Application of HuR and coding gene thereof in preparation of medicine for treating senile deafness

Abstract

The invention discloses an application of HuR and a coding gene thereof in preparation of a medicine for treating senile deafness, belongs to the technical field of gene engineering, and provides an application of the HuR coding gene in preparation of a medicine for preventing or treating senile deafness, and the nucleotide sequence of the HuR coding gene is shown as SEQ ID NO.1. According to the application of the HuR in preparation of the medicine for preventing or treating the senile deafness, the amino acid sequence of the HuR is shown as SEQ ID NO.2. The invention discovers the application of HuR in preventing or treating age-related hearing loss for the first time. The HuR coding gene is packaged by an adeno-associated virus (AAV) and then is injected into the inner ear through a round window, so that the HuR coding gene is widely overexpressed in various cells of the inner ear, the occurrence of the senile deafness can be resisted or delayed, and a theoretical basis is provided for clinically preventing and improving the hearing loss of a patient with the senile deafness.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of HuR and its encoding gene in the preparation of drugs for treating presbycusis. Background Art

[0002] Age-related hearing loss (ARHL), also known as presbycusis, is one of the most common and complex chronic diseases in the elderly population, and its pathogenesis is unknown. In China, deafness is one of the most common disabilities, and the population with presbycusis has accounted for more than half of the deaf population. Presbycusis not only seriously affects the quality of life of patients, but also brings huge economic burdens and pressures to society. Therefore, presbycusis is not only a medical problem but also a social problem. However, developing more effective treatment strategies for ARHL requires a comprehensive understanding of its underlying mechanisms.

[0003] The RNA-binding protein HuR is a classical 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 senescence inducer P21, and the degradation of HuR successfully prevents cardiac senescence. In addition, HuR and its stress granules can promote osteogenesis during bone aging. However, the potential mechanism of HuR in ARHL remains unclear. Summary of the Invention

[0004] To solve the above technical problems, the present invention proposes the application of HuR and its encoding gene in the preparation of drugs for treating presbycusis. After the overexpression of the HuR encoding gene in inner ear cells, it can effectively delay the occurrence of presbycusis from the source without any toxic and side effects, and can be used as an effective target for the prevention and treatment of presbycusis.

[0005] To achieve the above object, the present invention provides the application of the HuR encoding gene in the preparation of drugs for preventing or treating presbycusis, and the nucleotide sequence of the HuR encoding gene is as shown in SEQ ID NO.1.

[0006] Preferably, the drug prevents or treats presbycusis by overexpressing the HuR encoding gene, increasing the expression of HuR or enhancing the activity of HuR; the amino acid sequence of HuR is as shown in SEQ ID NO.2.

[0007] The present invention also provides an overexpression adeno-associated virus vector, which includes the HuR encoding gene; the nucleotide sequence of the HuR encoding gene is as shown in SEQ ID NO.1.

[0008] The present invention also provides the use of the overexpressing adeno-associated virus vector in the preparation of a medicament for preventing or treating presbycusis. The overexpressing adeno-associated virus vector prevents or treats presbycusis by overexpressing the HuR-encoding gene.

[0009] The present invention also provides the use of HuR in the preparation of a medicament for preventing or treating presbycusis. The amino acid sequence of the HuR is as shown in SEQ ID NO.2.

[0010] Preferably, by increasing the expression of HuR or enhancing the activity of HuR, the loss of hair cells is reduced, and the number of senescent hair cells is decreased, thereby preventing or treating presbycusis.

[0011] The present invention also provides a medicament for preventing or treating presbycusis, which comprises one of HuR, a HuR expression promoter or a HuR activity promoter; the amino acid sequence of the HuR is as shown in SEQ ID NO.2.

[0012] Preferably, the medicament prevents or treats presbycusis by increasing the expression of HuR or enhancing the activity of HuR, reducing the loss of hair cells, and decreasing the number of senescent hair cells.

[0013] Preferably, the medicament further comprises a pharmaceutically acceptable excipient.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects:

[0015] The present invention discovers for the first time the use of HuR in preventing or treating age-related hearing loss. After being packaged by adeno-associated virus (AAV), the HuR-encoding gene is injected into the inner ear through the round window, enabling it to be widely overexpressed in various types of inner ear cells, which can resist or delay the occurrence of presbycusis, thereby providing a theoretical basis for preventing and improving hearing loss in presbycusis patients in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0017] Figure 1For the construction of the HuR expression vector and the establishment and analysis of the treatment model for presbycusis in SAMP8 mice. Among them, A is the schematic diagram of the construction mode of the HuR expression vector. In the figure, AAV-ie-HA represents the empty vector, and AAV-ie-HuR-HA represents the HuR overexpression vector. B is the schematic diagram of rescuing the hearing of SAMP8 mice by round window injection of AAV-ie-HuR. C is the comparison chart of the transfection efficiency of AAV-ie-CON and AAV-ie-HuR. In the figure, the green fluorescence represents the cells with effective transfection marked, the red fluorescence represents phalloidin, and the scale bar is 50 μm. D is the statistical chart of the percentage of supporting cells labeled with HA in AAV-ie-CON and AAV-ie-HuR. In the figure, "ns" represents the significance analysis. E is the statistical chart of the percentage of hair cells labeled with HA in AAV-ie-CON and AAV-ie-HuR. In the figure, "ns" represents the significance analysis;

