Application of RNA binding protein HuR and its gene as target in screening drugs for treating presbycusis

Abstract

This invention discloses the application of the RNA-binding protein HuR and its gene as a target in screening drugs for the treatment of age-related hearing loss. Belonging to the field of genetic engineering technology, it provides the application of the RNA-binding protein HuR gene as a therapeutic target in screening drugs for the prevention or treatment of age-related hearing loss. Specifically, this invention utilizes Atoh1-Cre and Pax2-Cre mice to knock out HuR in the inner ear hair cells and the entire cochlea, respectively. It was found that the absence of HuR in hair cells induces stereocilia degeneration, accelerates hair cell aging, and consequently leads to early-onset hearing loss. These results indicate that HuR plays an important role in maintaining stereocilia homeostasis, delaying hair cell aging, and preventing age-related hearing loss, providing a therapeutic target for the prevention and improvement of hearing loss in patients with age-related hearing loss in clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to the application of RNA-binding protein HuR and its gene as targets in screening drugs for the treatment of age-related hearing loss. Background Technology

[0002] Age-related hearing loss (ARHL), also known as presbyopia, is one of the most common and complex chronic diseases among the elderly, with its pathogenesis remaining unclear. In my country, hearing loss is one of the most common disabilities, with presbyopia accounting for more than half of all hearing loss cases. ARHL not only severely impacts patients' quality of life but also imposes a significant economic burden and pressure on society. Therefore, ARHL is not only a medical problem but also a social one. However, developing more effective treatment strategies for ARHL requires a comprehensive understanding of its underlying mechanisms.

[0003] The RNA-binding protein HuR is a classic regulator of RNA metabolism. Recent studies have shown that HuR plays an important role in age-related diseases. In cardiac tissue, HuR enhances the expression of the aging inducer P21, and HuR degradation successfully halts cardiac aging. Furthermore, HuR and its stress particles can promote osteogenesis during bone aging. However, the potential mechanism of action of HuR in ARHL remains unclear. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes the application of RNA-binding protein HuR and its gene as targets in screening drugs for the treatment of age-related hearing loss. It has been found that RNA-binding protein HuR and its gene play an important role in maintaining stereocilia homeostasis, delaying hair cell aging, and preventing age-related hearing loss, providing therapeutic targets for the prevention and improvement of hearing loss in patients with age-related hearing loss in clinical practice.

[0005] To achieve the above objectives, this invention provides the application of the RNA-binding protein HuR gene as a therapeutic target in screening drugs for the prevention or treatment of age-related hearing loss.

[0006] Preferably, the nucleotide sequence of the RNA-binding protein HuR gene is the nucleotide sequence obtained by sequentially linking SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0007] Preferably, the drug prevents or treats age-related hearing loss by increasing the expression of the RNA-binding protein HuR gene, maintaining the homeostasis of the cilia, and delaying hair cell aging.

[0008] The present invention also provides a medicament for the prevention or treatment of age-related hearing loss, the medicament comprising an expression promoter of the RNA-binding protein HuR gene; the nucleotide sequence of the RNA-binding protein HuR gene is shown as the nucleotide sequence obtained by sequentially linking SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0009] This invention also provides the application of RNA-binding protein HuR as a therapeutic target in screening drugs for the prevention or treatment of age-related hearing loss, wherein the RNA-binding protein HuR is obtained by expressing the RNA-binding protein HuR gene; the nucleotide sequence of the RNA-binding protein HuR gene is shown as the nucleotide sequence obtained by sequentially linking SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10; the amino acid sequence of the RNA-binding protein HuR is shown as SEQ ID NO.11.

[0010] Preferably, the drug maintains the homeostasis of the radiocilia and delays hair cell aging by increasing the expression or activity of the RNA-binding protein HuR, thereby preventing or treating age-related hearing loss.

[0011] The present invention also provides a medicament for the prevention or treatment of age-related hearing loss, the medicament comprising an RNA-binding protein HuR expression promoter or an RNA-binding protein HuR activity promoter; the amino acid sequence of the RNA-binding protein HuR is shown in SEQ ID NO. 11.

