CN117899196A - Application of 14-3-3zeta protein or YWHAZ gene in the treatment of corneal injuries - Google Patents

Abstract

The present invention relates to the field of biomedical technology, and in particular to the use of 14-3-3zeta protein or YWHAZ gene in the treatment of corneal damage. The present invention finds that 14-3-3zeta protein can promote corneal epithelial cell proliferation, migration and inhibit apoptosis, thereby repairing damaged cornea. Therefore, by providing 14-3-3zeta protein or a vector containing a nucleic acid translated into 14-3-3zeta protein, the concentration of 14-3-3zeta protein in local tissue is increased, thereby effectively promoting the rapid repair of damaged cornea.

Description

Application of 14-3-3zeta protein or YWHAZ gene in the treatment of corneal injuries

Technical Field

The present invention relates to the field of biomedical technology, and in particular to the application of 14-3-3zeta protein or YWHAZ gene expressing 14-3-3zeta protein in the treatment of corneal damage.

Background technique

The cornea is an important physiological structure of the eyeball, and its light transmittance is the basis of visual function. Good corneal transparency is closely related to the normal structure and function of the corneal epithelium, stroma, and corneal endothelium. Due to the special position of the cornea in the eye, it is susceptible to adverse external stimuli. When the corneal structure is damaged, the integrity of the corneal epithelium and basement membrane is destroyed, a large number of cytokines and chemokines are released, inducing inflammatory cell infiltration, prompting fibroblasts to transform into myofibroblasts, and ultimately reducing corneal transparency, causing decreased vision or even blindness. The treatment of corneal injuries includes both medication and surgery. Currently widely used therapeutic drugs such as growth factors, steroids, vitamin C, collagenase inhibitors, and autologous serum eye drops have the limitations of a single mechanism of action or difficulty in preparation. Among them, improper use of steroid drugs may cause increased intraocular pressure in patients and delay corneal wound healing. Common surgical methods include limbal stem cell transplantation, corneal transplantation and artificial cornea implantation, which can restore vision to a certain extent in patients with severe corneal damage. However, the first two surgical methods are difficult to be widely carried out due to the shortage of human corneal donors and the high incidence of immune rejection after transplantation worldwide. The latter has poor long-term efficacy due to various complications such as post-prosthesis membrane formation and infectious endophthalmitis. Therefore, it is of great significance to explore an efficient and safe preparation to promote corneal damage repair.

14-3-3zeta is a naturally occurring protein in the human body, encoded by the YWHAZ gene. It participates in post-translational modification of proteins and intracellular transport, and can affect a variety of physiological and pathological processes by regulating multiple signaling pathways. It has rich biological functions and is expected to bring new directions for the treatment of corneal damage.

Summary of the invention

The present invention proves that 14-3-3zeta protein helps promote corneal epithelial cell proliferation and migration and inhibits apoptosis, thereby promoting the repair of corneal damage. Therefore, the present invention provides the use of 14-3-3zeta protein or YWHAZ gene expressing 14-3-3zeta protein in the treatment of corneal damage.

In order to achieve the above-mentioned object of the invention, the technical solution provided by the present invention is as follows:

The present invention provides application of 14-3-3zeta protein or YWHAZ gene expressing 14-3-3zeta protein in the treatment of corneal damage.

In the present invention, the 14-3-3zeta protein acts on corneal epithelial cells to promote the repair of corneal damage.

In the present invention, the 14-3-3zeta protein repairs the cornea by promoting the proliferation and migration of corneal epithelial cells and inhibiting their apoptosis.

In the present invention, the 14-3-3zeta protein promotes the repair of corneal damage by acting on one or more signaling pathways including PI3K-AKT, extracellular matrix-receptor interaction, focal adhesion, Hippo, and AMPK.

In the present invention, the YWHAZ gene promotes the repair of corneal damage by regulating the PI3K-AKT signaling pathway in corneal epithelial cells.

The present invention also provides a medicine for treating corneal damage, characterized in that the medicine contains a therapeutically effective amount of 14-3-3zeta protein.

In the present invention, the drug is a hydrogel containing 14-3-3zeta protein.

The present invention also provides a medicine for treating corneal damage, wherein the medicine contains a vector of nucleic acid translated into 14-3-3zeta protein.

