Use of miR-5588-3p inhibitors

Abstract

This invention discloses the application of a miR-5588-3p inhibitor, belonging to the field of biomedical technology. Studies have found that miR-5588-3p expression is abnormally elevated in the plasma of epilepsy patients. Administration of the miR-5588-3p inhibitor 72 hours before constructing a tree shrew epilepsy model can effectively reduce the seizure severity, seizure rate, and mortality rate in epileptic tree shrews, and alleviate brain pathological damage. The miR-5588-3p inhibitor described in this invention is used to prepare drugs for treating epilepsy, and its nucleotide sequence is as follows: GCUGGCAUUAGUGGGACUU. This invention can effectively reduce the seizure rate and mortality rate in epileptic tree shrews, effectively prolong the clonic latency and seizure latency, shorten the seizure duration, and effectively reduce the seizure severity. The miR-5588-3p inhibitor can significantly reduce hippocampal neuronal damage.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a miR-5588-3p inhibitor. Background Technology

[0002] Epilepsy is a chronic neurological disorder caused by sudden, abnormal, and excessive electrical discharges in the brain's neurons. Its main clinical manifestations are sudden or temporary abnormalities in consciousness, sensation, motor function, autonomic nervous system function, or mental and behavioral disturbances. The occurrence and development of epilepsy are caused by the combined effects of multiple factors, and its pathogenesis involves gene mutations, altered neuronal excitability, neurotransmitter imbalances, synaptic abnormalities, and inflammatory responses.

[0003] Currently, epilepsy treatment mainly includes antiepileptic drug therapy, surgical treatment, diet, and vagus nerve modulation therapy. However, approximately 30% of patients are diagnosed with drug-resistant epilepsy, and the cause remains unknown. Worse still, no drug can effectively prevent seizures or epileptic progression. Therefore, there is an urgent need to explore new drug targets and develop novel drugs that can delay or prevent seizures, inhibit epileptic progression, and reduce epilepsy-related complications.

[0004] MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs in eukaryotes, approximately 17–25 bp in length. They have been shown to play a crucial role in gene regulation and post-transcriptional regulation of gene expression in various cellular pathways and systems. Research on the function and mechanisms of miRNAs is of great significance to human health, as miRNA dysregulation is associated with many diseases. Therefore, utilizing miRNAs for disease diagnosis and treatment has become a research hotspot in recent years, providing new avenues for developing innovative therapies and diagnostic tools. Discovering molecular biomarkers that can distinguish epilepsy patients from healthy subjects and other neurological diseases allows for earlier and more accurate diagnosis and appropriate treatment. miRNAs, as regulators of gene expression, play a key role in the progression of epilepsy and hold promise as therapeutic targets for the disease. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to identify miRNA biomarkers related to epilepsy treatment. The inventors of this invention, in their research on the pathogenesis of epilepsy, discovered that miR-5588-3p expression is abnormally elevated in the plasma of epilepsy patients. Studies have shown that administering a miR-5588-3p inhibitor 72 hours before establishing a tree shrew epilepsy model can effectively reduce the seizure severity, seizure rate, and mortality rate in epileptic tree shrews, and alleviate brain pathological damage in epileptic tree shrews.

[0006] The miR-5588-3p inhibitor described in this invention is used to prepare a drug for treating epilepsy, and its nucleotide sequence is as follows: GCUGGCAUUAGUGGGACUU.

[0007] The beneficial effects of this invention are:

[0008] The miR-5588-3p inhibitor described in this invention can effectively reduce the seizure rate and mortality of epileptic tree shrews, effectively prolong the clonic latency and seizure latency of epileptic tree shrews, shorten the seizure duration of epileptic tree shrews, effectively reduce the seizure severity of epileptic tree shrews, and significantly reduce hippocampal neuronal damage. Attached Figure Description

[0009] Figure 1 The expression levels of miR-5588-3p in the plasma of healthy controls and patients with epilepsy were measured. #### This indicates that compared with the control group, p < 0.0001).

