Use of prex1, mmp10, bst2 and tmem70 in the molecular classification or prognosis of keloids

Abstract

The application relates to the technical field of biological detection, and provides application of PREX1, MMP10, BST2 and TMEM70 in molecular typing or prognosis of keloids. Immunohistochemical staining is carried out on the keloids removed by clinical operation, and it is found that MMP10, BST2 and TMEM70 have the highest pathological scores in patients with good prognosis, PREX1 has the highest score in patients with poor prognosis, and BST2 has the lowest score in patients with medium prognosis. The keloids typed by HMECS are accompanied by more pain, itching, color abnormality, blood vessel abnormality, higher Vancouver Scar Scale (VSS) scores and higher recurrence rates, which indicates that the patients have poorer prognosis. The above results indicate that the combined application of PREX1, MMP10, BST2 and TMEM70 can guide the molecular typing or prognosis evaluation of keloids. Therefore, the application provides refined diagnosis or prognosis for keloids, provides a basis for personalized treatment of the keloids, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to the application of PREX1, MMP10, BST2, and TMEM70 as biomarkers for keloids. More specifically, it relates to the combined application of PREX1, MMP10, BST2, and TMEM70 in molecular subtyping or prognostic assessment of keloids, enabling precise diagnosis or prognosis of keloid patients. Background Technology

[0002] Keloids are a common fibroproliferative skin disease, a benign skin tumor that typically occurs after skin injury caused by genetic or pathological factors. It is generally accepted that fibroblasts and myofibroblasts, which form the extracellular matrix (ECM), are the main cell types involved in wound healing remodeling and keloid formation. However, wound healing is a complex process, with angiogenesis playing a crucial role in tissue repair. Studies have shown that vascular endothelial cells and their extensive interactions with surrounding cells appear to play a vital inducing role in scar pathogenesis. Abnormal vascular endothelial cell function and their interactions with immune cells and fibrosis-associated cells may lead to inflammation and ECM deposition in abnormal scars, increasing the risk of keloid formation. Although various anti-angiogenic keloid treatment strategies have been developed, such as laser therapy and radiation therapy, they have had limited effectiveness in reducing keloid recurrence rates. Therefore, we hypothesize that abnormal vascular regulation may be the basis for keloid development during wound healing, but the underlying pathological mechanisms are not fully understood, and current treatment strategies still lack precise targeted drugs.

[0003] Keloid formation is the result of multiple gene interactions: the BST2 protein encoded by the BST2 gene can promote the adhesion of late circulating endothelial progenitor cells to damaged endothelial cells, thereby promoting angiogenesis; TMEM70 encodes a mitochondrial inner membrane protein that can promote the in situ assembly of ATP synthase proton channels, improve oxygen content in the wound microenvironment, and may represent better neovascularization and less collagen deposition, thereby improving scar prognosis; the MMP10 protein encoded by the MMP10 gene has been shown to participate in the VEGF-induced angiogenesis pathway; PREX1 encodes a Rac guanine nucleotide exchange factor that integrates chemotactic signals, and studies have shown that it may be a key mediator of vascular barrier disruption and participate in tumor necrosis factor (TNF)-α-induced endothelial barrier dysfunction.

[0004] In summary, PREX1, MMP10, BST2, and TMEM70 may play important roles in the development and progression of keloids. However, the combined effect of PREX1, MMP10, BST2, and TMEM70 remains unclear, and there are no literature reports on their use as novel molecular subtyping markers for keloids to guide prognosis and personalized treatment. Summary of the Invention

[0005] This invention addresses the aforementioned problems. The first objective is to provide the application of PREX1, MMP10, BST2, and TMEM70 in combination as refined diagnostic or prognostic markers for keloids. The second objective is to provide the application of PREX1, MMP10, BST2, and TMEM70 in combination in the preparation of a molecular typing or prognostic diagnostic kit for keloids, which classifies keloids into low-maturity vascular endothelial cell subtypes (LMECS), intermediate-maturity vascular endothelial cell subtypes (MMECS), and high-maturity vascular endothelial cell subtypes (HMECS).

[0006] The research process of this invention is as follows:

[0007] The inventors conducted integrated bioinformatics analysis on single-cell sequencing and tissue sequencing data of keloids from publicly available databases. By combining genes related to vascular endothelial cell fate differentiation and differentially expressed genes related to clinical prognosis, they obtained a key set of genes related to vascular endothelial cell fate differentiation. Further investigation revealed that PREX1, MMP10, BST2, and TMEM70 are associated with both vascular endothelial cell differentiation and the clinical outcome of keloids, suggesting that the combined application of PREX1, MMP10, BST2, and TMEM70 can guide the molecular subtyping of keloids.

