Application of UHRF1 inhibitor in preparation of medicine for treating tissue fibrosis after spinal cord injury

Abstract

The invention relates to application of a UHRF1 inhibitor in preparation of a medicine for treating tissue fibrosis after spinal cord injury, and belongs to the technical field of biological medicine. The invention proves that the expression of UHRF1 can be increased due to spinal cord injury; compared with mice of a false operation group, the mice of which the UHRF1 is specifically knocked down in the fibroblasts have the advantages that fibrosis caused by spinal cord injury is obviously reduced, nerve regeneration is promoted, and the motor function is obviously improved. In the aspect of mechanism, the knock-down of UHRF1 leads to the reduction of the number of proteins combined on a PPM1A gene promoter, the weakening of the transcription inhibition effect and the promotion of the expression of the PPM1A gene, while the PPM1A can inhibit the phosphorylation of Smad2 / 3 and inhibit the conduction of a TGF-beta1 / Smads pathway, thereby inhibiting tissue fibrosis.

Description

Technical Field

[0001] The invention relates to application of a UHRF1 inhibitor in preparing a medicine for treating tissue fibrosis after spinal cord injury, and belongs to the technical field of biomedicine. Background Art

[0002] Spinal cord injury (SCI) is a serious trauma to the central nervous system, with high morbidity, high disability rate, and low age of onset. There are about 1 million new cases of spinal cord injury every year in the world. Currently, there is a lack of effective treatment for the disease, and varying degrees of limb paralysis bring heavy burdens to patients and their families. Therefore, there is a huge social demand for the recovery of neurological function after spinal cord injury.

[0003] In the early stage of spinal cord injury, wound closure requires the recruitment of fibroblasts and the deposition of fibrotic extracellular matrix, but they ultimately lead to the formation of persistent scar tissue. The mainstream view at home and abroad is that the formation of fibrous scars at the tissue level in the unbalanced microenvironment after spinal cord injury is a key pathological event, and mature fibrous scar tissue is the main reason for preventing axon regeneration and the inability of new axons to cross the injured area. Accurate and targeted regulation of the fibrous scar formation process after spinal cord injury to achieve axon regeneration and recovery of neurological function has great therapeutic significance.

[0004] Activation of the TGF-β1 / Smads pathway can cause fibroblast proliferation, differentiation and secretion of extracellular matrix-related proteins, ultimately leading to the occurrence of fibrotic diseases. TGF-β1 first binds to the TβRII receptor on the cell membrane and phosphorylates it. TβRI is recruited by the phosphorylated TβRII, combined into a multimer and activated. The activated TβRI phosphorylates Smad2 / 3 in the cell to form p-Smad2 / 3, which separates from the receptor and forms a complex with Smad4 to enter the cell nucleus, where it regulates the transcription of target genes and produces biological effects. At the same time, Smad6 and 7 can antagonize the signals of p-Smad2 / 3 and Smad4, playing a negative feedback regulatory role (Chen Xiqi, Zhang Xiaoshuang, Zhou Yongkun, Zhu Yong. Research progress of TGF-β1 / Smads signaling pathway in fibrotic diseases [J]. Chinese Journal of Integrated Traditional and Western Medicine Surgery, 2021, 27(2): 351-354. DOI: 10.3969 / j.issn.1007-6948.2021.02.037). Chinese patent document CN104232640A (application number CN201410320910.8, application date 2014-07-07) discloses a siRNA molecule that inhibits the expression of the Smad4 gene. The siRNA molecule can specifically bind to the complementary sequence of the mRNA coding region of the Smad4 gene, and efficiently silence the Smad4 gene by degrading mRNA, thereby performing post-transcriptional regulation on the expression of the Smad4 gene. The change in the expression of Smad4 directly affects the TGF-β1 / Smads pathway, thereby affecting the proliferation and fibrosis of joint fibroblasts, and ultimately has a positive impact on the treatment of joint diseases related to joint fibrosis such as frozen shoulder.

