Use of an inhibitor of UHRF1 in the preparation of a medicament for treating tissue fibrosis after spinal cord injury

Abstract

The present invention relates to the use of an inhibitor of UHRF1 in the preparation of a medicament for treating tissue fibrosis after spinal cord injury, belonging to the technical field of biomedicine. The present invention has confirmed that spinal cord injury can lead to an increase in the expression of UHRF1; compared with the mice in the sham operation group, the mice with specific knockdown of UHRF1 in fibroblasts significantly reduced the fibrosis caused by spinal cord injury and promoted nerve regeneration, and the motor function was significantly improved. In terms of mechanism, the knockdown of UHRF1 led to a decrease in the number of its proteins bound to the promoter of the PPM1A gene, resulting in a weakened transcriptional inhibitory effect, promoting the expression of the PPM1A gene, and PPM1A can inhibit the phosphorylation of Smad2 / 3 and inhibit the conduction of the TGF-β1 / Smads pathway, thereby inhibiting tissue fibrosis.

Description

Technical Field

[0001] The present invention relates to the application of an inhibitor of UHRF1 in the preparation of a medicament for treating tissue fibrosis after spinal cord injury, and belongs to the field of biomedical technology. Background Art

[0002] Spinal cord injury (SCI) is a severe trauma to the central nervous system, characterized by a high incidence rate, a high disability rate, and a low onset age. There are approximately 1 million new cases of spinal cord injury worldwide every year. Currently, there is a lack of effective treatment methods for this disease, and varying degrees of limb paralysis bring a heavy burden to patients and their families. Therefore, there is a huge social demand for the restoration of neurological function after spinal cord injury.

[0003] In the initial 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 imbalanced microenvironment after spinal cord injury is a key pathological event, and mature fibrous scar tissue is the main reason for preventing axonal regeneration and the inability of newly formed axons to cross the injury area. Precise and targeted regulation of the fibrous scar formation process after spinal cord injury to achieve axonal regeneration and neurological function restoration has great therapeutic significance.

[0004] Activation of the TGF-β1 / Smads pathway can cause the proliferation, differentiation of fibroblasts and the 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 phosphorylated TβRII, combines into a multimer and is activated. Activated TβRI phosphorylates Smad2 / 3 in the cell to form p-Smad2 / 3. After separating from the receptor, it forms a complex with Smad4 and enters the nucleus, regulating the transcription of target genes in the nucleus and generating 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 XQ, Zhang XS, Zhou YK, Zhu Y. Research progress of TGF-β1 / Smads signaling pathway in fibrotic diseases [J]. Chinese Journal of Integrated Traditional and Western 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 July 7, 2014) discloses an siRNA molecule that inhibits the expression of the Smad4 gene. This 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 regulating the post-transcriptional expression of the Smad4 gene. Changes in the expression level of Smad4 directly affect the TGF-β1 / Smads pathway, thereby affecting the proliferation and fibrosis of articular fibroblasts, and ultimately having 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 the occurrence and development of tumors. In addition, Demin Cheng et al. explored the process of UHRF1 affecting 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 lung fibrosis treatment potential through regulation of fibroblast activation. JCI Insight. 2022 Nov 22;7(22):e162831. doi: 10.1172 / jci.insight.162831 IF: 6.3 B1. 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 processes are completely different.

[0006] There is currently no relevant report on the UHRF1 on the TGF-β1 / Smads pathway. There is also no relevant research on the regulatory role of UHRF1 in the fibrosis process after spinal cord injury. Therefore, clarifying the role of UHRF1 in the fibrosis process after spinal cord injury, providing new therapeutic targets and experimental basis for the treatment of spinal cord injury, and designing simple and efficient drug preparations have very important 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, providing a new molecular target for the treatment of spinal cord injury.

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

[0009] The use of an inhibitor of UHRF1 in the preparation of a drug for treating tissue fibrosis after spinal cord injury.

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

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

[0012] More preferably, the inhibitor of UHRF1 weakens the binding of UHRF1 protein to the promoter region of the 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 inhibitor of UHRF1 includes siRNA that interferes with the expression of UHRF1, or a virus packaged with shRNA that interferes with the expression of UHRF1.

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

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

[0016] More 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 as shown in SEQ ID NO.1; shUHRF1 is shRNA that interferes with the expression of UHRF1.

