Use of cartilage acidic protein 1 in preparation of reagent for diagnosing lung adenocarcinoma or evaluating prognosis of lung adenocarcinoma

Abstract

The application belongs to the field of biological medicine, and relates to application of chondroitin acid protein 1 in preparation of a reagent for diagnosing lung adenocarcinoma or evaluating lung adenocarcinoma prognosis. The application screens CRTAC1 from a GEO database through bioinformatics analysis and a machine learning algorithm, and the CRTAC1 is significantly lowly expressed in lung adenocarcinoma tissues, and an AUC reaches 0.972. Verification of a GEPIA database shows that low expression of the CRTAC1 is significantly related to poor prognosis such as advanced pathological stages, and a patient with high expression has better prognosis. The CRTAC1 blocks epithelial-mesenchymal transition through inhibition of an EGF / ErbB signal path, and remodels a tumor immune microenvironment through inhibition of M2 type macrophage polarization. A mouse lung metastasis model proves that overexpression of the CRTAC1 significantly reduces lung metastasis nodules. The application provides a new molecular marker and a drug target for early diagnosis, prognosis evaluation and precise treatment of lung adenocarcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a biomarker for the diagnosis or prognostic assessment of lung adenocarcinoma. Background Technology

[0002] Lung cancer is the leading cause of cancer incidence and death worldwide. According to the International Agency for Research on Cancer (IARC) 2024's "Global Cancer Statistics Report 2022," approximately 2.48 million new cases of lung cancer were diagnosed globally in 2022, with 1.817 million deaths, accounting for 18.7% of all cancer deaths and ranking first among all cancer causes. Lung adenocarcinoma (LUAD), the most common histological subtype of lung cancer, accounts for more than 50% of non-small cell lung cancers, and its incidence and mortality rates are continuously rising globally. Due to insufficient early diagnosis and chemotherapy resistance during treatment, approximately 70% of patients are diagnosed at an advanced stage, with a 5-year survival rate of only about 30%, seriously threatening global human health.

[0003] Recurrence and metastasis of lung cancer cells are the main causes of treatment failure. Currently, commonly used diagnostic markers for lung adenocarcinoma include carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1), and neuron-specific enolase (NSE). However, these markers have significant limitations in sensitivity and specificity, making them insufficient for early diagnosis. In terms of prognostic assessment, the TNM staging system remains the primary basis, but patients at the same stage show significant differences in prognosis, indicating a need for more precise molecular markers to guide individualized treatment. Therefore, there is an urgent need to find new, highly sensitive, and highly specific diagnostic markers and therapeutic targets for lung adenocarcinoma to improve patient prognosis and quality of life.

[0004] Cartilage acidic protein 1 (CRTAC1) is a secreted glycoprotein with a structure comprising a tight β-propeller core, a transthyretin-like (TR) domain, and multiple calcium, potassium, and sodium ion binding sites and functional domains. Initially identified in cartilage tissue, CRTAC1 is also widely distributed in brain, eye, nerve, and lung tissues, exhibiting high evolutionary conservation. A recent 2024 study used X-ray crystallography (1.6 Å resolution) to resolve the complete three-dimensional structure of CRTAC1, clarifying its unique three-domain folding feature. This protein also contains an integrin chain-like domain, an FG-GAP motif, an RGD motif, an ASPIC domain, an UnvB domain, and an EGF-like calcium-binding motif, suggesting a crucial role in mediating cell-cell or cell-matrix interactions.

[0005] In recent years, the relationship between CRTAC1 and tumors has gradually become a hot topic in cancer research. Studies have shown that downregulation of CRTAC1 expression in urothelial carcinoma can promote tumor invasiveness and lead to poor prognosis; in non-small cell lung cancer, CRTAC1 overexpression can induce cancer cell proliferation. 2+ CRTAC1-dependent apoptosis-promoting signaling and AKT1 degradation significantly enhance the sensitivity of tumor cells to cisplatin treatment. However, systematic studies on CRTAC1 in lung adenocarcinoma are still relatively lacking, and its potential as a diagnostic biomarker, prognostic indicator, and therapeutic target for lung adenocarcinoma has not been fully elucidated. In particular, the expression characteristics of CRTAC1 in lung adenocarcinoma, its correlation with clinicopathological features, its impact on tumor biological behavior, and its molecular mechanisms all lack in-depth research and experimental validation.

