Application of LIN28B in regulation and control of cochlea supporting cell reprogramming and hair cell regeneration

Abstract

The invention relates to the technical field of biology, and discloses application of LIN28B in regulation and control of cochlea supporting cell reprogramming and hair cell regeneration. The overexpression LIN28B promotes cochlea supporting cells to activate cell plasticity or the overexpression LIN28B induces the supporting cells to regenerate hair cells in an mTOR-dependent non-mitosis mode. According to the application of the LIN28B in regulation and control of cochlea supporting cell reprogramming and hair cell regeneration, after overexpression of the LIN28B, P5 mouse cochlea supporting cells are promoted to reactivate cell plasticity, and hair cell regeneration is achieved on the cochlea organ level and the cochlea explant level; meanwhile, a brand new method is provided for realizing plastic reactivation of cochlea supporting cells in mammals and regeneration of hair cells, 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 application of LIN28B in regulating cochlear supporting cell reprogramming and hair cell regeneration. Background Art

[0002] Hearing loss is called "invisible disability" and seriously affects people's cognition, learning and life. Hearing loss mainly includes two factors: genetic factors and environmental factors, of which more than 60% is caused by genetic factors.

[0003] The auditory system consists of the auditory organ, auditory nerve, and auditory center. The integrity of the structure and function of the auditory organ is crucial to the formation of hearing. In mammals, cochlear hair cells are only produced during the developmental stage. The loss of cochlear hair cells due to genetic or environmental reasons is the main cause of deafness. However, in non-mammalian vertebrates such as birds and fish, hair cell damage will induce adjacent supporting cells to directly transdifferentiate into hair cells or re-enter the cell cycle to divide and form new hair cells and supporting cells. The cochlear supporting cells of adult mammals lack the ability to regenerate hair cells, but the cochlear supporting cells of perinatal mice still maintain a certain degree of regenerative stemness. In the immature cochlea of ​​perinatal mice, supporting cells can regenerate new hair cells as progenitor cells, but this regenerative ability is lost as they mature.

[0004] LIN28B (Lin-28 Homolog B) is a highly conserved RNA-binding protein across species, and its mRNA structure has a site complementary to let-7 microRNA. LIN28B can enhance the transcription and translation of growth-promoting target genes and block the growth-inhibiting effect of let-7 microRNA, thereby promoting the growth, proliferation and reprogramming of stem cells.

[0005] Reactivating the plasticity of cochlear supporting cells and regenerating hair cells in mammals has important implications for the treatment of hearing loss. Summary of the invention

[0006] The purpose of the present invention is to provide the application of LIN28B in regulating the reprogramming of cochlear supporting cells and the regeneration of hair cells. By overexpressing LIN28B, 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 new method is provided for how to achieve the reactivation of cochlear supporting cell plasticity and regeneration of hair cells in mammals, which has important theoretical reference significance for the clinical treatment of hearing loss.

[0007] To achieve the above objectives, the present invention provides the use of LIN28B in regulating the reprogramming of cochlear supporting cells.

[0008] The present invention also provides application of LIN28B in regulating hair cell regeneration.

[0009] Furthermore, overexpression of LIN28B prompted cochlear supporting cells to activate cellular plasticity or induced supporting cells to regenerate hair cells in an mTOR-dependent non-mitotic manner.

[0010] The present invention also provides a method for regulating the reprogramming of cochlear supporting cells or the regeneration of hair cells by LIN28B, characterized in that it comprises the following steps:

[0011] Step 1, cochlear organoid culture;

[0012] Step 2, overexpression of LIN28B gene;

[0013] Step 3, cochlear explant culture;

[0014] Step 4, EdU proliferation assay;

[0015] Step 5, immunofluorescence staining.

[0016] Furthermore, the specific operation of step 1 is: taking the cochlear basement membrane of P5 premouse mice, separating the cochlear epithelial tissue after enzyme digestion, digesting and blowing away the cochlear epithelial tissue, filtering through a 35 μm filter to obtain a single-cell suspension, mixing the single-cell suspension with an equal volume of matrix gel, and dropping it into a preheated 4-well plate to establish cochlear organoid culture.

