Use of klk6, tpsb2, mmp1 and foxc2 in combination in the molecular subtyping or prognosis of keloids

Abstract

The application relates to the technical field of biological detection, and provides application of KLK6, TPSB2, MMP1 and FOXC2 in molecular typing or prognosis of keloids. Immunohistochemical staining is carried out on the keloids removed by clinical operation, and it is found that KLK6 and TPSB2 have the highest pathological score in patients with poor prognosis, MMP1 has the highest pathological score in patients with the best prognosis, FOXC2 has the highest score in patients with medium prognosis, the keloids of the HFIC typing have greater thickness, higher hardness and higher scores of the Vancouver Scar Assessment Scale, and the patients have poorer prognosis, and the above results indicate that the combined application of KLK6, TPSB2, MMP1 and FOXC2 can guide the molecular typing or prognosis evaluation of the keloids. Therefore, the application provides fine diagnosis or prognosis for the 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 KLK6, TPSB2, MMP1, and FOXC2 as markers for keloids. More specifically, it relates to the combined application of KLK6, TPSB2, MMP1, and FOXC2 in the molecular subtyping or prognosis of keloids, enabling precise diagnosis or prognosis of keloid patients. Background Technology

[0002] Keloids are a fibrous skin disease caused by abnormal deposition of collagen and extracellular matrix following deep skin damage due to trauma, burns, or surgery, affecting approximately 4.5-16% of the global population. [1 - 3] The resulting skin appearance and functional impairments can severely impact a patient's quality of life and mental and physical health. [4,5] Currently, the specific mechanisms of keloid formation remain unclear, and precise prevention and treatment strategies are lacking, leading to frequent recurrence in some patients after surgery. Fibroblasts are the most common cell type in the body's connective tissue, exhibiting high heterogeneity and dynamism. They possess the ability to produce, deposit, and remodel the extracellular matrix, playing a central role in tissue repair and fibrosis. [6] Therefore, fibroblasts, as key cells in scar formation and fibrosis, are also a popular research target in clinical practice seeking to improve wound healing and reduce fibrosis.

[0003] KLK6 belongs to the serine protease family and is commonly used as a biomarker of inflammation. Studies have shown that targeted overexpression of KLK6 in the skin can increase skin inflammation through the PAR1 axis, leading to severe systemic psoriasis-like dermatitis. [7] Furthermore, KLK6 acts on immune cells, increasing the expression of pro-inflammatory factors and decreasing the level of anti-inflammatory factors, thus shifting the immune system towards a pro-inflammatory response and exacerbating neuroinflammation and paralytic neurological deficits in patients with multiple sclerosis. [8] Therefore, it may regulate the immune microenvironment of skin injury repair, promote excessive deposition of extracellular matrix, and thus affect the wound repair process, leading to the formation of keloids and fibrosis. TPSB2 is also a serine protease closely related to inflammatory responses and fibrosis. Recent studies have found that TPSB2 can enhance fibroblast function, promote collagen synthesis and secretion, and activate the TGF-β signaling pathway, thereby leading to peritoneal adhesions, pulmonary fibrosis, liver fibrosis, and renal fibrosis. [9-13] This suggests that TPSB2 may participate in the formation and development of keloids through both pro-inflammatory effects and the promotion of matrix synthesis. MMP1 is a typical matrix metalloproteinase that can effectively disrupt the extracellular matrix.

[14] In vitro and in vivo studies have shown that MMP1 can reduce inflammation, promote wound healing, and reduce scar formation; furthermore, increasing the expression of MMP1 in keloid fibroblasts can effectively enhance collagen degradation, suggesting a potential therapeutic effect on keloids. [15-17] FOXC2 is a member of the Forkhead transcription factor family. It has a dual role in wound repair: it can promote wound repair by inducing angiogenesis and EMT, but it can also lead to excessive angiogenesis and extracellular matrix formation, thereby promoting scar formation. [18-21] .

[0004] In summary, KLK6, TPSB2, MMP1, and FOXC2 may play important roles in the development and progression of keloids. However, the combined effect of KLK6, TPSB2, MMP1, and FOXC2 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 KLK6, TPSB2, MMP1, and FOXC2 in combination as refined diagnostic or prognostic markers for keloids. The second objective is to provide the application of KLK6, TPSB2, MMP1, and FOXC2 in combination in the preparation of a molecular subtyping or prognostic diagnostic kit for keloids, which classifies keloids into low fibroblast infiltration type (LFIC), medium fibroblast infiltration type (MFIC), and high fibroblast infiltration type (HFIC).