[0018] Figure 2ABR audiometry results and basilar membrane spreading immunofluorescence staining results of mice treated with AAV virus. Among them, A is the statistical result of pure tone thresholds and Click thresholds at each frequency of ABR audiometry in mice. In the figure, CON represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, AAV-ie-HuR represents the AAV-ie-HuR injection group, and "*" represents the significance analysis compared with AAV-ie-CON. B is a representative ABR waveform diagram of mice at P90. In the figure, CON represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, AAV-ie-HuR represents the AAV-ie-HuR injection group, and the colored line represents the detected lowest threshold level. C is the staining result of the basilar membrane spreading of mice. In the figure, CON represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, AAV-ie-HuR represents the AAV-ie-HuR injection group, the green fluorescence represents Myo7A, the red fluorescence represents phalloidin, and the scale bar is 50 μm. D is the statistical analysis of the hair cell loss rate of mice. In the figure, CON represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, AAV-ie-HuR represents the AAV-ie-HuR injection group, and "*" and "ns" represent the significance analysis compared with AAV-ie-CON. E is the staining result of the aging marker of the basilar membrane spreading of mice. In the figure, CON represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, AAV-ie-HuR represents the AAV-ie-HuR injection group, the green fluorescence represents P21, the red fluorescence represents Myo7A, and the scale bar is 20 μm. F is the statistical analysis of the aging marker of the basilar membrane spreading of mice. In the figure, CON represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, AAV-ie-HuR represents the AAV-ie-HuR injection group, and "*" and "ns" represent the significance analysis compared with AAV-ie-CON. Detailed implementation manners

[0019] The various exemplary implementation manners 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, characteristics, and implementation schemes of the present invention.

[0020] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0022] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0023] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0024] Example 1

[0025] 1. Construction of HuR overexpression vector:

[0026] Design an AAV2 inverted terminal repeat (ITR-flanked) construct containing a CAG promoter, a HuR coding gene, an HA sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence, and an SV40 poly(A) sequence. This construct is packaged into the AAV-ie capsid to prepare AAV-ie-HuR. The AAV-ie capsid is a synthetic capsid that shows strong transduction efficiency in various types of cells in the inner ear without causing significant hearing loss.

[0027] Nucleotide sequence of the HuR-encoding gene, SEQ ID NO.1: atgtctaatggttatgaagaccacatggcggaagactgcaggga tgacattgggagaacgaatttaattgtcaactacctccctcagaacatgacccaagaggaactacgaagtctgttcagcagcattggcgaggttgaatctgcaaagcttattcgggataaagtagcaggacacagcttgggctacggttttgtgaactatgtgactgcaaaagatgcagagagagcaatcagcacactgaacggcttgagactccagtccaaaaccattaaggtgtcatatgctcgcccaagctcagaggtcatcaaagatgccaacttatacatcagtgggctcccaaggaccatgacacagaaggatgtggaagacatgttttctcggtttgggcgaatcatcaactccagggtccttgtggatcagaccacaggtttgtccagaggggttgcctttatccggtttgacaaacggtcagaagcagaagaggcaattaccagtttcaatggtcataaacccccaggttcctccgagcccatcacagtgaagtttgcagccaatcccaaccagaacaaaaacatggctctcctctcgcagctgtaccactcgcctgctaggcggtttggaggccctgtacaccaccaggcacagagattcaggttctcccctatgggtgtagatcacatgagtgggatttctggtgtcaatgtccccggcaatgcttcctcgggctggtgcatcttcatctacaaccttgggcaagacgccgatgaggggatcctctggcagatgtttggcccctttggtgcagttaccaatgtgaaagtgattcgtgatttcaacaccaacaagtgcaaagggtttggttttgtgaccatgacaaactatgaagaagctgcaatggccatagcaagtctgaacggctaccgcctgggggacaaaattttacaggtttccttcaaaaccaacaagtcccacaaa。.

