Application of protein PRDM16 in preparation of lung adenocarcinoma serum diagnosis and / or prognosis evaluation product and related product

Abstract

The invention discloses application of protein PRDM16 in preparation of lung adenocarcinoma serum diagnosis and / or prognosis evaluation products and related products, and belongs to the technical field of biological medicine. Through TCGA public database analysis, molecular biology experiments and clinical serum sample result verification, it is found that PRDM16 expression in lung adenocarcinoma is obviously lower than that of healthy people, the prognosis of people with lower PRDM16 expression is poorer, ROC curve analysis results show that PRDM16 has good discrimination and specificity as a lung adenocarcinoma diagnosis marker and a prognosis evaluation marker, and the PRDM16 has good application prospects in lung adenocarcinoma diagnosis and prognosis evaluation. The results show that the PRDM16 as the lung adenocarcinoma marker has good diagnosis and prognosis evaluation values, and has important significance on clinical diagnosis and prognosis evaluation of lung adenocarcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of PRDM16 protein in the diagnosis and prognosis evaluation of lung adenocarcinoma and related products. Background Art

[0002] Lung cancer is the most common cause of cancer death, causing huge social and medical burdens worldwide. Non-small cell lung cancer accounts for about 85% of lung cancer, and lung adenocarcinoma (LUAD) is its most important histological subtype, with the characteristics of insidious onset, rapid onset, difficult diagnosis, and poor prognosis. Although the diagnostic technology of LUAD has developed rapidly, most patients still have bottlenecks in diagnosis due to the hidden nature of the disease and the difficulty of collecting tissue samples. Similarly, since drug resistance will inevitably occur in the later stage of drug treatment, some patients cannot monitor the efficacy of drugs and the progression of the disease in a timely manner, resulting in delays in timely evaluation of the disease and changes in treatment plans, which ultimately affects the patient's prognosis. Studies have shown that the 5-year survival rate of lung adenocarcinoma is less than 20%, and the survival rate after metastasis is less than 3%. Therefore, it is of great clinical significance to find a simple, effective, reproducible, less invasive, sensitive and specific marker for early diagnosis and prognosis evaluation of lung adenocarcinoma. It can assist doctors in early identification of the disease, assessment of severity, and monitoring of drug efficacy in clinical practice.

[0003] Compared with traditional imaging and histopathological diagnostic methods, liquid biopsy, as a non-invasive diagnostic technology, has good application prospects in tumor detection. It has the advantages of safety, simplicity, good repeatability, and non-invasiveness. Among them, serum samples are easy to obtain, covering most of the advantages mentioned above, and using it as a carrier for marker detection is an ideal choice. At present, CEA (full name carcinoembryonic antigen, Carcinoembryonic Antigen), CA125 (a glycoprotein from coelomic epithelial cells, often used as a tumor marker, Carbohydrate Antigen 125), CYFRA21-1 (acidic soluble keratin fragment, mainly distributed in epithelial cells, is the main component of cell structural proteins, Cytokeratin 19FragmentAntigen 21-1), neuron-specific enolase, squamous cell carcinoma antigen, etc. are common serum tumor markers used in clinical diagnosis of lung cancer and disease progression and prognosis evaluation, among which CEA and CA125 are the most commonly used. It is worth noting that since both of the above are elevated in various pathological types of lung cancer, their specificity for the diagnosis and prognosis assessment of lung adenocarcinoma is not ideal.

[0004] The PRDM (PRDI-BF1 and RIZ homology domain containing) family is a gene family with 19 members, including PR domains and several zinc finger structures of transcriptional regulatory proteins, which are involved in a wide range of biological processes in the human body, including the regulation of proliferation and differentiation, cell cycle progression, and the maintenance of immune cell homeostasis. PRDM family members have been shown to be associated with the occurrence, invasion, and metastasis of a variety of tumors, and these functions strongly indicate their potential use as diagnostic, therapeutic, and prognostic assessment targets in cancer management. PRDM16 is a member of the PRDM family, and studies have shown that its expression is closely related to the malignancy of renal cancer and colorectal cancer. However, there are currently no reports on PRDM16 as a marker for the detection and prognostic assessment of lung adenocarcinoma. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide the use of protein PRDM16 in the preparation of serum diagnosis and / or prognosis evaluation products for lung adenocarcinoma and related products.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide the use of protein PRDM16 in the preparation of serum diagnosis and / or prognosis evaluation products for lung adenocarcinoma and related products.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention discloses the use of serum marker PRDM16 in the preparation of serum diagnostic products, wherein the functions of the product are at least one of the following a1) to a2):

[0010] a1) Perform serum diagnosis of lung adenocarcinoma;

[0011] a2) To evaluate the prognosis of lung adenocarcinoma;

[0012] The products include serum diagnostic reagents or kits.

