Application of SPTBN1 as target spot in treatment of GJB2-related sensorineural hearing loss

Abstract

The invention provides application of SPTBN1 as a target spot in treatment of sensorineural hearing loss related to GJB2, and belongs to the technical field of sensorineural hearing. Comprising the following steps: constructing a stably transfected cell line: culturing HEK293T cells in a humidifying incubator with temperature of 37 DEG C, 95% of air and 5% of CO2 by using a DMEM (Dulbecco Modified Eagle Medium) culture medium added with 10% of fetal calf serum and 1% of double antibodies, and performing cell passage when the cells grow to a confluence degree of 80-90%; performing IP-MS analysis: purifying a protein compound by using ProteinA / G immunoprecipitation magnetic beads, taking part of extracted protein as input, then adding 2 [mu] ganti-Cx26 antibody into the residual protein extract, slightly blowing, beating and mixing, and performing turnover shaking table incubation at 4 DEG C overnight; carrying out immunofluorescence observation on co-localization of the Cx26 and the SPTBN1; constructing a stably transfected cell line for expressing the WT-Cx26 and the Mut-Cx26; and carrying out co-immunoprecipitation (Co-IP) verification: constructing a stably transfected cell line for expressing the WT-Cx26. According to the invention, limitations of treatment modes such as relatively low complete protein targeting and short treatment time window are supplemented through gene therapy, and a new insight, a new target spot and a new treatment are provided for GJB2-related hearing loss.

Description

Technical Field

[0001] The present invention relates to the field of sensorineural hearing technology, and in particular to the application of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss. Background Art

[0002] In my country, gap junction beta 2 (GJB2) gene mutation is the most common cause of non-syndromic hereditary deafness, accounting for about 18%. Gap junction protein 26 (Connexin26, Cx26) encoded by the GJB2 gene is the most abundant gap junction in the cochlea, and the hearing loss caused by it is diverse, ranging from mild to extremely severe. At present, the main intervention measures for GJB2-related hereditary hearing loss in clinical practice are wearing hearing aids and implanting cochlear implants. However, there are certain surgical risks in the implantation process of cochlear implants, which are prone to wound infection and skull hematoma; they can cause sequelae such as dizziness, scars, poor sound discrimination ability, and trigeminal neuralgia; and cochlear implants are easy to lose, and implantation and subsequent maintenance and cleaning are expensive, which brings a heavy economic burden to individuals and society. In summary, it is crucial to develop drug treatments for GJB2-related sensorineural hearing loss.

[0003] Studies have shown that Cx26 is a gap junction channel that needs to be transported to the cell membrane to function normally. Some Cx26 mutants exhibit membrane transport defects, causing the mutants to be retained in various intracellular structures, thereby preventing them from being transported to the cell membrane to function as channels. For example, the Cx26 mutant p.Gly12Arg associated with keratitis-ichthyosis-deafness (KID) syndrome is retained in the perinuclear region and cannot be transported to the cell-cell junction. Immunofluorescence results showed that the Cx26 mutant p.Arg184Pro colocalized with the Golgi resident protein GM130, confirming that p.Arg184Pro was retained in the Golgi and could not be transported to the cell membrane. This suggests that the function of the mutant may be restored by promoting the re-transport of mutants retained in the cytoplasm to the cell membrane, thereby alleviating the patient's hearing loss.

[0004] Many point mutants or truncation mutants of Cx26 exhibit normal structure and function, but cannot be transported to the cell membrane. This observation suggests that providing a complete Cx26 protein may not be important, and promoting the re-transport of mutants retained in the cytoplasm to the cell membrane may restore the function of the mutant and alleviate hearing loss. At present, there are few reports on the membrane transport pathway of Cx26 and the key regulatory proteins in the membrane transport process. It has brought great challenges to promote the re-transport of Cx26 mutants to the cell membrane to rescue hearing loss by regulating the key proteins in the Cx26 membrane transport process. Therefore, this application provides the use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss to meet the needs. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide the application of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss to solve the problem that there are few reports on the membrane transport pathway of Cx26 and the key regulatory proteins in the membrane transport process. This poses a huge challenge to promote the re-transport of Cx26 mutants to the cell membrane to rescue hearing loss by regulating the key proteins in the membrane transport process of Cx26.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A method for treating GJB2-related sensorineural hearing loss using SPTBN1 as a target, comprising:

[0008] Construction of stable cell lines: HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% double antibody in a humidified incubator at 37°C, 95% air and 5% CO2. When the cells grew to 80-90% confluence, the cells were passaged.