[0012] The present invention also provides the application of the RNA-binding protein HuR gene in the preparation of a mouse model of age-related hearing loss, wherein the nucleotide sequence of the RNA-binding protein HuR gene is shown as the nucleotide sequence obtained by sequentially linking SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0013] Preferably, knocking out the mouse cochlear RNA-binding protein HuR gene reduces the expression of RNA-binding protein HuR, induces degeneration of the stereocilia, accelerates hair cell aging, and thus leads to age-related hearing loss; the amino acid sequence of the RNA-binding protein HuR is shown in SEQ ID NO.11.

[0014] The present invention also provides a method for constructing a mouse model of age-related hearing loss, comprising knocking out the mouse cochlear RNA-binding protein HuR gene, wherein the nucleotide sequence of the RNA-binding protein HuR gene is shown in the nucleotide sequence obtained by sequentially connecting SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects:

[0016] This invention discloses the application of the RNA-binding protein HuR and its gene as targets in screening drugs for the treatment of age-related hearing loss. This invention is the first to discover the age-related dynamic expression pattern of HuR in the cochlear cells of aged mice and non-human primates (cynomolgus monkeys). Using Atoh1-Cre and Pax2-Cre mice, HuR was knocked out in the inner ear hair cells and the entire cochlea, respectively. It was found that the absence of HuR in hair cells induced stereocilia degeneration, accelerated hair cell aging, and consequently led to premature hearing loss. These results indicate that HuR plays an important role in maintaining stereocilia homeostasis, delaying hair cell aging, and preventing age-related hearing loss, providing a therapeutic target for the prevention and improvement of hearing loss in patients with age-related hearing loss in clinical practice. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This diagram illustrates the dynamic expression of HuR in the mouse cochlea. Figure a shows the scRNA-seq procedure and subsequent data analysis of the C57BL / 6J mouse cochlea. Figure b shows the expression level of HuR mRNA in cochlear cells of C57BL / 6J mice at different ages. Figure c shows the expression level of HuR mRNA in hair cells of mice at different ages. Figure d shows the expression level of HuR mRNA in spiral ganglion neurons of mice at different ages. Figure e shows the expression level of HuR mRNA in intermediate cells of the stria vascularis of mice at different ages. Figure f shows the expression level of HuR mRNA in marginal cells of the stria vascularis of mice at different ages. Figure g shows the RT-qPCR analysis of HuR in the cochlea of ​​1, 5, and 12-month-old C57BL / 6J mice. mRNA expression results: h is the result of Western blot detection of HuR protein expression in the cochlea of ​​1-, 5-, and 12-month-old C57BL / 6J mice; i is the result of HuR expression analysis in the cochlea of ​​1-, 5-, and 12-month-old C57BL / 6J mice; j is the immunofluorescence staining analysis of HuR expression in the cochlea of ​​C57BL / 6J mice on day 0 after birth (scale bar: 50 μm); k is the immunofluorescence staining image of HuR in the cochlea of ​​1- and 12-month-old C57BL / 6J mice (scale bar: 10 μm). In the figure, 1M represents 1 month old, 2M represents 2 months old, 5M represents 5 months old, 12M represents 12 months old, 15M represents 15 months old, OC represents organ of Corti, SV represents stria vascularis, and SG represents spiral ganglion.

[0019] Figure 2 The expression of HuR in the cochlear hair cells of cynomolgus monkeys is shown in the following figures: a) Immunofluorescence staining of HuR in cochlear hair cells of young and old cynomolgus monkeys; b) The ratio of HuR signal intensity in the cytoplasm to the nucleus of outer cochlear hair cells of young and old cynomolgus monkeys; c) The ratio of HuR signal intensity in the cytoplasm to the nucleus of inner cochlear hair cells of young and old cynomolgus monkeys.

[0020] Figure 3 These are the hearing test results for mice, where 'a' represents Atoh1-HuR with hair cell knockout of HuR. - / - Pax2-HuR knockout of HuR in mice and the entire cochlea - / - A schematic diagram of mouse construction, where b represents WT and Atoh1-HuR. - / - and Pax2-HuR - / -Representative immunofluorescence staining images of HuR in mouse cochlea, c represents WT and Atoh1-HuR. - / - and Pax2-HuR - / - ABR threshold of mice at P30 (30 days after birth), d = WT, Atoh1-HuR - / - and Pax2-HuR - / - The ABR threshold of mice at P45, e is WT, Atoh1-HuR - / - and Pax2-HuR - / - ABR threshold of mice at P60, f being WT, Atoh1-HuR - / - and Pax2-HuR - / - Representative ABR waveforms of mice at P60, where g represents WT and Atoh1-HuR. - / - and Pax2-HuR - / - ABR threshold of mice at P90, h is WT, Atoh1-HuR - / - and Pax2-HuR - / - ABR I-wave amplitude in mice at P6090dB SPL, where i represents WT, Atoh1-HuR - / - and Pax2-HuR - / - In mice, the ABR wave I latency at P6090 dB SPL was WT, Atoh1-HuR. - / - and Pax2-HuR - / - The DPOAE threshold of mice at P60, where k is WT and Atoh1-HuR. - / - and Pax2-HuR - / - The DPOAE threshold in mice at P90;

