Application of TRIM71 in regulation and control of inner ear supportive cell proliferation and hair cell regeneration

Abstract

The invention relates to the technical field of biology, and discloses application of TRIM71 in regulation and control of inner ear supportive cell proliferation and hair cell regeneration. The overexpression TRIM71 promotes the dedifferentiation of cochlea supporting cells to progenitor cell-like cells, increases the mitosis and regeneration potential of the cochlea supporting cells, and regulates and controls the regeneration of hair cells. According to the application of the TRIM71 in regulation and control of inner ear supporting cell proliferation and hair cell regeneration, cell plasticity of cochlea supporting cells of a P5 mouse is reactivated through overexpression of the TRIM71, and hair cell regeneration is achieved on the cochlea organ level and the cochlea explant level; meanwhile, a method for reactivating cochlea supporting cell plasticity and regenerating hair cells in mammals is provided, and the method has important theoretical reference significance for clinical treatment of hearing loss.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of TRIM71 in regulating the proliferation of inner ear supporting cells and the regeneration of hair cells. Background Art

[0002] Mechanosensory hair cells in the auditory sensory organ, called the cochlea, are essential for sound perception. In mammals, auditory hair cells cannot regenerate, and their loss or dysfunction is the leading cause of deafness in humans. In non-mammalian vertebrates, such as birds and fish, damaged or lost hair cells are replenished by their surrounding supporting cells through both mitotic and non-mitotic mechanisms.

[0003] Hair cells and supporting cells are derived from pre-embryonic sensory cells that express SOX2 and Jagged1, and the fate of hair cells is determined by the transcription factor ATOH1, which is both necessary and sufficient for hair cell formation. In mice, hair cells form at a specific stage of embryonic development, but many genetic programs associated with hair cell formation remain active after birth. For example, early postnatal mouse cochlear supporting cells can re-enter the cell cycle, proliferate, and form hair cells under specific conditions.

[0004] TRIM71 and LIN28B are evolutionarily conserved targets of let-7 miRNAs, which enhance the regenerative potential of cochlear supporting cells by promoting dedifferentiation. Studies have found that TRIM71 and LIN28B can also enhance the plasticity of cochlear supporting cells. TRIM71's RNA binding activity is essential for its promotion of supporting cell dedifferentiation, while its ubiquitin ligase activity is not essential. Hmga2 is a common target gene of TRIM71 and LIN28B, which is important for supporting cells / "These findings provide new insights into the mechanisms of cochlear hair cell regeneration and offer potential new strategies for treating hearing loss," said Dr. Summary of the invention

[0005] The purpose of the present invention is to provide an application of TRIM71 in regulating the proliferation of inner ear supporting cells and the regeneration of hair cells. By overexpressing TRIM71, the cochlear supporting cells of P5 mice are prompted to reactivate cell plasticity, and the regeneration of hair cells is achieved at the level of cochlear organoids and cochlear explants. At the same time, a method for reactivating the plasticity of cochlear supporting cells and regenerating hair cells in mammals is provided, which has important theoretical reference significance for the clinical treatment of hearing loss.

[0006] To achieve the above objectives, the present invention provides the use of TRIM71 in regulating the proliferation of inner ear supporting cells and the regeneration of hair cells.

[0007] Furthermore, overexpression of TRIM71 promoted the dedifferentiation of cochlear supporting cells into progenitor-like cells, increased the mitosis and regenerative potential of cochlear supporting cells, and regulated the regeneration of hair cells.

[0008] Furthermore, TRIM71 is a target gene of LIN28B, and the TRIM71-LIN28B target gene Hmga2 is used to regulate the self-renewal of supporting cells and the formation of hair cells.

[0009] The present invention also provides a method for regulating inner ear supporting cell reprogramming by TRIM71, comprising the following steps:

[0010] Step 1, overexpression of TRIM71 gene;

[0011] Step 2, organoid culture;

[0012] Step 3, cochlear explant culture;

[0013] Step 4, immunohistochemical analysis;

[0014] Step 5, EdU proliferation assay.