[0010] Figure 2 This is a comparison of HE staining changes in hippocampal neural tissue after tree shrew modeling in the experiment of this invention (200×).

[0011] Figure 3 This is a comparison image (400×) of immunofluorescence staining of NeuN-positive neurons in the hippocampus region of tree shrews from different groups in this invention.

[0012] Figure 4 This is a comparison of the average fluorescence intensity ratios of NeuN-positive neurons in the hippocampus region of tree shrews in different groups according to the present invention. Detailed Implementation

[0013] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0014] 1. Expression characteristics and clinical significance of miR-5588-3p in patients with epilepsy

[0015] 1.1 Main reagents and consumables

[0016] Reagent Name company miRcute miRNA Extraction and Isolation Kit Heavenly Root Chloroform Xilong Science Anhydrous ethanol Xilong Science miRNA cDNA First-Strand Synthesis Kit Heavenly Root miRNA Quantitative Real-Time Detection Kit (SYBR Green) Heavenly Root

[0017] 1.2 Main Instruments

[0018] Instrument Name company Vortex mixer SCILOGEX High-speed refrigerated centrifuge SCILOGEX Handheld centrifuge SCILOGEX 96-well plate centrifuge Qilinbell water bath Jintan Science and Technology Quantitative PCR instrument Roche

[0019] 1.3 Experimental Methods

[0020] 1.3.1 Sample Collection

[0021] Plasma samples were collected from 5 patients with epilepsy and 5 healthy volunteers (control group) (a total of 10 subjects) as study subjects. Plasma sample preparation method: Approximately 4 mL of fasting peripheral venous blood was collected from the subjects. After collection, the plasma samples were stored in sodium citrate anticoagulant tubes, centrifuged (3000 rpm, 4℃, 10 min), and the plasma was immediately stored in a -80℃ freezer for later use.

[0022] 1.3.2 Primer Design and Synthesis

[0023] The corresponding sequence was obtained from the NCBI database, and the miR-5588-3p primer was designed. The primer was synthesized by Shanghai Sangon Biotech Co., Ltd. The primer sequence is as follows: miR-5588-3p Forward: 5'-AAGTCCCACTAATGCCAGC-3'.

[0024] 1.3.3 miRNA extraction

[0025] Take 200 µL of plasma sample and extract miRNA from the plasma sample using the miRcute miRNA extraction and isolation kit.

[0026] 1.3.4 miRNA reverse transcription

[0027] The reverse transcription system was performed according to the miRNA cDNA first-strand synthesis kit instructions.

[0028] Element volume 2×miRNA RT Reaction Buffer 10 μL miRNA RT Enzyme Mix 2 μL miRNA samples 8 μL

[0029] Bring the total volume of the three reagents to 20 µL, gently shake, centrifuge for 3-5 s to mix the reactants, incubate at 42°C for 60 min, then at 95°C for 3 min to obtain the first strand of reverse-transcribed cDNA. Immediately place on ice or store at -20°C.

[0030] 1.3.5 qPCR detection of target gene expression

[0031] Following the instructions of the miRNA fluorescence quantitative detection kit, add the following reagents to each well of a 96-well PCR plate in the order of sample loading:

[0032] Element volume 2×miRcute Plus miRNA PreMix (SYBR&ROX) 5 μL Forward Primer 0.2 μL Reverse Primer (10 µM) 0.2 μL miRNA first-strand cDNA 1 μL <![CDATA[ddH2O]]> 3.6 μL Total system 10 µL

[0033] The above system was thoroughly mixed, briefly centrifuged, and then placed in a LightCycler 96 instrument for detection. Amplification signals were collected. The following were the quantitative PCR conditions: pre-denaturation 95℃, 600s; 95℃: 5s, 60℃: 30s, 72℃: 30s, 45 cycles; melting curve analysis: 95℃: 15s; 60℃: 30s; 95℃: 1s. U6 was selected as an internal control. For the analysis of relative expression levels of the target gene, 2...-△△Ct The method is used for calculation.