[0005] The protein encoded by the UHRF1 gene is an important protein involved in epigenetic regulation and plays an important role in cell cycle regulation and tumor development. In addition, Demin Cheng et al. explored how UHRF1 affects the process of pulmonary fibrosis by directly regulating the proliferation-related gene beclin 1 in lung fibroblasts (Cheng D, Wang Y, Li Z, Xiong H, Sun W, Xi S, Zhou S, Liu Y, Ni C. Liposomal UHRF1 siRNA shows lungfibrosis treatment potential through regulation of fibroblast activation. JCI Insight. 2022 Nov 22; 7(22): e162831. doi: 10.1172 / jci.insight.162831 IF: 6.3B1. PMID: 36166308; PMCID: PMC9746815). Pulmonary fibrosis is a chronic progressive interstitial lung disease, while spinal cord injury is an acute destructive disease caused by violence, and their pathogenesis and fibrosis process are completely different.

[0006] There are no reports on the effect of UHRF1 on the TGF-β1 / Smads pathway. There is no research on the regulatory effect of UHRF1 on the fibrosis process after spinal cord injury. Therefore, clarifying the role of UHRF1 in the fibrosis process after spinal cord injury, providing new targets and experimental basis for the treatment of spinal cord injury, and designing simple and efficient drug preparations are of great clinical significance. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides the use of an inhibitor of UHRF1 in the preparation of a drug for treating tissue fibrosis after spinal cord injury, thereby providing a new molecular target for the treatment of spinal cord injury.

[0008] The technical solution of the present invention is as follows:

[0009] Use of UHRF1 inhibitors in the preparation of drugs for treating tissue fibrosis after spinal cord injury.

[0010] Preferably according to the present invention, the UHRF1 inhibitor can inhibit the tissue fibrosis pathway TGF-β1 / Smads pathway by inhibiting the expression of UHRF1, inhibit Smad2 / 3 phosphorylation, and reduce the degree of tissue fibrosis after spinal cord injury.

[0011] Preferably according to the present invention, the UHRF1 inhibitor increases the expression level of protein serine / threonine phosphatase PPM1A by inhibiting the expression of UHRF1, thereby inhibiting tissue fibrosis.

[0012] Further preferably, the UHRF1 inhibitor weakens the binding of UHRF1 protein to the promoter region of protein serine / threonine phosphatase PPM1A gene by inhibiting the expression of UHRF1, thereby promoting the transcription of PPM1A.

[0013] Preferably according to the present invention, the UHRF1 inhibitor comprises siRNA interfering with UHRF1 expression, or a virus packaged with shRNA interfering with UHRF1 expression.

[0014] Further preferably, the nucleotide sequence of the siRNA is as shown in SEQ ID NO.3.

[0015] Further preferably, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.2.

[0016] Further preferably, the virus is adeno-associated virus type 9, and the viral vector is pAV-mFSP1-GFP-mir30-shUHRF1, wherein mFSP1 is a fibroblast-specific promoter, and its nucleotide sequence is shown in SEQ ID NO.1; shUHRF1 is shRNA that interferes with UHRF1 expression.

[0017] A drug for treating tissue fibrosis after spinal cord injury, comprising an inhibitor of UHRF1.

[0018] Preferably according to the present invention, the UHRF1 inhibitor comprises siRNA interfering with UHRF1 expression, or a virus packaged with shRNA interfering with UHRF1 expression.

[0019] Further preferably, the nucleotide sequence of the siRNA is as shown in SEQ ID NO.3.

[0020] Further preferably, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.2.

[0021] Further preferably, the virus is adeno-associated virus type 9, and the viral vector is pAV-mFSP1-GFP-mir30-shUHRF1, wherein mFSP1 is a fibroblast-specific promoter, and its nucleotide sequence is shown in SEQ ID NO.1; shUHRF1 is shRNA that interferes with UHRF1 expression.

[0022] Beneficial effects:

[0023] 1. The present invention confirms that spinal cord injury can lead to increased expression of UHRF1; compared with mice in the sham operation group, mice with specific knockdown of UHRF1 in fibroblasts significantly reduced fibrosis caused by spinal cord injury and promoted nerve regeneration, and motor function was significantly improved. In terms of mechanism, the knockdown of UHRF1 leads to a decrease in the number of its proteins bound to the promoter of the PPM1A gene, resulting in a weakening of its transcriptional inhibition effect, which promotes the expression of the PPM1A gene. PPM1A can inhibit the phosphorylation of Smad2 / 3 and the conduction of the TGF-β1 / Smads pathway, thereby inhibiting tissue fibrosis.