In the present invention, the drug repairs the cornea by increasing the 14-3-3zeta protein content in local tissues.

According to the present invention, 14-3-3zeta protein can promote corneal epithelial cell proliferation, migration and inhibit apoptosis, and 14-3-3zeta protein can also affect signaling pathways such as PI3K-AKT, extracellular matrix-receptor interaction, focal adhesion, Hippo and AMPK. Therefore, by providing 14-3-3zeta protein or a vector containing a nucleic acid translated into 14-3-3zeta protein, the 14-3-3zeta protein content in local tissue is increased, thereby effectively promoting the rapid repair of damaged cornea, and this method does not require complicated surgery or immunosuppression.

BRIEF DESCRIPTION OF THE DRAWINGS

Next, in the description of the drawings and embodiments, for convenience, the term “human corneal epithelial cell line (Humancorneal epithelial )” is abbreviated as “HCE-T”; the term “adeno-associated virus (Adeno-associated Virus)” is abbreviated as “AAV”; and the term “corneal epithelial cells (Corneal epithelial cells)” is abbreviated as “CECs”.

FIG1 is a graph showing the expression level of 14-3-3zeta protein in mouse corneal tissue at different time points in normal state and after alkali burn;

FIG2 is a graph showing the effect of overexpression of 14-3-3zeta protein in mouse corneal tissue on corneal damage and repair;

FIG3 is a graph showing the effect of overexpression of 14-3-3zeta protein on the proliferation and migration of HCE-T cells;

Figures 4 and 5 are graphs showing the effect of knocking down the expression of 14-3-3zeta protein on the proliferation and migration of HCE-T cells;

Figures 6 to 8 are mRNA-seq analysis diagrams showing that 14-3-3zeta protein is involved in regulating various biological processes and signaling pathways of HCE-T cells;

FIG9 is a validation diagram of differentially expressed genes in the PI3K-AKT signaling pathway in HCE-T cells;

FIG. 10 is a diagram showing fluorescent staining of cornea observed by frozen section and the expression level of YWHAZ mRNA.

Detailed ways

Hereinafter, the present invention will be described in detail according to exemplary embodiments, but the present invention is not limited to these embodiments. The present invention is embodied in the following various forms, but should not be construed as being limited to the exemplary embodiments set forth herein. Therefore, the detailed description and embodiments of the present invention will convey the scope of the present invention to those of ordinary skill in the art and are construed as being within the scope of the present invention.

Example 1 Animal experiments show that 14-3-3zeta protein can promote corneal damage repair

1. Experimental methods

(1) Construction of mouse corneal injury model

Take a 2mm diameter filter paper, soak it in a 0.5 N sodium hydroxide solution, place the soaked filter paper in the center of the mouse cornea and leave it for 30 seconds, then rinse the eye surface with saline containing 0.9% NaCl for 1 minute. This method is a classic method for constructing a mouse corneal alkali burn model, which is a common cause of corneal injury and can cause damage to the corneal structure and function.

(2) Subconjunctival injection of drugs in mice

Adeno-associated virus vectors (AAV-14-3-3zeta) carrying nucleic acid translated into 14-3-3zeta and empty adeno-associated virus vectors (AAV-NC) were used for local ocular injection to upregulate the expression levels of YWHAZ gene and 14-3-3zeta protein in mouse cornea. Specifically, 7 μL of AAV-14-3-3zeta with a titer of 1×10 ¹² was injected into the conjunctiva of mice using a 33-gauge needle, and the negative control group was injected with 7 μL of AAV-NC with a titer of 1×10 ¹² under the conjunctiva of mice. AAV-14-3-3zeta vector and AAV-NC vector were purchased from Shanghai Hanheng Gene Technology Co., Ltd. The basic structure of AAV-14-3-3zeta is AAV2/9- CMV-Ywhaz-3flag-Zsgreen, and the basic structure of AAV-NC is AAV2/9-CMV-Zsgreen.