[0013] Preferably, PRDM16 in serum is measured by enzyme-linked immunosorbent assay.

[0014] Preferably, the serum marker PRDM16 is significantly under-expressed in lung adenocarcinoma serum.

[0015] Further preferably, the level of PRDM16 in serum is correlated with TNM stage, tumor diameter, lymph node metastasis and distant metastasis.

[0016] Further preferably, the serum marker PRDM16 level is associated with worse 3-year overall survival rate.

[0017] Further preferably, the serum marker PRDM16 shows specific low expression in lung adenocarcinoma tissue and is associated with a low survival rate.

[0018] The present invention also discloses the use of a reagent for detecting the expression of serum marker PRDM16 protein in preparing a product, wherein the product has at least one of the following functions a1) to a2):

[0019] a1) Perform serum diagnosis of lung adenocarcinoma;

[0020] a2) To evaluate the prognosis of lung adenocarcinoma;

[0021] The products include serum diagnostic reagents or kits.

[0022] Preferably, the serum marker PRDM16 is significantly under-expressed in lung adenocarcinoma serum.

[0023] The present invention also discloses a product for diagnosing lung adenocarcinoma and / or evaluating the prognosis of lung adenocarcinoma, wherein the product can diagnose lung adenocarcinoma and / or evaluate the prognosis of lung adenocarcinoma by examining the expression level of PRDM16 in serum / lung tissue; the product comprises reagents, a kit, and a chip.

[0024] Preferably, the chip comprises a gene chip and a protein chip; the kit comprises a gene detection kit and a protein immunoassay kit.

[0025] The present invention also discloses a medicine for treating lung adenocarcinoma, characterized in that the medicine comprises a promoter of the PRDM16 gene or a product of PRDM16 gene expression.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention verified the results of the analysis of the TCGA public database, molecular biology experiments and clinical serum samples and found that the expression of PRDM16 in lung adenocarcinoma was significantly lower than that in healthy people, and the lower the PRDM16 expression, the worse the prognosis. The ROC curve analysis results showed that PRDM16 had good discrimination and specificity as a diagnostic marker and prognostic evaluation marker for lung adenocarcinoma. The above results indicate that PRDM16 has good diagnostic and prognostic evaluation value as a lung adenocarcinoma marker, which is of great significance for the clinical diagnosis and prognostic evaluation of lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The expression analysis of PRDM16 in pan-cancer and non-small cell lung cancer in the online database. A is the mRNA expression analysis of PRDM16 in pan-cancer in the TIMER database; B and C are the mRNA expression analysis of PRDM16 in lung adenocarcinoma and lung squamous cell carcinoma in the GEPIA database, respectively.

[0029] Figure 2 Survival curve analysis of PRDM16 in non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma in GEPIA database and Kaplan-Meierplotter database. AC is the survival curve analysis of PRDM16 in non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma in lung adenocarcinoma in GEPIA database; DF is the survival curve analysis of PRDM16 in non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma in lung adenocarcinoma in Kaplan-Meierplotter database.

[0030] Figure 3 Figure 4 is the correlation analysis between PRDM16 expression level and clinical characteristics of lung adenocarcinoma in the TCGA database. AF is the correlation analysis between PRDM16 expression and age, gender, stage, T, N, and M stage, respectively.

[0031] Figure 4 The expression levels of PRDM16 mRNA and protein in lung adenocarcinoma cell lines are analyzed. A is the mRNA expression analysis of PRDM16 in cell lines; B is the protein immunoblot and statistical analysis of PRDM16 in cell lines.

