Use of an agent that inhibits expression of an ADNP gene or activity of an ADNP protein in the preparation of a medicament for treating renal fibrosis

Abstract

This invention discloses the application of a reagent that inhibits ADNP gene expression or ADNP protein activity in the preparation of drugs for treating renal fibrosis, belonging to the field of biomedical technology. The application of the reagent that inhibits ADNP gene expression or ADNP protein activity in the preparation of drugs for treating renal fibrosis. This invention effectively knocks down or knocks out the ADNP gene in renal tubular epithelial cells, objectively investigates the effects of ADNP deficiency on a series of phenotypes in HK-2 cells, and confirms that ADNP can inhibit EMT and renal fibrosis in HK-2 cells. Drugs that target and knock down or knock out the ADNP gene in human kidneys can be developed to delay the progression of renal fibrosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a reagent that inhibits ADNP gene expression or ADNP protein activity in the preparation of drugs for treating renal fibrosis. Background Technology

[0002] The global prevalence of chronic kidney disease (CKD) is approximately 10%–14%, with an absolute number of about 843.6 million (see, for example, JAGER KJ, KOVESDY C, LANGHAM R, et al. A single number for advocacy and communication - worldwide more than 850 million individuals have kidney diseases [J]. Kidney Int, 2019, 96(5): 1048-50). As renal fibrosis worsens in CKD patients, a significant proportion will progress to end-stage renal disease (ESRD), requiring dialysis or kidney transplantation for renal replacement therapy to sustain life.

[0003] The causes of chronic kidney disease (CKD) are diverse, with common clinical causes including internal medicine conditions such as hypertension, diabetes, and cardiovascular disease. In addition, obstructive nephropathy (ON), an important type of surgical disease, is also a significant contributing factor to CKD. Both CKD induced by internal medicine conditions and ON are characterized by significant inflammation in the renal tubulointerstitial region, which further leads to renal fibrosis. Renal fibrosis refers to the pathological process of excessive deposition of extracellular matrix (ECM) in the kidneys, forming scar tissue. It is a common pathological feature of chronic progressive kidney disease, mainly manifested as inflammatory cell infiltration, myofibroblast activation, ECM accumulation, capillary network loss, glomerular sclerosis, tubular atrophy, and decreased renal function.

[0004] Following kidney injury, early ECM deposition may aid in tissue repair. In cases of mild injury, ECM is absorbed during the repair process; however, in chronic CKD-related injuries, ECM deposition becomes uncontrolled and accumulates continuously. In chronic injury, the renal tubules respond to various damages through cell cycle arrest, senescence, and partial epithelial-mesenchymal transition (EMT). The common result is the large-scale production and secretion of extracellular vesicles, chemokines, pro-inflammatory factors, growth factors, and pro-fibrotic factors, inducing the expression of stromal cell proteins such as Tenasin C (TNC) and fibrillin 1 (FBN1), gradually forming a "fibrotic niche" anchored around the ECM network. As fibrosis progresses, renal blood flow decreases, tissue perfusion is insufficient, hypoxia worsens, glomerular filtration rate (GFR) declines, renal tubular water and electrolyte processing is impaired, and urinary protein excretion increases, leading to gradual deterioration of renal function and ultimately, end-stage renal disease (ESRD).

[0005] Persistent renal fibrosis is a characteristic pathological change in the progression of CKD to ESRD; therefore, inhibiting renal fibrosis is a key strategy to delay the progression of CKD to ESRD. Drugs such as renin-angiotensin system blockers, sodium-glucose cotransporter 2 inhibitors, glucagon-like peptide-1 receptor agonists, tolvaptan, and feneperidone have been shown to delay the progression of CKD to some extent. Although cell experiments and animal model experiments have confirmed that these drugs have a certain inhibitory effect on renal fibrosis, they were not initially developed to target the pathological process of renal fibrosis, and the clinical evidence for their anti-fibrotic efficacy is still insufficient; their therapeutic effects in humans require further validation.

[0006] Researchers have developed a series of drugs to slow the progression of chronic kidney disease (CKD) by identifying key targets in the mechanisms of renal fibrosis. Transforming growth factor-β (TGF-β) is a core regulator in the process of renal fibrosis, and antibodies and inhibitors targeting TGF-β (such as fresolimab and phenidone) have entered clinical trials. As a downstream effector of TGF-β, connective tissue growth factor (CTGF) can induce fibroblast activation and ECM deposition. Studies have shown that the anti-CTGF monoclonal antibody FG-3019 can reduce urinary microalbumin levels in patients with diabetic nephropathy, but related clinical trials have been terminated for various reasons. Methylbardoxazolonone is a nuclear factor E2-related factor 2 activator that can improve the estimated glomerular filtration rate (eGFR) in patients with various kidney diseases, including diabetic nephropathy, IgA nephropathy, and focal segmental glomerulosclerosis. However, a phase III clinical trial found that this drug not only failed to effectively reduce the risk of progression to ESRD or cardiovascular death in patients with diabetic nephropathy, but also increased the incidence of cardiovascular events.

[0007] Despite significant progress in research targeting key pathways and targets in the process of renal fibrosis, current clinical treatments for slowing the progression of CKD remain very limited, and no specific anti-renal fibrosis drugs have been approved. Therefore, exploring new potential targets in the renal fibrosis process of CKD patients and studying corresponding intervention strategies are of great clinical significance for improving the prognosis of CKD patients.

[0008] Epigenetics refers to the heritable alteration of biological traits through gene modification without changing the DNA nucleic acid sequence. Given that genetics and epigenetics are deep-seated and reversible mechanisms regulating renal fibrosis, identifying CKD-related epigenetic regulatory factors and exploring targeted intervention strategies holds promise for providing new therapeutic avenues to slow CKD progression.

[0009] In recent years, advancements in technologies such as whole-epigenome association studies have spurred research into regulating and delaying renal fibrosis. However, the drugs currently reported are still in the cell and animal experimental stages and have not yet entered clinical translation. Furthermore, the main mechanisms of action of current potential epigenetic regulatory drugs are concentrated on the regulation of DNA methylation, histone methylation, histone acetylation, and non-coding RNA, lacking effective therapeutic strategies that target specific transcription factors or chromatin regulatory factors. Summary of the Invention

[0010] To address the technical problems existing in the prior art, this invention provides the application of a reagent that inhibits ADNP gene expression or ADNP protein activity in the preparation of a drug for treating renal fibrosis. The technical solution is as follows:

[0011] Application of reagents that inhibit ADNP gene expression or ADNP protein activity in the preparation of drugs for treating renal fibrosis.