[0015] Furthermore, the specific operation of step 1 is: using a lentiviral vector to introduce the TRIM71 gene into cochlear supporting cells, constructing a lentiviral expression vector containing the TRIM71 gene, co-transfecting it with the packaging plasmid into HEK 293T cells to produce viral particles, and then infecting cochlear epithelial cells to achieve overexpression of the TRIM71 gene.

[0016] Furthermore, the specific operation of step 2 is: take the mouse cochlear basement membrane before P5, separate the cochlear epithelial tissue after enzyme digestion, digest the cochlear epithelial tissue with TrypLE solution and blow it apart, filter it through a 35μm filter to obtain a single cell suspension, mix the single cell suspension with an equal volume of Matrigel, and drop it into a preheated 4-well plate to establish cochlear organoid culture.

[0017] Furthermore, the specific operation of step 3 is as follows: the cochlea is isolated from wild-type mice at the P2 stage, the cochlea is isolated in a sterile PBS solution, and after removing the lateral wall, the cochlear apex and the spiral ganglion, the remaining cochlear sensory epithelium is transferred to a SPI-Pore membrane filter for suspension culture.

[0018] Furthermore, the specific operations of step 4 are as follows: fix the isolated cochlear tissue or cultured organoids or explants in 4% paraformaldehyde for 30 min, permeabilize and fix for 30 min, block with 0.25% Triton X-100 / 10% fetal bovine serum for 30 min, use Myo7a antibody to label hair cells, use Sox2 antibody to label supporting cells, and EdU staining to label proliferating cells and indicate the origin of newborn hair cells.

[0019] Furthermore, the specific operations of step 5 are: adding EdU reagent to the organoid culture medium, detecting the cell proliferation level after EdU staining, adding EdU to the cochlear explant culture medium, and detecting the regeneration mode of new hair cells formed by supporting cells based on the EdU staining results.

[0020] The advantages and positive effects of the use of TRIM71 in regulating inner ear supporting cell proliferation and hair cell regeneration are:

[0021] 1. The present invention promotes the reactivation of cell plasticity of P5 mouse cochlear supporting cells by overexpressing TRIM71, thereby achieving the regeneration of hair cells at the cochlear organoid and cochlear explant levels.

[0022] 2. The present invention provides a method for reactivating the plasticity of cochlear supporting cells and regenerating hair cells in mammals, which has important theoretical reference significance for the clinical treatment of hearing loss.

[0023] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of culturing organoids from P2 and P5 mice on the regeneration of cochlear supporting cells to form hair cells in the embodiments of the present invention is a schematic diagram of the conditional knockout strategy of the mouse Trim71 gene;

[0025] Figure 2 A is the experimental scheme of BF, TetO-Cre; R26 rtTA*M2 :Trim71 f / f Cochlear epithelial cells of P2 mice (Trim71KO) and littermates lacking the TetO-Cre transgene (wild type, WT) were cultured in expansion medium in the presence of doxycycline (dox). B is a bright field (BF) image of WT and Trim71 KO organoids after 6 days of culture. Scale bar = 400 μm. C is the colony formation efficiency of B (WT n = 4, Trim71 KO n = 5, two independent experiments). D is the organoid diameter, WT n = 4, Trim71KO n = 5. E is a high-magnification single-plane confocal image of EdU (red) and Hoechst (blue) labeled WT and Trim71 KO organoids. A single EdU pulse was given on day 7, and EdU incorporation was analyzed 1.5 h later. Scale bar = 25 μm. F is the EdU in E. + The percentage of cells (n = 15 in WT, n = 11 in Trim71KO, three independent experiments);