[0032] Figure 5 The above figure shows the expression level of PRDM16 mRNA and protein in clinical lung adenocarcinoma tissue samples. A shows the mRNA expression of PRDM16 in lung adenocarcinoma tissue; B shows the representative immunohistochemical image of PRDM16 in lung adenocarcinoma tissue.

[0033] Figure 6 Serum PRDM16 protein expression levels in patients with different clinical pathological characteristics of lung adenocarcinoma. A is the expression analysis of PRDM16 in the serum of healthy people and patients with lung adenocarcinoma, and BH are the expression level analysis of PRDM16 in different ages, genders, smoking history, early and late stages, T, N, and M stages.

[0034] Figure 7 This is a univariate analysis of PRDM16 in clinical serum samples and clinical characteristics in patients with lung adenocarcinoma.

[0035] Figure 8The ROC curves of PRDM16 in clinical serum samples for distinguishing lung adenocarcinoma from healthy people and early and late stage lung adenocarcinoma patients. A is the ROC curve of PRDM16 for distinguishing lung adenocarcinoma from healthy people; B is the ROC curve of PRDM16 for distinguishing lung adenocarcinoma patients in stages I-II and III-IV; C is the ROC curve of PRDM16 region and tumor markers for distinguishing lung adenocarcinoma patients in stages I-II and III-IV; D is the ROC curve of PRDM16 region combined with tumor markers for distinguishing lung adenocarcinoma patients in stages I-II and III-IV.

[0036] Fig. 9 The ROC curves of PRDM16 and tumor markers in clinical serum samples predicting the 3-year overall survival rate of patients with lung adenocarcinoma. Among them, A is the survival analysis of serum PRDM16 in lung adenocarcinoma; B is the ROC curve of PRDM16 predicting the 3-year overall survival rate of patients with lung adenocarcinoma; C is the ROC curve of tumor markers predicting the 3-year overall survival rate of patients with lung adenocarcinoma; D is the ROC curve of PRDM16 combined with tumor markers predicting the 3-year overall survival rate of patients with lung adenocarcinoma. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0038] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0039] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0040] The present invention uses the TIMER database to analyze the expression of PRDM16 in pan-cancer and identifies that PRDM16 is lowly expressed in non-small cell lung cancer. Subsequently, the expression of PRDM16 in lung adenocarcinoma and lung squamous cell carcinoma is differentially analyzed and the prognostic analysis is performed in the TCGA database, thereby clarifying that PRDM16 is specifically lowly expressed in lung adenocarcinoma and is associated with poor prognosis and clinical stage, suggesting that the protein encoded by this gene may be a potential recognition marker and prognostic evaluation marker for lung adenocarcinoma. However, its protein expression level in lung adenocarcinoma tissue and its diagnostic and prognostic evaluation value need to be further verified.

[0041] Furthermore, by extracting cellular RNA and total protein from the normal bronchial epithelial cell line BEAS-2B and the lung adenocarcinoma cell lines A549 and PC9, RT-qPCR and protein immunoblotting experiments were performed to confirm that PRDM16 mRNA and protein levels were significantly low expressed in lung adenocarcinoma cells.

[0042] Furthermore, cDNA chips of 14 lung adenocarcinoma and adjacent normal tissues were purchased, and tissue samples of 10 lung adenocarcinoma patients were collected from the Department of Respiratory Medicine and Department of Thoracic Surgery of the First Affiliated Hospital of Xi'an Jiaotong University School of Medicine. Adjacent normal tissues were used as controls, and RNA was extracted and paraffin sections were made from the above samples. RT-qPCR and IHC were used to identify the expression of PRDM16 mRNA and protein levels in lung adenocarcinoma tissues and their correlation with clinical characteristics, respectively.

[0043] Furthermore, serum of patients with lung adenocarcinoma was collected from the Department of Respiratory Medicine and Department of Thoracic Surgery, the First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, and serum collected from the Physical Examination Department of the same medical center was used as a control healthy population. 2 mL of venous blood was collected on an empty stomach in the morning of the second day after the diagnosis of all patients with lung adenocarcinoma. After standing at 4°C for 2 h, the blood was centrifuged at 3000 rpm for 20 min, and the supernatant was collected and stored in an ultra-low temperature refrigerator at -80°C.