[0009] IP-MS analysis: Protein A / G immunoprecipitation magnetic beads were used to purify the protein complex, and part of the extracted protein was used as input. Then 2 μg of anti-Cx26 antibody was added to the remaining protein extract, gently pipetted to mix, and incubated on a shaker at 4°C overnight;

[0010] Immunofluorescence observation of colocalization of Cx26 and SPTBN1: Stable cell lines expressing WT-Cx26 and Mut-Cx26 were constructed;

[0011] Co-immunoprecipitation (Co-IP) validation: A stable cell line expressing WT-Cx26 was constructed and Co-IP was performed using ProteinA / G immunoprecipitation magnetic beads;

[0012] Construction of SPTBN1 knockdown cell line: Cells were seeded into six-well plates at a density of 10^5 cells per well. Transfection was performed when cell confluence reached 50-60%. The transfection system was configured in RNase-free EP tubes.

[0013] Immunofluorescence detection of Cx26 transported to the cell membrane: cells were fixed with 4% paraformaldehyde at room temperature for 0.5 h, blocked in phosphate buffered saline (PBS) containing 1% fetal bovine serum (BSA) and 0.1% TritonX-100 solution for 15 min, and then blocked in PBS containing 1% BSA at room temperature for 45 min;

[0014] Western blotting was used to quantitatively detect Cx26 translocated to the cell membrane: proteins were extracted using RIPA lysis buffer containing 1% PMSF.

[0015] Optionally, the construction of the stable cell line includes preparing Cx26 red fluorescent protein (Cx26-RFP) and Cx26 mutants p.Ser199Phe-RFP, p.Glu187_Val226del-RFP and p.Leu79del-RFP recombinant viral plasmids and their auxiliary packaging agent vector plasmids, extracting high-purity, endotoxin-free recombinant plasmids and auxiliary plasmids, inoculating cells into 10-cm culture dishes, and transfecting when cell fusion reaches 80%. Fresh culture medium is replaced 1-2 hours before transfection, and a sterile 1.5 ml EP tube is taken, and 1 ml serum-free DMEM culture medium, 10 μg DNA, 10 μl lentivirus mixture and 60 μl transfection reagent are added to form a reaction system.

[0016] Optionally, the mixture is placed at room temperature for 18-20 minutes and then evenly added to the culture dish. After transfection for 18-20 hours, the culture medium is replaced with fresh 15 ml serum-free DMEM culture medium. After 48 hours, the supernatant is aspirated into a centrifuge tube and centrifuged at 4°C 500g for 5 minutes. The supernatant is filtered through a 0.45μm filter and transferred to a new centrifuge tube and stored at -80°C. When the cells are infected, the cells are inoculated into a 6-well plate. When the cell fusion reaches 60%, the collected virus solution is added to the cells. The culture medium is replaced 24 hours after infection, and the expression of the target protein is detected 96 hours after the culture medium is replaced.

[0017] Optionally, the IP-MS analysis includes magnetic bead pretreatment: 20 μl of magnetic beads are aspirated, washed with washing buffer for 2 minutes, and then magnetically separated using a magnetic stand, the washing step is repeated three times, the antigen-antibody complex is added to the pretreated magnetic beads, and the mixture is incubated overnight on a 4°C inverting shaker to allow the magnetic beads to bind to the antigen-antibody complex, the binding product is magnetically separated, the supernatant is aspirated as output, 50 μl of 1xSDS-PAGE protein sample loading buffer is added to the washed product, the mixture is mixed evenly, and the mixture is heated at 95°C for 5 minutes to elute the antigen, and magnetic separation is performed after heating, and the supernatant is used as the IP product.

[0018] Optionally, the purified protein complex is enzymatically cleaved to cut it into peptides, and after liquid chromatography separation, the peptides are placed in a mass spectrometer for analysis, and the proteins from which the peptides are derived are identified by detecting the mass-to-charge ratio (m / z) and secondary fragment information. By comparing and statistically analyzing the mass spectrometry data, candidate proteins with significant interactions with the target protein are screened out, and functional annotation and bioinformatics analysis are performed. The IP-MS results suggest that SPTBN1 and Cx26 interact with each other, and in the cell line stably expressing wild-type Cx26, Cx26 and SPTBN1 are clearly co-localized.

[0019] Optionally, the immunofluorescence observation of the co-localization of Cx26 and SPTBN1 is performed by constructing a stable cell line expressing WT-Cx26 and Mut-Cx26, fixing the cells with 4% paraformaldehyde at room temperature for 0.5 h, blocking them in phosphate buffered saline (PBS) containing 1% fetal bovine serum (BSA) and 0.1% TritonX-100 solution for 15 min, and then blocking them in PBS containing 1% BSA at room temperature for 45 min, incubating them with anti-Cx26 and anti-SPTBN1 antibodies at 4°C overnight, washing the samples three times with PBS, and using AlexaFluor488donkeyanti-mouseIgG and AlexaFluor647donkeyanti-rabbitIgG were incubated at room temperature for 2 h, and the samples were washed three times with PBS. Images were acquired using a laser scanning confocal microscope (Nikon). Immunofluorescence results showed that WT-Cx26 and SPTBN1 were obviously colocalized, but Mut-Cx26 and SPTBN1 were not colocalized. In cells stably expressing wild-type Cx26, Cx26 and SPTBN1 interacted with each other.