[0026] Figure 3A is the experimental scheme of BD, the organoids are as follows Figure 2 A is shown for establishment, expansion, and culture in differentiation medium for up to 6 days. A is RT-qPCR detection of Trim71 and its precursor cell (Hmag2 and Fat3) and hair cell-specific (Atoh1, Gfi1, and Pou4f3) mRNAs in control and Pou4f3 organoids on day 10 (n=4 in WT, n=5 in Trim71 KO, two independent experiments). B is RT-qPCR detection of Trim71 and hair cell-specific (Atoh1, Gfi1, and Pou4f3) mRNAs in WT and Trim71 KO organoids after 2 days of differentiation (n=3, two independent experiments). C is high-magnification single-plane confocal images of MYO7A and SOX2 immunostaining for WT and Trim71 KO organoids after 6 days of differentiation. Newborn hair cells co-express Atoh1-nGFP (green), MYO7A (red), and SOX2 (magenta). Scale bar = 25 μm. D is RT-qPCR detection of Trim71 and hair cell-specific (Atoh1, Gfi1, and Pou4f3) mRNAs in WT and Trim71 KO organoids after 10 days of differentiation. RT-qPCR detection of Trim71 and hair cell-specific (Atoh1, Gfi1, and Pou4f3) mRNA in KO organoids (n=3, two independent experiments);

[0027] Figure 4 The experimental scheme for the effect of overexpression of TRIM71 in P5 mouse organoids on the regeneration of cochlear supporting cells to form hair cells in the examples of the present invention, the cochlear epithelial cells of P5 Atoh1-GFP transgenic mice were transduced with a lentivirus expressing a control (Ctrl) or a lentivirus co-expressing mCherry and full-length (TRIM71) or mutant TRIM71 protein (ΔRING, ΔCoiled Coil, ΔNHL), and cultured in differentiation medium for 2 days after amplification;

[0028] Figure 5 Bright field (BF), red (mCherry), and green (Atoh1-nGFP) fluorescence images of organoid culture after 2 days of differentiation, scale bar = 400 μm;

[0029] Figure 6 for Figure 5 Atoh1-nGFP + mCherry + percentage of organoids;

[0030] Figure 7 RT-qPCR for Atoh1 mRNA in organoids;

[0031] Figure 8High-magnification single-plane confocal images of organoids immunostained for MYO7A (magenta), Atoh1-GFP (green) and MYO7A (magenta) mark newborn hair cells, and MCherry (red) marks transduced cells, scale bar = 20 μm;

[0032] Fig. 9 A in Figure 8 MYO7A in each organoid + mCherry + Quantification of cells, B is RT-PCR detection of Myo7a mRNA expression in organoids;

[0033] Fig.10 Experimental protocol for the effect of LIN28B-TRIM71 target Hmga2 on the regeneration of supporting cells to form hair cells in P5 mouse cochlear explants in the present example, P2 Atoh1-GFP transgenic mouse cochlear epithelial cells were transduced with lentivirus expressing scrambled shRNA (control, Ctrl) or lentivirus expressing shRNA of endogenous Hmga2 and cultured as indicated;

[0034] Fig.11 A in the middle is the colony formation efficiency of Hmga2 knockout and control organoids after 8 days of culture, and B is the organoid diameter of Hmga2 knockout and control organoids after 8 days of culture;

[0035] Fig.12 For RT-qPCR analysis of the expression of hair cell ( Aoth1 and Pou4f3 ) and progenitor cell-specific ( Trim71 ) genes in Hmga2 knockout and control cultures, Hmga2 expression was analyzed to confirm knockout, and plotted are the fold differences and mean SD between the control and Hmga2 shRNA groups;

[0036] Fig.13 A is a low-power bright field (BF) and green fluorescence image (Atoh1-nGFP) of Hmga2 shRNA knockdown and control organoid culture after 2 days of differentiation. Scale bar = 400 μm. B is the Atoh1-nGFP in A. + Quantification of organoids. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0038] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0039] Unless otherwise defined, the instruments, reagents, materials, etc. involved in the present invention are all conventional instruments, reagents, materials, etc. already in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the present invention, unless otherwise specified, are all conventional experimental methods, detection methods, etc. already in the prior art.