[0008] Next, differential gene expression analysis was performed to construct a new molecular subtype of keloids: low-maturity vascular endothelial cell subtype (LMECS), intermediate-maturity vascular endothelial cell subtype (MMECS), and high-maturity vascular endothelial cell subtype (HMECS). The results showed that MMP10, BST2, and TMEM70 were significantly highly expressed in LMECS, PREX1 was significantly highly expressed in HMECS, and BST2 was significantly lowly expressed in MMECS. Clinical correlation analysis confirmed that HMECS corresponds to the worst clinical prognosis, while LMECS corresponds to the best clinical prognosis.

[0009] Further immunohistochemical staining of surgically excised keloids revealed that MMP10, BST2, and TMEM70 scored highest in patients with good prognosis, PREX1 scored highest in patients with the worst prognosis, and BST2 scored lowest in patients with moderate prognosis. These results suggest that the combined use of PREX1, MMP10, BST2, and TMEM70 can guide the molecular subtyping of keloids.

[0010] Meanwhile, this invention performs clinical relevance scoring on immunohistochemical staining pathology scores and patient clinicopathological characteristics and prognosis. The results show that HMECS-type keloids are accompanied by more pain, itching, abnormal color, vascular abnormalities, higher Vancouver Scar Assessment Scale (VSS) scores, and higher recurrence rates, indicating a worse prognosis for patients. This suggests that the combined application of PREX1, MMP10, BST2, and TMEM70 can be used for prognostic assessment of keloids.

[0011] Based on the above research, the specific technical solution of the present invention is as follows:

[0012] In a first aspect, the present invention provides the application of a reagent for detecting a combined proteome in the preparation of molecular typing or prognostic reagents or kits for keloids, wherein the combined proteome is a combination of PREX1, MMP10, BST2 and TMEM70 proteins.

[0013] Preferably, the keloid molecular typing or prognostic reagent is a reagent for detecting the expression levels of PREX1, MMP10, BST2 and TMEM70 in biological samples; the keloid molecular typing or prognostic kit contains reagents for detecting the expression levels of PREX1, MMP10, BST2 and TMEM70 in biological samples.

[0014] Based on the expression levels of PREX1, MMP10, BST2, and TMEM70 in biological samples, keloids were classified into three subtypes: low-maturity vascular endothelial cells (LMECS), intermediate-maturity vascular endothelial cells (MMECS), and high-maturity vascular endothelial cells (HMECS). MMP10, BST2, and TMEM70 were significantly overexpressed in the low-maturity vascular endothelial cell subtype, PREX1 was significantly overexpressed in the high-maturity vascular endothelial cell subtype, and BST2 was significantly underexpressed in the intermediate-maturity vascular endothelial cell subtype.

[0015] Further optimization involves using reagents selected from those used in immunohistochemistry to detect the expression levels of PREX1, MMP10, BST2, and TMEM70 proteins in biological samples.

[0016] Further preferably, the biological sample is obtained from the subject's skin tissue, specifically paraffin-embedded tissue of a patient's keloid scar.

[0017] In a second aspect, the present invention provides a molecular typing or prognostic kit for keloids, the kit comprising reagents for detecting the expression levels of PREX1, MMP10, BST2 and TMEM70 in biological samples.

[0018] Preferably, the kit is a kit for detection using immunohistochemistry, containing antibodies that specifically recognize human PREX1, human MMP10, human BST2, and human TMEM70, as well as immunohistochemical reagents.

[0019] In a third aspect, the present invention provides a detection method for the above-described reagent kit for detecting PREX1, MMP10, BST2, and TMEM70 in keloid tissue, the specific steps of which are as follows:

[0020] 1. Dewaxing to water

[0021] (1) 4μm thick paraffin sections, 4 consecutive sections for each keloid tissue, baked in an oven at 60℃ for 1 hour;

[0022] (2) Xylene treatment for 10 min × 3 times;

[0023] (3) Wash with 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol and double-distilled water in sequence for 5 minutes each.

[0024] 2. Antigen retrieval

[0025] (1) Treat with a methanol solution containing 3% H2O2 for 20 min;

[0026] (2) Wash with double-distilled water for 5 minutes × 3 times;

[0027] (3) Antigen retrieval (high-pressure retrieval):

[0028] Place the slices in a pressure cooker containing EDTA alkaline repair solution (2mM EDTA, 2mM Na2HPO4), boil under pressure for 2 minutes, and then let cool naturally to room temperature.

[0029] 3. Primary antibody incubation

[0030] (1) Wash with PBS for 5 min;

[0031] (2) Cover the tissue with 50 μl of 1% BSA and block at 37°C for 30 min;

[0032] (3) Remove the blocking solution, add 50 μl of PREX1, MMP10, BST2 or TMEM70 primary antibody diluted according to the ratio to each of the four serial slices, and incubate overnight at 4°C in a humidified chamber.