[0020] Optionally, the co-immunoprecipitation (Co-IP) validation includes constructing a stable cell line expressing WT-Cx26, using ProteinA / G immunoprecipitation magnetic beads for Co-IP, using part of the extracted protein as input, and then adding 2 μg of anti-Cx26 antibody to the remaining protein extract, gently pipetting to mix, incubating on a 4°C flip shaker overnight, magnetic bead pretreatment: aspirating 20 μl of magnetic beads, washing with washing buffer for 2 minutes, and then performing magnetic separation with a magnetic stand, repeating the washing step three times, adding the antigen-antibody complex to the pretreated magnetic beads, and incubating on a 4°C flip shaker overnight to allow the magnetic beads to bind. The antigen-antibody complex was bound, and the binding product was separated magnetically. The supernatant was taken as the output. 50 μl 1xSDS-PAGE protein sample loading buffer was added to the washed product. After mixing evenly, it was heated at 95°C for 5 minutes to elute the antigen. After heating, magnetic separation was performed and the supernatant was used as the IP product. Western blotting was used to detect Cx26 and SPTBN1 in the input, output, and IP products. The Co-IP results confirmed that SPTBN1 interacted with Cx26. After knocking down SPTBN1 using siRNA, the Cx26 on the cell membrane was significantly reduced.

[0021] Optionally, the construction of the SPTBN1 knockdown cell line is carried out by seeding cells into a six-well plate at a density of 10^5 cells per well, transfection is performed when the cell fusion reaches 50-60%, and a transfection system is configured in an RNase-free EP tube, 125μl DMEM medium and 5μl Lipofectamine3000 are mixed, and gently pipetted to mix as system 1, 125μl DMEM medium and 5μl SPTBN1 siRNA or negative control siRNA are mixed, and gently pipetted to mix as system 2, and allowed to stand for 5 minutes, system 1 and system 2 are fully mixed, allowed to stand for 15-20 minutes, and then evenly added to the cell wells containing 1ml DMEM medium, 6-8h after transfection, the medium is replaced with a complete medium containing 1% FBS, 96 hours after the medium is replaced, and the difference in protein expression levels is detected by Western blotting, and the Western blotting results show that the use of siRNA#1 can effectively knock down SPTBN1, and the knockdown efficiency exceeds 50%.

[0022] Optionally, the immunofluorescence detection of Cx26 transported to the cell membrane includes fixing the cells with 4% paraformaldehyde at room temperature for 0.5 h, blocking in phosphate buffered saline (PBS) containing 1% fetal bovine serum (BSA) and 0.1% TritonX-100 solution for 15 min, and then blocking in PBS containing 1% BSA at room temperature for 45 min, incubating with anti-Cx26 and anti-SPTBN1 antibodies at 4°C overnight, washing the sample three times with PBS, incubating with AlexaFluor488donkeyanti-mouseIgG and AlexaFluor647donkeyanti-rabbitIgG at room temperature for 2 h, washing the sample three times with PBS, and acquiring images using a laser scanning confocal microscope (Nikon). The immunofluorescence results show that Cx26 on the cell membrane is significantly reduced after knocking down SPTBN1.

[0023] Optionally, the Western blotting quantitative detection of Cx26 transported to the cell membrane uses RIPA lysis buffer containing 1% PMSF to extract proteins, and equal amounts of protein samples are separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PVDF membrane. The PVDF membrane is blocked with 5% skim milk powder at room temperature for 1 hour, and then incubated with anti-Cx26 and anti-SPTBN1 antibodies at 4°C overnight, and then incubated at room temperature for 1.5 hours, developed using an ultrasensitive electrochemiluminescence (ECL) reaction kit, and measured using ImageJ software. The protein level was normalized with the P-cadherin (membrane protein) or GAPDH (total protein) level in the corresponding lane. The Western blotting results quantitatively showed that Cx26 on the cell membrane was significantly reduced after knocking down SPTBN1.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects:

[0025] In the above scheme, co-immunoprecipitation-mass spectrometry revealed that SPTBN1, which encodes βII Spectrin, interacts with Cx26. Previous studies have shown that SPTBN1 is closely related to the transport of membrane proteins. In mouse and human cardiomyocytes, SPTBN1 can be co-immunoprecipitated with a variety of membrane proteins (including Na+ / K+ATPase, Na+ / Ca2+exchanger, αII Spectrin) and cytoskeletal components (such as actin). In addition, knockout of SPTBN1 leads to reduced transport of RyR2 and αⅡspectrin on the mouse cardiomyocyte membrane, leading to severe arrhythmias. These evidences indicate that SPTBN1 may regulate the transport of membrane proteins by affecting the localization of the cytoskeletal framework and plasma membrane protein complexes. The above conclusions suggest that SPTBN1 may be related to the normal membrane localization of Cx26, and the inability of Cx26 mutants to transport to the cell membrane may be related to their inability to bind normally to SPTBN1. By regulating SPTBN1, we can promote the re-transport of Cx26 mutants to the cell membrane, rescue hearing loss, regulate the membrane transport process of Cx26, promote the re-transport of mutants retained in cells to the cell membrane, and rescue GJB2-related hearing loss. This breaks through the limitations of previous treatment methods such as more sequelae and heavy economic burden of cochlear implants, and low targeting and short treatment time window of supplementing complete proteins through gene therapy, providing new insights, new targets, and new treatments for GJB2-related hearing loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, further serve to explain the principles of the invention and to enable those skilled in the relevant art to make and use the invention.