[0021] Figure 4 To determine the effect of HuR gene knockout in mice on cochlear hair cells, where a represents WT and Atoh1-HuR. - / - and Pax2-HuR - / - Representative HE staining images of mouse hair cells, spiral ganglia, and stria vascularis, with a scale bar of 50 μm. OC represents the organ of Corti, SGN represents spiral ganglion neurons, SV represents stria vascularis, and b represents WT and Atoh1-HuR. - / - and Pax2-HuR - / - Mean density of spiral ganglion cells in mice, c = WT, Atoh1-HuR - / - and Pax2-HuR - / - Mean thickness of stria vascularis in mice, d for P60 and P90 WT, Atoh1-HuR - / - and Pax2-HuR - / -Representative staining image of mouse basement membrane, scale bar at 50 μm, e indicates P60WT, Atoh1-HuR - / - and Pax2-HuR - / - Percentage of hair cells lost in mice, f = P90 WT, Atoh1-HuR - / - and Pax2-HuR - / - Percentage of hair cells lost in mice;

[0022] Figure 5 To determine the effect of HuR gene knockout in mice on the stereocilia tuft, where a represents WT and Atoh1-HuR. - / - and Pax2-HuR - / - Pseudocolor scanning electron micrographs of single hair cell bundles in mice at P60 and P90. In the images, magenta represents the highest row of cilia, blue represents the middle row, yellow represents the shortest row, orange arrows point to fused cilia, green arrows point to broken cilia, and magenta arrows point to internalized cilia. The scale bar is 5 μm. b represents WT and Atoh1-HuR. - / - and Pax2-HuR - / - The number of outer hair cells in mice with disordered p60 cilia, c being WT, Atoh1-HuR - / - and Pax2-HuR - / - The number of outer hair cells in mice at P60 cilia fusion, d = WT, Atoh1-HuR - / - and Pax2-HuR - / - The number of outer hair cells in mice with internalized P60 cilia, e being WT, Atoh1-HuR - / - and Pax2-HuR - / - The number of outer hair cells in mice with P60 epicilia deficiency, f being WT, Atoh1-HuR - / - and Pax2-HuR - / - The number of outer hair cells in mice with disordered p90 cilia, g = WT, Atoh1-HuR - / - and Pax2-HuR - / - The number of outer hair cells in mice with P90 epicilia deficiency, h being WT, Atoh1-HuR - / - and Pax2-HuR - / - Comparative images of the "V"-shaped three-dimensional ciliary bundles at the base of the mouse cochlea, scale bar is 5μm, i represents WT, Atoh1-HuR - / - and Pax2-HuR - / - The number of outer hair cells at the base of the mouse cochlea in a V-shaped stereocilia tract with an angle >110°, j being P60 WT, Atoh1-HuR - / - and Pax2-HuR - / -Transmission electron microscopy image of mouse outer hair cells, scale bar is 2μm. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Example 1

[0029] 1. Dynamic expression of HuR in the mouse cochlea:

[0030] A high-throughput single-cell RNA sequencing (scRNA-seq) atlas of the C57BL / 6J mouse cochlea was constructed to analyze the expression of HuR in inner ear hair cells, spiral ganglion neurons, and stria vascularis cells. Immunofluorescence staining was also used to detect the expression and localization of HuR in inner ear hair cells of mice and cynomolgus monkeys of different ages.

[0031] The nucleotide sequence of the RNA-binding protein HuR gene, accession number NM010485, is shown as the nucleotide sequence obtained by sequentially linking SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10. The amino acid sequence of the RNA-binding protein HuR is shown in SEQ ID NO.11.