[0028] The amino acid sequence of HuR, SEQ ID NO.2: MSNGYEDHMAEDCRDDIGRTNLIVNYLPQNMTQE ELRSLFSSIGEVESAKLIRDKVAGHSLGYGFVNYVTAKDAERAISTLNGLRLQSKTIKVSYARPSSEVIKDANLYISGLPRTMTQKDVEDMFSRFGRIINSRVLVDQTTGLSRGVAFIRFDKRSEAEEAITSFNGHKPPGSSEPITVKFAANPNQNKNMALLSQLYHSPARRFGGPVHHQAQRFRFSPMGVDHMSGISGVNVPGNASSGWCIFIYNLGQDADEGILWQMFGPFGAVTNVKVIRDFNTNKCKGFGFVTMTNYEEAAMAIASLNGYRLGDKILQVSFKTNKSHK。

[0029] As Figure 1 shown in A, it is a schematic diagram of the construction of the HuR overexpression vector.

[0030] 2. Establish a treatment model for senile deafness in SAMP8 mice:

[0031] SAMP8 mice exhibit progressive hearing loss similar to that of human senile deafness. Therefore, wild-type SAMP8 mice at 3 days postnatal (P3) were selected and divided into three groups of equal number, namely the untreated control group (CON), the AAV-ie-CON injection group (AAV-ie-CON), and the AAV-ie-HuR injection group (AAV-ie-HuR). AAV-ie-HuR or AAV-ie-CON was injected into the left cochlea of neonatal (P3) SAMP8 mice through the round window. The AAV titer was 2.0×10 12 GC / mL, and the volume of virus injected into each ear was 1.0 μL. The cochleae of the mice were taken 7 days after virus injection to analyze the transfection efficiency of the virus. HA positive staining and quantitative analysis showed that AAV-ie-HuR transduced inner ear cells with a very high efficiency.

[0032] 3. Audiological evaluation:

[0033] (1) Hearing detection: The auditory brainstem response (ABR) of AAV-injected SAMP8 mice was detected, and the hearing threshold levels of the untreated control group (CON), the AAV-ie-CON injection group (AAV-ie-CON), and the AAV-ie-HuR injection group (AAV-ie-HuR) were compared. The lower the hearing threshold of each frequency band of the ABR in the HuR overexpressing mice, the better the hearing condition.

[0034] (2) Basilar membrane whole-mount staining: Cochlear basilar membranes of mice in the untreated control group (CON), AAV-ie-CON injection group (AAV-ie-CON), and AAV-ie-HuR injection group (AAV-ie-HuR) were prepared and subjected to immunofluorescence staining. The loss of hair cells in mice of the untreated control group (CON), AAV-ie-CON injection group (AAV-ie-CON), and AAV-ie-HuR injection group (AAV-ie-HuR) was compared using myosin VIIA (Myo7A), phalloidin, and 4',6-diamidino-2-phenylindole (DAPI). The number of senescent hair cells in mice of the untreated control group (CON), AAV-ie-CON injection group (AAV-ie-CON), and AAV-ie-HuR injection group (AAV-ie-HuR) was compared using Myo7A, cyclin-dependent kinase inhibitor 1A (P21), and DAPI. The number of hair cells was observed by green fluorescence (488 nm) in the results of basilar membrane whole-mount immunofluorescence staining.

[0035] ① Basilar membrane whole-mount preparation: Sampling and fixation: The cochleas of mice were sampled and placed in 4% paraformaldehyde at 4°C overnight; washing: Washed 3 times with PBS, 5 min each time; decalcification: The cochleas were placed in EDTA washing solution (10%) at room temperature for 5 h; washing: Washed 3 times with PBS, 5 min each time; The basilar membrane was carefully dissected under a dissecting microscope and cut into 3 segments and placed in PBS.

[0036] ② Immunofluorescence staining permeabilization: Permeabilized with 0.3% Triton X-100 at room temperature for 15 min; washing: Washed 3 times with PBS, 5 min each time; blocking: Blocked with 10% goat serum at room temperature for 1 h; primary antibodies (rabbit-derived Myo7A 1:400, rabbit-derived HA 1:400, rabbit-derived P21 1:400) were incubated overnight on a shaker at 4°C; washing: Washed 3 times with PBS, 5 min each time; secondary antibody (goat anti-rabbit, 1:400) was incubated at room temperature in the dark for 1 h; washing: Washed 3 times with PBS, 5 min each time; 488-labeled phalloidin (green, dilution ratio 1:3000) was incubated at room temperature in the dark for 15 min; washing: Washed 3 times with PBS, 5 min each time; Observed the loss of hair cells after mounting with DAPI.

[0037] 4. Result determination:

[0038] By evaluating the effect of overexpressing HuR on delaying presbycusis in the cochleas of mice in the above manner, it can be determined that overexpressing HuR can effectively delay the onset of presbycusis.