[0017] Furthermore, the specific operation of step 2 is: Col1a-TRE-LIN28B mice are hybridized with R26-M2-rtTA mice, and hybrid mice with a dual genotype background are obtained through identification, and LIN28B gene overexpression is induced by feeding the mice with water containing Dox.

[0018] Furthermore, the specific operation of step 3 is: dissecting the hybrid mouse cochlea in a sterile PBS solution, retaining the basement membrane wrapped around the modiolus of the cochlea, trimming the bottom end of the modiolus flat with tweezers, and placing it upright on the SPI-Pore membrane for suspension culture.

[0019] Furthermore, the specific operations of step 4 are: adding EdU reagent to the organoid culture medium, and 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] Furthermore, the specific operation of step 5 is: using Myo7a antibody to mark hair cells, using Sox2 antibody to mark supporting cells, and EdU staining to mark proliferating cells and indicate the source of new hair cells.

[0021] The advantages and positive effects of the application of LIN28B in regulating cochlear supporting cell reprogramming and hair cell regeneration of the present invention are:

[0022] 1. The present invention promotes the reactivation of cell plasticity of P5 mouse cochlear supporting cells by overexpressing the LIN28B gene, thereby achieving the regeneration of hair cells at the cochlear organoid and cochlear explant levels.

[0023] 2. The present invention provides a new method for reactivating the plasticity of cochlear supporting cells and regenerating hair cells in mammals, and has important theoretical reference significance for the clinical treatment of hearing loss.

[0024] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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, wherein A is a schematic diagram of cochlear organoid culture, B is a bright field image of cochlear organoids cultured from P2 and P5 mice after 5 days of proliferation, the scale bar is 400 μm, C is the number of organoids formed, and D is the size of the formed organoids;

[0026] Figure 2 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, wherein A is the EdU staining to detect the proliferating cells in P2 and P5 cochlear organoids (n=8), and B is the statistical proliferation ratio of the organoids in A;

[0027] Figure 3 A in the middle is the bright field and green fluorescence images of P2 and P5 mouse cochlear organoids, scale bar 50 μm, B is the ratio of Atoh-nGFP organoids;

[0028] Figure 4 A is a confocal image of P2 and P5 organoids after 10 days of differentiation. The newly formed hair cells express Atoh1-nGFP (green) and MYO7A (magenta). The scale bar is 50 μm. B is the proportion of MYO7A (purple) cells in each organoid in A (n=6).

[0029] Figure 5 : The effect of overexpression of LIN28B in P5 mouse organoids on the regeneration of cochlear supporting cells to form hair cells in an embodiment of the present invention, wherein A is a schematic diagram of cochlear organoid culture, B is a bright field image of P5Atoh1-nGFP; iLIN28B mouse cochlear organoids proliferated for 7 days and 13 days, the scale bar is 400 μm, C is the number of organoids formed, and D is the size of the formed organoids;

[0030] Figure 6 A in the middle is RT-qPCR detection of Sox2 mRNA expression levels in control and LIN28B overexpressing organoids after 7 and 13 days of proliferation, and B is EdU staining to detect proliferating cells in cochlear organoids (n=6);

[0031] Figure 7 A stands for statistics Figure 6 The proliferation ratio of B organoids, B is Figure 6 BSox2 high EDU + 、Sox2 low EDU + 、Sox2 high EDU - 、Sox2 low EDU - The proportion of cells in the organoid;

[0032] Figure 8 A is the bright field and green fluorescence images of cochlear organoids in the control and LIN28B overexpression groups, with a scale bar of 400 μm. B is the proportion of Atoh1-nGFP organoids at 3, 5, and 8 days of differentiation in A. C is the RT-qPCR detection of Atoh1, Myo7a, and Pou4f3 mRNA expression levels in the control and LIN28B overexpression group organoids at 5 days of differentiation.