[0044] The PRDM16 (Gene ID: 63976, also known as MEL1) described in the present invention is a member of the PRDM family and plays an important role in many physiological and pathological processes, such as cell differentiation, proliferation, apoptosis and immune microenvironment. Previous studies have confirmed that it plays an important role in the progression and metastasis of cancer.

[0045] In one embodiment, the present invention performed a differential analysis of the expression level of PRDM16 in pan-cancer using the TIMER online database and found that PRDM16 was significantly lowly expressed in both lung adenocarcinoma and lung squamous cell carcinoma in small cell lung cancer.

[0046] The Cancer Genome Atlas (TCGA) is a project jointly initiated by the National Cancer Institute and the National Human Genome Research Institute of the United States. It has molecularly characterized more than 20,000 primary tumors and matched normal samples of 33 tumors. It includes multiple types of sequencing data including genome, transcriptome, proteome, epigenomic data, and also integrates biological clinical sample information.

[0047] The Kaplan-Meier plotter database is able to evaluate the correlation between the expression of all genes (mRNA, miRNA, protein and DNA) and survival in 35k+ samples from 21 tumor types. Applied statistical tools include Cox proportional hazard regression and calculation of false discovery rate. KM-plotter performs 20,000 analyses per day and is an effective reference for discovering and validating survival biomarkers.

[0048] The present invention integrates the expression profiles of lung adenocarcinoma and lung squamous cell carcinoma in the TCGA database online through the GEPIA database, and the groups are normal tissue adjacent to lung adenocarcinoma patients (n=59) and lung adenocarcinoma (n=483) and normal tissue adjacent to lung squamous cell carcinoma (n=50) and lung squamous cell carcinoma (n=486).

[0049] In one embodiment, the results of differential analysis of the GEPIA online database showed that PRDM16 was significantly low expressed in both lung squamous cell carcinoma and lung adenocarcinoma.

[0050] In one embodiment, the present invention found through survival analysis of the GEPIA database that the median survival of the group with low PRDM16 expression in lung adenocarcinoma was lower than that of the group with high PRDM16 expression, while the median survival of the group with low PRDM16 expression in lung squamous cell carcinoma was higher than that of the group with high PRDM16 expression, which is contrary to the effect of low PRDM16 expression as a tumor suppressor gene on prognosis. As mentioned above, PRDM16 is lowly expressed only in lung adenocarcinoma and is associated with poor prognosis.

[0051] In one embodiment, the present invention performed a differential analysis of the relationship between PRDM16 and the prognosis of non-small cell lung cancer using the Kaplan-Meier plotter database and found that low expression of PRDM16 was significantly correlated with the poor prognosis of lung adenocarcinoma, but had no significant correlation with the prognosis of lung squamous cell carcinoma.

[0052] In one of the embodiments, the expression spectrum and clinical information of lung adenocarcinoma were downloaded from the TCGA official website, and the data were sorted using Perl and R languages. A total of 535 cases of lung adenocarcinoma and 59 cases of adjacent normal tissues were obtained. Clinical correlation analysis found that the specifically downregulated PRDM16 gene expression was related to the clinical Stage stage and N stage. The higher the stage, the lower the PRDM16 expression.

[0053] In one embodiment, RNA and total protein extraction, RT-qPCR and protein immunoblotting experiments were performed on lung adenocarcinoma cell lines A549 and PC9 and normal bronchial pulmonary epithelial cells BEAS-2B, and it was found that the expression of PRDM16 in lung adenocarcinoma cell lines was significantly lower than that in normal cells.

[0054] The present invention also used clinical tissue samples to verify the analysis results in the TCGA database and found that the mRNA and protein levels of PRDM16 in clinical lung adenocarcinoma tissue samples were significantly downregulated compared with adjacent normal tissues.

[0055] The present invention also used clinical serum samples to verify the analysis results of the TCGA database and the detection results in clinical tissue samples, and found that the serum PRDM16 level in patients with lung adenocarcinoma was significantly lower than that in the control healthy population. PRDM16 expression was not related to gender, age and smoking, but was related to TNM stage, tumor diameter, lymph node metastasis and distant metastasis.