[0032] (1) Establishment and analysis of high-throughput scRNA-seq map of the cochlea of ​​C57BL / 6J mice.

[0033] ① Take 1-month, 2-month, 5-month, 12-month and 15-month-old mice, 5 males and 5 females in each group, and mix cochlear samples from mice of the same age and sex for scRNA-seq.

[0034] ② The expression of RNA-binding proteins, including HuR, in various cell types of the mouse inner ear was analyzed in scRNA-seq maps.

[0035] (2) Immunofluorescence staining was used to detect the expression and localization of HuR in the inner ear cells of mice and cynomolgus monkeys.

[0036] ① Paraffin embedding and sectioning: Sampling and fixation: Mice were euthanized by cervical insemination, and the cochlea was removed and placed in 4% PFA overnight at 4°C; Washing: Washed three times with PBS, 5 min each time; Decalcification: The cochlea was placed in 10% EDTA solution at room temperature until the bone softened; Dehydration: The cochlea was placed in 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol (twice), each time for 5 min; Clearing: The cochlea was placed in ethanol:xylene at a ratio of 2:1 and 1:1, respectively. After 5 minutes in each of the mixed solutions, place the cochlea in xylene for about 3 minutes until transparent; paraffin infiltration: place the cochlea in mixed solutions of xylene:paraffin at a ratio of 2:1 and 1:1 for 20 minutes each, then place it in three different paraffin solutions for 30 minutes, 45 minutes, and 1 hour each; embedding: prepare an embedding box, pour in paraffin, place the cochlea in the box and wait for solidification to complete the embedding; sectioning: section the embedded cochlea to a thickness of 5 μm, collect the sections from the appropriate positions, and bake the sections in an oven at 37°C overnight.

[0037] ② Immunofluorescence staining of paraffin sections: Dewaxing: Place the sections in two different xylenes I and II for 20 min each for dewaxing; Rehydration: Place the sections in 100% ethanol, 100% ethanol, 95% ethanol, 80% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and distilled water for 3 min each; Antigen retrieval: Place the sections in sodium citrate retrieval solution in a boiling water bath for 15 min; Washing: Wash 3 times with PBS, 5 min each time; Blocking: Block with 10% goat serum at room temperature for 1 h, then incubate overnight at 4℃ with primary antibodies (rabbit HuR 1:400, mouse Myo7A 1:400, mouse parvoprotein antigen 1:2000); Washing: Wash 3 times with PBS, 5 min each time; Incubate with secondary antibodies (goat anti-rabbit 1:400, goat anti-mouse 1:400) at room temperature in the dark for 1 h; Washing: Wash 3 times with PBS, 5 min each time; Observe HuR expression after mounting with DAPI.

[0038] ③ Separation of the cochlear basilar membrane: Sampling and fixation: Sampling was performed on the cochleas of mice and cynomolgus monkeys and placed in 4% PFA overnight at 4°C; Washing: Washed 3 times with PBS for 5 min each time; Decalcification: The cochlea was placed in EDTA washing solution (10%) at room temperature until the bone softened; Washing: Washed 3 times with PBS for 5 min each time; The basilar membrane was carefully dissected under a dissecting microscope and cut into three segments: top, middle and bottom, and placed in PBS.

[0039] ④ Immunofluorescence staining: Permeabilization: 0.3% Triton X-100 permeabilization at room temperature for 15 min; Washing: PBS wash 3 times, 5 min each time; Blocking: 10% goat serum, room temperature for 1 h; Primary antibody (rabbit HuR 1:400) incubated overnight on a shaker at 4℃; Washing: PBS wash 3 times, 5 min each time; Secondary antibody (goat anti-rabbit, 1:400) incubated at room temperature in the dark for 1 h; Washing: PBS wash 3 times, 5 min each time; Mount with DAPI.