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

[0007] The inventors conducted integrated bioinformatics analysis on single-cell sequencing data and tissue sequencing data of keloids in publicly available databases. By taking the intersection of genes related to fibroblast fate differentiation and differentially expressed genes related to clinical prognosis, they obtained a set of key genes related to fibroblast fate differentiation. They further discovered that KLK6, TPSB2, MMP1, and FOXC2 are related to both fibroblast differentiation and the clinical outcome of keloids, suggesting that the combined application of KLK6, TPSB2, MMP1, and FOXC2 can guide the molecular subtyping of keloids.

[0008] A new molecular subtyping system for keloids was constructed: low fibroblast infiltration (LFIC), medium fibroblast infiltration (MFIC), and high fibroblast infiltration (HFIC), and differential gene expression analysis was performed. Results showed that KLK6 and TPSB2 were significantly highly expressed in HFIC, MMP1 was significantly highly expressed in LFIC, and FOXC2 was significantly highly expressed in MFIC. Clinical correlation analysis confirmed that HFIC corresponded to the worst clinical prognosis, while LFIC corresponded to the best clinical prognosis.

[0009] Further immunohistochemical staining of surgically excised keloids revealed that KLK6 and TPSB2 had the highest pathological scores in patients with poor prognosis, MMP1 had the highest score in patients with the best prognosis, and FOXC2 had the highest score in patients with moderate prognosis. These results suggest that the combined use of KLK6, TPSB2, MMP1, and FOXC2 can guide the molecular subtyping of keloids.

[0010] Meanwhile, this invention performs clinical relevance scoring on immunohistochemical staining pathological scores and patient clinicopathological characteristics and prognosis. The results showed that HFIC-type keloids were thicker, harder, and had higher scores on the Vancouver Scar Assessment Scale, with worse prognoses, suggesting that the combined application of KLK6, TPSB2, MMP1, and FOXC2 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 KLK6, TPSB2, MMP1 and FOXC2 proteins.

[0013] Preferably, the keloid molecular typing reagent is a reagent for detecting the expression levels of KLK6, TPSB2, MMP1, and FOXC2 in biological samples; the keloid molecular typing kit contains reagents for detecting the expression levels of KLK6, TPSB2, MMP1, and FOXC2 in biological samples.

[0014] Based on the expression levels of KLK6, TPSB2, MMP1, and FOXC2 in biological samples, keloids were classified into three subtypes: low fibroblast infiltration (LFIC), medium fibroblast infiltration (MFIC), and high fibroblast infiltration (HFIC). KLK6 and TPSB2 were highly expressed in the high fibroblast infiltration subtype, MMP1 was highly expressed in the low fibroblast infiltration subtype, and FOXC2 was highly expressed in the medium fibroblast infiltration subtype.

[0015] Further optimization involves using reagents selected from those used in immunohistochemistry to detect the expression levels of KLK6, TPSB2, MMP1, and FOXC2 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 KLK6, TPSB2, MMP1 and FOXC2 in biological samples.

[0018] Preferably, the kit is a detection kit using immunohistochemistry, containing antibodies that specifically recognize human KLK6, human TPSB2, human MMP1, and human FOXC2, as well as immunohistochemical reagents.

[0019] In a third aspect, the present invention provides a method for detecting the above-mentioned reagent kit for detecting KLK6, TPSB2, MMP1 and FOXC2 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 10min × 3 times;

[0023] (3) Pass the solution through 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol and double distilled water in sequence for 5 minutes each.

[0024] 2. Antigen retrieval

[0025] (1) 3% H2O2 methanol solution 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 KLK6, TPSB2, MMP1 or FOXC2 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] (1) Pass the sample through double-distilled water, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, carbolic acid, and xylene in sequence for 5 minutes each. Add resin to seal the slide.

[0042] 7. Analysis

[0043] Expression scores of KLK6, TPSB2, MMP1, and FOXC2 were interpreted under a microscope.

[0044] In a fourth aspect, the invention provides the application of reagents for detecting the expression levels of KLK6 and TPSB2 in the preparation of a drug screening kit for keloids with high fibroblast infiltration.