[0034] 1.3.6 Statistical Analysis

[0035] The analysis and comparison were performed using GraphPad Prism 9.0.0. The two independent samples t-test was used to compare between groups, and P < 0.05 was considered statistically significant.

[0036] 1.4 Experimental Results

[0037] The results are as follows Figure 1 As shown, compared with healthy volunteers (control group), the expression level of miR-5588-3p in the plasma of epilepsy patients was significantly increased (p<0.0001).

[0038] 2. Neuroprotective effect of miR-5588-3p inhibitors on tree shrew epilepsy

[0039] 2.1 Laboratory Animals

[0040] Eighteen male Burmese tree shrews of grade CL, weighing 120-150 g, were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK(Yunnan)K2023-0003] and housed in the general animal facility of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK(Yunnan)K2021-0003]. They were housed in stainless steel cages at a room temperature of (24±2)℃, relative humidity of (50±5)%, and a light / dark cycle of 12h / 12h.

[0041] 2.2 Main Reagents

[0042] Reagent Name company Phrixotoxin-1 alomone Paraformaldehyde recovery Hematoxylin and eosin (HE) staining kit Solarborg NeuN antibody abcam Goat Anti-Rabbit IgG H&L (DyLight® 488) abcam Fluorescent mounting medium (containing DAPI) Zhongshan Jinqiao ZLI-9557 Blocking sheep serum Zhongshan Jinqiao ZLI-9021

[0043] 2.3 Main Instruments

[0044] Instrument Name company Rotary slicer Leica, Germany Organizing the paver Leica, Germany Paraffin embedding machine Leica, Germany Freezer tray Leica, Germany Automatic dehydrator Wuhan Junjie Organizing sheet baking machine Wuhan Junjie Fluorescence microscopy OLYMPUS BX53 Electric heating drying oven thermocenter

[0045] 2.4 Preparation of drugs and reagents

[0046] Phrixotoxin-1 was prepared by dissolving Phrixotoxin-1 in physiological saline to prepare a solution with a concentration of 10 μg / mL and a pH of 7.4.

[0047] 2.5 Experimental Methods

[0048] 2.5.1 Animal Model Making

[0049] Experimental Groups:

[0050] ① Control group: 2 μL of 0.9% saline was injected into the hippocampus via stereotactic brain imaging, and an equal volume of 0.9% saline was injected intraperitoneally 72 hours later;

[0051] ② Model group: 2 μL of 0.9% saline was injected into the hippocampus via stereotactic brain imaging, followed by an intraperitoneal injection of 4 μg / kg Phrixotoxin-1 72 h later to establish a tree shrew epilepsy model;

[0052] ③ miR-5588-3p inhibitor group: 2 μL of a 1.0 nmol miR-5588-3p inhibitor was injected into the hippocampus via stereotactic injection, followed by an intraperitoneal injection of 4 μg / kg Phrixotoxin-1 72 h later to establish a tree shrew epilepsy model. The self-designed miR-5588-3p inhibitor sequence is as follows: GCUGCAUUAGUGGGACUU, synthesized by Shanghai Sangon Biotech Co., Ltd.

[0053] 2.5.2 Stereotactic Injection of the Brain

[0054] (1) Fixing the tree shrew: The tree shrew was anesthetized by intraperitoneal injection of 3% sodium pentobarbital at 2 μL / g body weight. After complete anesthesia, the hair on the cranial face of the tree shrew was removed with animal clippers and fixed on the stereotaxic device. The ear rod was inserted into the tree shrew's ear canal and the left and right ear rods were balanced so that the line connecting the two ears of the tree shrew was in the same straight line as the ear rod. The tree shrew's incisors were pried open with tweezers and the upper incisors were locked in the hole of the adapter. The nose rod locking screw was pressed down and the height of the incisor clamp was adjusted up and down, and the position of the adapter was adjusted back and forth to keep the cranial face of the tree shrew horizontal. The screw was locked so that the tree shrew's head could not move. According to the tree shrew's body size, the tree shrew's body was raised with foam on the base of the stereotaxic device so that the head and body were kept horizontal to prevent the tree shrew's breathing from being blocked.