[0027] Figure 1 Schematic diagram of the immunofluorescence results showing that WT-Cx26 and SPTBN1 are obviously co-localized (left: representative image of WT-Cx26 and SPTBN1 co-localization; right: local magnified image).

[0028] Figure 2 Schematic diagram of the lack of co-localization between Mut-Cx26 and SPTBN1 (left: mutant p.Ser199Phe has no co-localization with SPTBN1; middle: mutant p.Glu187_Val226del has no co-localization with SPTBN1; right: mutant p.Leu79del has no co-localization with SPTBN1. The bottom is a partial enlarged view of the above picture);

[0029] Figure 3 Schematic diagram of Co-IP confirming the interaction between SPTBN1 and Cx26;

[0030] Figure 4Schematic diagram of Western blotting results showing that siRNA #1 can effectively knock down SPTBN1 (A Western blotting detection of SPTBN1 expression after knocking down SPTBN1 using siSPTBN1; B Statistical graph of SPTBN1 expression level after knocking down using siSPTBN1 #1, the knockdown efficiency of siSPTBN1 #1 exceeds 50%, **p<0.01; C Statistical graph of SPTBN1 expression level after knocking down using siSPTBN1 #2; D Statistical graph of SPTBN1 expression level after knocking down using siSPTBN1 #3);

[0031] Figure 5 Schematic diagram showing that immunofluorescence results showed that Cx26 on the cell membrane was significantly reduced after knocking down SPTBN1 (A: In the control group, SPTBN1 was abundantly expressed, and Cx26 was transported to the cell membrane to form gap junction plaques; B: In the control group, Cx26 was transported to the cell membrane to form gap junction plaques; a partial enlarged view of Figure CB; D: After knocking down SPTBN1, the fluorescence intensity of SPTBN1 was significantly reduced, and Cx26 could not be transported to the cell membrane to form gap junction plaques; E: After knocking down SPTBN1, Cx26 could not be transported to the cell membrane to form gap junction plaques; a partial enlarged view of Figure FE);

[0032] Figure 6 Schematic diagram showing that the Western blotting results showed that the Cx26 on the cell membrane was significantly reduced after knocking down SPTBN1 (A After using siSPTBN1#1, the expression levels of SPTBN1 and Cx26 were detected by Western blotting; B After using siSPTBN1#1, the statistical graph of SPTBN1 expression level (*p<0.05); C After using siSPTBN1#1, the statistical graph of Cx26 expression level (*p<0.05)).

[0033] As shown in the figure, in order to clearly implement the structure of the embodiment of the present invention, specific structures and devices are marked in the figure, but this is only for illustrative purposes and is not intended to limit the present invention to the specific structure, device and environment. According to specific needs, ordinary technicians in this field can adjust or modify these devices and environments. DETAILED DESCRIPTION

[0034] The following is a detailed description of the application of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss provided by the present invention in conjunction with the accompanying drawings and specific embodiments. At the same time, it is explained here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments, and those skilled in the art may also adopt other alternative methods for some known technologies; and the drawings are only for a more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0035] It should be noted that the references to "one embodiment", "an embodiment", "an exemplary embodiment", "some embodiments" and the like in the specification indicate that the embodiments described may include specific features, structures or characteristics, but not every embodiment may include the specific features, structures or characteristics. In addition, when a specific feature, structure or characteristic is described in conjunction with an embodiment, it should be within the knowledge of a person skilled in the art to implement such feature, structure or characteristic in conjunction with other embodiments (whether or not explicitly described).

[0036] In general, a term can be understood, at least in part, from its use in context. For example, depending, at least in part, on the context, the term "one or more" as used herein can be used to describe any feature, structure, or characteristic in the singular sense, or can be used to describe a combination of features, structures, or characteristics in the plural sense. Additionally, the term "based on" can be understood as not necessarily intended to convey an exclusive set of factors, but can instead, depending, at least in part, on the context, allow for the presence of other factors that are not necessarily explicitly described.

[0037] It will be understood that the meanings of “on,” “over,” and “above” in the present invention should be interpreted in the broadest manner, so that “on” not only means “directly on” something, but also includes the meaning of being “on” something with intervening features or layers therebetween, and “on” or “over” not only means “on” or “above” something, but also includes the meaning of being “on” or “above” something with no intervening features or layers therebetween.