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

[0018] Preferably according to the present invention, the inhibitor of UHRF1 includes siRNA that interferes with the expression of UHRF1, or a virus packaged with shRNA that interferes with the expression of UHRF1.

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

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

[0021] More 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 as shown in SEQ ID NO.1; shUHRF1 is shRNA that interferes with the expression of UHRF1.

[0022] Beneficial effects:

[0023] 1. The present invention has confirmed that spinal cord injury can lead to an increase in the expression of UHRF1; compared with the mice in the sham operation group, the mice with specific knockdown of UHRF1 in fibroblasts significantly reduced the fibrosis caused by spinal cord injury, promoted nerve regeneration, and significantly improved motor function. In terms of mechanism, the knockdown of UHRF1 led to a decrease in the number of its proteins bound to the promoter of the PPM1A gene, resulting in a weakened transcriptional inhibition effect, promoting the expression of the PPM1A gene, and 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 with specific knockdown of UHRF1 in fibroblasts. This vector is appropriately selected in terms of the basic vector, target fragment, insertion position, etc. Animal experiments have confirmed that it can significantly inhibit the fibrosis process, enhance axon regeneration, improve motor function recovery, and has good application prospects.

[0025] 3. The present invention first proposes that UHRF1 can regulate the TGF-β1 / Smads pathway, laying a good foundation for future research on other disease models. Description of the Drawings

[0026] Figure 1 It is a bar chart of the expression level of UHRF1 detected by RNA-seq of spinal cord tissue.

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

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

[0029] Figure 4 It is a tissue immunofluorescence picture of spinal cord tissue.

[0030] Figure 5 It is a bar chart of the mRNA expression levels of fibrosis indexes acta2, col1a1, and fn1 after knockdown of UHRF1 in spinal cord tissue.

[0031] Figure 6 It is a bar chart of the mRNA expression levels of axon regeneration indexes ngf, bdnf, and gdnf after knockdown of UHRF1 in spinal cord tissue.

[0032] Figure 7 It is a curve of the BMS evaluation score of mice after knockdown of UHRF1 in spinal cord tissue.

[0033] Figure 8Bar graph of the mRNA expression levels of fibrosis markers acta2, col1a1, and fn1 after knocking down UHRF1 in meningeal fibroblasts.

[0034] Figure 9 Western Blot detection of P-Smad2 / 3 protein after TGF-β1 stimulation in meningeal fibroblasts with UHRF1 knocked down.

[0035] Figure 10 Analysis diagram of the binding of UHRF1 to the promoter of the PPM1A gene in CUT&TAG sequencing.

[0036] Figure 11 CHIP-qRCR verification result diagram of the binding of UHRF1 protein to the promoter of the PPM1A gene.

[0037] Figure 12 Bar graph of the mRNA expression level of PPM1A after knocking down UHRF1 in meningeal fibroblasts.

[0038] Figure 13 Western Blot detection of PPM1A protein after knocking down UHRF1 in meningeal fibroblasts.

[0039] Figure 14 Bar graph of the mRNA expression levels of fibrosis markers acta2, col1a1, and fn1 after knocking down PPM1A in meningeal fibroblasts. Detailed implementation methods

[0040] The technical solutions of the present invention will be further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. Reagents and consumables involved in the embodiments are all ordinary commercially available products without special instructions; steps and experimental operations involved in the embodiments are all conventional technical operations in the art without special instructions.