[0024] 2. The present invention constructs an adeno-associated virus vector for fibroblast-specific UHRF1 knockdown. The vector is appropriately selected in terms of the basic vector, target fragment and insertion position. Animal experiments have confirmed that it can significantly inhibit the process of fibrosis, enhance axon regeneration, and improve motor function recovery, and has good application prospects.

[0025] 3. This invention proposes for the first time that UHRF1 can regulate the TGF-β1 / Smads pathway, laying a good foundation for future research on other disease models. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a bar graph showing the expression level of UHRF1 detected by RNA-seq in spinal cord tissue.

[0027] Figure 2 It is a bar graph of the mRNA expression level of UHRF1 in spinal cord tissue.

[0028] Figure 3 This is a Western Blot detection image of UHRF1 protein in spinal cord tissue.

[0029] Figure 4 These are immunofluorescence images of spinal cord tissue.

[0030] Figure 5 This is a bar graph showing the mRNA expression levels of fibrosis indicators acta2, col1a1, and fn1 after knocking down UHRF1 in spinal cord tissue.

[0031] Figure 6 This is a bar graph showing the mRNA expression levels of axon regeneration indicators ngf, bdnf, and gdnf after knocking down UHRF1 in spinal cord tissue.

[0032] Figure 7 This is the BMS evaluation score curve of mice after knocking down UHRF1 in spinal cord tissue.

[0033] Figure 8This is a bar graph of the mRNA expression levels of fibrosis indicators acta2, col1a1, and fn1 after knocking down UHRF1 in meningeal fibroblasts.

[0034] Fig. 9 This is a Western Blot detection of P-Smad2 / 3 protein after knocking down UHRF1 in meningeal fibroblasts and stimulating it with TGF-β1.

[0035] Fig.10 This is an analysis diagram of the binding between UHRF1 and PPM1A gene promoter in CUT&TAG sequencing.

[0036] Fig.11 This is the CHIP-qRCR verification result of UHRF1 protein binding to the PPM1A gene promoter.

[0037] Fig.12 Bar graph showing the mRNA expression level of PPM1A after knockdown of UHRF1 in meningeal fibroblasts.

[0038] Fig.13 This is a Western Blot detection of PPM1A protein after knocking down UHRF1 in meningeal fibroblasts.

[0039] Fig.14 This is a bar graph of the mRNA expression levels of fibrosis indicators acta2, col1a1, and fn1 after knocking down PPM1A in meningeal fibroblasts. DETAILED DESCRIPTION

[0040] The technical scheme of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and consumables involved in the embodiments, unless otherwise specified, are all common commercially available products; the steps and experimental operations involved in the embodiments, unless otherwise specified, are all conventional technical operations in the art.

[0041] Example 1: Increased expression of UHRF1 in fibroblasts after spinal cord injury

[0042] 1.1. Construction of spinal cord injury model

[0043] Clean the operating table and sterilize the required surgical instruments by high-pressure steam sterilization in advance. Prepare sterile surgical drapes, sutures, suture needles, and iodine. Fast the mice 8 hours before surgery to avoid vomiting or aspiration during surgery. Prepare 3% pentobarbital solution with distilled water and inject the mice intraperitoneally at a dose of 30 mg / kg for anesthesia. No response to clamping the limbs indicates complete anesthesia. Shave the hair on the back of the mouse to expose the surgical area. Fix the mouse on the operating table in a prone position to ensure that the position of the mouse is stable during surgery. Use iodine cotton balls to disinfect the surgical area to reduce the risk of contamination during surgery. Spread a disposable sterile surgical drape, make a 1.5 cm incision along the midline of the back at the T9 segment of the spine, and bluntly separate the muscles and fascia layer by layer until the spinous process is exposed. Perform T9 laminectomy under a stereomicroscope using fiber forceps to expose the spinal cord. The mice in the sham group were not treated for spinal cord injury after laminectomy. The injured area was cleaned with sterile saline, the surgical incision was sutured layer by layer with sterile sutures, and disinfected with iodine cotton balls. The spinal cord of the mice in the SCI group was struck by a pneumatic spinal cord injury striker (68100, RWD, Shenzhen, China) with a strike speed of 1m / s, a strike depth of 2mm, and a striker residence time of 1s. The cleaning, suturing, and disinfection steps and operations were the same as those in the sham group. The mice were placed on a heating blanket for 2-3 hours and placed in a cage after they woke up (the mice had free access to food and water in the cage). To prevent urinary tract infection in mice, the mice were artificially squeezed twice a day to help urinate until they were killed.