[0038] Additionally, spatially relative terms such as "under," "beneath," "lower," "above," "upper," and the like may be used herein for descriptive convenience to describe the relationship of one element or feature to another element or features, as shown in the accompanying drawings. Spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the accompanying drawings. The device may be oriented in other ways, and the spatially relative descriptors used herein may be similarly interpreted accordingly.

[0039] like Figures 1 to 6 As shown, the embodiments of the present invention provide the use of SPTBN1 as a target in treating GJB2-related sensorineural hearing loss, including:

[0040] Construction of stable cell lines: HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% double antibody in a humidified incubator at 37°C, 95% air and 5% CO2. When the cells grew to 80-90% confluence, cell passage was performed;

[0041] IP-MS analysis: Protein A / G immunoprecipitation magnetic beads were used to purify the protein complex. A portion of the extracted protein was used as input, and then 2 μg of anti-Cx26 antibody was added to the remaining protein extract, gently pipetted to mix, and incubated on a shaker at 4°C overnight;

[0042] Immunofluorescence observation of colocalization of Cx26 and SPTBN1: Stable cell lines expressing WT-Cx26 and Mut-Cx26 were constructed;

[0043] Co-immunoprecipitation (Co-IP) validation: A stable cell line expressing WT-Cx26 was constructed and Co-IP was performed using ProteinA / G immunoprecipitation magnetic beads;

[0044] Construction of SPTBN1 knockdown cell line: Cells were seeded into six-well plates at a density of 10^5 cells per well. Transfection was performed when cell confluence reached 50-60%. The transfection system was configured in RNase-free EP tubes.

[0045] Immunofluorescence detection of Cx26 transported to the cell membrane: cells were fixed with 4% paraformaldehyde at room temperature for 0.5 h, blocked in phosphate buffered saline (PBS) containing 1% fetal bovine serum (BSA) and 0.1% TritonX-100 solution for 15 min, and then blocked in PBS containing 1% BSA at room temperature for 45 min;

[0046] Western blotting was used to quantitatively detect Cx26 translocated to the cell membrane: proteins were extracted using RIPA lysis buffer containing 1% PMSF.

[0047] The construction of the stable cell line includes preparing Cx26 red fluorescent protein (Cx26-RFP) and Cx26 mutants p.Ser199Phe-RFP, p.Glu187_Val226del-RFP and p.Leu79del-RFP recombinant virus plasmids and auxiliary packaging agent vector plasmids, extracting high-purity, endotoxin-free recombinant plasmids and auxiliary plasmids, inoculating cells into a 10-cm culture dish, and transfecting when the cell fusion reaches 80%. Fresh culture medium is replaced 1-2 hours before transfection, and a sterile 1.5 ml EP tube is taken, and 1 ml serum-free DMEM culture medium, 10 μg DNA, 10 μl lentivirus mixture and 60 μl transfection reagent are added to form a reaction system.

[0048] The mixture was placed at room temperature for 18-20 minutes and then evenly added to the culture dish. After transfection for 18-20 hours, the culture medium was replaced with fresh 15 ml serum-free DMEM culture medium. After 48 hours, the supernatant was aspirated into a centrifuge tube and centrifuged at 4°C 500g for 5 minutes. The supernatant was filtered through a 0.45μm filter and transferred to a new centrifuge tube and stored at -80°C. When cells were infected, the cells were inoculated into a 6-well plate. When the cell fusion reached 60%, the collected virus solution was added to the cells. The culture medium was replaced 24 hours after infection, and the expression of the target protein was detected 96 hours after the culture medium was replaced.

[0049] The IP-MS analysis includes magnetic bead pretreatment: 20 μl of magnetic beads are drawn, washed with washing buffer for 2 minutes, and then magnetically separated using a magnetic stand, the washing step is repeated three times, the antigen-antibody complex is added to the pretreated magnetic beads, incubated overnight on a 4°C inverting shaker to allow the magnetic beads to bind to the antigen-antibody complex, magnetically separate the binding product, draw the supernatant as output, add 50 μl of 1xSDS-PAGE protein sample loading buffer to the washed product, mix well, heat at 95°C for 5 minutes to elute the antigen, perform magnetic separation after heating, and use the supernatant as the IP product.

[0050] The purified protein complex is enzymatically cleaved to be cut into peptides, and after liquid chromatography separation, the peptides are placed in a mass spectrometer for analysis, and the protein from which the peptides are derived is identified by detecting the mass-to-charge ratio (m / z) and secondary fragment information, and candidate proteins with significant interactions with the target protein are screened out by comparison and statistical analysis of mass spectrometry data, and functional annotation and bioinformatics analysis are performed. The IP-MS results suggest that SPTBN1 and Cx26 interact with each other. In the cell line stably expressing wild-type Cx26, Cx26 and SPTBN1 are clearly co-localized ( Figure 1-2 ).