Among them, the nucleic acid sequence of AAV-14-3-3zeta is shown in the following table:

Sequence 1: AAV vector carries a nucleic acid sequence that translates into mouse 14-3-3zeta protein ATGGATAAAAATGAGCTGGTGCAGAAGGCCAAGCTGGCCGAGCAGGCAGAGCGATATGATGACATGGCAGCCTGCATGAAGTCTGTCACTGAGCAGGGAGCTGAGCTGTCGAATGAGGAGAGAAACCTTCTCTCTGTTGCTTATAAAAACGTTGTAGGAGCCCGTAGGTCATCGTGGAGGGTCGTCTCAAGTATTGAGCAGAAGACGGAAGGTGCTGAGAAAAAGCAGCAGATGGCTCGAGAATACAGAGAGAAGATCGAGACGGAGCTGCGTGACATCTGCAACGATGTACTGTCTCTTTTGGAAAAGTTCTTGATCCCCAATGCTTCGCAACCAGAAAGCAAAGTCTTCTATTTGAAAATGAAGGGT GACTACTACCGTTACTTGGCCGAGGTTGCTGCTGGTGATGACAAGAAAGGAATTGTGGACCAGTCACAGCAAGCATACCAAGAAGCATTTGAAATCAGCAAAAAGGAGATGCAGCCGACACACCCCATCAGACTGGGTCTGGCCCTCAACTTCTCTGTGTTCTATTACGAGATCCTGAACTCCCCAGAGAAAGCCTGCTCTCTTGCAAAAACAGCTTTCGATGAAGCCATTGCTGAACTTGATACATTAAGTGAAGAGTCGTACAAAGACAGCACGCTAATAATGCAGTTACTGAGAGACAACTTAACATTGTGGACATCGGATACCCAAGGAGATGAAGCAGAAGCAGGAGAAGGAGGGGAAAATTAA

(3) Corneal fluorescein sodium staining

Immediately, 1 day, and 2 days after alkali burn modeling, the mouse ocular surface was stained with sodium fluorescein solution, and observed and photographed using a slit lamp microscope equipped with a cobalt blue filter. The fluorescein-positive area was analyzed and counted.

(4) Corneal opacity score

Immediately and 7 days after alkali burn modeling, the corneal opacity was observed and evaluated using a slit lamp microscope, with complete transparency as 0 points, mild opacity as 1 point, moderate opacity as 2 points, unclear iris and pupil details but visible outlines as 3 points, and unclear iris and pupil outlines as 4 points.

(5) HE staining

At 7 and 14 days after alkali burn, the eyeballs of mice were removed and fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with HE. The corneal structure was observed under an optical microscope.

(6) Immunofluorescence staining

The eyeballs of the mice in the experimental and control groups were frozen and fixed in 4% paraformaldehyde solution for 10 minutes, then immersed in 0.2% Triton X-100 solution for 3 minutes, blocked with goat serum for 30 minutes at room temperature, and then incubated with primary antibodies 14-3-3zeta (Product No.: NBP1-31325, Novus Biologicals) or Ki67 (Product No.: ab16667, Abcam) at 4°C overnight, washed 3 times with PBS (i.e., phosphate buffered saline), and then incubated with fluorescein-conjugated secondary antibodies at room temperature for 2 hours. Mounting medium containing DAPI (4',6-diamidino-2-phenylindole) (Product No.: ab104139, Abcam) was dropped on the sample and covered with a coverslip, and the slices were observed and photographed under a confocal microscope.

(7) qRT-PCR

Total RNA of the samples was isolated using Trizol and resuspended in DEPC water. The obtained RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). qRT-PCR was performed in the CFX96 PCR system using iTaq Universal SYBR Green Supermix reagent (Bio-Rad). The expression level of sample mRNA was expressed as 2-ΔΔCt.

(8) Western Blot

After BCA quantification, equal amounts of protein were taken for electrophoresis and electrotransfer. The nitrocellulose membrane after electrotransfer was removed, blocked, and incubated with primary antibody overnight. The primary antibody was discarded, washed 3 times with TBST solution, and HRP-conjugated secondary antibody was added, and incubated on a shaker at room temperature for 2 hours. The secondary antibody was discarded, washed 3 times with TBST solution, and developed using ECL ultra-sensitive luminescent solution and ChemiDoc™ MP imaging system (Bio-Rad).