[0040] Mammalian cochlear hair cells usually cannot regenerate after loss, while the cochlear supporting cells of perinatal mice have a certain potential for hair cell regeneration. However, the regenerative ability of these cells decreases rapidly with maturity, especially in P5 (5th day after birth) mouse cochlear organoids, where they have lost the ability to regenerate hair cells. Studies have found that reactivating the RNA-binding protein LIN28B can restore the hair cell regeneration potential of P5 mouse cochlear supporting cells. The present invention found that TRIM71, a target gene of LIN28B, is a new factor that can enhance the plasticity of supporting cells. TRIM71 can increase the mitosis and regeneration potential of cochlear supporting cells by promoting the dedifferentiation of cochlear supporting cells into progenitor-like cells, and successfully achieves efficient regeneration of hair cells. In addition, the present invention also reveals that the RNA binding activity of TRIM71 is essential for its enhancement of supporting cell plasticity, while its ubiquitin ligase activity is not essential. It was found that the TRIM71-LIN28B target gene Hmga2 is essential for the self-renewal of supporting cells and the formation of hair cells.

[0041] Example 1

[0042] The present invention provides a method for regulating the reprogramming of inner ear supporting cells by the TRIM71 gene and its application in hair cell regeneration. The method for regulating the reprogramming of inner ear supporting cells by the TRIM71 gene comprises the following steps:

[0043] Step 1, overexpression of TRIM71 gene:

[0044] Select a suitable expression vector, such as pCDH-EF1a-eFFly-T2A-mCherry (Addgene, no.104833), to construct a lentiviral vector for overexpression of TRIM71. Then clone the HA tag sequence of the 3xHA-tagged human TRIM71 gene and its mutants (such as ΔRING, ΔCoiled-Coil, ΔNHL) into the above vector to replace the original eFFly sequence. Use restriction enzymes XbaI and EagI-HF for cloning. Confirm the correct insertion of the TRIM71 gene sequence by DNA sequencing. HEK 293T cells are used for packaging of lentiviral vectors. The constructed TRIM71 overexpression vector and packaging plasmids (such as pMD2.G and psPAX2) are co-transfected into HEK 293T cells. After 48 to 72 hours, the cell culture supernatant containing lentivirus containing viral particles is collected. The viral particles are concentrated by ultracentrifugation. Mouse cochlear epithelial cells were infected with lentiviral particles and cochlear organoids were cultured. The organoids were cultured for proliferation for 10 days and differentiation for 2 days, thereby achieving overexpression of the TRIM71 gene.

[0045] Step 2, cochlear organoid culture:

[0046] The regenerative capacity of perinatal mouse cochlear hair cells gradually decreases with development and maturity, and P2 and P5 mice were used to culture cochlear organoids. The specific steps are as follows: the cochlea was dissected in a sterile PBS solution and the cochlear basement membrane was taken. At room temperature, the cochlear basement membrane was digested with dispase and collagenase at a final concentration of 1 mg / mL for 8 minutes, and then the digestion was terminated with a DMEM / F12 solution containing 10% FBS. After standing at room temperature for 30 minutes, the cochlear epithelium was separated with a disposable injection needle. The cochlear epithelium was placed in a 1.5 mL centrifuge tube, TrypLE solution was added, and incubated at 37°C for 20 minutes. After terminating digestion and blowing cells, the cell suspension was filtered through a 35μm filter to obtain a single cell suspension. Take an equal amount of cell suspension and mix it evenly with Matrigel at a ratio of 1:1, and drop it into a preheated four-well plate. Place it in a 37°C incubator for 20 minutes, and add culture solution to each culture well to immerse the Matrigel after the Matrigel is completely solidified.

[0047] Cochlear organoids were cultured in proliferation medium (DMEM / F12, N-2supplement, B-27supplement, EGF, FGF2, CHIR99021, VPA, 616452, penicillin) for 10 days, and then changed to differentiation medium (DMEM / F12, N-2supplement, B-27supplement, CHIR99021, LY411575) for 6 days. Atoh1-nGFP (MGI: 3703598) transgenic mice can specifically mark cochlear hair cells with GFP green fluorescence. When cochlear organoids were cultured with this mouse, the formation of hair cells could be indicated by green fluorescence during the differentiation stage.

