Intervertebral disc related transcription factor and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a transcription factor TAF13 related to intervertebral disc nucleus pulposus cell senescence and application of the transcription factor TAF13 in an intervertebral disc degeneration biomarker. The transcription factor TAF13 has specific expression in intervertebral disc degeneration, can be used as a novel molecular marker for disease diagnosis, can also be used as a potential drug action target, can greatly relieve hair growth and development of diseases, and has great significance in diagnosis and treatment of human intervertebral disc degeneration and senescence related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a transcription factor related to the senescence of nucleus pulposus cells in intervertebral disc degeneration and its application. Background Art

[0002] The functional degradation of the nucleus pulposus tissue caused by senescence is one of the main reasons for the occurrence and development of intervertebral disc degenerative diseases. The structure of the intervertebral disc is relatively complex, with the nucleus pulposus (NP) as the center, surrounded by the annulus fibrosus (AF), and sealed by the upper and lower cartilage endplates, having functions such as buffering external impacts, increasing the flexibility of the spine, reducing external pressure, and maintaining the overall stability of the spine. There is a close relationship between the senescence of nucleus pulposus cells (NPC) and intervertebral disc degeneration (IVDD). Multiple factors, including genetics, age, DNA damage, mitochondrial dysfunction, and autophagy abnormalities, can all promote irreversible cell cycle arrest, oxidative stress damage, and senescence-associated secretory phenotype (SASP) in nucleus pulposus cells. With the senescence of nucleus pulposus cells, their ability to secrete ECM components such as collagen, proteoglycan, and glycosaminoglycan decreases, further affecting the height and stability of the intervertebral disc, and ultimately leading to necrosis and atrophy of the intervertebral disc tissue. In addition, senescent nucleus pulposus cells also release chemotactic factors and pro-inflammatory factors (such as IL-1β, IL-6), thereby changing the inflammatory microenvironment inside the nucleus pulposus.

[0003] Transcription factors play an important role in gene expression regulation and have important impacts on cell life activities and the development and health of organisms. Transcription factors (TF) are a class of protein molecules that can specifically bind to specific sequences upstream of the 5` end of genes, thereby ensuring the expression of target genes at a specific intensity in a specific time and space. Current research has found that TF can specifically recognize downstream target genes and then together construct a complex of transcription (TF) or transcriptional co-regulatory factor (TC) and DNA, thus playing an important role in regulating the activation or inhibition of various biological processes. TF also plays a crucial role in a series of key biological processes such as cell cycle regulation and DNA repair, may be involved in the regulation of cell growth and differentiation, and has an important regulatory role in the occurrence and development of diseases. These characteristics of TF make it possible to become a new biomarker for clinical diagnosis and prognosis, as well as a potential therapeutic target for diseases.

[0004] Abnormal expression of TF widely exists in aspects such as bone formation and remodeling, redox and mitochondrial function of skeletal muscle, differentiation and development of bone marrow cells, and osteoclast differentiation, and is involved in the pathogenesis and regulation of bones and joints. According to the above characteristics, TF is expected to become a molecular index for the diagnosis and prognosis evaluation of intervertebral disc degenerative diseases and a potential therapeutic target. To sum up, developing a transcription factor that can provide the above uses is a technical problem that those skilled in the art urgently need to solve. TAF13 is a known transcription factor, and its sequence has been publicly disclosed. However, its role in the process of intervertebral disc degeneration has not been fully studied. Through in-depth research, the present invention has discovered the specific expression pattern of TAF13 in intervertebral disc degeneration, so it can be used as a biomarker for diagnosing intervertebral disc degeneration. Summary of the Invention

[0005] The purpose of this application is to provide a transcription factor related to intervertebral discs and its applications, aiming to solve the problems existing in the prior art.

[0006] The embodiment of this application provides a transcription factor TAF13 related to the senescence of intervertebral disc nucleus pulposus cells and the application of the transcription factor TAF13 as a biomarker for intervertebral disc degeneration.

[0007] Furthermore, the transcription factor TAF13 serves as a molecular index for the early diagnosis, disease monitoring, and prognosis evaluation of intervertebral disc degeneration.