[0056] The present invention performs ROC curve analysis on the serum PRDM16 level of patients with lung adenocarcinoma. The area under the curve of PRDM16 for distinguishing lung adenocarcinoma from healthy people is 0.804, and its specificity is 83.33%. In addition, analysis of patients with early and late TNM stages found that PRDM16 was 0.759 in distinguishing stage I-II patients, stage III-IV patients and healthy people, and its sensitivity was as high as 80.85%. The ROC curve analysis results show that serum PRDM16 protein has good diagnostic efficacy in the diagnosis of lung adenocarcinoma.

[0057] The present invention performed univariate analysis on serum PRDM16 levels and clinical characteristics of patients with lung adenocarcinoma and found that low serum PRDM16 expression is a risk factor affecting the prognosis of patients with lung adenocarcinoma.

[0058] The present invention found through survival analysis of the 3-year overall survival rate of patients with lung adenocarcinoma that patients with low serum PRDM16 expression had a worse prognosis, verifying the results in the TCGA database. ROC curve analysis showed that the area under the curve of serum PRDM16 for predicting the 3-year overall survival rate of overall lung adenocarcinoma patients was 0.727, and its sensitivity was 88.24%. In addition, when serum PRDM16 combined with CEA and CA125 was used to predict the 3-year overall survival rate of patients with lung adenocarcinoma, the area under the curve was 0.822, and its sensitivity was as high as 92.86%. The above results show that serum PRDM16 protein has a good predictive efficacy in the prognosis assessment of lung adenocarcinoma.

[0059] In one embodiment, 95 lung adenocarcinoma patients and 30 healthy subjects were used for serum clinical sample validation.

[0060] In one embodiment, the detection of PRDM16 protein level is determined by enzyme-linked immunosorbent assay (ELISA).

[0061] Prism 9 and R (4.2.2) software were used for statistical analysis and drawing in the above specific examples. Mean ± standard deviation and median (interquartile range) represent normal and non-normal distribution measurement data, respectively. Independent sample t test was used for comparison between two groups, ANOVA one-way analysis of variance was used to compare sample differences between multiple groups, and the significance of categorical variable data was tested using χ 2 ROC curve analysis was used to evaluate the efficacy of PRDM16 in early diagnosis and prognosis evaluation of LUAD, and KM survival curve was used to evaluate the effect of PRDM16 expression on the prognosis of LUAD patients.

[0062] Example 1 Pan-cancer analysis of the TIMER database and the GEPIA database identified the expression of PRDM16 in non-small cell lung cancer and its correlation with prognosis

[0063] Combined with the literature and previous database search results, the present invention believes that PRDM family member PRDM16 may be a new marker for the diagnosis and prognosis evaluation of lung adenocarcinoma. Further evaluation of its value in clinical applications has important clinical practical significance for the diagnosis, treatment, disease severity and prognosis evaluation of lung adenocarcinoma.

[0064] Example 1

[0065] Pan-cancer analysis of the TIMER database and the GEPIA database identified the expression of PRDM16 in non-small cell lung cancer and its correlation with prognosis.

[0066] Combined with the literature and previous database search results, the present invention believes that PRDM family member PRDM16 may be a new marker for the diagnosis and prognosis evaluation of lung adenocarcinoma. Further evaluation of its value in clinical applications has important clinical practical significance for the diagnosis, treatment, disease severity and prognosis evaluation of lung adenocarcinoma.

[0067] 1. Differential analysis of PRDM16 in pan-cancer

[0068] The present invention firstly performed differential analysis on the expression of PRDM16 in pan-cancer using the TIMER online database and found that Figure 1 As shown, PRDM16 was downregulated in 13 tumor types including lung adenocarcinoma and lung squamous cell carcinoma.