[0048] Step 3, cochlear explant culture:

[0049] The mouse cochlea was dissected in a sterile PBS solution, the basement membrane was kept wrapped around the modiolus, the bottom of the modiolus was trimmed flat with forceps, and finally it was suspended on the SPI-Pore membrane for culture. The P2 mouse cochlear sensory epithelial cells were isolated by microdissection to establish cochlear explants. The cochlear explants were suspended in culture medium (DMEM / F12, N-2 supplement, B-27 supplement, EGF, ampicillin), and the culture medium was switched to 5×10 10 GC / mL of medium containing Anc80-TRIM71 or Anc80-Ctrl AAV particles. After 48 h of incubation, the medium was replaced and LY411575 was added. The medium was replaced every 2 days.

[0050] Step 4, EdU proliferation assay:

[0051] EdU reagent was added to the organoid culture medium, and the cell proliferation level was detected after EdU staining. EdU was added to the cochlear explant culture medium, and the regeneration mode of new hair cells formed by supporting cells was detected according to the EdU staining results. + Myo7a + The hair cells are derived from direct transdifferentiation of supporting cells, while Sox2 + Myo7a + EDU + Hair cells are formed by supporting cells re-entering the cell cycle and undergoing transdifferentiation.

[0052] Step 5, Immunofluorescence staining:

[0053] Myo7a antibody was used to label hair cells, Sox2 antibody was used to label supporting cells, and EdU staining was used to label proliferating cells and to indicate the source of new hair cells. For organoid samples, the organoid cell suspension was mixed with Matrigel before staining, and then re-plated on the coverslip. Subsequent staining was performed after Matrigel solidified. During the staining process of cochlear explants, the explants were retained on the SPI-Pore membrane. After staining, the explants were transferred from the membrane to the slide with a disposable syringe and then sealed. All immunofluorescence staining samples were imaged under a laser scanning confocal microscope.

[0054] Step 6, data statistical analysis:

[0055] Graphpad Prism 8.0 was used for statistical analysis of the data, and the differences were considered statistically significant when the p value was < 0.05.

[0056] Example 2 Results Analysis

[0057] Figure 1 The effect of culturing organoids from P2 and P5 mice on the regeneration of cochlear supporting cells to form hair cells. Trim71 is highly expressed in undifferentiated cochlear epithelial cells, but rapidly decreases at the beginning of cell differentiation and is almost undetectable after birth. To determine whether Trim71 is essential for supporting cells to re-enter the cell cycle and form new hair cells, Trim71 was knocked out in P2 cochlear organoid cultures. The experimental results are shown in Figure 2. Figure 1-Figure 3 It can be seen that Figure 1 Describes a Trim71floxed mouse model in which Cre-mediated recombination can knock out exon 4. Demonstrates that the Trim71 floxed mouse carries R26 rtTA*M2 series and TetO-Cre transgenesis, made possible using doxycycline-mediated Cre induction. Figure 2 Middle A: Shows how to induce Trim71 loss by doxycycline (DOX), and the settings of the control group and Trim71 KO group used in the experiment. Figure 2 Middle BD: Organoid formation efficiency and organoid size in control and Trim71 KO cultures were analyzed and the results showed no defects in organoid formation or growth in the absence of Trim71. Figure 2 Middle E and F: show the results of a 1.5h EdU pulse experiment after 7 days of expansion, indicating that the cell proliferation rate of Trim71 KO organoids did not change significantly compared with the control group. Figure 3 A and B analyze the effects of Trim71 deficiency on cochlear progenitor cells and supporting cells / The effects of Trim71 on the hair cell-forming ability of cells were investigated and it was found that Trim71 KO organoids expressed hair cell fate-inducing genes at levels that were on average two-fold lower than those of control organoids. Figure 3 Middle C shows that after 6 days of differentiation, most Atoh1-nGFP cells in control organoids co-expressed the hair cell-specific protein MYO7A, while Atoh1-nGFP cells in Trim71 KO organoids lacked MYO7A expression. These results indicate that Trim71 is essential for the self-renewal of cochlear anterior sensory cells and the formation of hair cells, especially playing an active role in the reprogramming of supporting cells into progenitor-like cells and subsequent hair cell formation. At later, perinatal stages, Trim71 is not essential for self-renewal.

