WT and miR-375 mimic into HEK-293 cells with Lipofectamine 3000, the luciferase activity of HEK-293 cells was decreased significantly, while after co-transfection of Sfrp1-WT and miR-375 inhibitor, the luciferase activity of HEK-293 cells was increased. Besides, after transfection of NC mimics, miR-375 mimic, NC inhibitor (NC inh) or miR-375 inhibitor (miR-375 inh) into neurons with Lipofectamine 3000, respectively, abnormal expression of Sfrp1 and β-catenin was observed. Western blotting further showed that overexpression of miR-375 significantly inhibited the expression of Sfrp1 in neurons and promoted the expression of β-catenin, while the low expression of miR-375 showed the opposite effect (Fig. 4C). It is worth noting that Uca1 overexpression significantly promoted the expression of Sfrp1, while suppressing the expression of β-catenin. Interestingly, miR-375 overexpression significantly reversed the effect of high expression of Uca1 on the expression of Sfrp1 and β-catenin (Fig. 4D). Together, these results suggest that up-regulation of Uca1 promoted Sfrp1 expression through miR-375 to regulate WNT/β-catenin pathway. Finally, this study explored whether Sfrp1 is involved in the regulation of abnormal proliferation of neural progenitors by Uca1. Epileptic mice were injected with Ad-Uca1 or/and shSfrp1 three days after kainic acid administration. The protein levels of SFRP1 and β-catenin was monitored by western blotting. As shown in Fig. 5A, Uca1 overexpression promoted the expression of Sfrp1 and suppressed the expression of β-catenin. After knockdown of Sfrp1 by shRNA in vivo, the expression of Sfrp1 was significantly reduced, while the expression of β-catenin was increased. Not only that, Sfrp1 knockdown reversed the effect of UCA1 on the expression of Sfrp1 and β-catenin. In addition, functional analysis showed that Uca1 overexpression inhibited the abnormal proliferation of neural progenitors, while sh-Sfrp1 showed the opposite effect, and Sfrp1 knockdown reversed the inhibitory effect of Uca1 on abnormal neuronal proliferation (Fig. 5B). Taken together, these results indicate that Uca1 reduces the abnormal proliferation of neural progenitors in epileptic mice by regulating the expression of Sfrp1.

DISCUSSION
TLE is one of the most common types of intractable epilepsy, characterized by periodic seizures and unpredictability (Han et al., 2018). Long-term recurrent seizures or epileptic status in the developmental stage can cause many cognitive disorders, such as impairment of learning, memory, language, etc (Jones-Gotman et al., 1997;Boling 2018;Buck & Sidhu 2020). Studies have shown that epileptic seizures are closely related to the abnormalities of hippocampal structure and function (Perkins et al., 2017). Epilepsy can cause the abnormal enhancement of hippocampal neurogenesis which is closely related to cognitive function . Therefore, it is very important to study the effect of epilepsy on hippocampal neurogenesis and early intervention. In this study, we successfully constructed kainic acid-induced epileptic mice model, and explored the role and potential molecular mechanism of lncRNA Uca1 in epileptic mice. The results found a new molecular mechanism of Uca1 in epilepsy, that is, the overexpression of Uca1 could significantly inhibit the abnormal proliferation of hippocampal neurons by WNT/β-catenin pathway via regulating Sfrp1 expression. Our findings provided a basis for early intervention of epilepsy.
Epilepsy is closely related to the abnormal regulation of lncRNAs (Qiao et al., 2018;Villa et al., 2019). It was found that 384 or 279 lncRNAs were significantly deregulated in pilocarpine or KA-induced epilepsy mouse models (Lee et al., 2015). Uca1 is an oncogene and plays an important role in the development of tumors. Notably, Uca1 has been proved to promote the proliferation and differentiation of neural stem cells (Liu et al., 2015). In epilepsy, Geng et al., found that Uca1 inhibited the apoptosis of hippocampal neurons, thus inhibiting the brain injury caused by epilepsy (Geng et al., 2018). The study of Wang et al revealed that Uca1 inhibited the activation of hippocampal astrocytes and improved the learning and memory ability of epilepsy rats, and had a protective effect on neuronal damage caused by KA. Further study clarifies that the role of Uca1 in epilepsy rats may be achieved by regulating the JAK/STAT signaling pathway (Wang et al., 2020). In addition, Yu et al., found that the expression of Uca1 in the SD rat epilepsy model established by lithium chloride and pilocarpine was also down-regulated. Uca1 overexpression can inhibit the epileptic inflammation by regulating the miR203/MEF2C/NF-κB axis (Yu et al., 2020). Consistent with the above results, this study constructed a KAinduced mouse epilepsy model and found that Uca1 is low-expressed in epileptic mice. And the low expression of Uca1 caused abnormal proliferation of hippocampal neurons in epileptic mice. After forced overexpres-sion of Uca1, the abnormal proliferation of hippocampal neurons was suppressed. Further mechanistic analysis showed that Uca1 could be targeted to downstream miR-375 to promote the expression of Sfrp1, thereby inhibiting the activation of WNT/β-catenin signaling pathway. Overall, this study considers Uca1 as a potential target for clinical treatment of epilepsy.