[0069] 2. Prognostic survival analysis of PRDM16 in pan-cancer

[0070] In order to further clarify the specificity of PRDM16 in lung adenocarcinoma, its prognosis in non-small cell lung cancer was analyzed through the GEPIA database and the Kaplan-Meierplotter database, and it was found that the low expression of PRDM16 was significantly correlated with both lung adenocarcinoma and lung squamous cell carcinoma. It is worth noting that although PRDM16 is lowly expressed in lung squamous cell carcinoma, the prognostic analysis results show that high expression of PRDM16 is associated with poor prognosis of this tumor type. This contradictory result suggests that PRDM16 may function as both an oncogene or a tumor suppressor gene in this disease. The expression difference of PRDM16 in lung adenocarcinoma and its relationship with prognosis are consistent with expectations, indicating that among the tumor types that can be retrieved in the database, PRDM16 is only associated with poor prognosis of lung adenocarcinoma in non-small cell lung cancer, and it may be used as a specific marker for the diagnosis and prognosis evaluation of lung adenocarcinoma. The analysis results of the Kaplan-Meierplotter database showed that low expression of PRDM16 was only associated with poor prognosis of lung adenocarcinoma, but not with lung squamous cell carcinoma. As Figure 2 As shown, AC represents the survival curves of PRDM16 in non-small cell lung cancer (A in the figure), lung adenocarcinoma (B in the figure), and lung squamous cell carcinoma (C in the figure) in the GEPIA database, and DF represents the survival curves of PRDM16 in non-small cell lung cancer (D in the figure), lung adenocarcinoma (E in the figure), and lung squamous cell carcinoma (F in the figure) in the Kaplan-Meierplotter database.

[0071] Example 2

[0072] Clinical relevance analysis of PRDM16 in lung adenocarcinoma in the TCGA database

[0073] 1. Research Tools

[0074] (1)TCGA database

[0075] The Cancer Genome Atlas (TCGA) is a project jointly initiated by the National Cancer Institute and the National Human Genome Research Institute of the United States. It has molecularly characterized more than 20,000 primary tumors and matched normal samples of 33 tumors. It includes multiple types of sequencing data including genome, transcriptome, proteome, epigenome and other omics data, and also integrates biological clinical sample information. All data collected and processed by TCGA in this study are available in the Genomic Data Sharing Resource (https: / / portal.gdc.cancer.gov).

[0076] (2) GEPIA database

[0077] Gene Expression Profiling Interactive Analysis (GEPIA) is a TCGA data analysis and visualization website that can directly analyze the expression differences and prognosis of genes in cancer in the TCGA database.

[0078] (3) R language

[0079] R language was developed by Ross Ihaka and Robert Gentleman of the University of Auckland, New Zealand in 1993. It is a programming language for statistical computing and data analysis. It is easy to use, flexible and extensible. The statistics, analysis and graphics of this study were based on various R packages of R software version 4.2.2.

[0080] 2. Research subjects

[0081] The LUAD gene expression profile and clinical sample information were downloaded from the TCGA-GDC platform, and the gene expression matrix of 594 samples (including 535 cancer tissue samples and 59 adjacent normal tissue samples) was obtained after integration using the Perl language, of which 522 LUAD patients had clinical information (including age, gender, T, N, M, Stage, survival status and time). Samples with incomplete clinical parameters or lack of prognostic data were excluded from the subsequent statistical analysis.

[0082] Table 1 Clinical baseline data of 522 patients with lung adenocarcinoma in the TCGA database

[0083]

[0084]

[0085] 3. Correlation analysis between PRDM16 and clinical characteristics of lung adenocarcinoma patients in the TCGA database

[0086] The present invention analyzes the expression of PRDM16 in different clinical characteristics of patients with lung adenocarcinoma and finds that PRDM16 is associated with lymph node metastasis and stage. The higher the stage, the lower the expression of PRDM16, suggesting that PRDM16 may be used to evaluate the disease progression or stage of lung adenocarcinoma. Figure 3 .

[0087] Example 3

[0088] Expression and clinical correlation analysis of PRDM16 in lung adenocarcinoma cell lines and clinical tissue samples

[0089] The present invention has confirmed in Examples 1 and 2 that PRDM16 may be a potential diagnostic and prognostic assessment biomarker for lung adenocarcinoma. In order to further clarify the expression and role of PRDM16 in lung adenocarcinoma, the present invention verified the results in the TCGA database using lung adenocarcinoma cell lines A549 and PC9 and clinical tissue samples.

[0090] 1. PRDM16 is lowly expressed in lung adenocarcinoma cell lines

[0091] A549 and PC9 are commonly used research tools in molecular biology experiments related to lung adenocarcinoma, while BEAS-2B is often used as a control for normal bronchial epithelial cells. The present invention conducted RT-qPCR and protein immunoblotting experiments on the above three cells and found that the mRNA and protein levels of PRDM16 were significantly reduced in lung adenocarcinoma cell lines compared with BEAS-2B. Figure 4 As shown, A and B are the mRNA expression analysis of PRDM16 in cell lines; C and D are the protein immunoblot images and statistical analysis images of PRDM16 in cell lines.