[0044] 1.2 Transcriptomics sequencing

[0045] The mice in the sham group and the SCI group were anesthetized by intraperitoneal injection of 3% pentobarbital solution on the 7th day after injury and then killed, and the spinal cords at the injured site were obtained for transcriptomic sequencing. The transcriptomic sequencing service was provided by Lianchuan Biotechnology Co., Ltd. The specific steps are as follows: use TRIzol reagent (thermofisher, 15596018) to separate and purify the RNA of the total sample according to the operation plan provided by the manufacturer; then use NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) to control the amount and purity of the total RNA and use Bioanalyzer 2100 (Agilent, CA, USA) to detect the integrity of the RNA; the concentration>50ng / μL, RIN value>7.0, and total RNA>1μg meet the requirements of downstream experiments; use oligo(dT) magnetic beads (DynabeadsOligo(dT), cat.25-61005, Thermo Fisher, USA) to specifically capture the mRNA containing Poly A (polyadenylic acid) through two rounds of purification; the captured mRNA is subjected to high temperature conditions using a magnesium ion fragmentation kit (NEBNextR Magnesium RNA Fragmentation Kit) Module, cat.E6150S, USA) for fragmentation and reacted at 94°C for 5-7 minutes; the fragmented RNA was synthesized into cDNA by reverse transcriptase (Invitrogen SuperScriptTMII ReverseTranscriptase, cat.1896649, CA, USA); then E. coli DNA polymeraseI (NEB, cat.m0209, USA) and RNase H (NEB, cat.m0297, USA) were used for duplex synthesis to convert the DNA and RNA composite duplex into DNA duplex, and dUTP Solution (Thermo Fisher Scientific) was incorporated into the duplex. Fisher, cat. R0133, CA, USA), the ends of the double-stranded DNA were filled to be blunt ends, and an A base was added to each end to enable it to be connected to the adapter with a T base at the end, and the fragment size was screened and purified using magnetic beads; the second strand was digested with UDG enzyme (NEB, cat. m0280, MA, US), and then PCR (pre-denaturation at 95°C for 3 minutes, denaturation at 98°C for 15 seconds for a total of 8 cycles, annealing to 60°C for 15 seconds, extension at 72°C for 30 seconds, and finally 72°C for 5 minutes) was used to form a library with a fragment size of 300 bp ± 50 bp (strand-specific library); finally, using Illumina NovaseqTM 6000 (LC BioTechnology CO., Ltd.Hangzhou, China) performed double-end sequencing according to standard operations, and the sequencing mode was PE150; after obtaining the sequencing data, the data was first filtered to obtain high-quality sequencing data (CleanData), and then the high-quality sequencing data was aligned to the reference genome of the species in this project, and gene expression quantification, GSEA analysis, gene difference analysis, enrichment analysis, etc. were performed.

[0046] RNA-seq data showed that Figure 1 Compared with the sham group, the mRNA level of UHRF1 in the SCI group mice was significantly increased on the 7th day after SCI.

[0047] 1.3. Preliminary experimental verification

[0048] The mice in the sham group and the SCI group were anesthetized by intraperitoneal injection of 3% pentobarbital solution on the 7th day after spinal cord injury, and the spinal cords at the injured site were used to detect the expression levels of UHRF1 mRNA and protein and their cellular localization by qPCR, Western Blot and immunofluorescence.

[0049] The qPCR results showed that Figure 2 The results were consistent with those of RNA-seq detection. Compared with the sham group, the mRNA level of UHRF1 in the SCI group mice was significantly increased on the 7th day after spinal cord injury.

[0050] Western Blot and immunofluorescence assay results Figure 3 and Figure 4 , which confirmed that the protein expression level of UHRF1 reached a peak on the 7th day after SCI, and that UHRF1 protein was highly co-localized with COL1A1-labeled fibroblasts in the spinal cord tissue.