[0033] 4. Secondary antibody incubation

[0034] (1) Remove the primary antibody and wash with PBS for 5 min × 4 times;

[0035] (2) Add 50 μl of HRP-labeled secondary antibody diluted according to the ratio and incubate at 37℃ for 30-45 min;

[0036] 5. Color rendering, blue reflection

[0037] (1) Wash with PBS for 5 min × 4 times;

[0038] (2) Develop color with DAB (1:50) for 3-10 minutes until a brick-red precipitate appears, then stop the color development with double-distilled water.

[0039] (3) Counterstain with hematoxylin for 10 min, differentiate with hydrochloric acid and alcohol, rinse with tap water for 20-30 min to reverse blue staining.

[0040] 6. Dehydration sealing sheet

[0041] The slides were sequentially treated with double-distilled water, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, carbolic acid, and xylene for 5 minutes each, and then sealed with resin.

[0042] 7. Analysis

[0043] Expression scores of PREX1, MMP10, BST2 and TMEM70 were interpreted under a microscope.

[0044] The beneficial protections and effects of this invention are as follows:

[0045] This invention provides a novel molecular subtyping marker for keloids using a combination of PREX1, MMP10, BST2, and TMEM70, offering significant guidance for prognostic assessment and personalized treatment of keloids. Immunohistochemical staining of surgically excised keloids revealed that MMP10, BST2, and TMEM70 scored highest in patients with good prognoses, while PREX1 scored highest in patients with poor prognoses. Keloids classified as HMECS were associated with increased pain, itching, abnormal color, vascular abnormalities, higher Vancouver Scar Assessment Scale (VSS) scores, and higher recurrence rates, indicating a worse prognosis. These results suggest that the combined application of PREX1, MMP10, BST2, and TMEM70 can guide molecular subtyping or prognostic assessment of keloids. This invention provides refined diagnosis and prognosis for keloids, laying the foundation for personalized treatment and possessing broad clinical application prospects. Attached Figure Description

[0046] Figure 1 This is a set of genes related to the key vascular endothelial cell fate differentiation.

[0047] Figure 2 To propose a new molecular subtyping of keloids based on a set of key genes related to vascular endothelial cell fate and differentiation;

[0048] Figure 3 To analyze the clinical relevance and predictive value of the new molecular subtypes of keloids;

[0049] Figure 4 Immunohistochemical staining and clinical relevance scoring were performed on a series of keloid samples. A represents the immunohistochemical results, and B represents the clinical relevance analysis of molecular subtypes (pain, itching, color abnormalities, vascular abnormalities, recurrence rate, and Vancouver Scar Assessment Scale score). Detailed Implementation

[0050] The following examples and experimental cases further illustrate the present invention and should not be construed as limiting the invention. The examples do not include detailed descriptions of conventional methods, such as PCR methods, methods used to construct vectors and plasmids, methods for inserting protein-encoding genes into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention, and the preferred embodiments and materials described herein are for illustrative purposes only.

[0052] Example 1: Identifying a set of key vascular endothelial cell fate and differentiation-related genes by integrating single-cell sequencing data from keloids and tissue sequencing data.

[0053] We performed integrative biological analysis on single-cell sequencing and tissue sequencing data of keloids in our database to identify gene sets related to vascular endothelial cell fate differentiation and clinical prognosis. Then, we obtained the key gene set related to vascular endothelial cell fate differentiation by taking the intersection of these gene sets. The results showed that PREX1, MMP10, BST2, and TMEM70 are associated with both vascular endothelial cell differentiation and clinical outcomes of keloids, suggesting that the combined application of PREX1, MMP10, BST2, and TMEM70 can guide the molecular subtyping of keloids. Figure 1 ).

[0054] Example 2: A novel molecular subtyping of keloids based on a set of key vascular endothelial cell fate and differentiation related genes.

[0055] Based on key genes related to vascular endothelial cell fate and differentiation, we constructed a new molecular subtyping of keloids using consensus clustering analysis: cluster 1, cluster 2, and cluster 3. Differential gene expression analysis revealed that MMP10, BST2, and TMEM70 were highly expressed in cluster 3, PREX1 was highly expressed in cluster 2, and BST2 was lowly expressed in cluster 1. This result suggests that the combined application of PREX1, MMP10, BST2, and TMEM70 can guide the molecular subtyping of keloids. Figure 2 ).

[0056] Example 3: Clinical Correlation Analysis and Prognostic Value of New Molecular Subtypes of Keloids

[0057] Principal component analysis and clinical relevance analysis of the molecular subtyping revealed that 76% of the samples in cluster 3 were healthy skin samples, while only 24% were keloid samples. In contrast, keloids accounted for a higher proportion in clusters 1 and 2 (83% and 68%, respectively), confirming that our molecular subtyping has clinical guiding value.