2. Experimental results

(1) Expression of 14-3-3zeta protein in normal cornea and corneal injury mouse model cornea

Figure 1A shows the immunofluorescence staining results of 14-3-3zeta protein in normal mouse cornea and 7 days after corneal alkali burn (green is 14-3-3zeta, blue is DAPI, scale bar: 30 μm). Under normal conditions, 14-3-3zeta protein is mainly expressed in the corneal epithelium of mice. On the 7th day after corneal alkali burn, the immunofluorescence staining results showed that 14-3-3zeta protein was still mainly expressed in the corneal epithelium, and the expression of the protein increased in the corneal stroma and endothelium.

Figure 1B shows the expression levels of YWHAZ mRNA in normal mouse cornea and cornea at different time points after alkali burn. qRT-PCR results showed that YWHAZ mRNA levels increased on the 4th and 7th days after alkali burn and returned to normal levels on the 14th day.

Figures 1C and 1D show the expression levels of 14-3-3zeta protein in normal mouse cornea and at different time points after corneal alkali burn (* p < 0.05, ** p < 0.01, *** p < 0.001). Western Blot results showed that the level of 14-3-3zeta protein gradually increased in the first 7 days after corneal alkali burn and returned to the baseline level on the 14th day.

(2) Upregulating 14-3-3zeta protein expression to promote corneal wound healing

FIG2A shows the delivery of AAV vectors by subconjunctival injection in mice. FIG10A shows that Zsgreen green fluorescence can be observed in the cornea through frozen sections 3 weeks after injection (scale bar: 100 μm), confirming that the AAV vector successfully transfected the corneal tissue.

Figure 10B shows the expression of YWHAZ gene in local tissues of mice after AAV transfection, and the qRT-PCR results show that the expression of YWHAZ gene is upregulated. Figures 2B and 2C show the Western Blot results of 14-3-3zeta protein in corneal tissue, and the results confirm that 14-3-3zeta protein in corneal tissue was successfully upregulated 3 weeks after subconjunctival injection of AAV-14-3-3zeta or AAV-NC.

Figures 2D and 2E show the corneal epithelial defect in the corneal alkali burn model. The results of sodium fluorescein staining showed that the corneal epithelium was significantly defective after injury. On the first and second days after injury, the corneal fluorescein-positive area of the mice in the AAV-14-3-3zeta group was significantly lower than that in the AAV-NC group.

Figures 2F and 2G show the corneal opacity scores in the corneal alkali burn model. The results showed that on the 7th day after injury, the corneal opacity score of the AAV-14-3-3zeta group was significantly lower than that of the AAV-NC group.

Figures 2H and 2I show the number of Ki67 cells on the first day after corneal alkali burn. The immunofluorescence staining results showed (scale bar: 30 μm) that on the first day after injury, the number of Ki67 (i.e., proliferating cell nuclear protein) positive cells in the AAV-14-3-3zeta group increased significantly compared with the AAV-NC group, suggesting that upregulation of 14-3-3zeta can promote the proliferation of corneal epithelial cells in mice after injury.

Figures 2J and 2K show the HE staining results of corneal tissues on the 7th and 10th days after corneal alkali burn. The results showed (scale bar: 50 μm) that on the 7th and 14th days after injury, the thickness of corneal regenerated epithelium in the AAV-14-3-3zeta group was greater than that in the AAV-NC group, and the morphology of the corneal epithelium and stroma in the AAV-14-3-3zeta group was more regular, and the connection between the corneal epithelial basal cells and the Bowman's membrane (i.e., the anterior elastic layer of the cornea) was tighter.

The above experimental results show that 14-3-3zeta protein can promote the repair of corneal damage.

Example 2 Cell experiments show that 14-3-3zeta protein can promote corneal damage repair

1. Experimental methods

(1) Culture and transfection of corneal epithelial cells

Human corneal epithelial cell line (HCE-T) was cultured in DMEM/F-12 medium with 6% fetal bovine serum and 1% penicillin-streptomycin in a cell incubator. The incubator conditions were set at 37°C and 5% CO ₂ .

(2) Cell transfection

HCE-T cells were transfected with 14-3-3zeta overexpression plasmid (from Suzhou Genema Co., Ltd.) and 14-3-3zeta siRNA (from Shanghai Hanheng Gene Technology Co., Ltd.) to up-regulate or down-regulate the 14-3-3zeta level in cells. The specific operation method is as follows:

a. Inoculate HCE-T cells into the well plate and culture them in a cell incubator overnight;

b. The next day, when the cell confluence reached 70%, the cells were transfected with 14-3-3zeta overexpression plasmid and negative control plasmid using Lipofectamine 3000 (product from ThermoFisher Scientific); or the cells were transfected with 14-3-3zeta siRNA and negative control siRNA using Lipofectamine RNAiMAX (product from Thermo Fisher Scientific).

