Application of PRTFDC1 in treatment and prognosis evaluation of testicular germ cell tumor

Abstract

The invention relates to application of PRTFDC1 in treatment and prognosis evaluation of testicular germ cell tumor (TGCT). According to the invention, through a large amount of researches, the expression level of PRTFDC1 is found to be a factor highly associated with TGCT. By detecting the expression level of PRTFDC1 in the body of a TGCT patient, the tumor tension condition in the body of the patient can be judged and reasonably predicted, so that a personalized treatment scheme is provided, and the clinical treatment effect is improved; the prognosis of the patient can be reasonably evaluated, and a reasonable and effective guiding effect is provided for treatment and rehabilitation. In general, the key function of the PRTFDC1 in TGCT is defined, a new thought is provided for acquisition of related target spots, treatment of related diseases and the like in follow-up research, a clear direction is provided for follow-up medicine research and development, clinical treatment and the like, and the PRTFDC1 has extremely high social value and market application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology and relates to the application of PRTFDC1 in the treatment and prognosis evaluation of testicular germ cell tumors. Background Art

[0002] Testicular germ cell tumor (TGCT) is one of the most common tumors in adolescents and young men aged 15 to 39 years. Although the 5-year survival rate of TGCT is over 94%, the number of new cases has been increasing over the past few decades. The occurrence of TGCT may be associated with a variety of high-risk factors, including cryptorchidism, hypospadias, family history of TGCT, testicular degeneration syndrome, and the effects of endogenous and exogenous estrogens. In addition, external environmental factors such as diagnostic radiation exposure to the lower abdomen and pelvis are also believed to increase the risk of TGCT.

[0003] TGCT originates from germ cells and can present in a variety of different histological types, such as seminoma, non-seminoma, and mixed TGCT. Among them, seminoma is the most common type of TGCT, accounting for about 50% of all cases, while non-seminoma mainly includes embryonal carcinoma, teratoma, yolk sac tumor, choriocarcinoma, and trophoblastic tumor. Depending on the different histological types and the extent of tumor involvement, TGCT uses different treatments, such as surgery, chemotherapy, radiotherapy, or combined therapy. Seminoma is highly sensitive to radiotherapy and chemotherapy, and this high sensitivity is closely related to the good prognosis of its patients. In contrast, non-seminoma and mixed TGCT have relatively poor prognosis because they contain different tissue components. For example, teratoma is extremely insensitive to radiotherapy and chemotherapy; choriocarcinoma easily invades blood vessels and can cause multiple distant metastases throughout the body when the primary tumor is not obvious.

[0004] Currently, serum tumor markers and the International Germ Cell Cancer Collaborative Group (IGCCG) prognostic model are mainly used to predict the prognosis of TGCT patients and monitor their recurrence and metastasis. Serum tumor markers include α-fetoprotein (AFP), human chorionic gonadotropin subunit β (β-HCG), and lactate dehydrogenase (LDH). It has been reported that about 20% of non-seminoma patients and about 80% of seminoma patients have false-negative results in the detection of these biomarkers. In addition, patients with chronic liver disease, liver damage, and liver cancer may have elevated AFP, leading to false-positive results, which suggests the limitations of these markers in predicting the prognosis of TGCT. The IGCCCG prognostic model is to comprehensively evaluate factors such as histological type, primary site, metastatic site, and serum tumor markers to divide patients into good, intermediate, and poor prognoses to guide the selection of standard treatment and first-line chemotherapy regimens. However, this model has limitations. The IGCCCG prognostic model based on large-scale statistical data may not be applicable to all patients, especially some special types and TGCT patients who have failed first-line chemotherapy, and cannot fully reflect the specific situation of individual patients. Therefore, it is particularly urgent and necessary to conduct in-depth research on tumor heterogeneity, combine bioinformatics and multi-omics analysis methods, explore and identify new biomarkers, and further explore and develop new prognostic biomarkers related to TGCT with higher sensitivity and specificity, which will help provide more accurate prognostic assessment and support clinical decision-making. Summary of the invention

[0005] The purpose of the present invention is to solve the problems existing in the prior art, thereby providing a target that is highly related to the occurrence and development of testicular germ cell tumors, namely PRTFDC1. By using PRTFDC1 as a marker, it can be used to effectively predict the occurrence and development of testicular germ cell tumor diseases, thereby screening out the high-risk population for testicular germ cell tumors, so as to carry out reasonable early prevention, significantly reduce the probability and severity of the disease, and reduce damage to human health.

[0006] In order to solve the above technical problems, the present invention is implemented through the following technical solutions.

[0007] In a first aspect, the present invention provides use of a reagent for detecting the expression level of PRTFDC1 in the preparation of a product for auxiliary diagnosis and / or prognosis evaluation of testicular germ cell tumors.

[0008] Preferably, the reagent for detecting the expression level of PRTFDC1 includes primers for detecting the expression level of PRTFDC1 gene and / or a reagent for detecting the content of PRTFDC1 protein.