[0012] Optionally, the reagent for inhibiting ADNP gene expression or ADNP protein activity includes: siRNA as shown in SEQ ID No. 1 or SEQ ID No. 2, or sgRNA as shown in any one of SEQ ID No. 7 or SEQ ID No. 9.

[0013] Optionally, the reagent that inhibits ADNP gene expression or ADNP protein activity inhibits TGF-β-induced epithelial-mesenchymal transition.

[0014] A pharmaceutical composition for treating renal fibrosis, the pharmaceutical composition comprising: an agent that inhibits ADNP gene expression or ADNP protein activity.

[0015] Optionally, the reagent for inhibiting ADNP gene expression or ADNP protein activity includes: siRNA as shown in SEQ ID No. 1 or SEQ ID No. 2, or sgRNA as shown in any one of SEQ ID No. 7 or SEQ ID No. 9.

[0016] Optionally, the reagent that inhibits ADNP gene expression or ADNP protein activity inhibits TGF-β-induced epithelial-mesenchymal transition.

[0017] A reagent for inhibiting ADNP gene expression or ADNP protein activity, said reagent comprising: siRNA as shown in SEQ ID No. 1 or SEQ ID No. 2, or sgRNA as shown in any one of SEQ ID No. 7 or SEQ ID No. 9.

[0018] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0019] This invention successfully knocked down or eliminated the ADNP gene in HK-2 cells by designing two siRNAs (siRNA1 and siRNA2) and two sgRNAs (sgRNA1 and sgRNA3), effectively inhibiting the expression of EMT and fibrosis-related protein markers. This provides an effective method for knocking down or eliminating the ADNP gene in HK-2 cells, offering a reliable intervention target and approach for the treatment of renal fibrosis. The successful knockdown and elimination of the ADNP gene in HK-2 cells by siRNA1 / 2 and sgRNA1 / 2 serve as key evidence demonstrating their anti-EMT and anti-fibrotic effects.

[0020] This invention effectively knocks down or knocks out the ADNP gene in renal tubular epithelial cells, objectively investigating the effects of ADNP deficiency on a series of phenotypes in HK-2 cells, and confirming that ADNP can inhibit EMT and renal fibrosis in HK-2 cells. Drugs that target and knock down or knock out the ADNP gene in human kidneys can be developed to delay the progression of renal fibrosis. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a graph showing the time effect detection of siRNA knockdown of the ADNP gene in HK-2 cells provided in Example 1 of the present invention, where β-actin refers to β-actin.

[0023] Figure 2 This is a graph showing the effect and quantitative analysis of ADNP knockdown on TGF-β-induced EMT-related protein expression in HK-2 cells, as provided in Example 1 of this invention; wherein, Figure 2 Image A shows the bands representing the expression of ADNP, FN, COL1A1, and E-cadherin proteins in cells of each group, as detected by Western blot. Figure 2 Figure B is a quantitative analysis chart of the expression levels of various proteins, n=3. ns: No statistically significant difference;

[0024] Figure 3 This is a figure showing the effect of ADNP-KO on TGF-β-induced EMT-related protein expression in HK-2 cells, as provided in Example 1 of this invention; wherein, Figure 3 Image A shows the initial screening results of ADNP protein expression in four candidate ADNP-KO cell lines (Western blot image). Figure 3Figure B is a graph showing the verification and quantitative analysis of the knockout effect of ADNP-KO1 and ADNP-KO2 cells; Figure 3 The graph in section C shows the effect of ADNP knockout on the expression of EMT-related proteins in HK-2 cells under TGF-β stimulation, and the quantitative statistical results. (n=3) ns: No significant difference;

[0025] Figure 4 This is a figure showing the effect of ADNP overexpression on TGF-β-induced EMT-related protein expression in HK-2 cells, as provided in Example 1 of this invention; wherein, Figure 4 Figure A shows the Western blot results and quantitative analysis of ADNP protein in ADNP-OE and Ctr group cells; Figure 4 Image B shows the Western blot bands of ADNP, FN, COL1A1 and E-cadherin proteins in cells of the ADNP-OE group and Ctr group under TGF-β induction. Figure 4 Figure C is a quantitative analysis of the results of Western blot detection of ADNP, FN, COL1A1, and E-cadherin proteins in cells of the ADNP-OE group and the Ctr group under TGF-β induction, n=3. There was no significant difference in ns. Detailed Implementation

[0026] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0027] In embodiments of the present invention, words such as "examplely" and "for example" are used to indicate that something is being described as an example, illustration, or illustration. Any embodiment or design described as "example" in the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the word "example" is intended to present the concept in a concrete manner. Furthermore, in embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one.

[0028] In this invention, ADNP refers to Activity-dependent neuroprotective protein.

[0029] Our team previously performed proteomic analysis on obstructed kidney tissues from 11 UPJO patients, 8 normal human kidney tissues, 6 kidney tissues from mice with unilateral ureteral obstruction (UUO), and 6 kidney tissues from mice in the sham-operated group, identifying 1469 differentially expressed proteins. Using a data mining system, we analyzed the expression characteristics of transcription-related factors (TRFs) in the kidneys of UPJO patients and UUO mice, finding that the expression of activity-dependent neuroprotective protein (ADNP) was significantly upregulated in the kidney tissues of UPJO patients and UUO mice compared to normal human kidney tissues and sham-operated mouse kidney tissues.

[0030] ADNP is a neuroprotective protein with nine zinc finger domains and one typical homeobox domain. It functions as a transcription factor and is widely expressed throughout the body, including in the kidneys. Studies have shown that ADNP can directly bind to specific gene loci on chromosomes to regulate gene expression, and can also regulate chromatin accessibility by forming chromatin regulatory complexes. Therefore, ADNP is not only a transcription factor but also a chromatin regulator. Given the high expression of ADNP as an epigenetic regulator in obstructed kidney tissue, the inventors hypothesize that ADNP may play a role in the progression of renal fibrosis. Currently, research on ADNP mainly focuses on embryonic development, neural differentiation, and tumors; its specific functions and related mechanisms remain unclear. Therefore, exploring the role and mechanism of ADNP in renal fibrosis may provide insights into identifying potential intervention targets for renal fibrosis.

[0031] Given the current lack of specific drugs to inhibit renal fibrosis, this invention suppresses renal fibrosis by knocking down or knocking out the ADNP gene in renal tubular epithelial cells, thereby inhibiting EMT in these cells. This invention provides a methodological approach for knocking down and knocking out the ADNP gene and demonstrates that both ADNP knockdown and ANDP knockdown significantly inhibit the expression of EMT and fibrosis-related proteins. It also demonstrates that targeted knockdown or knockout of ADNP can lead to the development of drugs for treating renal fibrosis.