[0006] Therefore, in-depth research into the expression characteristics, clinical significance, and biological functions of CRTAC1 in lung adenocarcinoma is of profound clinical significance for developing novel precision diagnosis and treatment strategies. Summary of the Invention

[0007] This invention addresses the shortcomings of traditional lung adenocarcinoma diagnostic biomarkers in terms of sensitivity and specificity, as well as the limitations of existing therapeutic targets in predicting chemotherapy and immunotherapy responses. It proposes the application of chondroitin 1 in the preparation of reagents for diagnosing or assessing the prognosis of lung adenocarcinoma.

[0008] The technical solution of this invention is implemented as follows:

[0009] In a first aspect, the present invention provides a diagnostic biomarker for lung adenocarcinoma, the diagnostic biomarker including CRTAC1.

[0010] Preferably, the coding gene sequence of CRTAC1 can be found in NCBI Gene ID: 55118, with its mRNA sequence number NM_018058.4 and protein sequence number NP_060528.3. The CRTAC1 protein consists of an N-terminal signal peptide, a β-propeller core domain, a TTR-like domain, and a C-terminal functional domain, with a molecular weight of approximately 52 kDa.

[0011] Secondly, the present invention provides the application of the above-mentioned CRTAC1 as a prognostic marker for lung adenocarcinoma.

[0012] Preferably, CRTAC1 expression levels in lung adenocarcinoma patients are significantly lower than in adjacent normal tissues, and its low expression is closely associated with poor prognosis (including shorter overall survival and disease-free survival), advanced pathological stage, cancer cell metastasis, and higher tumor mutation burden. Therefore, CRTAC1 can serve as an independent prognostic marker for lung adenocarcinoma patients, while high expression indicates a favorable prognosis.

[0013] Thirdly, this invention provides the application of reagents for detecting CRTAC1 expression levels in the preparation of kits for the diagnosis or prognostic assessment of lung adenocarcinoma.

[0014] Preferably, the reagent includes antibodies, primers, probes, or combinations thereof that specifically recognize CRTAC1 and can be used for immunohistochemistry (IHC), Western blotting, qPCR, ELISA, or liquid biopsy detection.

[0015] Fourthly, the present invention also provides a medicament for treating lung adenocarcinoma, comprising a promoter that increases CRTAC1 expression and a pharmaceutically acceptable carrier.

[0016] CRTAC1 exerts its anti-cancer effect through a dual pathway of "cellular autonomy and microenvironment regulation": within tumor cells, CRTAC1 directly inhibits the invasive and metastatic ability of cancer cells by suppressing the EGF / ErbB signaling pathway, blocking epithelial-mesenchymal transition (EMT) and cytoskeleton remodeling; within the tumor microenvironment, CRTAC1 indirectly inhibits tumor progression by inhibiting the polarization of M0 macrophages into M2 tumor-associated macrophages (TAMs), remodeling the immunosuppressive microenvironment. Therefore, CRTAC1 can serve as a therapeutic target molecule for lung adenocarcinoma, used in the development of targeted drugs or immunomodulatory agents. Preferably, the expression promoter includes a CRTAC1 gene overexpression vector, recombinant CRTAC1 protein, a CRTAC1 small molecule agonist, or a demethylating agent, used to restore CRTAC1 expression levels in lung adenocarcinoma, inhibit tumor metastasis, and enhance chemosensitivity.

[0017] The present invention has the following beneficial effects:

[0018] 1. High diagnostic value: This invention is the first to systematically elucidate that CRTAC1 can serve as a diagnostic biomarker for lung adenocarcinoma. Studies show that, compared with normal tissues, CRTAC1 is significantly downregulated in tumor tissues of lung adenocarcinoma patients (both mRNA and protein levels are reduced), with an area under the ROC curve (AUC) of 0.972, demonstrating extremely high diagnostic accuracy.