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

[0042] 1.1. Construction of spinal cord injury model

[0043] Clean the operating table and sterilize the required surgical instruments in advance by autoclaving. Prepare sterile surgical drapes, suture threads, suture needles, and povidone iodine. Fast the mice 8 hours before the operation to avoid vomiting or aspiration during the operation. Prepare a 3% pentobarbital solution with distilled water and inject the mice intraperitoneally for anesthesia according to the dosage standard of 30 mg / kg. The absence of response to clamping of the four limbs indicates complete anesthesia. Shave the hair on the back of the mice to expose the surgical area. Fix the mice in the prone position on the operating table to ensure the stability of the mice's position during the operation. Disinfect the surgical area with povidone iodine cotton balls to reduce the risk of contamination during the operation. Place a disposable sterile surgical drape, make a 1.5-cm incision at the T9 segment of the spine along the midline of the back, and bluntly separate the muscle and fascia layer by layer until the spinous process is exposed. Perform a T9 laminectomy under a stereomicroscope using fiber forceps to expose the spinal cord. The mice in the Sham group were not subjected to spinal cord injury after laminectomy, the injury site was washed with sterile saline, the surgical incision was sutured layer by layer with sterile suture threads, and disinfected with povidone iodine cotton balls. The mice in the SCI group were struck on the spinal cord with a pneumatic spinal cord injury impactor (68100, RWD, Shenzhen, China) at a striking speed of 1 m / s, a striking depth of 2 mm, and a residence time of the impactor of 1 s. The washing, suturing, and disinfection steps and operations were the same as those in the Sham group. Place the mice on a heating blanket for 2-3 hours, and place them in a breeding cage after they wake up (the mice can freely obtain food and water in the cage). To prevent urinary tract infection in mice, manually squeeze the bladder of the mice twice a day to help them urinate until the mice are sacrificed.

[0044] 1.2 Transcriptomic sequencing

[0045] The mice in the Sham group and the SCI group were anesthetized by intraperitoneal injection of 3% pentobarbital solution and sacrificed on the 7th day after injury. The spinal cord at the injury site was taken for transcriptomic sequencing. The transcriptomic sequencing service was provided by Lianchuan Biotechnology Co., Ltd. The specific steps were as follows: TRIzol reagent (Thermofisher, 15596018) was used to isolate and purify the RNA of the total sample according to the operation protocol provided by the manufacturer; then NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) was used to quality control the quantity and purity of the total RNA, and the integrity of the RNA was detected by Bioanalyzer 2100 (Agilent, CA, USA); a concentration > 50 ng / μL, RIN value > 7.0, and total RNA > 1 μg were required to meet the downstream experiments; oligo(dT) magnetic beads (Dynabeads Oligo(dT), cat. 25-61005, Thermo Fisher, USA) were used to specifically capture the mRNA with PolyA (polyadenylic acid) through two rounds of purification; the captured mRNA was fragmented using a magnesium ion fragmentation kit (NEBNext® Magnesium RNA Fragmentation Module, cat. E6150S, USA) at high temperature, and the reaction was carried out at 94 °C for 5 - 7 minutes; the fragmented RNA was reverse transcribed into cDNA by the action of reverse transcriptase (Invitrogen SuperScript™ II Reverse Transcriptase, cat. 1896649, CA, USA); then E. coli DNA polymerase I (NEB, cat. m0209, USA) and RNase H (NEB, cat. m0297, USA) were used for second-strand synthesis to convert the double-stranded complex of these DNA and RNA into a double-stranded DNA. At the same time, dUTP Solution (Thermo Fisher, cat. R0133, CA, USA) was incorporated into the second strand to fill in the ends of the double-stranded DNA to form blunt ends, and then an A base was added to each end to enable it to be ligated to an adapter with a T base at the end, and magnetic beads were used to screen and purify the fragment size; the second strand was digested with UDG enzyme (NEB, cat. m0280, MA, US), and then through 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), a library (strand-specific library) with a fragment size of 300 bp ± 50 bp was formed; finally, illumina Novaseq™ 6000 (LC BioTechnology CO., Ltd.(Hangzhou, China) It was subjected to paired-end sequencing according to the standard operation, and the sequencing mode was PE150; after obtaining the sequencing data off the machine, the data off the machine was first filtered to obtain high-quality sequencing data (Clean Data), 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 differential analysis, enrichment analysis, etc. were performed.

[0046] The RNA-seq data showed that, as Figure 1 , compared with the sham operation group (sham group), the mRNA level of UHRF1 in the SCI group of mice was significantly increased on the 7th day after spinal cord injury.

[0047] 1.3. Preliminary experiment verification

[0048] The mice in the Sham group and the SCI group were sacrificed after intraperitoneal injection of 3% pentobarbital solution for anesthesia on the 7th day after spinal cord injury, and the spinal cord at the injury site was taken for qPCR, Western Blot and immunofluorescence to detect the expression level and cellular localization of UHRF1 mRNA and protein.