[0032] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0033] Unless otherwise specified, the experimental methods described in the following embodiments are conventional experimental methods well known to those skilled in the art, and are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Where specific conditions are not specified in the experimental methods, they are generally operated under conventional conditions.

[0034] Unless otherwise specified, all materials and reagents described in the following examples are commercially available.

[0035] Example 1

[0036] 1. Technical methods

[0037] 1.1 Knockdown of ADNP gene in HK-2 cells

[0038] To verify the functional role of ADNP in renal tubular cell EMT and renal fibrosis, it is necessary to knock down the ADNP gene in renal tubular cells using RNA interference (RNAi) technology. The specific steps are as follows:

[0039] (1) Preparation of siRNA working solution: ① After receiving the dry powder reagent, centrifuge briefly (4000 rpm, 30~60 s), then carefully and gently open the cap and add enzyme-free ultrapure water to dissolve and mix well. Prepare a 20 μm (i.e., 20 pmol / μL) solution, adding 50 μL of ultrapure enzyme-free water for every nmol; ② Take an enzyme-free EP tube, aliquot the prepared 20 μm stock solution, and store it in a -80℃ freezer for long-term storage, avoiding repeated freeze-thaw cycles. The siRNA sequences used in this experiment are shown in Table 1 and can be purchased from Sangon Biotech.

[0040] Table 1. siRNA sequences corresponding to ADNP gene knockdown in HK-2 cells.

[0041]

[0042] Among them, / dT / / dT / is a common design for chemical modification of siRNA in this field, which refers to adding two consecutive deoxythymidine residues to the 3' end of the sense and antisense strands of siRNA.

[0043] (2) HK-2 cell treatment: ① Take HK-2 cells in logarithmic growth phase (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CL-0109), discard the original culture medium in the culture flask, and wash the cell surface twice with sterile PBS buffer; ② Add an appropriate amount of 0.25% trypsin digestion solution, incubate at 37℃ for 2~3 min, observe cell shrinkage and increased intercellular spaces under an inverted microscope, then add MEM complete culture medium (purchased from EallBio) to terminate digestion; ③ Gently pipette the cells to prepare a single-cell suspension, count the cells with a cell counter, and then divide the suspension into 2×10⁻⁶ cells. 5 Cells / well were seeded at a density of 1.5 mL of MEM medium (without antibiotics) containing 10% FBS in each well.

[0044] (3) Lipofectamine™ RNAiMAX-mediated siRNA transfection of HK-2 cells: ① Prepare 3 enzyme-free EP tubes, labeled NC, siADNP-1, and siADNP-2 respectively; ② Add 1100 ml of Opti-MEM medium (purchased from GIBCO) (500 × 2.2 μL = 1100 μL) to each tube, then add the corresponding siRNA (2 × 2.2 μL = 4.4 μL) to each EP tube, gently vortex for 5 seconds to mix, and let stand at room temperature for 5 min; ③ Add Lipofectamine™ RNAiMAX transfection reagent (purchased from Thermo Fisher Scientific) (6 × 2.2 = 13.2 μL) to each EP tube, gently invert to mix, and let stand at room temperature for 20-25 min to allow the siRNA and transfection reagent to form a stable complex; ④ Slowly add the prepared siRNA-transfection reagent complex to the corresponding wells of a six-well plate (500 ml of Opti-MEM medium (purchased from GIBCO)). μL / well), after adding, gently shake the culture plate to distribute the complex evenly in the culture medium (the amount of transfection complex in each EP tube corresponds to two wells, because the experimental group needs TGF-β stimulation during the experiment, so the NC group and siADNP group each correspond to two wells); ⑤ Place the culture plate back in a 37℃, 5% CO2 constant temperature incubator for 36 h.

[0045] (4) Optimization of the time effect of siRNA knockdown of ADNP gene in HK-2 cells

[0046] To determine the optimal time for siRNA knockdown of the ADNP gene in HK-2 cells, this study included a negative control group (NC) and an ADNP siRNA treatment group (siADNP). ADNP protein expression levels were detected by Western blot at 24 h, 36 h, and 48 h post-transfection. Results showed that ADNP expression in the NC group did not change significantly within 24–48 h; however, in the siADNP group, ADNP expression gradually decreased with increasing transfection time, with the most significant knockdown effect observed at 48 h. For detailed results, please refer to [link to relevant documentation]. Figure 1 Therefore, this study determined that 48 hours is the optimal time for siRNA to knock down the ADNP gene in HK-2 cells.

[0047] 1.2 ADNP gene knockout in HK-2 cells

[0048] This study utilized CRISPR-Cas9 technology to target and knock out the ADNP gene in HK-2 cells. The specific steps are as follows:

[0049] 1.2.1 Construction and extraction of ADNP gene knockout plasmid for HK-2 cells

[0050] 1.2.2 Primer Design

[0051] The Gen ID of human ADNP, 23394, was obtained through NCBI query. This Gen ID was then entered into the website https: / / www.genscript.com.cn / tools / gRNA-design-tool to identify suitable target sites and predict and sequence possible off-target sequences, thus obtaining the sgRNA. The reverse complementary sequence was then quickly obtained using ApE software. Homologous sequences digested with BsmBI were added to both ends of the sequence to obtain the primer sequences. We selected the first four oligonucleotides (Oligonucleotide, Oligo) from the sgRNA design software as the sgRNA for this experiment. The primer sequences are shown in Table 2. They were synthesized by Shanghai Sangon Biotech Co., Ltd., and purified by PAGE.

[0052] Table 2 Human ADNP sgRNA sequences and corresponding primer sequences

[0053]

[0054] In this invention, four sgRNAs were designed during the experiment. This is because reagent experiments typically require at least 3-4 sgRNAs, as some sgRNAs may be off-target or fail to be knocked out. Ultimately, only sgRNA1 and sgRNA3 were screened out in this invention.

[0055] 1.2.3 Annealing of synthesized sgRNA primers

[0056] The synthesized sgRNA upstream and downstream primers were dissolved in ultrapure, enzyme-free water to a final concentration of 10 μM. Annealing samples were prepared according to Table 3. The PCR instrument annealing program was: 37℃ for 30 min, 95℃ for 5 min, followed by slow cooling at 2℃ / min until reaching 25℃.

[0057] Table 3 sgRNA primer annealing samples

[0058]

[0059] 1.2.4 Preparation of linearized lentiCRISPR v2 vector by enzyme digestion

[0060] Prepare single-enzyme digestion vectors using the reagents listed in Table 4. PCR run procedure: 37°C for 2 hours or overnight.