[0033] Fig. 9 A schematic diagram of cochlear explant culture showing the effect of overexpression of LIN28B on the regeneration of supporting cells to form hair cells in P5 mouse cochlear explants in an embodiment of the present invention;

[0034] Fig.10 Rapamycin treatment inhibits the formation of hair cells in the control and LIN28B-overexpressing cochlear explants. Shown are confocal images of the control and LIN28B-overexpressing cochlear explants, stained with MYO7A (magenta), Atoh1-nGFP (green), SOX2 (blue), and EdU (red), with white asterisks marking newly formed hair cells.

[0035] Fig.11 for Fig.10 Newly formed hair cells (MYO7A + Atoh1-nGFP + SOX2 + )’s statistical results. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0037] 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.

[0038] 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.

[0039] Example 1

[0040] The present invention provides the use of LIN28B gene in regulating the reprogramming of cochlear supporting cells and regulating the regeneration of hair cells. The method of LIN28B regulating the proliferation and differentiation of cochlear supporting cells comprises the following steps:

[0041] Step 1, cochlear organoid culture:

[0042] 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: dissect the cochlea in a sterile PBS solution to obtain the cochlear basement membrane. 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 DMEN / 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 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.

[0043] Cochlear organoids were cultured in proliferation medium (DMEM / F12, N2 supplement, B-27supplement, EGF, FGF2, CHIR99021, VPA, 616452, penicillin) for 10 days, and then changed to differentiation medium (DMEM / F12, N2 supplement, B-27supplement, CHIR99021, LY411575) for 10 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.

[0044] Step 2, overexpression of LIN28B gene:

[0045] Col1a-TRE-LIN28B (MGI: 5294612) mice were crossed with R26-M2-rtTA (MGI: 3798943) mice, and mice with a dual genotype background were identified. The mice can achieve overexpression of the LIN28B gene (iLIN28B) by Dox induction. The iLIN28B genotype mice were introduced into the Atoh1-nGFP genotype by crossing to mark hair cells. Control mice with Atoh1-nGFP; iLIN28B genotype background and without Col1a-TRE-LIN28B genotype at P5 were taken to culture cochlear organoids. The organoids were cultured for proliferation for 13 days and differentiation for 10 days, and Dox was added to the culture medium to induce overexpression of the LIN28B gene.

[0046] Step 3, cochlear explant culture:

[0047] First, the mouse cochlea was dissected in a sterile PBS solution, the basilar membrane was kept wrapped around the modiolus, the bottom of the modiolus was trimmed flat with tweezers, and finally it was placed on the SPI-Pore membrane for suspension culture.

[0048] Pregnant mice were fed Dox-containing water at E17.5 days, and samples were taken at P5 after birth to establish cochlear explants. Cochlear explants were suspended in culture medium (DMEM / F12, N2 supplement, B-27supplement, CHIR99021, LY411575), and EdU reagent was added during the culture process to mark proliferating cells. Samples were collected after 4 days of explant culture, immunofluorescence staining was performed, and imaging was performed under a laser scanning confocal microscope.

[0049] Step 4, EdU proliferation assay:

[0050] 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.

[0051] Step 5, Immunofluorescence staining:

[0052] 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.

[0053] Step 6, data statistical analysis:

[0054] 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.

[0055] Example 2 Results Analysis

[0056] Figure 1 This is the effect of culturing organoids from P2 and P5 mice on the regeneration of cochlear supporting cells to form hair cells in an example of the present invention. Figure 1 A is a schematic diagram of cochlear organoid culture. B is a bright field image of cochlear organoids cultured from P2 and P5 mice after 5 days of proliferation, with a scale of 400 μm. C is the number of organoids formed, which shows that the ability of mouse cochlear organoids to form gradually weakens after birth, with P2 being greater than P5. D is the size of the formed organoids, which also shows that the ability of mouse cochlear organoids to form gradually weakens after birth, with P2 being greater than P5.