[0019] 2. Precise prognostic assessment: Low CRTAC1 expression is significantly associated with advanced TNM stage and poor prognosis. Kaplan-Meier survival analysis showed that patients in the high CRTAC1 expression group had significantly better overall survival than those in the low expression group (TCGA cohort P=0.026; GSE31210 cohort P=0.042), confirming that CRTAC1 is an independent prognostic protective factor for lung adenocarcinoma.

[0020] 3. Novel Therapeutic Target: This invention reveals a novel network through which CRTAC1 exerts its anti-cancer effect via a dual mechanism of "intracellular ErbB pathway inhibition - microenvironmental immune remodeling". Overexpression of CRTAC1 can significantly inhibit the migration and invasion of lung adenocarcinoma cells and suppress M2 macrophage polarization, indicating that CRTAC1 plays the role of a tumor suppressor gene in the development of lung adenocarcinoma.

[0021] 4. Broad clinical application prospects: This invention provides novel molecular markers and drug targets for the early diagnosis, prognostic assessment and precision treatment of lung adenocarcinoma, and is expected to fill the gap in liquid biopsy and targeted therapy for lung adenocarcinoma. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The diagram shows the screening process and validation results for diagnostic biomarkers for lung adenocarcinoma. (A) Differentially expressed genes between lung adenocarcinoma tissue samples and adjacent normal tissue samples. (B) GO enrichment analysis of lung adenocarcinoma samples and adjacent normal tissue samples. (C) GSEA enrichment analysis of lung adenocarcinoma samples and adjacent normal tissue samples. (D) LASSO regression using cross-validation with a penalty coefficient λ to determine the optimal gene set. (E) The SVM-RFE algorithm using recursive feature elimination to screen the optimal discriminant gene set. (F) Taking the intersection of the two algorithms, seven candidate biomarkers were finally obtained.

[0024] Figure 2 Validation plots for CRTAC1 as a novel biomarker for lung adenocarcinoma; (A) Receiver operating characteristic (ROC) curves of seven candidate novel biomarkers for lung adenocarcinoma. (B) Expression levels of the seven candidate novel biomarkers for lung adenocarcinoma in lung adenocarcinoma. (C) Survival curves of the seven candidate novel biomarkers for lung adenocarcinoma.

[0025] Figure 3 The diagram shows the effect of CRTAC1 on the migration and invasion of lung adenocarcinoma cells; (A) shows the expression level of CRTAC1 in human lung adenocarcinoma cell lines. (B) shows the effect of CRTAC1 overexpression and knockdown on the migration and invasion of LUAD cells as detected by Tanswell assay.

[0026] Figure 4 Diagram illustrating the mechanism by which CRTAC1 inhibits the ErbB signaling pathway and the epithelial-mesenchymal transition.

[0027] Figure 5This is a diagram showing the regulation of macrophage polarization by CRTAC1 in the tumor microenvironment.

[0028] Figure 6 This diagram illustrates the in vivo inhibitory effect and mechanism of action of CRTAC1 on lung adenocarcinoma metastasis. (A) shows the construction of a mouse lung metastasis model. (B) shows the pathological examination of the mice. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0031] The main materials, reagents, and instruments used in this invention are as follows:

[0032] Cell lines: Human lung adenocarcinoma cell lines A549, H1299, H1975, LLC cells and mouse macrophages RAW264.7 were all derived from laboratory-preserved cell lines.

[0033] Main reagents: Fetal bovine serum (Lanzhou Rongye Biotechnology Co., Ltd.); 2×Taq Plus Master Mix II (Vazyme); Reverse transcription kit (Vazyme); HE staining solution kit (Servicebio); HiScript II Q Select RTSuperMix (Vazyme); SYBR Green qPCR reagent (Vazyme); DAB chromogenic reagent (Servicebio); DNA ladder (Takara); Agarose (Invitrogen); LV-CRTAC1, LV-CRTAC1-RNAi, negative control virus CON522, negative control virus CON351, HiTransGP virus transfer aid, HiTransGA virus transfer aid (Jikai Gene).