[0061] Table 4. Reagent components and volumes for preparing lentiCRISPR v2 single-enzyme digestion vectors

[0062]

[0063] 1.2.5 Plasmid separation by agarose gel electrophoresis

[0064] (1) Gel preparation: Prepare 1.5%~2% agarose gel, add nucleic acid dye, mix well, pour into gel preparation plate, insert comb and wait for gel to solidify.

[0065] (2) Sample loading: The experimental group sample consisted of 20 μL of lentiCRISPR v2 single-enzyme digested vector, and the control group sample consisted of 20 μL of vector without restriction endonucleases. 4 μL of 6×DNA loading buffer was added to each sample. The gel loading wells were placed sideways to the negative electrode (black electrode) of the electrophoresis tank. 1×TAE was added to the electrophoresis tank until the gel was completely submerged, and then the comb was slowly pulled out. 2 μL of 100 bp DNA ladder was added to the leftmost loading well, followed by 20 μL of each of the experimental and control group samples.

[0066] (3) Electrophoresis: Electrophoresis at a constant voltage of 120 V for 30~40 min. Stop electrophoresis when the bromophenol blue indicator band corresponding to the DNA Marker migrates to the lower part of the gel.

[0067] (4) Development: After electrophoresis, the target gene bands were observed and identified in the Bio-Rad gel imaging system.

[0068] (5) Gel block preparation and weight measurement: Agarose gel blocks containing the target DNA are precisely cut under UV gel cutter, and then placed into a pre-weighed sterile EP tube; weigh again and calculate the net weight of the gel, and use this weight as 1 gel volume (e.g., 100 mg gel corresponds to 100 μL volume).

[0069] (6) Gel dissolution: Add 3 times the gel volume of DNA gel dissolution buffer (Buffer DE-A) to the centrifuge tube, mix thoroughly, and incubate in a 75°C water bath. Invert the centrifuge tube every 2-3 minutes to mix until the gel block is completely melted. Buffer DE-A is a red solution, and the uniformity of the solution color can be used to determine whether the gel is completely dissolved.

[0070] (7) Preparation of binding buffer: Add 0.5 times the volume of Buffer DE-A DNA binding buffer (Buffer DE-B) to the above molten gel system, repeatedly invert the centrifuge tube to mix, at this time the color of the mixture should be uniform yellow, and then centrifuge briefly.

[0071] (8) Negative pressure adsorption of target DNA: Load the DNA preparation tube provided with the kit into the negative pressure pump interface and ensure a tight connection; transfer the mixture from the previous step into the DNA preparation tube, turn on the negative pressure pump and adjust the negative pressure value to -25~-30 inches of mercury, slowly drain the liquid in the tube, so that the target DNA is fully adsorbed onto the DNA binding membrane in the preparation tube.

[0072] (9) Washing impurities: After the liquid in the preparation tube is completely drained, add 500 μL of washing buffer (Buffer W1) and maintain negative pressure to drain; then add 700 μL of washing buffer (Buffer W2), drain, and repeat the washing step once.

[0073] (10) Removal of residual washing solution: Transfer the DNA preparation tube to the 2 mL centrifuge tube provided in the kit and centrifuge at 12000 g for 1 min at room temperature to completely remove the residual washing solution in the tube.

[0074] (11) Drying the conjugate membrane: Discard the waste liquid in the 2 mL centrifuge tube, transfer the preparation tube back to a new 1.5 mL centrifuge tube, keep the cap open and place at room temperature for 2-3 min to allow the residual ethanol on the conjugate membrane to evaporate completely.

[0075] (12) DNA elution and preservation: Add 25 μL of ultrapure enzyme-free water to the center of the conjugate membrane of the preparation tube to ensure that the liquid completely wets the conjugate membrane; tighten the cap of the tube, place it at room temperature for 2 min, centrifuge at 12000 g speed for 1 min, and collect the elution solution at the bottom of the centrifuge tube, which is the purified target DNA fragment.

[0076] 1.2.6 Ligation of sgRNA to enzyme digestion vector

[0077] The annealed sgRNA oligonucleotide fragment was ligated into the enzyme-digested linearized lentiCRISPR v2 vector to construct a recombinant expression plasmid. The specific steps are as follows:

[0078] (1) Preparation of the ligation reaction system: Prepare 10 μL of ligation reaction system according to Table 5.

[0079] Table 5. Connection reaction system (10 μL)

[0080]

[0081] (2) Mix the reaction system: Gently vortex the prepared reaction system for 5 to 10 seconds, and then centrifuge it briefly.

[0082] (3) Connection reaction conditions: Place the centrifuged reaction tube into the PCR instrument and set the reaction program to incubate at 16℃ for 2 h.

[0083] 1.2.7 Transformation of Stbl3 competent cells with recombinant plasmid

[0084] (1) Preparation of solid culture medium: Accurately weigh 1 g of tryptone, 0.5 g of yeast extract, 1.0 g of sodium chloride, and 1.5 g of agar powder, and add them to a 200 mL Erlenmeyer flask. Then add 100 mL of deionized water and stir well. After sealing the Erlenmeyer flask, place it in an autoclave (121℃, 103.4 kPa) and sterilize for 15 min. After sterilization, wait for the culture medium to cool to about 60℃, and under aseptic conditions, add 100 μL of ampicillin solution (stock concentration: 100 μg / μL, final concentration added to the culture medium: 100 µg / mL). Immediately and gently invert the Erlenmeyer flask 3-5 times. Slowly pour the mixed culture medium into 6 cm sterile petri dishes, adding about 20 mL of culture medium to each dish. After the culture medium has completely cooled and solidified, seal the edges of the petri dishes with sterile sealing film and store them upside down in a refrigerator at 4℃.

[0085] (2) Thawing and aliquoting of competent cells: Stbl3 competent cells (purchased from Beyotime) were taken out from the -80℃ freezer and quickly placed on ice to thaw naturally; after thawing, they were aliquoted into pre-cooled EP tubes, 50 μL per tube.

[0086] (3) Binding of ligation product to competent cells: Add 5 μL of ligation product to each tube of competent cells, gently tap the side wall of the centrifuge tube to mix, and then place on ice for 30 min.

[0087] (4) Heat shock and ice bath: Place the centrifuge tubes in a 42℃ constant temperature water bath for 60 seconds and then quickly transfer them to ice and let them stand for 2 minutes.

[0088] (5) Resuscitation culture: Add 600 μL of non-resistant LB liquid medium to the centrifuge tube and place it in a benchtop constant temperature shaker at 37℃ and 160 rpm for 45~60 min.