[0058] Further calculations of vascular endothelial cell scores for each subtype of our proposed molecular subtyping were performed to reflect vascular endothelial cell maturity. The results showed that cluster 3 had the lowest score, and the lower the score, the higher the proportion of healthy skin samples. Finally, a Sankey diagram was used to illustrate the molecular subtyping, keloid diagnosis, and diagnosis time corresponding to high and low vascular endothelial cell scores. Figure 3 Based on vascular endothelial cell scores and gene expression, we proposed a new classification of keloids: low-maturity vascular endothelial cell subtypes (cluster 3, LMECS, highly expressed by MMP10, BST2, and TMEM70 as markers); high-maturity vascular endothelial cell subtypes (cluster 2, HMECS, highly expressed by PREX1 as a marker); and intermediate-maturity vascular endothelial cell subtypes (cluster 1, MMECS, low expressed by BST2 as a marker). This molecular classification suggests that it has guiding significance for the clinical diagnosis and prognosis of keloids.

[0059] Example 4: Immunohistochemical staining and clinical relevance scoring of a series of keloid samples

[0060] We collected tissue samples and basic clinical information of 19 patients with clinical keloids. Immunohistochemical staining of paraffin specimens with PREX1, MMP10, BST2 and TMEM70 was performed. Professional pathologists were invited to score the expression of the above genes in vascular endothelial cells to identify the molecular subtype of keloids in the patients. Among the patients, 4 were HMECS, 4 were MMECS, and 11 were LMECS.

[0061] Immunohistochemical results showed that the expression levels of MMP10, BST2, and TMEM70 were highest in the LMECS group, the expression level of PREX1 was highest in the HMECS group, and the expression level of BST2 was significantly lower in the MMECS group. Figure 4 A). Further clinical relevance scoring was performed on the molecular subtypes corresponding to gene scoring. The results showed that 100% of keloids in the HMECS subtype were accompanied by pain, 75% by itching, and 100% by abnormal color and vascularity, with a recurrence rate as high as 75%. Furthermore, the mean score of the Vancouver Scar Assessment Scale for the HMECS subtype was 8.25, significantly higher than that of the LMECS group (5.23) and the MMECS group (6.88). Figure 4 B). The results further suggest that immunohistochemistry using PREX1, MMP10, BST2 combined with TMEM70 protein can diagnose a new molecular subtype of keloids and has clinical guiding significance.

[0062] The above experimental results show that detecting the expression of PREX1, MMP10, BST2, and TMEM70 proteins using immunohistochemistry can predict the clinical prognosis of keloid patients. High expression of MMP10, BST2, and TMEM70 proteins in keloid tissue indicates a better prognosis; low BST2 expression indicates a moderate prognosis; and high PREX1 expression indicates a poor prognosis, with keloids accompanied by more pain, itching, abnormal color, vascular abnormalities, and a higher recurrence rate and Vancouver Scar Assessment Scale (VSS) score. Therefore, detecting the expression of PREX1, MMP10, BST2, and TMEM70 as molecular subtyping markers can predict the clinical prognosis of keloids. Correspondingly, specific antibodies against PREX1, MMP10, BST2, and TMEM70, including monoclonal and polyclonal antibodies, can be used to prepare formulations or kits for keloid molecular subtyping, which is obvious to those skilled in the art.

[0063] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of reagents for detecting the expression levels of PREX1, MMP10, BST2 and TMEM70 in the preparation of molecular typing or prognostic assessment reagents or kits for keloids.

2. The application according to claim 1, characterized in that, Based on the expression levels of PREX1, MMP10, BST2, and TMEM70 in biological samples, keloids were classified into three subtypes: low-maturity vascular endothelial cells, medium-maturity vascular endothelial cells, and high-maturity vascular endothelial cells.

3. The application according to claim 2, characterized in that, MMP10, BST2, and TMEM70 were highly expressed in low-maturity vascular endothelial cell subtypes, PREX1 was highly expressed in high-maturity vascular endothelial cell subtypes, and BST2 was lowly expressed in intermediate-maturity vascular endothelial cell subtypes.

4. The application according to claim 1, characterized in that, The reagents used to detect the expression levels of PREX1, MMP10, BST2, and TMEM70 proteins in biological samples were selected from those used for immunohistochemical detection.

5. The application according to claim 2, characterized in that, The biological sample was obtained from the subject's skin tissue.

6. A molecular subtyping or prognostic assessment kit for keloids, characterized in that, This kit contains reagents for detecting the expression levels of PREX1, MMP10, BST2, and TMEM70 in biological samples. The reagents include antibodies that specifically recognize human PREX1, human MMP10, human BST2, and human TMEM70, as well as immunohistochemical reagents.

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