[0045] The method for drug screening using KLK6 and TPSB2 as biomarkers is as follows:

[0046] (A) Treat cells, tissues, or animals with high fibroblast infiltration keloids with candidate drugs;

[0047] (B) Detect the levels of KLK6 and TPSB2 or nucleic acid molecules encoding the protein in the cells, tissues or animals described;

[0048] (C) If the levels of KLK6 and TPSB2 or the nucleic acid molecules encoding the protein are lower than the levels before treatment with the candidate drug, it indicates that the candidate drug has the effect of treating keloids with high fibroblast infiltration by inhibiting KLK6 and TPSB2.

[0049] The beneficial protections and effects of this invention are as follows:

[0050] This invention provides a novel molecular subtyping marker for keloids using a combination of KLK6, TPSB2, MMP1, and FOXC2, particularly offering important guidance for prognostic assessment and personalized treatment of keloids with high fibroblast infiltration (HFIC, with KLK6 and TPSB2 as markers). Immunohistochemical staining of surgically excised keloids revealed that KLK6 and TPSB2 had the highest pathological scores in patients with poor prognosis, MMP1 had the highest score in patients with the best prognosis, and FOXC2 had the highest score in patients with moderate prognosis. Furthermore, HFIC-type keloids were thicker, harder, and had higher scores on the Vancouver Scar Assessment Scale, indicating a worse prognosis. These results suggest that the combined application of KLK6, TPSB2, MMP1, and FOXC2 can guide molecular subtyping or prognostic assessment of keloids. Therefore, this invention provides a refined diagnosis or prognosis for keloids, laying the foundation for personalized treatment and possessing broad clinical application prospects. Attached Figure Description

[0051] Figure 1 This is a set of genes related to key fibroblast fate and differentiation.

[0052] Figure 2 To propose a new molecular subtyping of keloids based on a set of key fibroblast fate and differentiation-related genes;

[0053] Figure 3 To analyze the clinical relevance and predictive value of the new molecular subtypes of keloids;

[0054] Figure 4 Prediction of small molecule drugs targeted by HFIC;

[0055] Figure 5 Immunohistochemical staining and clinical relevance scoring were performed on a series of keloid samples. A represents the immunohistochemical results, B represents the clinical relevance score of the molecular subtype, and C represents the Vancouver Scar Assessment Scale for the HFIC group. Detailed Implementation

[0056] 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.

[0057] 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.

[0058] Example 1: Identifying key fibroblast fate and differentiation-related gene sets by integrating single-cell sequencing data from keloids and tissue sequencing data.

[0059] We performed integrative biological analysis on single-cell sequencing and tissue sequencing data of keloids in our database to identify gene sets related to fibroblast fate and differentiation, as well as gene sets related to clinical prognosis. Then, we took the intersection of these gene sets to obtain a key set of genes related to fibroblast fate and differentiation. The results showed that KLK6, TPSB2, MMP1, and FOXC2 are associated with both fibroblast differentiation and the clinical outcome of keloids, suggesting that the combined application of KLK6, TPSB2, MMP1, and FOXC2 can guide the molecular subtyping of keloids. Figure 1 ).

[0060] Example 2: A novel molecular subtyping of keloids based on a set of key fibroblast fate and differentiation-related genes.

[0061] Based on key fibroblast fate and differentiation-related genes, we constructed a new molecular subtype of keloids using consensus clustering analysis: cluster 1, cluster 2, and cluster 3. Differential gene expression analysis revealed high expression of KLK6 and TPSB2 in cluster 3, high expression of MMP1 in cluster 1, and high expression of FOXC2 in cluster 2. This result suggests that the combined application of KLK6, TPSB2, MMP1, and FOXC2 can guide the molecular subtyping of keloids. Figure 2).

[0062] Example 3: Clinical Correlation Analysis and Prognostic Value of New Molecular Subtypes of Keloids

[0063] Principal component analysis and clinical relevance analysis were performed on the molecular subtypes. The results showed that 76% of the samples in cluster 1 were healthy skin samples, and 24% were keloid samples, while in cluster 3, 90% were keloid samples and 10% were healthy samples, confirming the clinical guiding value of our molecular subtypes. Further calculation of fibroblast scores for each subtype of our proposed molecular subtypes to reflect fibroblast infiltration revealed that cluster 1 had the lowest score, while cluster 3 had the highest score, and higher scores corresponded to fewer healthy skin samples. Finally, a Sankey diagram illustrated the molecular subtypes, keloid diagnosis, and diagnosis time corresponding to high and low fibroblast scores. Figure 3 Based on fibroblast scores and gene expression, we proposed a new classification of keloids: low fibroblast infiltration (cluster 1, LFIC, and MMP1 as markers), medium fibroblast infiltration (cluster 2, MFIC, and FOXC2 as markers), and high fibroblast infiltration (cluster 3, HFIC, KLK6, and TPSB2 as markers). This molecular classification suggests that it has guiding significance for the clinical diagnosis and prognosis of keloids.