[0008] Furthermore, the transcription factor TAF13 serves as a drug target for intervertebral disc degeneration.

[0009] The detection method of the transcription factor TAF13 in intervertebral disc degeneration includes: extracting RNA from a sample, obtaining cDNA through reverse transcription, then performing PCR amplification using specific primers, and detecting the expression level of TAF13 by qPCR.

[0010] The beneficial effects of the present invention are: The transcription factor TAF13 involved in the present invention has specific expression in intervertebral disc degenerative diseases, can be used as a new molecular biomarker for disease diagnosis, and can also be used as a potential drug target, which can greatly alleviate the occurrence and development of the disease, and has great significance for the diagnosis and treatment of human intervertebral disc degeneration and senescence-related diseases. Brief Description of the Drawings

[0011] Figure 1 For the differential analysis of intervertebral disc degenerated nucleus pulposus tissue and normal nucleus pulposus tissue.

[0012] (A) Volcano plot; (B) Heat map; (C) Differential ranking plot of DEGs between IVDD and the control group; (D) Venn diagram of intersection genes between the GSE70362 dataset and the CellAge database;

[0013] DEGs: Differentially Expressed Genes; SRGs: Senescence-Related Genes; DESRGs: Differentially Expressed Senescence-Related Genes; IVDD: Intervertebral Disc Degeneration

[0014] Figure 2 These are the enrichment analysis results of gene set enrichment analysis (GSEA).

[0015] Four significant gene set enrichment pathways (FDR < 0.25, adjusted p < 0.05).

[0016] (A) Oxidative phosphorylation; (B) Extracellular matrix receptor interaction; (C) Extracellular matrix structural components; (D) Mitochondrial protein complex-containing; (E) Collagen-containing extracellular matrix

[0017] FDR, False Discovery Rate

[0018] Figure 3 These are the expression and correlation analysis of differential genes in the nucleus pulposus tissue of intervertebral disc degeneration and normal nucleus pulposus tissue.

[0019] The expression levels of DESRGs in the IVDD group and the control group in the GSE70362 training set were visualized by box plots, *p < 0.05, **p < 0.01, ***p < 0.001;

[0020] Figure 4 These are the GO enrichment analysis of DESRGs.

[0021] Figures (A-C) and (D-F) represent biological process (BP), cellular component (CC), and molecular function (MF) respectively; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes

[0022] Figure 5 These are the KEGG enrichment analysis and protein interaction network of DESRGs.

[0023] (A) KEGG enrichment analysis pathway diagram; (B) PPI protein interaction network

[0024] Figure 6 These are the diagnostic values of the ROC curves of 6 Hub genes in the GSE70362 dataset.

[0025] ROC curve diagrams of BANF1 (A), TIMP3 (B), STC1 (C), TAF13 (D), DDIT4L (E), GDF15 (F);

[0026] Figure 7 These are the comparisons of the expression levels of 6 Hub genes in the GSE70362 dataset.

[0027] Expression levels of BANF1 (A), TIMP3 (B), STC1 (C), TAF13 (D), DDIT4L (E), and GDF15 (F);

[0028] ns, not significant;

[0029] Figure 8 Results of CCK-8 cell viability assays for NP cells and TBHP-induced senescent NP cells.

[0030] (A) Cell viability was evaluated by CCK-8 assay; data are presented as mean ± standard deviation (n = 3); (B) Target mRNA levels were determined by qPCR;

[0031] Figure 9 Results of qPCR detection of TAF13 gene expression in NP cells and TBHP-induced senescent NP cells.

[0032] (A) Construction of miRNA-gene networks; (B) Venn diagram of interacting compounds; (C) Intersection compounds of potential targets; Detailed implementation manners

[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0034] Construct a TBHP-induced senescence model of intervertebral disc nucleus pulposus cells (NPCs) to verify the difference in TAF13 expression between normal and degenerated intervertebral disc nucleus pulposus cells (NPCs).

[0035] 1. Obtaining data of normal and degenerated intervertebral disc nucleus pulposus tissues

[0036] Data of normal and degenerated intervertebral disc nucleus pulposus tissues were obtained from the NCBI database, namely GSE70362 and GSE147383 datasets, respectively, with Control:IVDD = 10:18. Senescence-related genes (SRGs) were obtained from the Senescence Database (https: / / genomics.senescence.info / ).