[0089] (6) Concentration and spreading of bacterial culture: Place the centrifuge tube at room temperature and centrifuge at 12,000 rpm for 1 min to allow the bacteria to precipitate at the bottom of the tube; discard the excess supernatant and retain 50-100 μL of supernatant, and gently resuspend the bacterial precipitate by pipetting; then spread the resuspended bacterial culture evenly on LB solid medium plates containing 100 μg / mL ampicillin resistance.

[0090] (7) Constant temperature incubation: Invert the plate and place it in a 37℃ constant temperature incubator overnight. Observe the growth of single colonies the next day.

[0091] 1.2.8 Screening for recombinant plasmid-positive colonies

[0092] (1) Preparation of liquid culture medium: Weigh 1 g of tryptone, 0.5 g of yeast extract, and 1 g of sodium chloride, add deionized water to a final volume of 100 mL, and stir thoroughly with a magnetic stirrer until all reagents are completely dissolved. Transfer the prepared culture medium to an Erlenmeyer flask, seal it, and place it in an autoclave for 15 min. After sterilization, remove the Erlenmeyer flask and place it in a laminar flow hood to cool naturally to room temperature. Under aseptic conditions, add 100 μL of ampicillin stock solution (the final concentration of ampicillin in the culture medium is 100 μg / mL), and gently invert the Erlenmeyer flask to mix.

[0093] (2) Colony selection and inoculation: Under aseptic conditions, the sterilized ampicillin-resistant LB liquid medium was dispensed into 12 mL sterile shaking tubes, with 3 mL dispensed into each tube. The LB solid medium plate after transformation of the recombinant plasmid was taken and, under aseptic conditions in a clean bench, 3 single colonies with regular morphology, clear edges and no contamination were selected (each colony corresponds to 1 shaking tube). The single colony was gently picked up with a 10 μL sterile pipette tip and inserted into the liquid medium in the corresponding shaking tube. The pipette tip was gently blown 3-5 times to fully disperse the colony in the medium.

[0094] (3) Shaking culture: Place the 12 mL shaking tube after inoculation in a constant temperature shaker, set the culture parameters to 37℃ and 160 rpm, and shake culture for 12~16 h.

[0095] (4) Recombinant plasmid sequencing verification: Under the aseptic conditions of a clean bench, 2 mL of bacterial culture was taken from each amplified bacterial culture, placed into a sterile centrifuge tube, and labeled. The clearly labeled bacterial culture samples were sent to Ribot Biotech for first-generation sequencing to verify whether the sgRNA fragment was correctly inserted into the lentiCRISPR v2 vector and whether there were any sequence mutations. The remaining bacterial culture was sealed and stored in a 4°C refrigerator for a short period of time to avoid contamination or excessive bacterial growth.

[0096] 1.2.9 Scale-up culture of positive bacterial suspension and plasmid extraction

[0097] (1) Preservation of bacteria: After the sequencing results are returned, the recombinant bacterial culture that confirms the correct insertion of the sgRNA fragment and the absence of sequence errors is the positive bacterial culture. The positive bacterial culture is first preserved and then expanded for plasmid extraction.

[0098] (2) Inoculation: Under the sterile environment of the ultra-clean workbench, the positive bacterial solution with correct sequencing was inoculated into an Erlenmeyer flask containing 100 mL of ampicillin selective LB liquid medium at a ratio of 1:100.

[0099] (3) Shaking amplification: Place the inoculated conical flask in a 37℃, 220 rpm benchtop constant temperature shaker and shake for 16~20 h to allow the bacteria to multiply in large quantities to obtain a sufficient amount of recombinant plasmid.

[0100] (4) Large-scale extraction of recombinant plasmids: Plasmids were extracted using the plasmid large-scale extraction kit from ZYMO according to the instructions. The plasmid concentration and purity were tested using a nucleic acid concentration analyzer. After passing the test, the plasmids were aliquoted and stored at -20℃ to avoid repeated freeze-thaw cycles.

[0101] 1.2.10 Lentiviral Packaging and 293T Cell Transfection

[0102] (1) Preparation of PEI transfection reagent: ① Accurately weigh 50 mg of PEI (purchased from Sigma-Aldrich) and place it in a 10 mL sterile centrifuge tube. Add physiological saline (0.9% NaCl) to make up to 10 mg / mL. Place the tube in a 50℃ water bath to aid dissolution. Gently shake the centrifuge tube during this period until the PEI is completely dissolved. ② Take an appropriate amount of 10 mg / mL PEI stock solution and dilute it with physiological saline to make a working solution of 1 mg / mL. After sterilization by filtration through a 0.22 μm sterile filter, dispense it into sterile EP tubes and store them sealed at -20℃ for later use. Avoid repeated freeze-thaw cycles.

[0103] (2) Preparation of plasmids for transfection: Prepare plasmids required for lentivirus packaging, including packaging plasmid psPAX2 (PAX2) (purchased from Addgene), envelope plasmid pMD2.G (VSVG) (purchased from Addgene), and target plasmids (lentiCRISPRv2 empty vector plasmid, lentiCRISPR v2-ADNP sgRNA recombinant plasmid, the latter containing four target sites ADNP-KO1, ADNP-KO2, ADNP-KO3, and ADNP-KO4); all plasmids must be of acceptable purity (OD260 / OD280 ratio 1.8~2.0) and free from nuclease contamination.

[0104] (3) Cell seeding: 18-20 h before transfection, 293T cells in logarithmic growth phase (purchased from Fenghui Biotechnology Co., Ltd.) were seeded into 6-well plates, with 5 × 10⁶ cells per well. 5 Add 2 mL of DMEM complete medium to each cell and incubate in a 37°C, 5% CO2 incubator. The cell confluence during transfection should be controlled at 30%~40% and should not exceed 60% to ensure the best transfection efficiency.

[0105] (4) Change the culture medium before transfection: 1-2 h before transfection, discard the original culture medium in each well of the 6-well plate, add 2 mL of DMEM basic culture medium to each well, and put it back into the 37℃, 5% CO2 incubator for continued culture.