Among them, the nucleic acid sequence of the 14-3-3zeta overexpression plasmid is shown in the following table:

Sequence 2: The nucleic acid sequence carried by the plasmid vector and translated into human 14-3-3zeta protein ATGGATAAAAATGAGCTGGTTCAGAAGGCCAAACTGGCCGAGCAGGCTGAGCGATATGATGACATGGCAGCCTGCATGAAGTCTGTAACTGAGCAAGGAGCTGAATTATCCAATGAGGAGAGGAATCTTCTCTCAGTTGCTTATAAAAATGTTGTAGGAGCCCGTAGGTCATCTTGGAGGGTCGTCTCAAGTATTGAACAAAAGACGGAAGGTGCTGAGAAAAAACAGCAGATGGCTCGAGAATACAGAGAGAAAATTGAGACGGAGCTAAGAGATATCTGCAATGATGTACTGTCTCTTTTGGAAAAGTTCTTGATCCCCAATGCTTCACAAGCAGAGAGCAAAGTCTTCTATTTGAAAATGAAAGGA GATTACTACCGTTACTTGGCTGAGGTTGCCGCTGGTGATGACAAGAAAGGGATTGTCGATCAGTCACAACAAGCATACCAAGAAGCTTTTGAAATCAGCAAAAAGGAAATGCAACCAACACATCCTATCAGACTGGGTCTGGCCCTTAACTTCTCTGTGTTCTATTATGAGATTCTGAACTCCCCAGAGAAAGCCTGCTCTCTTGCAAAGACAGCTTTTGATGAAGCCATTGCTGAACTTGATACATTAAGTGAAGAGTCATACAAAGACAGCACGCTAATAATGCAATTACTGAGAGACAACTTGACATTGTGGACATCGGATACCCAAGGAGACGAAGCTGAAGCAGGAGAAGGAGGGGAAAATTAA

The nucleic acid sequence of 14-3-3zeta siRNA is as follows:

Sequence 3: Human 14-3-3zeta siRNA positive strand GAUUGUCGAUCAGUCACAATT

Sequence 4: Human 14-3-3zeta siRNA antisense strand UUGUGACUGAUCGACAAUCTT

The negative control siRNA sequence is as follows:

Sequence 5: Negative control siRNA sequence sense strand UUCUCCGAACGUGUCACGUTT

Sequence 6: Negative control siRNA sequence antisense strand ACGUGACACGUUCGGAGAATT

(3) CCK-8 kit to detect cell activity

HCE-T cells were seeded into 96-well plates and transfected with 14-3-3zeta overexpression plasmid or 14-3-3zeta siRNA and its negative control the next day.

Immediately, 24 h, 36 h, 48 h, 60 h, and 72 h after transfection, the cell activity was detected using the CCK-8 kit, and the absorbance at 450 nm was measured using a microplate reader.

(3) EdU kit to detect cell proliferation

HCE-T cells were inoculated into a 48-well plate with a cell slide and transfected with 14-3-3zeta overexpression plasmid or 14-3-3zeta siRNA and its negative control the next day. After 48 hours of transfection, the cell slide was taken out and stained with EdU cell proliferation kit and DAPI. The fluorescence microscope was used to take pictures and count the number of EdU positive cells.

(4) Cell scratch test

HCE-T cells were seeded into 6-well plates and transfected with 14-3-3zeta overexpression plasmid or 14-3-3zeta siRNA and its negative control the next day. 24 hours after transfection, a straight line was drawn in the center of each well with the same force using a 1 ml pipette tip, and then washed twice with PBS buffer to wash away the detached cells, and DMEM/F-12 medium without fetal bovine serum was added, and photos were taken under a microscope. After culturing the cells in an incubator for 24 hours, photos were taken again at the same position as the first photo, and the area of the scratch was analyzed and calculated using ImageJ software.