[0039] As Figure 1As shown in B, it is a schematic diagram of rescuing the hearing of SAMP8 mice by injecting AAV-ie-HuR through the round window; as Figure 1 As shown in C, it is a comparison of the transfection efficiencies of AAV-ie-CON and AAV-ie-HuR. The green fluorescence in the figure is the effectively transfected cells labeled with HA, and the red fluorescence in the figure is phalloidin. It can be seen from the figure that both AAV-ie-CON and AAV-ie-HuR can effectively transfect supporting cells and hair cells in the entire cochlea; as Figure 1 In D and Figure 1 As shown in E, it shows the percentages of HA-labeled supporting cells and HA-labeled hair cells, indicating that both AAV-ie-CON and AAV-ie-HuR have very high transduction efficiencies in hair cells and supporting cells.

[0040] As Figure 2 As shown in A, it is the statistical results of the pure tone thresholds and Click thresholds at each frequency of ABR audiometry in mice. It can be seen that compared with the control group (CON), there is no significant difference in the thresholds at any frequency tested in the AAV-ie-CON group at P90, which proves the safety of the AAV vector and the surgical operation. However, injecting AAV-ie-HuR at P3 significantly reduced the ABR threshold; as Figure 2 As shown in B, it is the representative ABR waveform diagram of SAMP8 mice in the untreated control group (CON), the AAV-ie-CON injection group (AAV-ie-CON), and the AAV-ie-HuR injection group (AAV-ie-HuR) at P90. The colored line represents the detected lowest threshold level. It can be seen that the Click threshold of SAMP8 mice in the AAV-ie-HuR group decreased; as Figure 2 In C and Figure 2 As shown in D, it is the staining result of the basilar membrane spread and the statistical analysis result of the hair cell loss rate of SAMP8 mice in the untreated control group (CON), the AAV-ie-CON injection group (AAV-ie-CON), and the AAV-ie-HuR injection group (AAV-ie-HuR). The green fluorescence is Myo7A and the red fluorescence is phalloidin, showing that injecting AAV-ie-HuR through the round window effectively reduced the loss of hair cells in SAMP8 mice; as Figure 2 In E and Figure 2 As shown in F, it is the staining result of the aging biomarker of the basilar membrane spread and its statistical analysis result of SAMP8 mice in the untreated control group (CON), the AAV-ie-CON injection group (AAV-ie-CON), and the AAV-ie-HuR injection group (AAV-ie-HuR). The green fluorescence is P21 and the red fluorescence is Myo7A, showing that injecting AAV-ie-HuR through the round window significantly reduced the aging hair cells in SAMP8 mice.

[0041] The present invention has been experimentally verified that overexpression of HuR in inner ear cells of mice using AAV technology can effectively reduce senescent hair cells, thereby preventing or delaying the occurrence of presbycusis without any side effects, and has high reference value for the treatment of clinical presbycusis patients, and is suitable for popularization and application. Thus, HuR can be used as an effective target for the prevention and treatment of presbycusis, providing more treatment options for the prevention or treatment of presbycusis clinically, and having important social and economic values.

[0042] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of HuR-encoding gene in the preparation of a medicament for preventing or treating presbycusis, characterized in that, The nucleotide sequence of the HuR-encoding gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that The drug prevents or treats presbycusis by overexpressing the HuR-encoding gene, increasing the expression of HuR or enhancing the activity of HuR. The amino acid sequence of HuR is shown in SEQ ID NO.

2.

3. An adeno-associated virus vector with overexpression, characterized in that, The overexpressing adeno-associated virus vector comprises the HuR-encoding gene; the nucleotide sequence of the HuR-encoding gene is shown in SEQ ID NO.

1.

4. Use of the overexpressed adeno-associated virus vector as described in claim 3 in the preparation of a medicament for preventing or treating presbycusis, characterized in that, The overexpressing adeno-associated virus vector prevents or treats presbycusis by overexpressing the HuR-encoding gene.

5. Use of HuR in the preparation of a medicament for preventing or treating presbycusis, characterized in that, The amino acid sequence of HuR is shown in SEQ ID NO.

2.

6. The application according to claim 5, wherein By increasing the expression of HuR or enhancing the activity of HuR, the loss of hair cells is reduced, and the number of senescent hair cells is decreased, thereby preventing or treating presbycusis.

7. A drug for preventing or treating presbycusis, characterized in that, The drug comprises one of HuR, a HuR expression promoter or a HuR activity promoter; the amino acid sequence of HuR is shown in SEQ ID NO.

2.

8. The drug according to claim 7, wherein The drug prevents or treats presbycusis by increasing the expression of HuR or enhancing the activity of HuR, reducing the loss of hair cells, and decreasing the number of senescent hair cells.

9. The drug according to claim 7, wherein The drug further comprises a pharmaceutically acceptable excipient.

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