[0057] Figure 2 A is the detection of proliferating cells in P2 and P5 cochlear organoids by EdU staining (n=8), and B is the statistical analysis of the proliferation ratio of the organoids in A. Figure 2 This indicates that the proliferation ability of mouse cochlear organoids gradually weakens after birth, with P2 being greater than P5.

[0058] Figure 3 A shows the bright field and green fluorescence images of P2 and P5 mouse cochlear organoids, with a scale bar of 50 μm. B shows the proportion of Atoh-nGFP organoids, where GFP-positive cells indicate formed hair cells. Figure 3 This indicates that the ability of mouse cochlear organoids to regenerate hair cells gradually weakens after birth, with P2 being greater than P5.

[0059] Figure 4A in the middle is a confocal image of P2 and P5 organoids 10 days after differentiation. The newly formed hair cells express Atoh1-nGFP (green) and MYO7A (magenta). The scale bar is 50μm. B is the proportion of MYO7A (purple) cells in each organoid in A (n=6). Myo7a-positive cells are hair cells, and Atoh1-nGFP (green) are also indicated as hair cells, indicating that GFP fluorescence can be used to indicate the formation of hair cells. It also shows that the ability of mouse cochlear organoids to regenerate hair cells gradually weakens after the mouse is born, with P2 being greater than P5.

[0060] Figure 5 The effect of overexpression of LIN28B in P5 mouse organoids on the regeneration of cochlear supporting cells to form hair cells. A is a schematic diagram of cochlear organoid culture. B is a bright field image of P5 Atoh1-nGFP; iLIN28B mouse cochlear organoids proliferated for 7 and 13 days, with a scale bar of 400 μm. C is the number of organoids formed, and D is the size of the formed organoids. Figure 5 This indicates that overexpression of LIN28B can promote the formation ability of mouse cochlear organoids.

[0061] Figure 6 Figure 2 is the RT-qPCR detection of Sox2 mRNA expression levels in the control and LIN28B overexpression groups after 7 and 13 days of proliferation, indicating that after overexpression of LIN28B, the expression of the Sox2 gene first decreased and then increased. B is the EdU staining detection of proliferating cells in cochlear organoids (n=6).

[0062] Figure 7 A stands for statistics Figure 6 The proliferation ratio of B-type organs in the cochlear tissue was 4.34 %, indicating that overexpression of LIN28B significantly promoted the proliferation of cochlear organoids. Figure 6 Middle B Sox2 high EDU + 、Sox2 low EDU + 、Sox2 high EDU - 、Sox2 low EDU - The proportion of cells in the organoid.

[0063] Figure 8A in the middle shows the bright field and green fluorescence images of cochlear organoids in the control and LIN28B overexpression groups, with a scale bar of 400 μm. GFP fluorescence indicates hair cells, indicating that overexpression of LIN28B promotes the formation of hair cells. B shows the proportion of Atoh1-nGFP organoids in A at 3, 5, and 8 days of organoid differentiation, indicating that overexpression of LIN28B promotes the formation of hair cells. C shows the expression levels of Atoh1, Myo7a, and Pou4f3 mRNA in organoids in the control and LIN28B overexpression groups detected by RT-qPCR after 5 days of differentiation. Atoh1, Myo7a, and Pou4f3 are all hair cell-related genes, indicating that overexpression of LIN28B promotes the expression of hair cell-related genes and promotes the formation of hair cells.

[0064] Fig. 9 Schematic diagram of cochlear explant culture showing the effect of overexpression of LIN28B on the regeneration of supporting cells into hair cells in P5 mouse cochlear explants. Fig.10 Rapamycin treatment inhibits the formation of hair cells in control and LIN28B-overexpressing cochlear explants. Shown are confocal images of control and LIN28B-overexpressing cochlear explants, stained with MYO7A (magenta), Atoh1-nGFP (green), SOX2 (blue), and EdU (red). White asterisks mark newly formed hair cells. Fig.11 for Fig.10 Newly formed hair cells (MYO7A + Atoh1-nGFP + SOX2 + ) showed that overexpression of LIN28B could significantly promote the formation of new hair cells at the cochlear explant level, but the promoting effect of LIN28B was weakened after the mTOR signaling pathway was inhibited by Rapamycin. EdU negativity represented that the new hair cells did not go through the mitotic stage but were directly transdifferentiated from the existing supporting cells to form hair cells. In summary, overexpression of LIN28B regenerated hair cells by inducing supporting cells in an mTOR-dependent non-mitotic manner.