[0106] (5) Preparation of plasmid mixture: ① Plasmid ratio of transfection system: The total amount of plasmid in each transfection system is 2 μg, including 0.8 μg of psPAX2 (PAX2) plasmid, 0.4 μg of pMD2.G (VSVG) plasmid, and 0.8 μg of target plasmid. The specific grouping and plasmid composition are shown in Table 6; ② Calculation of plasmid volume: Calculate the required plasmid volume according to the concentration of each plasmid. See Table 7 for details; ③ Mixing operation: Since psPAX2 and pMD2.G are the basic plasmids shared by all groups, in order to improve the accuracy of sample addition, calculate the total amount of basic plasmid used according to 7 samples (1 sample from the Mock group, 1 sample from the V2 group, and 4 samples from the ADNP-KO group, for a total of 6 samples, with an extra sample volume reserved to avoid loss), that is, (0.25 μL VSVG + 0.44 μL PAX2) × 7 = 4.83 μL, and add it to 2.1 mL. Mix thoroughly in DMEM basal medium; then take 6 sterile EP tubes, aliquot 300 μL of the above mixture into each tube, and add the corresponding target plasmid to each tube (0.84 μL lentiCRISPR v2 plasmid for V2 group, the corresponding volume of recombinant plasmid for ADNP-KO group, and no target plasmid for Mock group), mix gently, and let stand at room temperature for 5 min.

[0107] Table 6 Components of plasmid mixture

[0108]

[0109] Table 7 Plasmid Volume

[0110]

[0111] (6) Preparation of PEI mixture: Calculate the amount of PEI to be used based on 7 sample volumes (4 μL × 7 = 28 μL). Add 28 μL of 1 mg / mL PEI working solution to 2.1 mL of DMEM basal medium, mix gently (vortex for 5~10 s), and let stand at room temperature for 5 min.

[0112] (7) Transfection complex formation: Gently mix the above plasmid mixture with the corresponding volume of PEI mixture (vortex for 5-10 s), and let stand at room temperature for 20-25 min to allow the plasmid and PEI to form a stable transfection complex (600 μL).

[0113] (8) Transfection loading: Gently aspirate the serum-free DMEM medium from each well of the 6-well plate, slowly add the prepared transfection complex to the corresponding well, gently shake the culture plate after adding to make the complex evenly distributed, and incubate in a 37°C, 5% CO2 incubator.

[0114] (9) Culture medium replacement and virus culture: After incubation for 4-6 h, discard the transfection mixture in each well, add 2 mL of fresh DMEM complete culture medium to each well, and continue to culture in a 37℃, 5% CO2 incubator for 48 h.

[0115] (10) Lentiviral Collection and Processing: After viral culture, the cell supernatant containing the virus was collected under sterile conditions. The specific procedures were as follows: ① Consumables preparation: 6 2 mL sterile syringes (needles removed), 6 1.5 mL sterile EP tubes (clearly marked according to the transfection well number to ensure one-to-one correspondence with the Mock group, V2 group, ADNP-KO1, ADNP-KO2, ADNP-KO3 and ADNP-KO4 group), 6 0.45 μm sterile filter membranes (fitted to the 2 mL syringes); ② Environmental preparation: All operations were performed in a biosafety cabinet. The biosafety cabinet was disinfected with ultraviolet light for 30 min in advance to ensure a sterile operating environment; ③ Take one 2 mL sterile syringe with the needle removed, insert it into the corresponding well of the 6-well plate, and slowly aspirate all the cell supernatant from the well; ④ Install the 0.45 μm sterile filter membrane at the syringe outlet, slowly push the syringe plunger, and inject the filtered cell supernatant into the corresponding marked sterile EP tube; pass through the 0.45 μm sterile filter membrane. μm filter membrane filtration can effectively remove cell debris and residual cells to obtain purified virus solution; ⑤ Complete the virus collection and filtration of all transfection wells in sequence according to the above steps. Replace one set of syringes and filter membranes for each sample to avoid cross-contamination; ⑥ If the collected virus solution needs to be used immediately, it can be stored on ice for a short period of time; if it is not used immediately, it should be quickly aliquoted into sterile EP tubes, labeled with the virus name and collection date, and stored in a -80℃ freezer for long-term storage to avoid repeated freeze-thaw cycles; ⑦ Waste disposal: All consumables that have come into contact with the virus (including used syringes, filter membranes, centrifuge tubes, etc.) must be collected in a dedicated biohazard waste container, sterilized by high-pressure steam at 121℃ and 103.4 kPa for 30 min, and then disposed of in accordance with medical waste regulations to prevent virus contamination.

[0116] 1.2.11 Lentiviral infection of HK-2 cells

[0117] (1) Cell plating: One day before infection, HK-2 cells in logarithmic growth phase (purchased from Pronosei Life Sciences Co., Ltd.) were plated at a concentration of 2 × 10⁶ cells / cells. 5 Cells were seeded at a density of approximately 50% per well in 6-well plates, with 2 mL of MEM complete culture medium (purchased from EallBio) added to each well. The plates were then incubated overnight in a 37°C, 5% CO2 incubator to ensure that the cell density reached approximately 50% at infection.

[0118] (2) Virus preparation: Take out the collected lentivirus solutions from the -80℃ freezer and quickly place them at room temperature to thaw naturally. During the thawing process, gently invert the EP tube 2-3 times to ensure that the virus solution is mixed evenly and avoid violent shaking that could damage the virus particles. After thawing, place it on ice for short-term storage.

[0119] (3) Viral infection: ① Take out the HK-2 cells incubated one day ago in a 6-well plate and observe the cell state under an inverted microscope to confirm that the cell density reaches 50% and the morphology is good; then make clear marks on the culture plate to identify the virus group corresponding to each well; ② Use a sterile pipette to remove the MEM complete culture medium in each well and add the virus stock solution to the corresponding well according to the marking (the virus solution yield per well of the 6-well plate is about 1~2 mL, and all of it is added at once to ensure the multiple of infection); ③ After adding the virus, gently shake the 6-well plate to make the virus solution evenly cover the cell surface, and incubate it in a 37℃, 5% CO2 constant temperature cell culture incubator.

[0120] (4) Culture medium replenishment and subsequent screening: ① After incubation for 6 h, add 500 μL of MEM complete culture medium to each well and continue to culture in a 37℃, 5% CO2 incubator for 48 h to provide nutritional support for the cells and promote viral integration. ② After 48 h of culture, screening can be performed using 1.1 μg / mL puromycin to obtain stably transfected HK-2 cell lines.

[0121] 1.2.12 Selection of stable transfected cells using puromycin

[0122] (1) Replacement of selection medium: 48 h after lentivirus transduction of HK-2 cells, in a biosafety cabinet, aspirate the complete MEM medium in each well of a 6-well plate and add 2 mL of MEM selection medium containing puromycin (1.1 μg / mL) (containing 10% FBS and 1% penicillin) to each well to ensure that the selection medium evenly covers the cell surface. Incubate in a 37°C, 5% CO2 constant temperature cell culture incubator.