[0040] like Figure 1 Figure a shows a schematic diagram of the scRNA-seq procedure and subsequent data analysis of the cochlea of ​​C57BL / 6J mice. Single cells were isolated from the cochlea of ​​C57BL / 6J mice at 1, 2, 5, 12, and 15 months of age for single-cell sequencing and cluster analysis. Figure 1 As shown in Figure b, the expression level of HuR mRNA in the cochlear cells of C57BL / 6J mice is upregulated with age; Figure 1 c, Figure 1 d, Figure 1 China and Figure 1As shown in figure f, HuR mRNA expression is found in inner ear hair cells (HC), spiral ganglion neurons (SGN), intermediate cells (IC), and limbic cells (MC). The expression levels of HuR mRNA in the cochlea of ​​C57BL / 6J mice in HC, SGN, IC, and MC all increase with age. Figure 1 As shown in Figure g, RT-qPCR analysis of HuR mRNA expression in the cochlea of ​​1-month-old, 5-month-old, and 12-month-old C57BL / 6J mice revealed that, consistent with single-cell sequencing results, HuR mRNA levels increased with age. Figure 1 h and Figure 1 As shown in Figure i, Western blot analysis of HuR expression in the cochlea of ​​1-month-old, 5-month-old, and 12-month-old C57BL / 6J mice revealed that HuR protein expression levels increased with age. Figure 1 As shown in Figure j, immunofluorescence staining was used to analyze the expression of HuR in the cochlea of ​​day 0 (P0)C57BL / 6J mice. Myo7A was used as the marker, and cell albumin antigen was used to label spiral ganglion cells. The results showed that HuR was strongly expressed in mouse cochlear hair cells, supporting cells, stria vascularis, spiral ganglion cells, and vestibular cells. Figure 1 As shown in Figure k, HuR immunofluorescence staining in the cochlea of ​​1-month-old and 12-month-old C57BL / 6J mice shows that the cytoplasmic localization of HuR in hair cells increases with age.

[0041] like Figure 2 The image shows the expression of HuR in the cochlear hair cells of non-human primates (cynomolgus monkeys). Figure 2 In the middle a, the immunofluorescence staining of HuR in the cochlear hair cells of young and old cynomolgus monkeys shows an increase in the cytoplasmic localization of HuR in the outer hair cells. Figure 2 b and Figure 2 In the figure, c represents the fluorescence intensity ratio of HuR signal in the outer hair cells (OHC) and inner hair cells (IHC) of young and old cynomolgus monkeys, respectively. It can be seen that the fluorescence intensity ratio of the cytoplasm to the nucleus of the outer hair cells is significantly increased, while there is no significant change in the inner hair cells.

[0042] 2. Construct mouse models of HuR knockout specifically in hair cells and the entire cochlea:

[0043] ①HuR flox / flox Mice were mated with Atoh1-cre and Pax2-cre mice to obtain mice with the HuR gene specifically knocked out in inner ear hair cells (Atoh1-HuR). - / - ) and mice with the HuR gene knocked out specifically in the entire cochlea (Pax2-HuR) - / - ).

[0044] ② Immunofluorescence staining of paraffin sections was used to confirm that HuR was present in Atoh1-HuR. - / - Mouse hair cells and Pax2-HuR - / - The entire cochlea of ​​the mouse was missing.

[0045] like Figure 3 a and Figure 3 As shown in b, Figure 3 In the middle, 'a' represents the inner ear hair cells (Atoh1-HuR). - / - ) and the entire cochlea (Pax2-HuR) - / - A schematic diagram of transgenic mice with the HuR gene knocked out. Figure 3 b is Atoh1-HuR - / - and Pax2-HuR - / - Representative immunofluorescence staining images of HuR in the mouse cochlea, confirming that HuR is present in Atoh1-HuR. - / - Mouse hair cells and Pax2-HuR - / - The entire cochlea of ​​the mouse was missing.

[0046] 3. Audiological assessment:

[0047] ① Hearing test: WT (wild-type mice) and Atoh1-HuR were tested. - / - and Pax2-HuR - / - The thresholds of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) in mice.

[0048] ② Morphological examination: WT and Atoh1-HuR samples were taken respectively. - / - and Pax2-HuR - / - Paraffin sections of mouse cochlea were prepared and stained with hematoxylin and eosin (HE) to observe whether any abnormalities were found in the various structures of the mouse cochlea.

[0049] HE staining: Dewaxing: Place the sections in xylene I and II for 20 min each for dewaxing; Rehydration: Place the sections in 100% ethanol, 100% ethanol, 95% ethanol, 80% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and distilled water for 3 min each; Place the sections in hematoxylin for about 5 min until the staining is adequate, then rinse with tap water for 3 min; Separate the sections with 0.5% hydrochloric acid alcohol for 3 s, then wash with tap water for 3 min; Place the sections in 70%, 80%, and 95% ethanol for 3 min each, then stain with eosin dye (alcohol-soluble) for about 3 min, then place the sections in 95% ethanol for 30 s and then in 100% ethanol for 2 min; Place the sections in xylene for 10 min, then mount with neutral resin and observe.