[0092] 2. PRDM16 is lowly expressed in lung adenocarcinoma tissue and is associated with tumor diameter and pathological grade

[0093] In order to further verify the results of bioinformatics analysis in clinical tissue samples, this experiment purchased cDNA chips from 10 paired lung adenocarcinoma patients and preliminarily analyzed the mRNA expression level of PRDM16 in tumor tissues and adjacent normal tissues of lung adenocarcinoma patients by RT-qPCR. Figure 5 As shown in Figure A, compared with the paired adjacent normal tissues, the expression of PRDM16 mRNA in primary tumors was significantly reduced (P<0.05). In addition, the present invention also collected tumor tissues from 10 patients with lung adenocarcinoma, and evaluated the expression level of PRDM16 protein in lung adenocarcinoma tissues by immunohistochemical staining. It was found that compared with adjacent normal tissues, the staining intensity of PRDM16 in tumor tissues was lower, confirming that the PRDM16 protein level was also significantly downregulated in lung adenocarcinoma, as shown in Figure 1. Figure 5 As shown in B.

[0094] Example 4

[0095] The diagnostic and prognostic value of serum PRDM16 in lung adenocarcinoma

[0096] The results of TCGA database analysis suggest that PRDM16 is lowly expressed in lung adenocarcinoma and is associated with poor prognosis. In addition, PRDM16 can significantly distinguish adjacent tumor tissues from primary tumor tissues. Based on this, the present invention further confirms the results in the database in lung adenocarcinoma cell lines and tissues, suggesting that the protein encoded by this gene may become a feasible tool for diagnosis and prognosis evaluation of lung adenocarcinoma. However, some patients with lung adenocarcinoma currently still face barriers in early diagnosis and prognosis evaluation due to difficulties in sample acquisition. Therefore, it is of great clinical significance to invent a simple, reproducible, sensitive and specific diagnostic and prognostic tool. Detection using serum as a carrier is an ideal choice. Therefore, the present invention verifies the above results by collecting clinical serum specimens and evaluates the value of serum PRDM16 in diagnosis and prognosis evaluation of lung adenocarcinoma.

[0097] The serum of 95 patients with lung adenocarcinoma and 30 healthy subjects was collected, and the ELISA method was used to detect the PRDM16 protein level in the serum. Table 2 lists the clinical characteristics of the subjects. It can be seen that there is no statistical difference in the baseline data of gender and age between lung adenocarcinoma and healthy subjects, and the data are comparable.

[0098] Table 2 Clinical characteristics of included patients

[0099]

[0100]

[0101] Note: HP, healthy people; LUAD, lung adenocarcinoma; *P<0.05, **P<0.01, ***P<0.001.

[0102] As shown in Table 3 and Figure 6As shown in the results, the serum PRDM16 level in LUAD patients was significantly lower than that in the healthy control group [2.306 (1.780, 2.612) ng / mL vs. 2.897 (2.419, 3.404) ng / mL], P<0.05. Further correlation analysis of clinical characteristics found that the expression of PRDM16 was not significantly correlated with gender, age, and smoking (P>0.05). This study also observed that the PRDM16 level in LUAD patients with TNM stage III-IV was significantly lower than that in stage I-II (2.129±0.578 ng / mL vs. 2.977±0.806 ng / mL, P<0.001). Similarly, the PRDM16 levels in patients with T2-T4, N1-N3, and M1 stages were lower than those in patients with T1, N0, and M0 stages (P<0.001). The above results are consistent with the previous bioinformatics analysis and tissue RT-qPCR clinical correlation analysis results. The three together confirmed that low expression of PRDM16 in LUAD tissue and serum was associated with worse Stage, T, N, and M stages.

[0103] Table 3 Serum PRDM16 expression levels in patients with lung adenocarcinoma

[0104]

[0105] Note: HP, healthy people; LUAD, lung adenocarcinoma; *P<0.05, **P<0.01, ***P<0.001.