[0037] 2. Screening of relevant key genes (Hub genes) by bioinformatics analysis

[0038] 2.1. Analysis of differentially expressed genes

[0039] The gene probe IDs were converted to gene symbols using R software. The expression matrix of the GSE70362 dataset was normalized using the "limma" package, and

[0040] 87 differentially expressed genes (DEGs) were obtained using the screening criteria of |log2(FC)| > 0.5 and p-value < 0.05, as shown in Figure 1 (A)- Figure 1 (D). There were 48 downregulated genes and 39 upregulated genes in the nucleus pulposus tissue of intervertebral disc degeneration compared with normal nucleus pulposus tissue. There were 8 intersection genes with aging, namely: STC1, TAF13, TIMP3, BANF1, GDF15, CITED2, DDIT4L, FAM129A.

[0041] Volcano plots, differential ranking plots, and heatmaps were drawn using "ggplot2" and "pheatmap". Genes with |log2(FC)| > 1 were marked in the differential ranking plot. Gene set enrichment analysis (GSEA) was performed using the "clusterProfiler" package and two gene sets ("c5.go.all.v2022.1.Hs.symbols.gmt gene ontology" and "c2.cp.kegg.v2022.1.Hs.symbols.gmt KEGG pathway database") to clarify the potential mechanisms of IVDD. As shown in Figure 2 (A)- Figure 2 (E), these DEGs were mainly involved in oxidative phosphorylation, extracellular matrix receptor interaction, extracellular matrix structural components, mitochondrial protein complexes, and collagen-containing extracellular matrix. The following criteria were used when screening DEGs: |log2(FC)| > 0.5, p < 0.05; meanwhile, the enriched pathways of GSEA were screened by FDR < 0.25 and p < 0.05.

[0042] 2.2. Differential expression analysis of aging-related Hub genes

[0043] The "VennDiagram" package in R software was used to draw the intersection of DEGs and SRGs, and 8 differentially expressed aging-related genes (DESRGs) were obtained. Figure 3 The expression levels and correlations of these 8 DESRGs in GSE70362 were shown, including 5 upregulated DESRGs and 3 downregulated DESRGs. By using the "clusterProfiler" and "pathview" packages to analyze the KEGG pathways and GO enrichment of DESRGs, the possible biological functions and enriched pathways of DESRGs were revealed.

[0044] Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, the possible biological functions and enriched pathways of DESRGs were revealed. GO analysis was divided into biological process (BP), cellular component (CC), and molecular function (MF). The biological process (BP) of DESRGs mainly focused on axial characteristics and positive regulation of the transmembrane receptor protein serine / threonine kinase signaling pathway, such as Figure 4 (A), Figure 4 (D) shown. In terms of cellular component (CC), DESRGs were mainly enriched in platelet dense granules and the lumen of platelet dense granules, such as Figure 4 (B), Figure 4 (E) shown. In terms of molecular function (MF), DESRGs were mainly enriched in protein C-terminal binding and BMP receptor binding, such as Figure 4 (C), Figure 4 (F) shown. KEGG analysis showed that DESRGs were mainly related to basal transcription factors and mitophagy in animals, such as Figure 5 (A) shown.

[0045] 2.3. Hub gene screening

[0046] The protein-protein interaction (PPI) network of DESRGs was analyzed through the STRING database, and then 12 algorithm scores of 6 genes (STC1, GDF15, TAF13, BANF1, DDIT4L, TIMP3) were obtained using the Cytohubba plugin in Cytoscape software (version 3.9.1). Figure 5 (B) shown, a total of 8 nodes and 4 edges were identified in the PPI network diagram. According to the expression of hub genes in the validation set GSE147383, the transcription factor TAF13 could be determined as a potential therapeutic target for intervertebral disc degeneration.

[0047] 2.4. Prediction of miRNA-Gene network and potential targeting compounds

[0048] The miRNet database was used to predict the interaction between diagnostic genes and miRNAs, and visualize the miRNA-gene regulatory network; the Comparative Toxicogenomics Database was used to construct a gene-drug interaction network to predict new potential targets for the synthesis of IVDD drugs.