[0009] Preferably, the primers for detecting the expression level of the PRTFDC1 gene are selected from one or more of the following primer pairs:

[0010] Primer pair 1: the upstream sequence is shown in SEQ ID NO: 1 (5'-GTTGGTGAAGAGAACATCCAG-3'), and the downstream sequence is shown in SEQ ID NO: 2 (5'-TCAGATCTCTGAAGTATTCATTG-3');

[0011] Primer pair 2: the upstream sequence is shown in SEQ ID NO: 3 (5'-TGTGGTGGGATATGCCTTAGA-3'), and the downstream sequence is shown in SEQ ID NO: 4 (5'-TCTGGGACTTTAGTGGTGAGAAT-3').

[0012] Preferably, the reagent for detecting the PRTFDC1 protein content is selected from anti-PRTFDC1 antibody (ProteinTech Group, Inc, 11986-1-AP)).

[0013] It should be understood that, in the context of the present invention, unless otherwise specified, the primers and / or primer pairs refer to PCR primers used to synthesize the cDNA chain of the PRTFDC1 gene in PCR, so as to detect the expression level of the PRTFDC1 gene mRNA. In addition to the primers, primer pairs and / or antibodies listed in the present invention, those skilled in the art are fully capable of designing corresponding primers, primer pairs, and antibodies according to the gene sequence of PRTFDC1 using conventional methods and means in the art including but not limited to molecular biology, and screening the designed primers, primer pairs, and antibodies by conventional experimental means, or obtaining them commercially, as long as they can achieve specific detection of the expression level of PRTFDC1.

[0014] The second aspect of the present invention provides the use of a PRTFDC1 inhibitor in the preparation of a medicament for preventing and / or treating testicular germ cell tumors.

[0015] Preferably, the PRTFDC1 inhibitor is selected from one or more of siRNA, sh RNA, and sgRNA designed based on the PRTFDC1 gene.

[0016] Preferably, the PRTFDC1 inhibitor is selected from siRNA designed based on the PRTFDC1 gene.

[0017] Preferably, the siRNA designed based on the PRTFDC1 gene can be purchased from Dharmacon (Lafayette, CO, USA).

[0018] It should be understood that, unless otherwise specified, in the context of the present invention, the PRTFDC1 inhibitor refers to a substance that can specifically down-regulate the expression level of PRTFDC1 and / or the transcription level of its mature mRNA and / or the expression level or activity of PRTFDC1 protein, such as a small molecule compound, antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA Erasers, Target Master and / or multi-target methods to down-regulate the expression level and / or activity of PRTFDC1. Those skilled in the art can design inhibitory agents that can inhibit the expression level of PRTFDC1 based on the gene sequence of PRTFDC1, or they can be obtained commercially, as long as they can achieve a reduction in the level and / or activity of PRTFDC1.

[0019] The third aspect of the present invention provides a kit for auxiliary diagnosis and / or prognosis evaluation of testicular germ cell tumors, comprising a reagent for detecting the expression level of PRTFDC1.

[0020] Preferably, the reagent for detecting the expression level of PRTFDC1 includes primers for detecting the expression level of PRTFDC1 gene and / or a reagent for detecting the content of PRTFDC1 protein.

[0021] The primers for detecting the expression level of the PRTFDC1 gene are selected from one or more of the following primer pairs:

[0022] Primer pair 1: the upstream sequence is shown in SEQ ID NO: 1 (5'-GTTGGTGAAGAGAACATCCAG-3'), and the downstream sequence is shown in SEQ ID NO: 2 (5'-TCAGATCTCTGAAGTATTCATTG-3');

[0023] Primer pair 2: the upstream sequence is shown in SEQ ID NO: 3 (5'-TGTGGTGGGATATGCCTTAGA-3'), and the downstream sequence is shown in SEQ ID NO: 4 (5'-TCTGGGACTTTAGTGGTGAGAAT-3').

[0024] Preferably, the reagent for detecting the PRTFDC1 protein content is selected from anti-PRTFDC1 antibody (ProteinTech Group, Inc, 11986-1-AP).

[0025] Preferably, the kit further comprises one or more of PCR enzyme, PCR buffer, dNTPs and fluorescent substrate.

[0026] Preferably, the fluorescent substrate is selected from Syber Green or a fluorescently labeled probe.

[0027] The fourth aspect of the present invention provides a pharmaceutical composition for preventing and / or treating testicular germ cell tumors, comprising a PRTFDC1 inhibitor and a pharmaceutically acceptable carrier.

[0028] Preferably, the PRTFDC1 inhibitor is selected from one or more of siRNA, sh RNA, and sgRNA designed based on the PRTFDC1 gene.

[0029] Preferably, the PRTFDC1 inhibitor is selected from siRNA designed based on the PRTFDC1 gene.

[0030] Preferably, the siRNA designed based on the PRTFDC1 gene can be purchased from Dharmacon (Lafayette, CO, USA).