[0106] In addition, the present invention also evaluated the value of serum PRDM16 protein level in the diagnosis of lung adenocarcinoma. The results are shown in Table 4 and Figure 7 As shown in the figure, the AUC of PRDM16 for distinguishing healthy people from LUAD patients was 0.804, and the sensitivity and specificity of diagnosing LUAD at a cutoff value of 4.285 ng / mL were 76.67% and 71.59%, respectively. Further analysis showed that the AUC of PRDM16 for distinguishing healthy controls from stage I-II patients was 0.715, with a sensitivity of 79.07% and a specificity of 56.67%, and the AUC for distinguishing healthy controls from stage III-IV patients was 0.889, with a sensitivity of 75.56% and a specificity of 90.00%. Through clinical serum sample analysis, we confirmed that PRDM16 protein can be used as a diagnostic marker for lung adenocarcinoma, and PRDM16 protein has good discrimination and specificity in the diagnosis of lung adenocarcinoma.

[0107] Figure 8 Univariate analysis found that, in addition to TNM stage, low serum PRDM16 expression was also a risk factor affecting the 3-year prognosis of patients with lung adenoma.

[0108] Table 4 Diagnostic value of serum PRDM16 in LUAD patients

[0109]

[0110] Note: HP, healthy people; LUAD, lung adenocarcinoma; *P<0.05, **P<0.01, ***P<0.001.

[0111] The present invention conducted a prognostic follow-up for all included patients after 3 years. The results showed that 61 LUAD patients survived, 17 died, and 17 were lost to follow-up. A total of 78 patients were finally used for survival analysis in this study. Survival analysis found that patients with low serum PRDM16 expression had a shorter survival time (P<0.001), which was consistent with the results of bioinformatics analysis. The present invention further analyzed the value of PRDM16 and tumor markers in assessing the prognosis of LUAD patients, such as Fig. 9 As shown in the figure, ROC curve analysis found that the AUC of serum PRDM16 for predicting the 3-year survival rate of LUAD patients was 0.727, with a sensitivity and specificity of 88.24% and 61.67%, respectively. When combined with CEA and CA125, the prediction sensitivity can be increased to 92.86%. The above results show that PRDM16 has the potential to become a prognostic assessment marker for LUAD patients.

[0112] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. The use of serum marker PRDM16 in the preparation of serum diagnostic products, characterized in that: The functions of the product are at least one of the following a1) to a2): a1) Perform serum diagnosis of lung adenocarcinoma; a2) To evaluate the prognosis of lung adenocarcinoma; The products include serum diagnostic reagents or kits.

2. The use according to claim 1, characterized in that The marker PRDM16 in serum was measured by enzyme-linked immunosorbent assay.

3. The use according to claim 1, characterized in that The serum marker PRDM16 was significantly down-expressed in the serum of lung adenocarcinoma.

4. The use according to claim 3, characterized in that The serum PRDM16 level was correlated with TNM stage, tumor diameter, lymph node metastasis and distant metastasis.

5. The use according to claim 1, characterized in that The serum marker PRDM16 showed specific low expression in lung adenocarcinoma tissues and was associated with poor survival rate.

6. Use of a reagent for detecting the expression of serum marker PRDM16 protein in preparing a product, characterized in that: The functions of the product are at least one of the following a1) to a2): a1) Perform serum diagnosis of lung adenocarcinoma; a2) To evaluate the prognosis of lung adenocarcinoma; The products include serum diagnostic reagents or kits.

7. A product for diagnosing lung adenocarcinoma and / or evaluating the prognosis of lung adenocarcinoma, characterized in that: The product can diagnose lung adenocarcinoma and / or perform prognosis assessment of lung adenocarcinoma by examining the expression level of PRDM16 in serum / lung tissue.

8. The product for diagnosing lung adenocarcinoma and / or evaluating the prognosis of lung adenocarcinoma according to claim 7, characterized in that: The products include reagents, test kits, and chips.

9. The product for diagnosing lung adenocarcinoma and / or evaluating the prognosis of lung adenocarcinoma according to claim 8, characterized in that: The chips include gene chips and protein chips; the kits include gene detection kits and protein immunoassay kits.

10. A drug for treating lung adenocarcinoma, characterized in that: The drug includes a promoter of the PRDM16 gene or a product of PRDM16 gene expression.

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