[0051] Example 2: In vivo functional experiments after knockdown of UHRF1

[0052] The construction of cell-specific UHRF1 knockdown adeno-associated virus type 9 (AAV9) was completed by Weizhen Biotechnology Co., Ltd. sh-UHRF1 group: vector pAV-mFSP1-GFP-mir30-shUHRF1, where mFSP1 is a fibroblast-specific promoter, shUHRF1 is a shRNA that knocks down UHRF1, and the virus titer is 5.3×10 13 viral genomes / mL; sh-NC group: vector pAV-mFSP1-GFP-mir30 with nonsense sequence inserted, viral titer was 4.7×10 13 viral genomes / mL.

[0053] Wherein, the nucleotide sequence of mFSP1 is shown in SEQ ID NO.1;

[0054] The nucleotide sequence of shUHRF1 is shown in SEQ ID NO.2.

[0055] AAV9 was injected intrathecally into mice in the sh-UHRF1 group and the sh-NC group using a microsyringe at a dose of 2 μL / mouse. The spinal cord injury model was established 3 weeks later. The mice were then divided into 4 groups: sh-UHRF1+SCI group, sh-UHRF1+sham group, sh-NC+SCI group, and sh-NC+sham group. The BMS score was used to evaluate the recovery of hindlimb motor function on days 0, 1, 3, 7, 14, 21, 28, and 35 after spinal cord injury. Then, the mice in the 4 groups were killed after intraperitoneal injection of 3% pentobarbital solution on day 35 for anesthesia. The spinal cord at the injury site was taken for qPCR to evaluate fibrosis indicators (acta2, col1a1, fn1) and nerve regeneration indicators (ngf, bdnf, gdnf).

[0056] BMS score: It is suitable for evaluating the recovery of motor function of mice after spinal cord injury. This method was improved and modified by Basso in 2006 based on the BBB score according to the motor characteristics of mice. It is a scoring method specifically for the hind limb function of mice, including the main scoring system and the secondary scoring system. Compared with the BBB score, the BMS scoring system is more sensitive and reliable.

[0057] The test was performed by two experienced researchers. The BMS main score ranged from 0 (no ankle movement) to 9 (completely normal motor ability), and the specific scoring details are shown in Table 1. BMS behavioral assessment was performed on days 0, 1, 3, 7, 14, 21, 28, and 35 after spinal cord injury.

[0058] The specific scoring steps are as follows:

[0059] ① One day before surgery, place normal mice in an open field to familiarize them with the environment;

[0060] ② BMS scoring began on the first day after surgery, and the observation period for BMS scoring of mice was 5 minutes;

[0061] ③ Record the video with a video recorder and analyze it on a computer, or give the score immediately after mastering the scoring rules;

[0062] ④Result analysis: The BMS score represents the state of recovery of hind limb motor function after spinal cord injury in mice. The higher the score, the better the recovery. A score of 9 is equivalent to that of normal animals.

[0063] Table 1. BMS scoring criteria

[0064]

[0065] annotation:

[0066] Mild: less than half activity;

[0067] Extensive: more than half of the activities;

[0068] Normal and powerful placement of the lower limbs: actively placing the paw on the ground, with the big toe and the last toe both in contact with the ground;

[0069] Supporting Weight: The hind legs must be raised high enough so that the base of the tail is off the ground and the knees do not touch the ground during walking;

[0070] Stepping: The hind legs are able to support the body weight when they initially leave the ground, step forward, and then regain the body weight when they contact the ground;

[0071] Good coordination: Each forelimb step is accompanied by a hindlimb step, and the hindlimbs step alternately. In order to be able to evaluate, the mouse must step continuously and at a constant speed and at least 3 times its body length. Pauses or hesitations during walking are not included. To test coordination, at least 3 times are required. If one of the 3 times is unsuccessful, it is considered uncoordinated;

[0072] Paw position: The paw is placed parallel to the body. If it is outward away from the body, it is external rotation, and if it is inward towards the body, it is internal rotation;

[0073] Severe trunk instability: The hind limbs show obvious postural instability during monitoring, such as extreme leaning, obvious swaying, and near falls.

[0074] The qPCR results showed that Figure 5 and Figure 6 In the chronic stage of spinal cord injury (35 days), compared with the control group (sh-NC+SCI group), the fibrosis-related indicators in the spinal cord tissue of the experimental group (sh-UHRF1+SCI group) were significantly reduced, while the levels of various neurotrophic factors were significantly increased, indicating that specific knockdown of UHRF1 in fibroblasts significantly reduced fibrosis caused by spinal cord injury and promoted nerve regeneration. The BMS score results also showed that Figure 7 As shown, the hindlimb motor function of the experimental group was significantly improved.