(5) TUNEL kit to detect cell apoptosis

HCE-T cells were inoculated into a 48-well plate with a cell slide, and transfected with 14-3-3zeta overexpression plasmid or 14-3-3zeta siRNA and its negative control the next day. After 48 hours of transfection, the cell slide was taken out and stained with TUNEL cell apoptosis kit and DAPI. Fluorescence microscope was used to take pictures and count the number of TUNEL-positive cells.

(6) mRNA-sequencing analysis

Total RNA from samples was extracted using the SMART-Seq ^® HT Kit.

The cDNA library was constructed using the TruSeq ^® RNA Sample Preparation Kit. The specific method is to perform polyA-containing RNA enrichment, fragmentation, first-strand cDNA synthesis, second-strand cDNA synthesis, end repair, 3' end addition of A bases, purification and enrichment on the purified total RNA, and finally establish a sequencing sample library.

Quantification was performed using a Qubit ^® 2.0 fluorometer and validation was performed using an Agilent 2100 Bioanalyzer to examine the quality and size of the libraries.

Sequencing was performed using the Illumina HiSeq X-ten platform.

The obtained raw data were preprocessed, i.e., rRNA sequences, primers, and low-quality data were removed. Hisat2 (version 2.0.4) was used for genome matching, and StringTie (version 1.3.0) was used to count the number of fragments of each gene after matching, and the FPKM value of each gene was calculated.

EdgeR was used to analyze differentially expressed genes among samples, and the threshold of difference was set to FDR ≤ 0.05 and the difference fold ≥ 2.

GO enrichment analysis and KEGG signaling pathway enrichment analysis were used to annotate the differentially expressed genes.

2. Experimental results

(1) Effect of upregulating 14-3-3zeta on HCE-T cell function

Figure 3A shows the expression of YWHAZ and CCND1 mRNA after plasmid transfection in HCE-T cells by qRT-PCR; Figures 3B and 3C show the expression of 14-3-3zeta and cyclin D1 proteins after plasmid transfection in HCE-T cells by Western Blot. The structure shows that overexpression plasmid transfection significantly upregulated the expression levels of YWHAZ gene and 14-3-3zeta protein in HCE-T cells. At the same time, the expression of cell cycle-related gene CCND1 and protein cyclin D1 increased.

Figure 3D shows the viability of HCE-T cells after plasmid transfection measured by CCK-8 method. The test results show that starting from 48 hours after overexpression plasmid transfection, the cell viability of the OE-14-3-3zeta group was significantly improved compared with the OE-NC group.

Figures 3E and 3F show the proliferation capacity of HCE-T cells after plasmid transfection determined by EdU (scale bar: 100 μm). The EdU detection results showed that the EdU positive cell rate in the OE-14-3-3zeta group was significantly higher than that in the OE-NC group.

Figures 3G and 3H show the migration ability of HCE-T cells after plasmid transfection measured by scratch assay (scale bar: 25 μm). The results of the scratch assay showed that the migration ability of cells in the OE-14-3-3zeta group was significantly higher than that in the OE-NC group.

The above experimental results show that overexpression of 14-3-3zeta protein can promote the proliferation and migration of HCE-T cells.

(2) Effect of downregulating 14-3-3zeta on HCE-T cell function

Figure 4A shows the expression of YWHAZ, CCND1 and caspase-3 mRNA after siRNA transfection in HCE-T cells by qRT-PCR. Figures 4B and 4C show the expression of 14-3-3zeta, cyclin D1 and caspase-3 proteins after siRNA transfection in HCE-T cells by Western Blot. The results showed that siRNA transfection significantly downregulated the expression levels of YWHAZ gene and 14-3-3zeta protein in HCE-T cells. At the same time, the expression of cell cycle-related gene CCND1 and protein cyclin D1 decreased, and the expression of apoptosis-related genes and protein caspase-3 increased.

Figure 5A shows the activity of HCE-T cells after siRNA transfection as determined by CCK-8. The CCK-8 test results showed that the cell activity of the si-14-3-3zeta group was significantly lower than that of the si-NC group starting from 24 hours after siRNA transfection.

Figures 5B and 5C show the proliferation capacity of HCE-T cells after siRNA transfection determined by EdU (scale bar: 100 μm). The EdU detection results showed that the EdU positive cell rate in the si-14-3-3zeta group was significantly lower than that in the si-NC group.