[0051] The immunofluorescence observation of the co-localization of Cx26 and SPTBN1, the construction of the stable cell line expressing WT-Cx26 and Mut-Cx26, the cells were fixed with 4% paraformaldehyde at room temperature for 0.5h, blocked in phosphate buffered saline (PBS) containing 1% fetal bovine serum (BSA) and 0.1% TritonX-100 solution for 15min, and then blocked in PBS containing 1% BSA at room temperature for 45min, incubated with anti-Cx26 and anti-SPTBN1 antibodies at 4°C overnight, the sample was washed three times with PBS, incubated with AlexaFluor488donkeyanti-mouseIgG and AlexaFluor647donkeyanti-rabbitIgG at room temperature for 2h, the sample was washed three times with PBS, and the image was collected using a laser scanning confocal microscope (Nikon). The immunofluorescence results showed that WT-Cx26 and SPTBN1 were obviously co-localized ( Figure 1 ), but Mut-Cx26 did not colocalize with SPTBN1 ( Figure 2 ), in cells stably expressing wild-type Cx26, Cx26 interacts with SPTBN1 ( Figure 4 ),

[0052] The co-immunoprecipitation (Co-IP) verification includes constructing a stable cell line expressing WT-Cx26, using ProteinA / G immunoprecipitation magnetic beads for Co-IP, using part of the extracted protein as input, and then adding 2μg anti-Cx26 antibody to the remaining protein extract, gently blowing to mix, incubating on a 4°C flip shaker overnight, magnetic bead pretreatment: aspirating 20μl magnetic beads, washing with washing buffer for 2 minutes, and then magnetically separating using a magnetic stand, the washing step is repeated three times, adding the antigen-antibody complex to the pretreated magnetic beads, incubating on a 4°C flip shaker overnight to allow the magnetic beads to bind to the antigen-antibody complex, magnetically separating the binding product, aspirating the supernatant as output, adding 50μl 1xSDS-PAGE protein sample loading buffer to the washed product, mixing evenly, heating at 95°C for 5 minutes to perform antigen elution, and performing magnetic separation after heating. The supernatant is used as the IP product, and Cx26 and SPTBN1 in the input, output, and IP product are detected by Western blotting. Co-IP results ( Figure 3 ) confirmed that there is an interaction between SPTBN1 and Cx26. After knocking down SPTBN1 using siRNA, Cx26 on the cell membrane was significantly reduced ( Figure 4-6 ).

[0053] The construction of the SPTBN1 knockdown cell line was carried out by seeding cells into a six-well plate at a density of 10^5 cells per well. When the cell fusion reached 50-60%, transfection was performed. A transfection system was configured in an RNase-free EP tube. 125μl of DMEM medium and 5μl of lipofectamine3000 were mixed and gently blown to mix as system 1. 125μl of DMEM medium and 5μl of SPTBN1 siRNA or negative control siRNA were mixed and gently blown to mix as system 2. The mixture was allowed to stand for 5 minutes. System 1 and system 2 were fully mixed and allowed to stand for 15-20 minutes. Then, the mixture was evenly added to the cell wells containing 1ml of DMEM medium. After transfection for 6-8h, the medium was replaced with a complete medium containing 1% FBS. After 96 hours of replacing the medium, the difference in protein expression levels was detected by Western blotting. The results of Western blotting ( Figure 4 ) showed that siRNA#1 could effectively knock down SPTBN1, with a knockdown efficiency of over 50%.

[0054] The immunofluorescence detection of Cx26 transported to the cell membrane includes fixing the cells with 4% paraformaldehyde at room temperature for 0.5 h, blocking in phosphate buffered saline (PBS) containing 1% fetal bovine serum (BSA) and 0.1% TritonX-100 solution for 15 min, and then blocking in PBS containing 1% BSA at room temperature for 45 min, incubating with anti-Cx26 and anti-SPTBN1 antibodies at 4°C overnight, washing the sample three times with PBS, incubating with AlexaFluor488donkeyanti-mouseIgG and AlexaFluor647donkeyanti-rabbitIgG at room temperature for 2 h, washing the sample three times with PBS, and acquiring images using a laser scanning confocal microscope (Nikon). The immunofluorescence results ( Figure 5 ) showed that the Cx26 on the cell membrane was significantly reduced after knockdown of SPTBN1.

[0055] The Western blotting quantitative detection of Cx26 transported to the cell membrane was performed by extracting proteins using RIPA lysis buffer containing 1% PMSF, and equal amounts of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk powder at room temperature for 1 hour, and then incubated with anti-Cx26 and anti-SPTBN1 antibodies at 4°C overnight, and then incubated at room temperature for 1.5 hours, and developed using an ultrasensitive electrochemiluminescence (ECL) reaction kit. The protein level was measured using ImageJ software and normalized with the level of P-cadherin (membrane protein) or GAPDH (total protein) in the corresponding lane. The Western blotting results ( Figure 6 ) Quantification showed that Cx26 on the cell membrane was significantly reduced after knockdown of SPTBN1.