[0055] (2) Micro-injection pump parameter settings: Turn on the power switch of the micro-injection pump, set the operation mode to first draw and then infuse, and set the drawing parameters: the volume of liquid drawn, the drawing time is 0.5 min; the interval between drawing and infusing is 0.5 min; and the infusing time is 5 min.

[0056] (3) Positioning reference point: After fixing the RN type injection needle (specification 10μL) on the holder, turn on the power switch of the digital positioning instrument, disinfect the skin surface of the tree shrew skull with iodine, make a longitudinal incision from the line connecting the two ears to the eyes to expose the skull, find the intersection of the interauricular line and the sagittal suture, i.e. the anterior fontanelle point, and use this point as the reference point O of the three-dimensional coordinate system. Move the three-dimensional manipulator arm to align its positioning point with the anterior fontanelle point, and press the "CLR" key of the digital display base X, Y, and Z axes to return to zero (desktop digital positioning instrument).

[0057] (4) Locating the target area: The stereotactic positioning parameters for the hippocampus were selected according to the "Stereotactic Atlas of Tree Shrew Brain": AP (Y-axis): 2.5mm; ML (X-axis): ±6.5mm; DV (Z-axis): -8mm (starting from the dura mater). That is, move forward 2.5mm along the Y-axis from point O, and then move left and right 6.5mm along the X-axis, marking the position with a marker; remove the cross arm, drill a hole in the skull, and expose the brain tissue.

[0058] (5) Experiment: After the micro-injection pump is turned on to draw the prepared composite solution, within 0.5 min, according to the determined value DV: 8 mm below the dura mater, the operating arm is moved downward to the target depth. After the injection is completed, the needle is left in place for 5 min, and then the needle is slowly withdrawn at a speed of 1.0 mm / min. The coordinate values ​​are set according to the method of locating the target area to carry out the injection experiment at another coordinate point. After the injection is completed, the RN-type injection needle is withdrawn, the injection hole is glued with bone glue, the wound is sutured and disinfected with povidone-iodine, and an appropriate amount of erythromycin ointment is applied to the wound. After the operation, penicillin (22000 IU / kg) is injected into the peritoneum.

[0059] 2.5.3 Assessment of Seizure Behavior

[0060] The Racine scale was used to assess the seizure behavior of tree shrews. Grade 0: no seizure behavior; Grade 1: rhythmic twitching of the corner of the mouth, facial muscles, or ears; Grade 2: more severe focal twitching; Grade 3: clonic jerking of the forelimbs, without standing or falling; Grade 4: generalized tonic-clonic jerking, with falling while standing or lying on one's side; Grade 5: generalized tonic-clonic seizure, with falling while supine; Grade 6: death. Tree shrews were observed continuously for 60 minutes. The seizure latency was recorded, including the time from drug administration to the onset of a Grade 3 seizure (clonic latency) and the time from drug administration to the onset of a tonic-clonic seizure (tonic latency). The seizure rate was also recorded.

[0061] 2.5.4 HE staining

[0062] Twenty-four hours after intraperitoneal injection, tree shrew tissue was harvested. Following anesthesia, the tree shrews were perfused sequentially with physiological saline and then 4% paraformaldehyde solution via the heart. The brain tissue was then fixed in 4% paraformaldehyde for 48 hours. The brain tissue was then dehydrated using a gradient of 75%, 85%, 90%, 95%, and 100% ethanol. After clearing in xylene, the dehydrated and cleared brain tissue was embedded in paraffin to displace the xylene, followed by paraffin embedding, sectioning, and subsequent hematoxylin and eosin (HE) staining.