[0064] Example 4: HFIC-targeted small molecule drug prediction

[0065] Cmap data analysis revealed that Ouabain is one of the top 10 inhibitors of HFIC. Literature review also confirmed that Ouabain can inhibit the classical fibrosis pathway TGFβ and suppress fibroblast differentiation into myofibroblasts, improve collagen synthesis in skin fibroblasts, and potentially improve the microenvironment to promote angiogenesis, showing promise as a potential drug for treating fibrotic diseases such as keloids. These data suggest that Ouabain may be used for personalized treatment of HFIC. Figure 4 ).

[0066] Example 5: Immunohistochemical staining and clinical relevance scoring of a series of keloid samples

[0067] Nineteen clinical keloid tissue samples and basic clinical information were collected. Immunohistochemical staining of paraffin-embedded specimens for KLK6, TPSB2, MMP1, and FOXC2 was performed. Professional pathologists were invited to score the expression of these genes in fibroblasts to determine the molecular subtype of keloids. Twelve patients were found to have HFIC, three to have MFIC, and four to have LFIC. Immunohistochemical results showed that MMP1 expression was highest in the LFIC group, FOXC2 expression was highest in the MFIC group, while KLK6 and TPSB2 expression were highest in the HFIC group. Figure 5 A). Further clinical relevance scoring based on gene scores and corresponding molecular subtypes showed that nearly 83% of keloids in the HFIC group had a thickness greater than 2 mm, far exceeding other groups; and the keloids in the HFIC group were significantly harder ( Figure 5 B); In addition, the mean score of the Vancouver Scar Assessment Scale in the HFIC group was 6.92, which was much higher than that in the LFIC group (4.75) and the MFIC group (5.33). Figure 5 C). The results further suggest that immunohistochemistry using KLK6, TPSB2, MMP1 combined with FOXC2 protein can diagnose a new molecular subtype of keloids and has clinical guiding significance.

[0068] The above experimental results show that detecting the expression of KLK6, TPSB2, MMP1, and FOXC2 proteins using immunohistochemistry can predict the clinical prognosis of keloid patients. When KLK6 and TPSB2 proteins are highly expressed in keloid tissue, the keloid is thicker and harder, the Vancouver Scar Assessment Scale score is higher, and the recurrence rate is higher. Therefore, detecting the expression of KLK6, TPSB2, MMP1, and FOXC2 as molecular subtyping markers can predict the clinical prognosis of keloids. Correspondingly, specific antibodies against KLK6, TPSB2, MMP1, and FOXC2, 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.

[0069] The references for this invention are as follows:

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[0091] 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 combined reagents for detecting the expression levels of KLK6, TPSB2, MMP1, and FOXC2 genes or proteins in the preparation of molecular typing reagents or kits for keloids. Based on the expression levels of KLK6, TPSB2, MMP1, and FOXC2 genes or proteins in biological samples, keloids were classified into three subtypes: low fibroblast infiltration, medium fibroblast infiltration, and high fibroblast infiltration. KLK6 and TPSB2 were highly expressed in the high fibroblast infiltration subtype, MMP1 was highly expressed in the low fibroblast infiltration subtype, and FOXC2 was highly expressed in the medium fibroblast infiltration subtype. The biological sample was obtained from the skin tissue of the subject being tested.

2. The application according to claim 1, characterized in that, The reagents used to detect the expression levels of KLK6, TPSB2, MMP1, and FOXC2 proteins in biological samples were selected from those used for immunohistochemical detection.

3. A molecular typing kit for keloids, characterized in that, This kit contains reagents for detecting the expression levels of molecular subtyping marker proteins for keloids in biological samples. These markers consist of KLK6, TPSB2, MMP1, and FOXC2. The reagent consists of antibodies that specifically recognize human KLK6, human TPSB2, human MMP1, and human FOXC2.

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