[0075] Example 3: In vitro functional experiments after knockdown of UHRF1

[0076] Take a suckling mouse within 7 days of birth, cut off the head of the suckling mouse under sterile conditions, separate the suckling mouse skull under a stereo microscope, take the meningeal tissue covering the skull and brain substance and cut it into pieces with tissue scissors. Add 0.125% trypsin and DNase, digest in a 37°C constant temperature water bath for 15 minutes, and shake once every 5 minutes. Use DMEM complete medium to terminate digestion. Centrifuge at 4°C 1000rpm for 5 minutes, resuspend, and filter to make a single cell suspension, and inoculate the single cell suspension in a 10cm cell culture dish containing DMEM complete medium (DMEM, 10% FBS, 1% double antibody) to obtain primary meningeal fibroblasts.

[0077] 3.1. Detection of fibrosis-related indicators:

[0078] After 3 days of cell culture, the cells were inoculated into 6-well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. The cells were divided into 4 groups: Si-NC CON, Si-NC TGF-β1, Si-UHRF1 CON, and Si-UHRF1 TGF-β1. When the cells grew to 50% to 60% using DMEM complete medium, siRNA transfection was performed. After 24 hours of continuous culture, TGF-β1 protein (working concentration was 10 ng / mL) was given to the TGF-β1 group, and the control group (CON group) was not treated. After another 24 hours of culture, the cells were collected to extract mRNA for qPCR experiments to detect fibrosis-related indicators (acta2, col1a1, fn1).

[0079] Wherein: the siRNA nucleotide sequence of the Si-UHRF1 group is shown in SEQ ID NO.3;

[0080] The siRNA of the Si-NC group is a nonsense sequence, and its nucleotide sequence is shown in SEQ ID NO.4.

[0081] The qPCR results showed that Figure 8 In primary meningeal fibroblasts cultured in vitro, fibrosis-related indicators in the experimental group (Si-UHRF1 TGF-β1) were significantly reduced compared with those in the control group (Si-NCTGF-β1).

[0082] 3.2. Detection of TGF-β1 / Smads pathway activation:

[0083] After 3 days of cell culture, the cells were inoculated into 6-well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. DMEM complete medium was used to culture the cells until the cells grew to 50%-60%, and then siRNA transfection was performed. The control group was Si-NC, and the experimental group was Si-UHRF1. The corresponding siRNA was the same as above. After 24 hours of continuous culture, serum-free medium was switched for starvation for 6 hours, and then TGF-β1 (working concentration was 10 ng / mL) was added to each well to stimulate the cells. Cell proteins were extracted at 0 min, 15 min, 30 min, and 60 min after the addition of TGF-β1, and related indicators of the TGF-β1 / Smads pathway were detected by Western Blot.

[0084] Western Blot results showed that Fig. 9 Knockdown of UHRF1 significantly reduced the expression levels of P-Smad2 and P-Smad3, confirming that knockdown of UHRF1 inhibited the activation of the TGF-β1 / Smads pathway.

[0085] Example 4: Searching for and validating downstream targets of UHRF1

[0086] 4.1.CUT&TAG sequencing:

[0087] CUT&TAG sequencing service is provided by Jingjie Biotechnology Co., Ltd. The cells used in the experiment are NIH / 3T3 mouse embryonic fibroblast cell line (Zhongqiao Xinzhou), which are divided into UHRF1-OP group and IgG group. The UHRF1-OP group uses anti-UHRF1 antibody as the primary antibody, while the IgG group uses IgG antibody as the primary antibody for negative control experiments. The specific experimental process is as follows:

[0088] (1) Extract the cell nucleus and bind it to magnetic beads: CUT&Tag usually uses unfixed fresh samples as starting materials, but can also use frozen samples, or samples that have been lightly fixed with formalin after the cell nucleus is extracted. After the cell nucleus is extracted, the cell nucleus is combined with concanavalin A magnetic beads (ConA magnetic beads). ConA magnetic beads can bind to glycoproteins on the cell membrane and adsorb to the cell, thereby improving the efficiency of cell experimental operations and reducing cell loss in subsequent experiments. After binding, digitonin is used to permeabilize the cell membrane (the principle is to bind to cholesterol molecules), providing the possibility for the entry of antibodies, etc.