[0063] Paraffin sections were dewaxed to water and stained with hematoxylin at room temperature for 3-5 minutes. They were then washed with tap water for 10 minutes to remove excess stain, differentiated with 1% hydrochloric acid alcohol for 2 seconds, rinsed with tap water for 10 minutes, and observed under a microscope after the cell nuclei turned blue-purple. They were then stained with eosin at room temperature for 5 minutes, dehydrated in graded alcohols, cleared in xylene, mounted with neutral resin, and the color development results were observed and photographed under a regular optical microscope.

[0064] 2.5.5 Immunofluorescence staining

[0065] Paraffin sections were dewaxed to water, then immersed in an antigen retrieval box containing citrate buffer (pH 6.0) in a microwave oven for antigen retrieval. After blocking with 5% goat serum for 1 hour, primary antibody (NeuN 1:100) was added and incubated overnight at 4°C. Then, 488-labeled goat anti-rabbit IgG (1:1000) was added and incubated at room temperature in the dark for 2 hours. The sections were then mounted with mounting medium containing DAPI, observed and photographed under a fluorescence microscope.

[0066] 2.5.6 Statistical Analysis

[0067] The analysis and comparison were performed using GraphPad Prism 9.0.0. One-way ANOVA was used for comparisons between groups, and LSD method was used for pairwise comparisons. A p-value < 0.05 was considered statistically significant.

[0068] 2.6 Experimental Results

[0069] 2.6.1 Incidence of epilepsy in each group of tree shrews

[0070] This experiment used a total of 18 tree shrews (n=6). The control group of tree shrews were normal, with no seizures or deaths. The seizure rate in the model group was 100.00%, and the mortality rate was 16.67%. The seizure rate in the miR-5588-3p inhibitor group was 33.33%, and the mortality rate was 0.00%. The results show that miR-5588-3p inhibitors can effectively reduce the seizure rate and mortality rate of epileptic tree shrews (Table 1).

[0071]

[0072] 2.6.2 Assessment of seizure behavior in tree shrews of each group

[0073] The seizure severity of tree shrew epilepsy was assessed using the Racine score. The results showed that, compared with the model group, the miR-5588-3p inhibitor group could effectively prolong the clonic latency and seizure latency of epileptic tree shrews and shorten the duration of seizures (Table 2).

[0074]

[0075] 2.6.3 Assessment of Seizure Severity in Tree Shrews of Each Group

[0076] The seizure severity of tree shrews was assessed using the Racine score. Results showed that all seizures in the model group were grade V or higher; in the miR-5588-3p inhibitor group, only 1, 3, and 2 tree shrews experienced grade II, III, and IV seizures, respectively. This indicates that the miR-5588-3p inhibitor can effectively reduce the seizure severity in epileptic tree shrews (Table 3).

[0077]

[0078] 2.6.4 HE staining results

[0079] HE results showed that in the control group, the hippocampal neurons of the tree shrew were neatly and densely arranged with clear edges, normal neuronal morphology, and uniform nuclear staining; in the model group, the hippocampal neurons of the tree shrew were disordered, with blurred boundaries, a large number of neurons showed shrinkage and necrosis, reduced cell volume, irregular cell morphology, and broken and dissolved nuclei; in the miR-5588-3p inhibitor group, hippocampal neuronal damage was significantly reduced, neurons were more neatly arranged, and only a small number of cells were necrotic.

[0080] 2.6.5 NeuN immunofluorescence staining results

[0081] Immunofluorescence staining results for NeuN showed that, compared with the control group, the average fluorescence intensity of NeuN in the model group was significantly reduced, indicating severe damage to neurons in the hippocampus of epileptic tree shrews; the average fluorescence intensity of NeuN in the miR-5588-3p inhibitor group was significantly higher than that in the model group (P < 0.0001), indicating that prior administration of miR-5588-3p inhibitor can effectively reduce neuronal damage in tree shrew epilepsy (P < 0.01). Figure 2 NeuN expression is highly neuron-specific and serves as a marker for mature neurons.

Claims

1. The application of miR-5588-3p inhibitors, characterized in that, The inhibitor is used to prepare a drug for treating epilepsy, and its nucleotide sequence is GCUGGCAUUAGUGGGACUU.

Images