[0123] (2) Regular replacement of screening medium: In order to maintain the effective screening concentration of puromycin, fresh MEM screening medium containing 1.1 μg / mL puromycin should be replaced every 2-3 days under sterile conditions.

[0124] (3) Cell survival status monitoring and screening endpoint determination: ① From the addition of the screening medium, observe and record the survival status, growth ratio and morphological changes of cells in each well daily under an inverted microscope, focusing on distinguishing between surviving adherent cells and floating dead cells; the optimal screening period for puromycin is 5-7 days, and continuous monitoring is required until the screening endpoint. ② After 7 days of screening, focus on observing the cell status of the Mock group. When almost all normal cells in the Mock group die (survival rate ≤5%), while there are still adherent and surviving cells in the experimental group, it indicates that the surviving cells have successfully integrated the lentiCRISPR v2 vector, reaching the screening endpoint.

[0125] (4) Stable cell line passage culture: During the passage culture process, puromycin (final concentration 1.1 μg / mL) needs to be continuously added to the complete culture medium to maintain the selection pressure and ensure the stability of the cell line.

[0126] (5) Identification of stable cell lines: The expression level of ADNP protein was detected by Western blot.

[0127] 1.3 Overexpression of ADNP gene in HK-2 cells

[0128] 1.3.1 Primer Design:

[0129] Log in to NCBI (https: / / www.ncbi.nlm.nih.gov / datasets / gene / 23410 / ) to retrieve and obtain the gene sequence of human ADNP, as shown in Table 8.

[0130] Table 8 ADNP gene sequence of HK-2 cells

[0131]

[0132] 1.3.2 PCR amplification of the ADNP target gene:

[0133] (1) Prepare a 50 μL reaction system according to Table 9. The order of adding each component is ddH2O, 2×PCR mix, ADNPcDNA template, and specific primers to avoid premature contact of enzymes with high concentrations of primers / templates, which may affect their activity.

[0134] Table 9 50 μL reaction system

[0135]

[0136] (2) Mixing and centrifuging the reaction system: Gently vortex the prepared PCR reaction system for 10-15 seconds to mix, and then centrifuge briefly.

[0137] (3) PCR amplification program: 94℃ for 4 min (pre-denaturation), then 94℃ for 30 s (denaturation), 55℃ for 30 s (annealing), 72℃ for 60 s for 30 cycles, and after the cycle, 72℃ for 10 min (extension), 4℃ (∞).

[0138] (4) Product preservation: After amplification, the PCR product is stored at 4°C for a short period of time. If long-term preservation or subsequent purification is required, it should be aliquoted and stored at -20°C to avoid repeated freeze-thaw cycles.

[0139] 1.3.3 Preparation of linearized vector pLVX-IRES-puro by enzymatic digestion

[0140] (1) Preparation of double enzyme digestion reaction system: Double digestion was performed using restriction endonucleases BamHI and EcoRI. A 20 μL reaction system was prepared according to the following ratio, and the specific components are shown in Table 10:

[0141] Table 10. Double enzyme digestion reaction system (20 μL)

[0142]

[0143] (2) Enzyme digestion reaction operation: ① Mix the system: Gently vortex the prepared double enzyme digestion reaction system for 5-10 seconds to mix, and then perform instantaneous centrifugation to collect the residual liquid on the tube wall to the bottom of the tube to ensure that the reaction components are evenly distributed; ② Enzyme digestion reaction conditions: Place the centrifuged reaction tube in a constant temperature metal bath, set the temperature to 37℃ and the incubation time to 2 h; ③ Reaction termination: After the enzyme digestion is completed, it can be placed in a 65℃ water bath for 10 min to inactivate the restriction endonuclease.

[0144] (3) Separation by agarose gel electrophoresis and purification by gel extraction after enzyme digestion: The process is the same as that for ADNP gene knockout. Please refer to the relevant description.

[0145] 1.3.4 Recombination reaction

[0146] (1) System preparation: Precisely prepare 10 μL of the recombinant reaction system in a sterile EP tube. The specific components are shown in Table 11.

[0147] Table 11 10 μL Recombinant Reaction System

[0148]

[0149] (2) Reaction operation: Gently vortex the prepared recombination reaction system for 5-10 s to mix it, then instantly centrifuge to collect the residual liquid on the tube wall to the bottom of the tube, and place the EP tube in a 50℃ constant temperature water bath for 5-15 min to ensure that the recombination reaction is fully carried out; after the incubation, place the EP tube on ice to cool for 5 min to terminate the reaction.

[0150] The subsequent processes, including recombinant plasmid transformation of competent cells (E.coli DH5α), positive colony screening and amplification by shaking, large-scale extraction of recombinant plasmid, lentivirus packaging, lentivirus collection and filtration, lentivirus infection of target cells, puromycin screening for stable cell lines, and identification of stable cell lines, are the same as those for ADNP gene knockout.

[0151] 2. Results

[0152] 2.1 ADNP knockdown inhibits TGF-β-induced EMT in HK-2 cells

[0153] To clarify the regulatory function of ADNP in TGF-β-induced EMT in HK-2 cells, this study included a negative control (NC) group, a group transfected with non-specific siRNA sequences, and an ADNP-targeting siRNA knockdown group (siADNP-1 and siADNP-2 correspond to different siRNA sequences). Six subgroups were further established based on the presence or absence of TGF-β stimulation: NC+PBS, siADNP-1+PBS, siADNP-2+PBS, NC+TGF-β, siADNP-1+TGF-β, and siADNP-2+TGF-β. The expression levels of ADNP and EMT-related proteins were detected and quantified using Western blot. Experimental results are shown below. Figure 2 .

[0154] The experimental results showed that, regardless of whether TGF-β stimulation was administered, ADNP protein expression in the siADNP-1 and siADNP-2 groups was significantly downregulated compared to the NC group (P<0.001 or P<0.0001), demonstrating that the ADNP knockdown effect was stable and significant. Compared with the PBS-treated NC group, ADNP protein expression was significantly upregulated in the NC+TGF-β group (P<0.001), while the expression of fibrosis-related proteins FN and COL1A1 was significantly upregulated (P<0.001), and the expression of the epithelial marker E-cadherin was significantly downregulated (P<0.001), suggesting that TGF-β successfully induced EMT in HK-2 cells. Compared with the NC+TGF-β group, the expression of FN and COL1A1 proteins in the siADNP-1+TGF-β and siADNP-2+TGF-β groups was significantly downregulated (P<0.01), while the expression of E-cadherin protein was significantly upregulated (P<0.01). Moreover, the regulatory trends of the two independent siRNAs were completely consistent, further verifying the reliability of the results.