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

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

[0051] Example 2: In vivo functional experiment after knocking down UHRF1

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

[0053] Among them, 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 of the sh-UHRF1 group and the sh-NC group was injected into the intratheca of mice using a microsyringe, and the injection dose was 2 μL per mouse. After 3 weeks, spinal cord injury was modeled. Subsequently, the mice were divided into 4 groups: sh-UHRF1+SCI group, sh-UHRF1+sham group, sh-NC+SCI group, and sh-NC+sham group. The recovery of hindlimb motor function was evaluated by BMS score in the 4 groups of mice at 0, 1, 3, 7, 14, 21, 28, and 35 days after spinal cord injury. Then, on the 35th day, the mice were anesthetized with 3% pentobarbital solution by intraperitoneal injection and sacrificed. The spinal cord at the injury site was taken for qPCR to evaluate the fibrosis indexes (acta2, col1a1, fn1) and nerve regeneration indexes (ngf, bdnf, gdnf).

[0056] BMS score: It is applicable to evaluate the recovery of motor function in 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 dedicated to the hindlimb function of mice, including a main scoring system and a 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 consists of 0 points (no ankle movement) to 9 points (completely normal motor ability). The specific scoring rules are shown in Table 1. BMS behavioral assessment was performed at 0, 1, 3, 7, 14, 21, 28, and 35 days after spinal cord injury.

[0058] The specific scoring steps are as follows:

[0059] ① One day before the operation, normal mice were placed in an open field to familiarize themselves with the environment;

[0060] ② BMS score started on the first day after the operation, and the observation period for the BMS score of mice was 5 minutes;

[0061] ③ After recording the video with a video recorder, it was analyzed with a computer, or the score could be given immediately after being proficient in the scoring rules;

[0062] ④ Result analysis: The level of the BMS score represents the state of the recovery of hindlimb motor function in mice after spinal cord injury. The higher the score, the better the recovery. When it reaches 9 points, it is equivalent to normal animals.

[0063] Table 1. BMS Scoring Rules

[0064]

[0065] Annotation:

[0066] Slight: Activity less than half;

[0067] Extensive: Activity greater than half;

[0068] Normal and strong placement of the lower limbs: Actively place the claws on the ground, with the big toe and the last toe both in contact with the ground;

[0069] Supporting gravity: The hind limbs must be lifted high enough so that the root of the tail is lifted off the ground, and the knees do not touch the ground during walking;

[0070] Stepping and walking: The hind limbs can support the body weight when initially leaving the ground, step forward, and then support the body weight again when touching the ground;

[0071] Good coordination: Each step of the forelimbs is accompanied by a step of the hind limbs, and the hind limbs alternate in stepping. In order to be evaluated, the mouse must step continuously and evenly and be at least 3 times its own body length. Pauses or hesitations during walking cannot be included. To detect coordination, at least 3 times are required. If one of the 3 times is unsuccessful, it is considered uncoordinated;

[0072] Claw position: The claws are placed parallel to the body. If they move outward away from the body, it is external rotation, and if they move inward towards the body, it is internal rotation;

[0073] Severe trunk instability: During monitoring, obvious postural instability is shown in the hind limbs, such as extreme tilting, obvious swaying, and approaching falling.

[0074] The qPCR results showed that, as 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 indexes in the spinal cord tissue of the experimental group (sh-UHRF1+SCI group) mice were significantly reduced, while the levels of various neurotrophic factors were significantly increased, suggesting that specific knockdown of UHRF1 in fibroblasts significantly reduced the fibrosis caused by spinal cord injury and promoted nerve regeneration. The BMS score results also showed that, as Figure 7 shown, the hind limb motor function of the experimental group was significantly improved.

[0075] Example 3: In vitro functional experiment after knocking down UHRF1

[0076] Newborn mice within 7 days after birth were taken. Under sterile conditions, the heads of the newborn mice were cut off. The skulls of the newborn mice were separated under a stereomicroscope. The meningeal tissues covering the skull and the brain parenchyma were taken and minced with tissue scissors. 0.125% trypsin and DNase were added and digested in a 37°C constant temperature water bath for 15 min, with shaking every 5 min. The digestion was terminated with DMEM complete medium. After centrifugation at 1000 rpm for 5 min at 4°C, resuspension, and filtration, a single-cell suspension was prepared. The single-cell suspension was inoculated into a 10 cm cell culture dish containing DMEM complete medium (DMEM, 10% FBS, 1% double antibody), and primary meningeal fibroblasts were obtained.