There is increasing evidence that miRNAs exhibit abnormal regulation in epilepsy (Jimenez-Mateos et al., 2011;Hu et al., 2012). These abnormally regulated miR-NAs mainly participate in the occurrence of epilepsy by regulating cell proliferation and migration, neuroinflammation and neuronal apoptosis (Karnati et al., 2015). MiR-375 is a widely studied miRNA that has been proved to participate in tumorigenesis (Wang et al., 2016;Kang et al., 2018). Studies have shown that The target binding sites of miR-375 and Sfrp1 was predicted by TargetScan. B. Luciferase activity was monitored by dual luciferase reporter assay. (p<0.0001) C. Neurons were transfected with NC mimic, miR-375 mimic, NC inhibitor or miR-375 inhibitor with Lipofectamine 3000 (Invitrogen), respectively. The protein levels of SFRP1 and β-catenin was monitored by western blotting. D. Neurons were co-transfected with Ad-vector and NC mimic or co-transfected with Ad-Uca1 and NC mimic or miR-375 mimic. (p<0.001) the expression of miR-375 was upregulated in the KAinduced SD epilepsy rat model (Henshall 2013), indicating that the abnormal expression of miR-375 may be related to the occurrence of epilepsy. However, the underlying molecular mechanism of the high expression of miR-375 for epilepsy remains unknown. In this study, a KA-induced epilepsy rat model was constructed, and the expression of miR-375 in the hippocampus tissues of epilepsy mice and normal mice was detected. Consistent with the above results, this study also found that miR-375 was highly expressed in KA-induced epilepsy mice. Further analysis showed that Uca1 targeted miR-375 and reduced the level of miR-375 in the hippocampus of epilepsy mice, which further inhibited the abnormal proliferation of hippocampal neurone. Tar-getScan analysis showed that Sfrp1 was also a target of miR-375. Correlation analysis showed that miR-375 was negatively correlated with the expression of Uca1 and Sfrp1.
SFRP1 is a negative regulator of WNT signaling and dose-dependently regulate the development of midbrain dopamine neurons (Kele et al., 2012). SFRP1 participates in the regulation of WNT/β-catenin pathway by suppressing the accumulation of β-catenin through a GSK-3 dependent mechanism, which interferes with the binding receptor of WNT and FRIZZLED protein (Kawano & Kypta 2003;Galli et al., 2006). The WNT/β-catenin pathway regulates hippocampal neurogenesis, synaptic division, and mitochondrial regulation, and is critical to the development and function of the central nervous system (Rubio et al., 2020). WNT/β-catenin signals modulate epileptic neurogenesis and neuronal death. It also plays a role in the susceptibility of epilepsy and the development of chronic epilepsy, and has been found to be a Epileptic mice were injected with Ad-Uca1 or/and shSfrp1 three days after kainic acid administration. A. The protein levels of SFRP1 and β-catenin was monitored by western blotting. (p<0.0001) B. The proliferation of neural progenitors in hippocampus was monitored by Brdu staining. (p<0.0001) promising antiepileptic target for the treatment of epilepsy in the future (Hodges & Lugo 2018). In this study, it was found that miR-375 could promote the activation of WNT/β-catenin pathway by targeting Sfrp1, thus promoting the abnormal proliferation of hippocampal neurone in epileptic mice. It is worth noting that UCA1 overexpression can inhibit the activation of WNT/βcatenin pathway and prevent epilepsy by reducing the level of miR-375 and alleviating the inhibitory effect of miR-375 on Sfrp1.
In conclusion, we found that Uca1 was highly expressed in epileptic mice and miR-375 was poorly expressed. Further studies have shown that Uca1 can promote the expression of Sfrp1 by reducing the level of miR-375, which further inhibits the abnormal proliferation of neural progenitors in epileptic mice by inhibiting the activation of the WNT/β-catenin pathway.