[0056] The present invention covers any substitution, modification, equivalent method and scheme made on the essence and scope of the present invention. In order to make the public have a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention, but those skilled in the art can fully understand the present invention without the description of these details. In addition, in order to avoid unnecessary confusion about the essence of the present invention, well-known methods, processes, procedures, components and circuits are not described in detail.

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for treating GJB2-related sensorineural hearing loss using SPTBN1 as a target, characterized in that: include: Construction of stable cell lines: HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% double antibody in a humidified incubator at 37°C, 95% air and 5% CO2. When the cells grew to 80-90% confluence, the cells were passaged. IP-MS analysis: Protein A / G immunoprecipitation magnetic beads were used to purify the protein complex, and part of the extracted protein was used as input. Then 2 μg of anti-Cx26 antibody was added to the remaining protein extract, gently pipetted to mix, and incubated on a shaker at 4°C overnight; Immunofluorescence observation of colocalization of Cx26 and SPTBN1: Stable cell lines expressing WT-Cx26 and Mut-Cx26 were constructed; Co-immunoprecipitation (Co-IP) validation: A stable cell line expressing WT-Cx26 was constructed and Co-IP was performed using ProteinA / G immunoprecipitation magnetic beads; Construction of SPTBN1 knockdown cell line: Cells were seeded into six-well plates at a density of 10^5 cells per well. Transfection was performed when cell confluence reached 50-60%. The transfection system was configured in RNase-free EP tubes. Immunofluorescence detection of Cx26 transported to the cell membrane: cells were fixed with 4% paraformaldehyde at room temperature for 0.5 h, blocked in phosphate buffered saline (PBS) containing 1% fetal bovine serum (BSA) and 0.1% TritonX-100 solution for 15 min, and then blocked in PBS containing 1% BSA at room temperature for 45 min; Western blotting was used to quantitatively detect Cx26 translocated to the cell membrane: proteins were extracted using RIPA lysis buffer containing 1% PMSF.

2. The use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss according to claim 1, characterized in that: The construction of the stable cell line includes preparing Cx26 red fluorescent protein (Cx26-RFP) and Cx26 mutants p.Ser199Phe-RFP, p.Glu187_Val226del-RFP and p.Leu79del-RFP recombinant virus plasmids and auxiliary packaging agent vector plasmids, extracting high-purity, endotoxin-free recombinant plasmids and auxiliary plasmids, inoculating cells into a 10-cm culture dish, and transfecting when the cell fusion reaches 80%. Fresh culture medium is replaced 1-2 hours before transfection, and a sterile 1.5 ml EP tube is taken, and 1 ml serum-free DMEM culture medium, 10 μg DNA, 10 μl lentivirus mixture and 60 μl transfection reagent are added to form a reaction system.

3. The use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss according to claim 2, characterized in that: The mixture was placed at room temperature for 18-20 minutes and then evenly added to the culture dish. After transfection for 18-20 hours, the culture medium was replaced with fresh 15 ml serum-free DMEM culture medium. After 48 hours, the supernatant was aspirated into a centrifuge tube and centrifuged at 4°C 500g for 5 minutes. The supernatant was filtered through a 0.45μm filter and transferred to a new centrifuge tube and stored at -80°C. When cells were infected, the cells were inoculated into a 6-well plate. When the cell fusion reached 60%, the collected virus solution was added to the cells. The culture medium was replaced 24 hours after infection, and the expression of the target protein was detected 96 hours after the culture medium was replaced.

4. The use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss according to claim 3, characterized in that: The IP-MS analysis includes magnetic bead pretreatment: 20 μl of magnetic beads are drawn, washed with washing buffer for 2 minutes, and then magnetically separated using a magnetic stand, the washing step is repeated three times, the antigen-antibody complex is added to the pretreated magnetic beads, incubated overnight on a 4°C inverting shaker to allow the magnetic beads to bind to the antigen-antibody complex, magnetically separate the binding product, draw the supernatant as output, add 50 μl of 1xSDS-PAGE protein sample loading buffer to the washed product, mix well, heat at 95°C for 5 minutes to elute the antigen, perform magnetic separation after heating, and use the supernatant as the IP product.

5. The use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss according to claim 4, characterized in that: The purified protein complex is enzymatically cleaved to cut it into peptides, and after liquid chromatography separation, the peptides are placed in a mass spectrometer for analysis, and the proteins from which the peptides are derived are identified by detecting the mass-to-charge ratio (m / z) and secondary fragment information, and candidate proteins with significant interactions with the target protein are screened out through comparison and statistical analysis of mass spectrometry data, and functional annotation and bioinformatics analysis are performed. The IP-MS results suggest that SPTBN1 and Cx26 interact with each other, and in the cell line stably expressing wild-type Cx26, Cx26 and SPTBN1 are clearly co-localized.