[0058] Figure 4 To investigate whether TRIM71 expression enhances the regeneration of cochlear supporting cells / hair cells in P5 mouse organoids. At P5, the cochlear epithelial cells (supporting cells and Cells) still generate some hair cells in organoid culture, but the rate of hair cell production is greatly reduced compared to earlier stages. To be able to monitor the dynamics of hair cell formation in culture, cochlear epithelial cells from Atoh1-nGFP transgenic mice were used, which express nuclear GFP in newborn hair cells. The results of the experiment are shown in Figure 2. Figure 4-Figure 9 It can be seen that in order to stimulate hair cell formation, organoids contain the GSK-3β inhibitor CHIR99021 (to activate Wnt signaling) and the γ-secretase inhibitor LY411575 (to inhibit Notch signaling). Figure 5 , Figure 6 As shown, after 2 days of differentiation, about 40% of TRIM71-expressing organoids contained newborn hair cell clusters (Atoh1-GFP cells), while only about 5% of control organoids contained newborn hair cells. Expression of TRIM71 protein lacking the RING domain (ΔRING) was as effective as the full-length TRIM71 protein in promoting hair cell formation. In contrast, TRIM71 proteins lacking the coiled-coil domain (ΔCoiled-Coil) or the NHL (ΔNHL) domain were ineffective in inducing hair cell formation. Figure 7 : The positive effect of TRIM71 on hair cell formation was confirmed by analyzing Atoh1 mRNA expression using RT-qPCR. It was found that the expression of full-length or RING domain-deficient TRIM71 protein resulted in a 4- to 8-fold increase in Atoh1 mRNA abundance compared with the control, whereas the Atoh1 mRNA level remained unchanged by the expression of NHL or coiled-coil domain-deficient TRIM71 protein compared with the control. Figure 8 and Fig. 9 : Using the hair cell-specific protein myosin VIIa (MYO7A). Fig. 9 Middle B: In addition, we found that TRIM71 expression increased Myo7a mRNA expression by more than threefold compared with the control group, while expression of TRIM71 protein lacking the NHL domain failed to increase Myo7a expression. In summary, the organoid culture experiments indicate that the ability of TRIM71 to bind RNA is important for enhancing the function of cochlear support cells and TRIM71 is essential for the cell's ability to undergo mitosis and form hair cells, whereas its ubiquitin ligase activity is dispensable.

[0059] Figure 10-13 To investigate the effect of the LIN28B-TRIM71 target Hmga2 on the regeneration of supporting cells into hair cells in P5 mouse cochlear explants. The TRIM71- and LIN28B-regulated gene Hmga2 encodes a chromatin structural protein that functions to maintain self-renewal capacity. To address the function of HMGA2 in hair cell regeneration, Fig.10 : We first examined whether Hmga2 is required for cell cycle re-entry, cell proliferation, and hair cell formation in cochlear organoid cultures. Fig.11 Middle A: To disrupt Hmga2 gene function, cochlear epithelial cells from P2 mice were transduced with lentivirus expressing short hairpin RNA (shRNA) targeting endogenous Hmga2. Control cultures were transduced with lentivirus expressing short hairpin RNA with a scrambled (scr) sequence. After 8 days of expansion, Hmga2 knockdown cultures contained an average of four times fewer organoids than control cultures. Fig.11 Middle B: The average organoid size in Hmga2 knockdown cultures, although slightly reduced, was not significantly different from control cultures. Fig.12 : RT-qPCR confirmed the reduction in Hmga2 expression in Hmga2 knockdown cultures and showed a significant decrease in the expression of Trim71 and Atoh1 compared with control cultures, indicating defects in reprogramming and hair cell fate induction, respectively. Fig.13 : Next, the ability of organoid cells to differentiate into hair cells in Hmga2 knockout and control organoid cultures was analyzed. It was found that the percentage of Atoh1-GFP organoids was significantly reduced in Hmga2 knockdown cultures compared with control cultures, indicating that Hmga2 is required for hair cell formation. Together, these results demonstrate the necessity of Hmga2 in cell cycle re-entry (organoid formation) and hair cell formation in neonatal stage cochlear organoid cultures, and the role of HMGA2 overexpression in restoring supporting cell / A deficiency in the ability of cells to re-enter the cell cycle and form hair cells late after birth.