[0034] Main instruments: 7500 fast qPCR instrument (Thermo Fisher); real-time quantitative PCR instrument (Thermo Fisher); flow cytometer (Beckman); conventional optical inverted microscope (Macody); multi-functional microplate reader (BMG); R software (version 4.2.0).

[0035] Example 1: Screening for novel biomarkers for lung adenocarcinoma CRTAC1 based on machine learning and bioinformatics

[0036] 1. Data Source and Differential Analysis: Lung adenocarcinoma datasets GSE32863 and GSE75037, totaling 131 lung adenocarcinoma samples and 131 adjacent normal tissue samples, were downloaded from the GEO database. Differential analysis was performed using the "limma" package in R software. The selection criteria were: P-value < 0.05 and |log2FC| > 1. A total of 140 differentially expressed genes were identified (68 upregulated and 72 downregulated). Figure 1 A).

[0037] 2. Functional enrichment analysis: GO and GSEA functional enrichment analyses were performed using the "clusterProfiler" package in R. GO enrichment showed that cellular components were significantly enriched in the sarcolemma, basolateral sarcolemma, and basement membrane, etc. Figure 1 B); GSEA enrichment results showed that it was mainly enriched in the ErbB signaling pathway, cell adhesion and cytoskeleton regulation pathway (B); Figure 1 C).

[0038] 3. Machine Learning Screening: LASSO regression and SVM-RFE algorithms were used to screen for potential novel biomarkers for lung adenocarcinoma. LASSO regression determined the optimal gene set through cross-validation using the penalty coefficient λ. Figure 1 D); The SVM-RFE algorithm uses recursive feature elimination to select the optimal set of distinguishing genes (D); Figure 1 E). Taking the intersection of the two algorithms, seven candidate markers were finally obtained: MMRN1, SPOCK2, CAV2, C9orf19, FLJ13909, CRTAC1, KIAA1324L ( Figure 1 F).

[0039] 4. Diagnostic Value Assessment: ROC curves were generated using mRNA expression levels from 131 samples to evaluate the diagnostic value of seven candidate biomarkers. Results showed that all seven biomarkers possessed good diagnostic efficacy, with CRTAC1 exhibiting an AUC of 0.972, demonstrating excellent diagnostic accuracy. Figure 2 A).

[0040] Example 2: Validating the clinical value of CRTAC1 using the GEPIA database

[0041] 1. Expression Level Validation: CRTAC1 was independently validated using the GEPIA2 database (http: / / gepia2.cancer-pku.cn / ). Analysis of CRTAC1 expression levels in the TCGA-LUAD cohort showed that CRTAC1 mRNA expression in lung adenocarcinoma tissues was significantly lower than in normal tissues (P<0.05). Figure 2 B).

[0042] 2. Survival Analysis: Kaplan-Meier survival analysis showed that patients in the high CRTAC1 expression group had significantly better overall survival than those in the low expression group (TCGA cohort P=0.026; GSE31210 cohort P=0.042), confirming that CRTAC1 is an independent prognostic protective factor for lung adenocarcinoma. Figure 2 C).

[0043] Example 3: Cellular-level validation of CRTAC1

[0044] 1. Western blot and qPCR analysis of human lung adenocarcinoma cell lines (A549, H1299, H1975) confirmed that CRTAC1 was lowly expressed in lung adenocarcinoma cells, with the lowest expression in A549 and H1299 cells and higher expression in H1975 cells. Figure 3 A).

[0045] 2. Transwell migration and invasion assays showed that CRTAC1 overexpression significantly inhibited the migration and invasion abilities of A549 cells. Figure 3 B).

[0046] Example 4: Transcriptome sequencing and the molecular mechanism of CRTAC1 repression of metastasis

[0047] Transcriptome sequencing and GSEA analysis showed that CRTAC1 overexpression inhibited the ErbB signaling pathway, cell adhesion, and cytoskeleton regulation pathways; molecular docking and Co-IP experiments confirmed that CRTAC1 binds to EGFR via the RGD motif, inhibiting the EGF / ErbB signaling pathway and blocking EMT processes. Figure 4 ).