6. The use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss according to claim 5, characterized in that: The immunofluorescence was used to observe the colocalization of Cx26 and SPTBN1. The stable cell lines expressing WT-Cx26 and Mut-Cx26 were constructed, and the cells were fixed with 4% paraformaldehyde at room temperature for 0.5 h, blocked in phosphate buffered saline (PBS) containing 1% fetal bovine serum (BSA) and 0.1% TritonX-100 solution for 15 min, and then blocked in PBS containing 1% BSA at room temperature for 45 min. Anti-Cx26 and anti-SPTBN1 antibodies were used to incubate at 4°C overnight, and the samples were washed three times with PBS and stained with Al exaFluor488donkeyanti-mouseIgG and AlexaFluor647donkeyanti-rabbitIgG were incubated at room temperature for 2 h, and the samples were washed three times with PBS. Images were acquired using a laser scanning confocal microscope (Nikon). Immunofluorescence results showed that WT-Cx26 and SPTBN1 were obviously colocalized, but Mut-Cx26 and SPTBN1 were not colocalized. In cells stably expressing wild-type Cx26, Cx26 and SPTBN1 interacted with each other.

7. The use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss according to claim 6, characterized in that: The co-immunoprecipitation (Co-IP) validation includes constructing a stable cell line expressing WT-Cx26, using ProteinA / G immunoprecipitation magnetic beads for Co-IP, using part of the extracted protein as input, then adding 2 μg of anti-Cx26 antibody to the remaining protein extract, gently pipetting to mix, incubating overnight on a 4°C flip shaker, pre-treating the magnetic beads: aspirating 20 μl of magnetic beads, washing with washing buffer for 2 minutes, and then performing magnetic separation with a magnetic stand, repeating the washing step three times, adding the antigen-antibody complex to the pre-treated magnetic beads, incubating overnight on a 4°C flip shaker, so that the magnetic beads and the antigen The antibody complex was bound, and the binding product was separated by magnetic separation. The supernatant was taken as the output. 50 μl 1xSDS-PAGE protein sample loading buffer was added to the washed product. After mixing evenly, it was heated at 95°C for 5 minutes to elute the antigen. After heating, magnetic separation was performed and the supernatant was used as the IP product. Western blotting was used to detect Cx26 and SPTBN1 in the input, output, and IP products. The Co-IP results confirmed that SPTBN1 interacted with Cx26. After knocking down SPTBN1 using siRNA, the Cx26 on the cell membrane was significantly reduced.

8. The use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss according to claim 7, characterized in that: The construction of the SPTBN1 knockdown cell line was carried out by seeding cells into a six-well plate at a density of 10^5 cells per well, and transfection was performed when the cell fusion reached 50-60%. A transfection system was configured in an RNase-free EP tube, 125μl DMEM culture medium and 5μl Lipofectamine3000 were mixed, and gently blown to mix as system 1, 125μl DMEM culture medium and 5μl SPTBN1 siRNA or negative control siRNA were mixed, and gently blown to mix as system 2, and allowed to stand for 5 minutes, and system 1 and system 2 were fully mixed, allowed to stand for 15-20 minutes, and then evenly added to the cell wells containing 1ml DMEM culture medium. After transfection for 6-8h, the culture medium was replaced with a complete culture medium containing 1% FBS. After 96 hours of replacing the culture medium, the difference in protein expression levels was detected by Western blotting. The results of Western blotting showed that the use of siRNA#1 can effectively knock down SPTBN1, and the knockdown efficiency exceeded 50%.

9. The use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss according to claim 8, characterized in that: The immunofluorescence detection of Cx26 transported to the cell membrane includes fixing the cells with 4% paraformaldehyde at room temperature for 0.5 h, blocking in phosphate buffered saline (PBS) containing 1% fetal bovine serum (BSA) and 0.1% TritonX-100 solution for 15 min, and then blocking in PBS containing 1% BSA at room temperature for 45 min, incubating with anti-Cx26 and anti-SPTBN1 antibodies at 4°C overnight, washing the sample three times with PBS, incubating with AlexaFluor488donkeyanti-mouseIgG and AlexaFluor647donkeyanti-rabbitIgG at room temperature for 2 h, washing the sample three times with PBS, and acquiring images using a laser scanning confocal microscope (Nikon). The immunofluorescence results show that Cx26 on the cell membrane is significantly reduced after knocking down SPTBN1.

10. The use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss according to claim 9, characterized in that: The Western blotting quantitative detection of Cx26 transported to the cell membrane used RIPA lysis buffer containing 1% PMSF to extract proteins, and equal amounts of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk powder at room temperature for 1 hour, and then incubated with anti-Cx26 and anti-SPTBN1 antibodies at 4°C overnight, and then incubated at room temperature for 1.5 hours, developed using an ultrasensitive electrochemiluminescence (ECL) reaction kit, and measured protein levels using ImageJ software, and normalized with the P-cadherin (membrane protein) or GAPDH (total protein) levels in the corresponding lanes. The Western blotting results quantitatively showed that Cx26 on the cell membrane was significantly reduced after knocking down SPTBN1.

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