[0050] ③ Basement membrane preparation staining: WT and Atoh1-HuR were stained separately. - / - and Pax2-HuR - / - Immunofluorescence staining was performed on the basement membrane of the mouse cochlea, and the loss of hair cells in WT and HuR-deficient mice was compared using myosin VIIA (Myo7A), phalloidin, and 4',6-diamidinyl-2-phenylindole (DAPI).

[0051] ④ Scanning electron microscopy of the basement membrane: P60 and P90 WT, Atoh1-HuR - / - and Pax2-HuR - / - The basilar membrane of the mouse cochlea was examined using scanning electron microscopy to observe whether the structure of the hair cells and cilia was abnormal.

[0052] ⑤ Transmission electron microscopy of the basement membrane: P60 and P90 WT, Atoh1-HuR - / - and Pax2-HuR - / - Transmission electron microscopy was used to examine the submicroscopic structure of hair cells and whether there were any abnormalities in organelles in the mouse cochlear basilar membrane.

[0053] The role of HuR in age-related hearing loss was evaluated using the methods described above.

[0054] like Figure 3 c, Figure 3 d, Figure 3 China and Figure 3 As shown in g, WT mice and HuR gene deletion mice were respectively observed at P30 ( Figure 3 c), P45 Figure 3 (middle d), P60 ( Figure 3 (e) and P90 ( Figure 3 The ABR thresholds of mice with the P30 HuR gene deletion were compared with those of age-matched WT mice. It can be seen that the ABR thresholds of P30 HuR gene deletion mice were not significantly different, indicating that their auditory system development was normal. P45 HuR gene deletion mice showed significantly elevated high-frequency ABR thresholds, P60 HuR gene deletion mice showed significantly elevated ABR thresholds across all frequencies, and P90 HuR gene deletion mice were nearly totally deaf. Figure 3 As shown in Figure f, this is a representative ABR waveform at P60 for WT mice and HuR gene-deleted mice under the same testing conditions. The colored line represents the lowest decibel heard. It can be seen that the ABR mixing threshold of HuR gene-deleted mice is significantly increased. Figure 3 h and Figure 3 As shown in Figure i, the ABR I-wave amplitude at P6090 dB SPL is (i = 0.05 m / s). Figure 3 (middle h) and incubation period ( Figure 3As shown in i), the amplitude and latency of wave I in HuR gene-deficient mice were not significantly different from those in WT mice; Figure 3 j and Figure 3 As shown in Figure k, the results show the P60 values ​​of WT mice and HuR gene deletion mice. Figure 3 (j) and P90 ( Figure 3 The DPOAE threshold of (k) shows that the DPOAE threshold is significantly increased across the entire frequency in P60 HuR gene deletion mice, and is close to total deafness in P90 HuR gene deletion mice.

[0055] like Figure 4 As shown, the deletion of the HuR gene accelerates the loss of cochlear hair cells, such as Figure 4 As shown in Figure a, these are representative HE-stained images of cochlear hair cells, spiral ganglia, and stria vascularis in WT mice and HuR gene-deficient mice. It can be seen that in these mice, only hair cells are lost, while the spiral ganglia and stria vascularis remain normal. Figure 4 b and Figure 4 As shown in Figure c, this represents the average number of spiral ganglion cells in WT mice and HuR gene-deleted mice. Figure 4 (b) and average thickness of striae ( Figure 4 Statistical analysis in c) showed that, compared with WT mice, HuR gene-deficient mice had no significant differences in spiral ganglion cell density and stria vascularis thickness; Figure 4 As shown in Figure d, these are representative images of basement membrane staining in P60 and P90 WT mice and HuR gene deletion mice. The images show that hair cell loss begins in P60 HuR gene deletion mice, and hair cell loss is severe in P90 HuR gene deletion mice. Figure 4 China and Figure 4 As shown in f, it is P60 ( Figure 4 (e) and P90 ( Figure 4 The percentage of hair cell loss in WT mice and HuR gene-deleted mice (f) shows that the hair cell loss rate in P60 HuR gene-deleted mice gradually increases from the apical gyrus to the basal gyrus, and the hair cell loss rate in P90 HuR gene-deleted mice is further aggravated, with almost all hair cells lost in the basal gyrus.