[0049] 2.5. Identification and validation of TAF13 biomarker

[0050] The “pROC” package in R software was used to perform ROC analysis on the data to evaluate the diagnostic value of 6 Hub genes in IVDD, and “ggplot2” was used for result visualization. The results showed that as Figure 6(A)- Figure 6 (F) As shown, except for TIMP3, the AUC values of all genes reached or exceeded 0.820, and the transcription factor TAF13 could be identified as a potential therapeutic target for intervertebral disc degeneration. Figure 7 (A)- Figure 7 (F) showed the gene expression levels in the GSE70362 dataset and were compared by the Mann-Whitney U test. Except for TIMP3, there were significant differences in gene expression levels between the IVDD group and the control group.

[0051] Taking the GSE147383 dataset as a validation set, the expression of Hub genes was verified. Only the expression level of TAF13 showed a statistically significant difference.

[0052] 3. NPC culture and senescence model construction

[0053] NP cells (NPCs) with good growth status were selected and cultured in DMEM / F12 complete medium (8123030, gibco), containing 10% fetal bovine serum (FBS, FS301 - 02, TransGen Biotech) and 1% penicillin and streptomycin (Sigma, USA) at 37 °C in a 5% CO 2 incubator. Cells were induced to senesce by treatment with 60 μM tert-butyl hydroperoxide (TBHP, MACKLIN) for 24 h. After the induction ended, they were continued to be cultured in DMEM / F12 complete medium for subsequent studies.

[0054] 4. CCK-8 assay

[0055] After induction with different concentrations of TBHP (0, 20, 40, 60, 80, 100 μM) for 24 h, cell viability was evaluated by CCK-8 assay (K1018, APExBIO), and the absorbance was read at a wavelength of 450 nm. The results were as Figure 8 (A) and Figure 8 (B) showed that after treatment of NP cells with 60 μM TBHP for 2 h, cell activity remained at a low level and the cell cycle was arrested.

[0056] 5. Quantitative real-time PCR

[0057] Total RNA was extracted from NPCs treated with 60 μm TBHP (B802372, Macklin, China) for 24 hours (IVDD group) and NPCs treated with normal saline for 24 hours (control group) using Trizol reagent; Complementary DNA (cDNA) was synthesized using PrimeScript RT MasterMix (RR036A, Takara); SYBR Green Premix (RR420A, Takara) was used for amplification. The operation method is detailed in the instruction manual.

[0058] Reaction conditions: 50°C for 2 min; 95°C for 10 min; (95°C for 15 s, 60°C for 60 s) for 40 cycles.

[0059] The primers are shown in Table 2.

[0060] Table 2. Primer sequence list

[0061] Name Sequence GAPDH-F 5'-GTCATCCATGACAACTTTGGTATC-3' GAPDH-R 5'-AGTAGAGGCAGGGATGATGTTCT-3' TAF12-F 5'-TGTATGGCTTTGGGATGAC-3' TAF13-R 5'-GCAAACTTCCTTGGTCCTT-3'

[0062] As Figure 9 (A)- Figure 9 (C) shows that the qPCR detection results show that the expression of TAF13 in TBHP-induced senescent NPCs is significantly higher than that in normal NPCs.

[0063] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claims involved.

Claims

1. A transcription factor TAF13 associated with aging of intervertebral disc nucleus pulposus cells, and the application of transcription factor TAF13 as a biomarker of intervertebral disc degeneration.

2. The use according to claim 1, characterized in that: Transcription factor TAF13 is used as a molecular marker for intervertebral disc degeneration, including early diagnosis, disease monitoring and prognosis evaluation.

3. The use according to claim 1, characterized in that: Transcription factor TAF13 as a drug target for intervertebral disc degeneration.

4. A method for detecting transcription factor TAF13 in intervertebral disc degeneration, characterized in that: include: RNA was extracted from the samples, cDNA was obtained by reverse transcription, PCR amplification was performed using specific primers, and the expression level of TAF13 was detected by qPCR.

Images