Figures 5D and 5E show the apoptosis level of HCE-T cells after siRNA transfection detected by TUNEL staining (scale bar: 50 μm). The results of TUNEL detection showed that the TUNEL positive cell rate in the si-14-3-3zeta group was significantly higher than that in the si-NC group.

Figures 5F and 5G show the migration ability of HCE-T cells after siRNA transfection determined by scratch assay (scale bar: 25 μm). The results of the scratch assay showed that the migration ability of cells in the si-14-3-3zeta group was significantly lower than that in the si-NC group.

The above experimental results show that knocking down 14-3-3zeta can inhibit the proliferation and migration of HCE-T cells and induce their apoptosis.

(3) 14-3-3zeta protein is involved in regulating the PI3K-AKT signaling pathway

Figure 6A shows a volcano plot of differentially expressed genes after HCE-T cells were transfected with si-14-3-3zeta and si-NC. The results of mRNA-seq analysis showed that a total of 2219 differentially expressed genes were found in the si-14-3-3zeta group compared with the si-NC group HCE-T cells, including 1026 up-regulated genes and 1193 down-regulated genes.

Figure 6B shows a GO enrichment analysis of differentially expressed genes. The results of GO enrichment analysis indicate that differentially expressed genes are mainly involved in biological processes such as tissue structure formation and development.

Figure 7 shows the KEGG signaling pathway enrichment analysis diagram of differentially expressed genes. The results of KEGG pathway enrichment analysis showed that 14-3-3zeta may affect signaling pathways such as PI3K-AKT, extracellular matrix-receptor interaction, focal adhesion, Hippo and AMPK.

Figure 8 shows some differentially expressed genes and their signal pathways. The analysis results further indicate that the YWHAZ gene may directly regulate the PI3K-AKT signaling pathway in corneal epithelial cells.

Figure 9A shows the mRNA levels of FGF5, ITGB1, PIK3R1 and PRKAA1 detected by qRT-PCR. Figures 9B and 9C show the protein levels of FOXO3a detected by Western Blot. By using qRT-PCR to verify some genes of the PI3K-AKT signaling pathway, the results showed that downregulating 14-3-3zeta protein can cause a decrease in the mRNA levels of FGF5, ITGB1, PIK3R1, PRKAA1 and a decrease in the protein level of FOXO3a.

The above experimental results show that the YWHAZ gene and 14-3-3zeta protein are involved in regulating the PI3K-AKT signaling pathway.

As determined in the above results, the 14-3-3zeta protein of the present invention can promote corneal epithelial cell proliferation, migration and inhibit apoptosis. Therefore, the application of 14-3-3zeta protein or YWHAZ gene expressing 14-3-3zeta protein in the treatment of corneal injury is beneficial to promote the repair of corneal injury without the need for complex surgery or immunosuppression.

It should be understood that the exemplary embodiments described herein should be considered in a descriptive sense only and not for limiting purposes. Descriptions of features or aspects in each embodiment should generally be considered as possible for similar features or aspects in other embodiments.

Claims (9)

1. Application of 14-3-3zeta protein or YWHAZ gene expressing 14-3-3zeta protein in the treatment of corneal injuries. 2. The use according to claim 1, characterized in that the 14-3-3zeta protein acts on corneal epithelial cells to promote the repair of corneal damage. 3. The use according to claim 2, characterized in that the 14-3-3zeta protein repairs the cornea by promoting the proliferation and migration of corneal epithelial cells and inhibiting their apoptosis. 4. The use according to claim 1 is characterized in that the 14-3-3zeta protein promotes the repair of corneal damage by acting on one or more signal pathways including PI3K-AKT, extracellular matrix-receptor interaction, focal adhesion, Hippo, and AMPK. 5. The use according to claim 1, characterized in that the YWHAZ gene promotes the repair of corneal damage by regulating the PI3K-AKT signaling pathway in corneal epithelial cells. 6. A drug for treating corneal damage, characterized in that the drug contains a therapeutically effective amount of 14-3-3zeta protein. 7. The drug according to claim 6, characterized in that the drug is a hydrogel containing 14-3-3zeta protein. 8. A drug for treating corneal damage, characterized in that the drug contains a vector of nucleic acid that translates into 14-3-3zeta protein. 9. The drug according to claim 8, characterized in that the drug repairs the cornea by increasing the content of 14-3-3zeta protein in local tissues.