[0056] like Figure 5 The image shows the degeneration of the stereocilia tufts in mice with the HuR gene deletion; as shown... Figure 5 As shown in Figure a, WT mice and Atoh1-HuR mice are used. - / - Mice and Pax2-HuR - / -Pseudocolor scanning electron microscopy images of single hair cell bundles in mice at P60 and P90 show three rows of stereocilia in the outer hair cells (highest row (magenta), middle row (blue), and shortest row (yellow)). Orange arrows point to fused stereocilia, green arrows to broken stereocilia, and magenta arrows to internalized stereocilia. This indicates severe stereocilia degeneration in HuR gene-deficient mice. Figure 5 b, Figure 5 c, Figure 5 d and Figure 5 As shown in e, this represents P60 static ciliary disorder ( Figure 5 (b) , integration ( Figure 5 c), internalization Figure 5 d) or missing ( Figure 5 The number of outer hair cells in (e) indicates that the proportion of disordered, fused, internalized, and absent outer hair cells in HuR gene-deficient mice is significantly increased; for example... Figure 5 f and Figure 5 As shown in g, this represents P90 static ciliary disorder ( Figure 5 f) or missing ( Figure 5 The number of outer hair cells in the middle (g) indicates that the proportion of disordered and missing outer hair cells in HuR gene-deficient mice is further increased; for example Figure 5 h and Figure 5 As shown in Figure i, compared with WT mice, Atoh1-HuR - / - and Pax2-HuR - / - The number of outer hair cells at the base of the mouse cochlea, characterized by a wider "V"-shaped three-dimensional ciliary bundle (>110°), was significantly increased; such as Figure 5 As shown in Figure j, transmission electron microscopy revealed that the outer hair cells of P60 WT mice had normal morphology, and Atoh1-HuR - / - and Pax2-HuR - / - The mouse's outer hair cells were severely degenerated, while the epidermal plate and mitochondria remained normal.

[0057] This invention has demonstrated that HuR is a negative regulator of ARHL in mice, playing a crucial role in the ARHL progression and providing a novel target for the prevention and treatment of ARHL. HuR expression in all cell types of the mouse inner ear increases with age, and increased cytoplasmic localization of HuR was observed in hair cells of aged mice and cynomolgus monkeys. Furthermore, HuR expression was correlated with Atoh1-Cre and Pax2-Cre, respectively. flox / flox Mice were bred to obtain mice in which HuR was knocked out in the cochlear hair cells or the entire cochlea. It was found that the absence of HuR in hair cells led to the degeneration of stereocilia, accelerated hair cell aging, and consequently triggered ARHL. These results indicate that HuR plays an important role in the development and progression of ARHL, thus providing a therapeutic target for the prevention and improvement of hearing loss in ARHL patients in clinical practice.

[0058] This invention confirms that HuR is persistently and extensively expressed in mouse inner ear cells and non-human primate (cynomolgus monkey) inner ear cells, and its expression gradually increases with age in all inner ear cells. Furthermore, the cytoplasmic localization of HuR in hair cells gradually increases with age. Further investigation in mouse models with HuR knockout in cochlear hair cells or the entire cochlea revealed that HuR deficiency in hair cells leads to disorganization of the stereocilia, accelerates hair cell aging, and ultimately causes age-related hearing loss. These results indicate that HuR plays a crucial role in the progression of age-related hearing loss and could serve as an effective target for the prevention and treatment of this condition.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The RNA binding protein HuR gene is applied in the preparation of presbycusis mouse model, characterized in that, The nucleotide sequence of the RNA-binding protein HuR gene is shown in the sequence obtained by linking SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 in sequence. By knocking out the mouse cochlear RNA-binding protein HuR gene, the expression of RNA-binding protein HuR is reduced, causing degeneration of the stereocilia, accelerating hair cell aging, and thus leading to age-related hearing loss; the amino acid sequence of the RNA-binding protein HuR is shown in SEQ ID NO.

11.

2. A method for constructing a mouse model of presbycusis, characterized by, The invention includes knockout of the mouse cochlear RNA-binding protein HuR gene, the nucleotide sequence of which is shown in the sequence obtained by linking SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 in sequence.

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