[0089] (2) Binding of primary and secondary antibodies: The primary antibody is a specific antibody designed for the target, and positive (UHRF1 antibody) and negative control (isotype control IgG) samples need to be added at the same time. After incubation with the primary antibody, the cells are quickly washed with a washing buffer containing digitonin, and then the cell nuclei are incubated with the secondary antibody.

[0090] (3) ChiTag transposase-binding antibody: Protein A / G-Tn5 system is generally used to achieve the binding of Tn5 transposase. Protein A / G has a high affinity for the Fc segment of IgG and is often used in the purification of IgG antibodies. Therefore, using Protein A / G can bring Tn5 transposase to the genomic site where the IgG secondary antibody binds.

[0091] (4) Tn5 transposase activation and fragmentation: by adding Mg 2+ The reaction solution enables Tn5 transposase to start working, breaking the DNA region bound by the target protein and connecting the library construction adapter carried to the DNA fragment.

[0092] (5) Sequencing library construction and high-throughput sequencing: Extract DNA, perform PCR amplification to construct the library, and perform high-throughput sequencing.

[0093] (6) Data analysis: including raw data quality control, reference genome alignment, Peak gene annotation, peak plot analysis and annotation, Motif analysis, GO annotation analysis, KEGG annotation analysis, etc.

[0094] CUT&TAG sequencing showed that Fig.10 Compared with the IgG group, the UHRF1-OP group had a significant binding peak in the promoter region of the PPM1A gene, suggesting that the UHRF1 protein can bind to the promoter region of the PPM1A gene.

[0095] 4.2.CHIP-qPCR detection:

[0096] ChIP assay was performed using ABclonal Ultrasonic ChIP Kit (RK20258). Briefly, NIH / 3T3 mouse embryonic fibroblasts were cultured in DMEM complete medium to a density of 70% in the CON and TGF-β1 groups, TGF-β1 protein (working concentration of 10 ng / mL) was given to the TGF-β1 group, and the control group (CON group) was not treated. After culturing for another 24 hours, the cells were harvested, cross-linked with 1% formaldehyde, and then 10× glycine was added at room temperature for 5 minutes. Then, after immunoprecipitation with IgG and anti-UHRF1 antibody at 4°C overnight under ultrasonic treatment, the cells were washed and collected in a centrifuge tube. Then, the immunoprecipitate was eluted and reverse cross-linked, followed by purification of DNA fragments for qPCR amplification.

[0097] qPCR test results, such as Fig.11 , which confirmed that the UHRF1 protein can bind to the promoter region of the PPM1A gene.

[0098] 4.3. Detection of PPM1A expression level after knockdown of UHRF1:

[0099] The extraction method of primary meningeal fibroblasts is as described above. After 3 days of cell culture, the cells were inoculated into 6-well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. The cells were divided into 4 groups: Si-NC CON, Si-NC TGF-β1, Si-UHRF1CON, and Si-UHRF1 TGF-β1. When the cells were cultured with DMEM complete medium to 50% to 60%, siRNA transfection was performed. The siRNA was the same as in Example 3. After 24 hours of continuous culture, the TGF-β1 group was given TGF-β1 protein (working concentration was 10 ng / mL), and the control group (CON group) was not treated. After another 24 hours of culture, the cells were collected, and the mRNA was extracted for qPCR experiments to detect the mRNA expression level of PPM1A, and the cell protein was extracted for Western Blot to detect the protein expression level of PPM1A.

[0100] UHRF1 is a classic epigenetic inhibitor. The increase of UHRF1 inhibits the transcription of PPM1A gene by binding to the promoter region. After knocking down UHRF1, the mRNA and protein levels of PPM1A increased significantly (e.g. Fig.12 , Fig.13 ), suggesting that PPM1A is a downstream target gene of UHRF1. PPM1A is the only Smad phosphatase among many protein serine / threonine phosphatases, which can inhibit the activation of Smad2 and Smad3 by cleaving the phosphorylated serine and threonine residues, thereby inhibiting the TGF-β1 / Smads pathway (Lin X, Duan X, Liang YY, Su Y, Wrighton KH, Long J, Hu M, Davis CM, Wang J, Brunicardi FC, Shi Y, Chen YG, Meng A, Feng XH. PPM1A functions as a Smadphosphatase to terminate TGFbeta signaling. Cell. 2006 Jun 2; 125(5): 915-28. doi: 10.1016 / j.cell.2006.03.044 IF: 45.5B1. PMID: 16751101; PMCID: PMC6309366.).