[0065] Mammalian cochlear hair cells cannot regenerate after damage, and perinatal mouse cochlear supporting cells have limited hair cell regeneration capacity. P5 mouse cochlear organoids lose the ability to regenerate hair cells. The present invention achieves efficient regeneration of hair cells in P5 mouse cochlear organoids and cochlear explants by overexpressing the LIN28B gene. It also reveals at the explant level that overexpression of LIN28B causes supporting cells to regenerate hair cells in an mTOR-dependent non-mitotic manner. The present invention successfully achieves the reactivation of the plasticity of P5 mouse cochlear supporting cells, and provides an effective method for promoting reprogramming and redifferentiation of terminally differentiated cells. The present invention is expected to achieve hair cell regeneration in mammals, providing a method and theoretical basis for the clinical treatment of hearing loss.

[0066] Therefore, the present invention adopts the above-mentioned LIN28B in regulating the reprogramming of cochlear supporting cells and the application in the regeneration of hair cells. By overexpressing LIN28B, the cochlear supporting cells of P5 mice are prompted to reactivate the cell plasticity, and the regeneration of hair cells is achieved at the level of cochlear organoids and cochlear explants. At the same time, it provides a new method for how to achieve the reactivation of the plasticity of cochlear supporting cells and regenerate hair cells in mammals, which has important theoretical reference significance for the clinical treatment of hearing loss.

[0067] 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 LIN28B in regulating the reprogramming of cochlear supporting cells.

2. Application of LIN28B in regulating hair cell regeneration.

3. The use according to claim 1 or 2, characterized in that: Overexpression of LIN28B causes cochlear supporting cells to activate cellular plasticity or overexpression of LIN28B induces supporting cells to regenerate hair cells in an mTOR-dependent non-mitotic manner.

4. A method for regulating cochlear supporting cell reprogramming or hair cell regeneration by LIN28B, characterized in that: The following steps are involved: Step 1, cochlear organoid culture; Step 2, overexpression of LIN28B gene; Step 3, cochlear explant culture; Step 4, EdU proliferation assay; Step 5, immunofluorescence staining.

5. The method according to claim 4, characterized in that The specific operations of step 1 are: The cochlear basal membrane of P5 mice was obtained, and the cochlear epithelial tissue was separated after enzyme digestion. The cochlear epithelial tissue was digested and blown away, and filtered through a 35μm filter to obtain a single-cell suspension. The single-cell suspension was mixed with an equal volume of matrix gel and dropped into a preheated 4-well plate to establish cochlear organoid culture.

6. The method according to claim 4, characterized in that The specific operation of step 2 is: Col1a-TRE-LIN28B mice are hybridized with R26-M2-rtTA mice, and hybrid mice with a dual genotype background are obtained after identification, and LIN28B gene overexpression is induced by feeding the mice with water containing Dox.

7. The method according to claim 4, characterized in that The specific operation of step 3 is: dissect the hybrid mouse cochlea in a sterile PBS solution, keep the basement membrane wrapped around the modiolus, trim the bottom end of the modiolus flat with tweezers, and place it upright on the SPI-Pore membrane for suspension culture.

8. The method according to claim 4, characterized in that The specific operations of step 4 are: adding EdU reagent to the organoid culture medium, and 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.

9. The method according to claim 4, characterized in that The specific operation of step 5 is: using Myo7a antibody to mark hair cells, using Sox2 antibody to mark supporting cells, and EdU staining to mark proliferating cells and indicate the source of new hair cells.

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