[0048] Example 5: Regulation of macrophage polarization in the tumor microenvironment by CRTAC1

[0049] Flow cytometry validation: In a mouse lung metastasis model, flow cytometry analysis showed that the proportion of M2 macrophages in the lung tissue of the CRTAC1 overexpression group was significantly reduced (P<0.05), further validating the above conclusions. Figure 5 ).

[0050] Example 6: Verification of the in vivo anti-tumor effect of CRTAC1 using a mouse lung metastasis model

[0051] 1. Animal experimental protocol: The experimental animals were 5-week-old female C57BL / 6N mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., ethics approval number: HUSOM2025-855. After one week of acclimatization culture, the mice were randomly divided into two groups (n=6 per group): control group and CRTAC1 overexpression group.

[0052] 2. Lung metastasis model construction: Logarithmic growth phase LLC cells were collected by trypsin digestion, centrifugation, and washing twice with PBS. The cells were then resuspended in ready-to-use sterile PBS and counted. The dosage was 1 × 10⁻⁶ cells per mouse. 6 Cells / 150 μL, injected via tail vein. Body weight changes were recorded every two days. Mice were sacrificed on day 20, and lung tissue was photographed and surface metastatic nodules were counted. Figure 6 A).

[0053] 3. Pathological examination: Lung tissue was fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin (HE) to observe lung metastases. Results showed that the number of lung metastatic nodules in the CRTAC1 overexpression group was significantly less than that in the control group (P<0.01). HE staining showed reduced tumor cell infiltration in the lung tissue, confirming that CRTAC1 significantly inhibited lung adenocarcinoma metastasis in vivo. Figure 6 B).

[0054] In summary, this invention, by integrating bioinformatics mining, machine learning algorithms, multi-center clinical validation, and in vitro and in vivo functional experiments, systematically elucidates for the first time the multiple values ​​of CRTAC1 as a diagnostic / prognostic biomarker and therapeutic target for lung adenocarcinoma. CRTAC1 exhibits specific low expression in lung adenocarcinoma, demonstrating extremely high diagnostic accuracy (AUC=0.972); its expression level is positively correlated with patient prognosis, making it an independent protective prognostic factor; overexpression of CRTAC1 significantly inhibits tumor metastasis through a dual mechanism of inhibiting the ErbB signaling pathway and M2 macrophage polarization. This invention provides novel molecular biomarkers and drug development targets for the precision diagnosis and treatment of lung adenocarcinoma, possessing significant clinical translational value.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of chondroitin acidic protein 1 (CRTAC1) in the preparation of reagents for diagnosing lung adenocarcinoma.

2. Application of chondroitin acidic protein 1 (CRTAC1) in the preparation of reagents for assessing the prognosis of lung adenocarcinoma.

3. The application according to claim 1 or 2, characterized in that: The coding gene for CRTAC1 has an NCBI Gene ID of 55118, an mRNA sequence number of NM_018058.4, and a protein sequence number of NP_060528.

3.

4. The application according to claim 3, characterized in that: The CRTAC1 exerts its anti-cancer effect by inhibiting the EGF / ErbB signaling pathway and / or inhibiting M2 macrophage polarization.

5. A kit for diagnosing or assessing the prognosis of lung adenocarcinoma, characterized in that: This includes antibodies, primers, probes, or combinations thereof that specifically recognize CRTAC1.

6. The reagent kit according to claim 5, characterized in that: It also includes standard reagents for immunohistochemistry, Western blotting, qPCR, ELISA, or liquid biopsy testing.

7. A drug for treating lung adenocarcinoma, characterized in that: It contains promoters that increase CRTAC1 expression and pharmaceutically acceptable carriers.

8. The medicament for treating lung adenocarcinoma according to claim 7, characterized in that: The promoter is the CRTAC1 recombinant protein.

9. The medicament for treating lung adenocarcinoma according to claim 7, characterized in that: The CRTAC1 gene overexpression vector.

10. The medicament for treating lung adenocarcinoma according to any one of claims 7-9, characterized in that: The lung adenocarcinoma is either A549 or H1299 cancer cells.

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