[0101] 4.4. Knockdown of PPM1A for detection of fibrosis-related indicators:

[0102] The extraction method of primary meningeal fibroblasts was as described above. After 3 days of cell culture, the cells were inoculated into 6-well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. The cells were divided into 4 groups: Si-NC CON, Si-NC TGF-β1, Si-PPM1ACON, and Si-PPM1A TGF-β1. When the cells grew to 50% to 60% using complete medium, siRNA was transfected. After 24 hours of continuous culture, TGF-β1 protein (working concentration was 10 ng / mL) was given to the TGF-β1 group, and the control group (CON group) was not treated. After another 24 hours of culture, the cells were collected to extract mRNA for qPCR experiments to detect fibrosis-related indicators (acta2, col1a1, fn1).

[0103] Among them, the siRNA nucleotide sequence of the Si-PPM1A group is shown in SEQ ID NO.5;

[0104] The siRNA of the Si-NC group is a nonsense sequence, and its nucleotide sequence is shown in SEQ ID NO.4.

[0105] The qPCR results showed that Fig.14 , which confirmed that knockdown of PPM1A significantly enhanced the expression of fibrosis-related genes when TGF-β1 was added simultaneously.

[0106] In summary, the present invention confirms that UHRF1 affects the fibrosis process caused by the TGF-β1 / Smads pathway by regulating the expression of the PPM1A gene.

Claims

1. Application of UHRF1 inhibitors in the preparation of drugs for treating tissue fibrosis after spinal cord injury.

2. The use according to claim 1, characterized in that The UHRF1 inhibitor can inhibit the tissue fibrosis pathway TGF-β1 / Smads pathway by inhibiting the expression of UHRF1, inhibit Smad2 / 3 phosphorylation, and reduce the degree of tissue fibrosis after spinal cord injury.

3. The use according to claim 1, characterized in that The UHRF1 inhibitor increases the expression level of protein serine / threonine phosphatase PPM1A by inhibiting the expression of UHRF1, thereby inhibiting tissue fibrosis.

4. The use according to claim 3, characterized in that The UHRF1 inhibitor weakens the binding of UHRF1 protein to the promoter region of protein serine / threonine phosphatase PPM1A gene by inhibiting the expression of UHRF1, thereby promoting the transcription of PPM1A.

5. The use according to claim 1, characterized in that The UHRF1 inhibitor includes siRNA that interferes with UHRF1 expression, or a virus packaged with shRNA that interferes with UHRF1 expression; Preferably, the nucleotide sequence of the siRNA is shown in SEQ ID NO.3; Preferably, the nucleotide sequence of the shRNA is shown as SEQ ID NO.

2.

6. The use according to claim 5, characterized in that The virus is adeno-associated virus type 9, and the viral vector is pAV-mFSP1-GFP-mir30-shUHRF1, wherein mFSP1 is a fibroblast-specific promoter, and its nucleotide sequence is shown in SEQID NO.1; shUHRF1 is shRNA that interferes with the expression of UHRF1.

7. A drug for treating tissue fibrosis after spinal cord injury, characterized in that: Including inhibitors of UHRF1.

8. The drug according to claim 7, characterized in that The UHRF1 inhibitor includes siRNA interfering with UHRF1 expression, or a virus packaged with shRNA interfering with UHRF1 expression.

9. The drug according to claim 8, characterized in that The nucleotide sequence of the siRNA is shown in SEQ ID NO.3; Preferably, the nucleotide sequence of the shRNA is shown as SEQ ID NO.

2.

10. The drug according to claim 8, characterized in that The virus is adeno-associated virus type 9, and the viral vector is pAV-mFSP1-GFP-mir30-shUHRF1, wherein mFSP1 is a fibroblast-specific promoter, and its nucleotide sequence is shown in SEQ ID NO.1; shUHRF1 is shRNA that interferes with UHRF1 expression.

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