[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, Si-UHRF1 TGF-β1. When the cells grew to 50% - 60% using DMEM complete medium, siRNA transfection was performed. After continuous culture for 24 hours, the TGF-β1 group was given TGF-β1 protein (working concentration: 10 ng / mL), and the control group (CON group) was not treated. After further culture for 24 hours, the cells were harvested to extract mRNA for qPCR experiments to detect fibrosis-related indicators (acta2, col1a1, fn1).

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

[0080] The siRNA in 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, as Figure 8 , in primary meningeal fibroblasts cultured in vitro, compared with the control group (Si-NC TGF-β1), the fibrosis-related indicators in the experimental group (Si-UHRF1 TGF-β1) were significantly reduced.

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

[0083] The cells were inoculated into 6-well plates 3 days after cell culture, placed in an incubator at 37 °C, 5% CO2 and saturated humidity, and cultured with DMEM complete medium. When the cells grew to 50% - 60%, 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 continuous culture for 24 hours, the cells were starved in serum-free medium for 6 hours, and then TGF-β1 (working concentration: 10 ng / mL) was added to each well to stimulate the cells. Cell proteins were extracted at the 0 min, 15 min, 30 min, and 60 min after the addition of TGF-β1, and the related indicators of the TGF-β1 / Smads pathway were detected by Western Blot.

[0084] The results of Western Blot detection showed that, as Figure 9 , knocking down UHRF1 significantly reduced the expression levels of P-Smad2 and P-Smad3, confirming that knocking down UHRF1 inhibited the activation of the TGF-β1 / Smads pathway.

[0085] Example 4: Search for downstream targets of UHRF1 and verify them

[0086] 4.1 CUT&TAG sequencing:

[0087] The CUT&TAG sequencing service was provided by PTMScan Biotechnology Co., Ltd. The cells used in the experiment were the NIH / 3T3 mouse embryonic fibroblast cell line (Zhongqiao Xinzhou). The groups were the UHRF1-OP group and the IgG group. The UHRF1-OP group used the anti-UHRF1 antibody as the primary antibody, while the IgG group used the IgG antibody as the primary antibody for the negative control experiment. The specific experimental procedure was as follows:

[0088] (1) Extract the cell nuclei and bind them to magnetic beads: CUT&Tag usually uses fresh, unfixed samples as the starting material, and can also use frozen samples, or samples that have been slightly fixed with formalin after cell nucleus extraction. After completing the extraction of the cell nuclei, the cell nuclei were bound to concanavalin A magnetic beads (ConA magnetic beads). ConA magnetic beads can bind to glycoproteins on the cell membrane, thereby adsorbing on the cells, improving the efficiency of cell experiment operations, and reducing cell loss in subsequent experimental processes. After binding, digitonin was 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 the primary antibody and the secondary antibody: The primary antibody is a specific antibody designed against the target. At the same time, positive (UHRF1 antibody) and negative control (isotype control IgG) samples need to be added. After incubation with the primary antibody, it was quickly washed with a washing buffer containing digitonin, and then the cell nuclei were incubated with the secondary antibody.

[0090] (3) ChiTag transposome-binding antibody: Generally, the Protein A / G-Tn5 system is 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 for the purification of IgG antibodies. Therefore, using Protein A / G can bring the Tn5 transposase to the genomic locus bound by the IgG secondary antibody.

[0091] (4) Activation and fragmentation of Tn5 transposase: By adding a reaction solution containing Mg 2+ , the Tn5 transposase begins to function, breaking the DNA region bound by the target protein while ligating the carried library adaptors to the DNA fragments.

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

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

[0094] CUT&TAG sequencing showed that, as Figure 10 , compared with the IgG group, there were significant binding peaks in the promoter region of the PPM1A gene in the UHRF1-OP group, suggesting that the protein of UHRF1 can bind to the promoter region of the PPM1A gene.