[0155] The above results indicate that knocking down the ADNP gene can effectively block TGF-β-induced EMT phenotypic transformation in HK-2 cells, confirming that ADNP is a key promoting factor in TGF-β-mediated EMT process.

[0156] 2.2 ADNP knockout inhibits TGF-β-induced EMT in HK-2 cells

[0157] This study utilized CRISPR-Cas9 technology to target and knock out the ADNP gene in HK-2 cells. Four sgRNAs corresponding to Oligo genes were designed, and after plasmid construction, cell transfection, and puromycin screening, four candidate ADNP-KO cell lines were obtained. The knockout effect was preliminarily verified by Western blot; the experimental results are shown below. Figure 3 .

[0158] The results showed that ADNP protein expression was most significantly downregulated in cell lines 1 and 3. (See details...) Figure 3 A. From Figure 3 As shown in Figure A, the first third of the four sgRNAs designed in this invention can effectively knock out ADNP. To further confirm the knockout stability, Western blot analysis and quantitative analysis were performed on these two cell lines again. Because subsequent experiments will use these two sgRNAs for verification, they were renamed ADNP-KO1 and ADNP-KO2. The results confirmed that ADNP expression has been stably lost, and they were named ADNP-KO1 and ADNP-KO2. See Figure A for details. Figure 3 B.

[0159] To clarify the regulatory role of ADNP in TGF-β-induced EMT in HK-2 cells, this study used empty vector-transfected cells as the negative control group (Ctr group) and ADNP stable knockout cell lines (ADNP-KO1 and ADNP-KO2) as the experimental groups. Both PBS treatment and TGF-β stimulation were set up. The expression changes of related molecules were detected by Western blot and protein quantification analysis. The results showed that, without TGF-β stimulation, the expression of ADNP in the ADNP-KO1 and ADNP-KO2 groups was significantly downregulated compared with that in the Ctr group, while the expression of EMT-related proteins such as FN, COL1A1, and E-cadherin showed no significant difference. Under TGF-β stimulation, the expression of ADNP in the Ctr group was significantly upregulated (P<0.01), while the expression levels of fibrosis-related proteins FN and COL1A1 were significantly increased (P<0.001 or P<0.0001), and the expression of the epithelial marker E-cadherin was significantly downregulated (P<0.0001), which further verified the induction effect of TGF-β on EMT in HK-2 cells. When TGF-β stimulation was applied to ADNP knockout cells, compared with the Ctr+TGF-β group, the expression of FN and COL1A1 in the ADNP-KO1+TGF-β and ADNP-KO2+TGF-β groups significantly decreased (P<0.01 or P<0.001), while the expression of E-cadherin significantly increased (P<0.05 or P<0.001). See details... Figure 3 C.

[0160] The above results are consistent with the conclusions of the previous ADNP knockdown experiment, further clarifying that ADNP is a promoting factor for TGF-β-induced EMT in HK-2 cells, and that targeted knockout of ADNP can effectively inhibit the TGF-β-induced transformation of HK-2 cells to the EMT phenotype.

[0161] 2.3 ADNP overexpression promotes TGF-β-induced EMT in HK-2 cells

[0162] To further clarify the positive regulatory role of ADNP in the EMT process of HK-2 cells, this study constructed an ADNP overexpression recombinant plasmid using the pLVX-IRES-pur0 vector. After lentiviral packaging and transfection into HK-2 cells, stable cell lines were obtained through puromycin selection (ADNP-OE group). Cells transfected with the empty vector served as a negative control (Ctr group). The experimental results are shown in [link to experimental results]. Figure 4 .

[0163] Western blot analysis and quantitative analysis showed that the expression level of ADNP protein in the ADNP-OE group was significantly upregulated compared with that in the Ctr group (P<0.05), indicating that the overexpression cell line was successfully constructed. See details... Figure 4 A.

[0164] To further elucidate the regulatory role of ADNP in EMT, this study used the Ctr and ADNP-OE groups as experimental groups, simultaneously setting up PBS treatment and TGF-β stimulation subgroups. Western blot was used to detect the protein expression levels of EMT-related markers. Without TGF-β stimulation, there were no significant differences in the expression of EMT-related proteins such as FN, COL1A1, and E-cadherin between the Ctr and ADNP-OE groups. After TGF-β stimulation, the expression of fibrosis-related proteins FN and COL1A1 was significantly upregulated in the Ctr group (P<0.01), while the expression of the epithelial marker E-cadherin was significantly downregulated (P<0.01), confirming that TGF-β successfully induced EMT in HK-2 cells. Compared to the Ctr+TGF-β group, the ADNP-OE+TGF-β group showed further significant upregulation of FN and COL1A1 expression (P<0.01), and further significant downregulation of E-cadherin expression (P<0.05). See details... Figure 4 B and C in the middle.

[0165] The above results indicate that overexpression of ADNP can significantly exacerbate the TGF-β-induced EMT phenotype in HK-2 cells, which contradicts the previous finding that "knockdown or knockout of ADNP can inhibit EMT," further confirming that ADNP is a key promoting factor in TGF-β-mediated EMT process.

[0166] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Application of reagents that inhibit ADNP gene expression or ADNP protein activity in the preparation of drugs for treating renal fibrosis.

2. The application according to claim 1, characterized in that, The reagents for inhibiting ADNP gene expression or ADNP protein activity include: siRNA as shown in SEQ ID No. 1 or SEQ ID No. 2, or sgRNA as shown in any one of SEQ ID No. 7 or SEQ ID No.

9.

3. The application according to claim 1, characterized in that, The reagent that inhibits ADNP gene expression or ADNP protein activity inhibits TGF-β-induced epithelial-mesenchymal transition.

4. A pharmaceutical composition for treating renal fibrosis, characterized in that, The pharmaceutical composition includes: a reagent that inhibits ADNP gene expression or ADNP protein activity.

5. The pharmaceutical composition according to claim 4, characterized in that, The reagents for inhibiting ADNP gene expression or ADNP protein activity include: siRNA as shown in SEQ ID No. 1 or SEQ ID No. 2, or sgRNA as shown in any one of SEQ ID No. 7 or SEQ ID No.

9.

6. The pharmaceutical composition according to claim 1, characterized in that, The reagent that inhibits ADNP gene expression or ADNP protein activity inhibits TGF-β-induced epithelial-mesenchymal transition.

7. A reagent for inhibiting ADNP gene expression or ADNP protein activity, characterized in that, The reagents include: siRNA as shown in SEQ ID No. 1 or SEQ ID No. 2, or sgRNA as shown in any one of SEQ ID No. 7 or SEQ ID No. 9.

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