[0060] Therefore, the present invention adopts the above-mentioned application of TRIM71 in regulating the proliferation of inner ear supporting cells and the regeneration of hair cells. By overexpressing TRIM71, the cochlear supporting cells of P5 mice are prompted to reactivate cell plasticity, and the regeneration of hair cells is achieved at the level of cochlear organoids and cochlear explants. At the same time, a method for reactivating the plasticity of cochlear supporting cells and regenerating hair cells in mammals is provided, which has important theoretical reference significance for the clinical treatment of hearing loss.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of TRIM71 in regulating inner ear supporting cell proliferation and hair cell regeneration.

2. The use according to claim 1, characterized in that: Overexpression of TRIM71 promotes the dedifferentiation of cochlear supporting cells into progenitor-like cells, increases the mitosis and regenerative potential of cochlear supporting cells, and regulates the regeneration of hair cells.

3. The use according to claim 1, characterized in that: TRIM71 is a target gene of LIN28B, and the TRIM71-LIN28B target gene Hmga2 is used to regulate the self-renewal of supporting cells and the formation of hair cells.

4. A method for regulating inner ear supporting cell reprogramming by TRIM71, characterized in that: The following steps are involved: Step 1, overexpression of TRIM71 gene; Step 2, organoid culture; Step 3, cochlear explant culture; Step 4, immunohistochemical analysis; Step 5, EdU proliferation assay.

5. The method according to claim 4, characterized in that The specific operation of step 1 is: using a lentiviral vector to introduce the TRIM71 gene into cochlear supporting cells, constructing a lentiviral expression vector containing the TRIM71 gene, co-transfecting it with the packaging plasmid into HEK 293T cells to produce viral particles, and then infecting cochlear epithelial cells to achieve overexpression of the TRIM71 gene.

6. The method according to claim 4, characterized in that The specific operation of step 2 is: take the mouse cochlear basement membrane before P5, separate the cochlear epithelial tissue after enzyme digestion, digest the cochlear epithelial tissue with TrypLE solution and blow it apart, filter it through a 35μm filter to obtain a single-cell suspension, mix the single-cell suspension with an equal volume of Matrigel, and drop it into a preheated 4-well plate to establish cochlear organoid culture.

7. The method according to claim 4, characterized in that The specific operation of step 3 is as follows: the cochlea is isolated from wild-type mice at the P2 stage, the cochlea is isolated in a sterile PBS solution, and after removing the lateral wall, the cochlear apex and the spiral ganglion, the remaining cochlear sensory epithelium is transferred to the SPI-Pore membrane filter for suspension culture.

8. The method according to claim 4, characterized in that The specific operation of step 4 is as follows: the isolated cochlear tissue or cultured organoids or explants are permeabilized and fixed in 4% paraformaldehyde for 30 minutes, blocked with 0.25% Triton X-100 / 10% fetal bovine serum for 30 minutes, hair cells are labeled with Myo7a antibody, supporting cells are labeled with Sox2 antibody, and EdU staining is used to label proliferating cells and to indicate the origin of newborn hair cells.

9. The method according to claim 4, characterized in that The specific operations of step 5 are: adding EdU reagent to the organoid culture medium, detecting the cell proliferation level after EdU staining, adding EdU to the cochlear explant culture medium, and detecting the regeneration mode of new hair cells formed by supporting cells based on the EdU staining results.

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