[0095] 4.2. CHIP-qPCR detection:

[0096] Perform ChIP assays using the ABclonal ultrasonic ChIP kit (RK20258). Briefly, NIH / 3T3 mouse embryonic fibroblasts were cultured in DMEM complete medium in the CON group and TGF-β1 group until the density reached 70%. The TGF-β1 group was treated with TGF-β1 protein (working concentration of 10 ng / mL), 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 overnight at 4°C with IgG and anti-UHRF1 antibody under sonication, the cells were washed and collected in a centrifuge tube. Then, the immunoprecipitate was eluted and reverse-cross-linked, and then the DNA fragments for qPCR amplification were purified.

[0097] The qPCR detection results, as Figure 11 , confirmed that the protein of UHRF1 can bind to the promoter region of the PPM1A gene.

[0098] 4.3. Detection of the expression level of PPM1A after knocking down UHRF1:

[0099] The extraction method of primary meningeal fibroblasts is as described above. After culturing the cells for 3 days, they were seeded 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 were cultured to 50%-60% confluence using DMEM complete medium, siRNA transfection was performed. The siRNA was the same as in Example 3. After continuing to culture for 24 hours, the TGF-β1 group was given TGF-β1 protein (working concentration: 10 ng / mL), and the control group (CON group) was not treated. After culturing for another 24 hours, the cells were harvested, and mRNA was extracted for qPCR experiments to detect the mRNA expression level of PPM1A, and cell proteins were extracted for Western Blot to detect the protein expression level of PPM1A.

[0100] UHRF1 is a classical epigenetic inhibitor. The elevation of UHRF1 inhibits the transcription of the PPM1A gene by binding to the promoter region of the PPM1A gene. After knocking down UHRF1, the mRNA and protein levels of PPM1A were significantly increased (as Figure 12 , Figure 13 ), suggesting that PPM1A is a downstream target gene of UHRF1. PPM1A is the only Smad phosphatase among numerous protein serine / threonine phosphatases, and it can inhibit the activation of Smad2 and Smad3 by cleaving phosphorylated serine and threonine residues on them, 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 Smad phosphatase to terminate TGF-beta signaling. Cell. 2006 Jun 2;125(5):915-28. doi:10.1016 / j.cell.2006.03.044 IF:45.5 B1. PMID:16751101; PMCID:PMC6309366.).

[0101] 4.4. Detection of fibrosis-related indicators after knocking down PPM1A:

[0102] The extraction method of primary meningeal fibroblasts is as described above. After culturing the cells for 3 days, they were seeded 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-PPM1A CON, Si-PPM1A TGF-β1. When the cells were cultured to 50%-60% confluence with complete medium, siRNA transfection was performed. After continuous culture for 24 hours, the TGF-β1 group was given TGF-β1 protein (working concentration: 10 ng / mL), and the control group (CON group) was not treated. After further culturing for 24 hours, the cells were harvested 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, as Figure 14 , it was confirmed that under the condition of adding TGF-β1 simultaneously, knocking down PPM1A significantly enhanced the expression of fibrosis-related genes.

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

Claims

1. Use of an inhibitor of UHRF1 in the preparation of a medicament for treating tissue fibrosis after spinal cord injury, wherein the inhibitor of UHRF1 comprises siRNA interfering with the expression of UHRF1, or a virus packaged with shRNA interfering with the expression of UHRF1; the nucleotide sequence of the siRNA is as shown in SEQ ID NO.3; the nucleotide sequence of the shRNA is as shown in SEQ ID NO.

2.

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

3. The application according to claim 1, characterized in that, The inhibitor of UHRF1 plays a role in inhibiting tissue fibrosis by increasing the expression level of the protein serine / threonine phosphatase PPM1A by inhibiting the expression of UHRF1.

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

5. The application according to claim 1, 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 as shown in SEQ ID NO.1; shUHRF1 is shRNA interfering with the expression of UHRF1.

6. A drug for treating tissue fibrosis after spinal cord injury, characterized in that, Comprising an inhibitor of UHRF1, wherein the inhibitor of UHRF1 comprises siRNA interfering with the expression of UHRF1, or a virus packaged with shRNA interfering with the expression of UHRF1; the nucleotide sequence of the siRNA is as shown in SEQ ID NO.3; the nucleotide sequence of the shRNA is as shown in SEQ ID NO.

2.

7. The drug according to claim 6, 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 as shown in SEQ ID NO.1; shUHRF1 is shRNA interfering with the expression of UHRF1.

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