[0031] A fifth aspect of the present invention provides the use of a PRTFDC1 inhibitor in the preparation of a drug for improving the immunotherapeutic activity of testicular germ cell tumors.

[0032] Preferably, the PRTFDC1 inhibitor is selected from one or more of siRNA, sh RNA, and sgRNA designed based on the PRTFDC1 gene.

[0033] Preferably, the PRTFDC1 inhibitor is selected from siRNA designed based on the PRTFDC1 gene.

[0034] Preferably, the siRNA designed based on the PRTFDC1 gene can be purchased from Dharmacon (Lafayette, CO, USA).

[0035] Preferably, the immunotherapy is selected from one or more of anti-PD-1 antibody therapy, anti-PD-L1 antibody therapy, and CA RT therapy.

[0036] A sixth aspect of the present invention provides the use of PRTFDC1 in screening drugs for the treatment of testicular germ cell tumors.

[0037] Due to the heterogeneity and complex classification of testicular germ cell tumors, the prognostic evaluation and therapeutic targets are still unclear. Therefore, it is of great significance to explore a new biomarker to comprehensively evaluate the prognosis and immunotherapy response of TGCT. The present invention collected data from TGCT patient samples and normal testicular samples from six cohorts, including 48 microarray data. The differentially expressed genes of TGCT were screened out by differential expression analysis, and combined with univariate Cox regression analysis, the prognostic marker PRTFDC1 (Pho sphoribosyl Transferase Domain Containing 1) of TGCT was identified. PRTFDC1 is a gene encoding a protein located at 10p12.1. The protein it encodes contains a phosphoribosyl transferase domain, and its function is still unclear. At present, there are relatively few studies on PRTFDC1, but some studies have shown that PRTFDC1 may play a certain role in tumor occurrence and development. The present invention uses multivariate Cox regression analysis and survival analysis to explore the value of PRTFDC1 in prognosis. In addition, functional enrichment analysis and immunohistochemical staining of PRTFDC1 in TGCT were also performed. Finally, the relationship between PRTFDC1 and somatic mutation, copy number variation, immune cell infiltration and immunotherapy response was studied. Through the above analysis, the present invention clarified that PRTFD C1 is an independent risk factor for poor prognosis of TGCT. Immunohistochemical analysis confirmed that PRTFDC1 protein is highly expressed in TGCT tissues. PRTFDC1 can activate carcinogenic pathways and inhibit immune-related pathways. Immunoanalysis showed that high expression of PRTFDC1 is associated with CD8 + Patients with low PRTFDC1 expression may respond better to CAR-T cell therapy and anti-PD-1 / PD-L1 immunotherapy. In summary, the present invention reveals the biological characteristics and clinical significance of PRTFDC1, indicating that it can be used as an effective and reliable biomarker for predicting the prognosis and immunotherapy response of TGCT.

[0038] Compared with the prior art, the present invention has the following technical effects:

[0039] (1) Through extensive research, the present invention has found that the expression level of PRTFDC1 is a factor that is highly correlated with testicular germ cell tumors; further, by detecting PRTFDC1 in the subject's body, an expression level index is obtained, thereby effectively predicting the probability of the subject suffering from TGCT, that is, the expression level of PRTFDC1 can be used as a biomarker for clinical auxiliary diagnosis of TGCT disease.

[0040] (2) By detecting the expression level of PRTFDC1 in TGCT patients, we can make a reasonable prediction of the tumor tension in the patients, thereby providing personalized treatment plans to improve clinical treatment effects; we can also make a reasonable assessment of the patient's prognosis and provide reasonable and effective guidance for treatment and rehabilitation.

[0041] (3) The present invention clarifies that PRTFDC1 can inhibit CD8 + T cell infiltration, leading to poor prognosis. By inhibiting the expression of PRTFDC1, the effect of immunotherapy in TGCT patients can be improved, and the potential of PRTFDC1 expression level as a predictor of immunotherapy response can be detected, highlighting the importance of PRTFDC1 in individualized treatment strategies. The present invention provides a sufficient theoretical basis for the disease progression and targeted treatment of testicular germ cell tumors, and provides a new idea for the later development of related drugs, clinical diagnosis and treatment, prognosis evaluation, and efficacy evaluation, which has great social significance and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of batch correction results for GSE3218 and GSE10783 (GPL96, GPL97) datasets.

[0043] Figure 2 Schematic diagram of the principal component analysis (PCA) results of the GSE3218-10783 (GPL96, GPL97) dataset.

[0044] Figure 3 Figure 2 is a heat map of differentially expressed genes between TGCT and normal tissues.

[0045] Figure 4 Figure 2 is a Venn diagram of up-regulated and down-regulated differentially expressed genes between GPL96 and GPL97 platforms.

[0046] Figure 5 Schematic diagram of the KEGG pathway enrichment analysis results of up-regulated and down-regulated differentially expressed genes.

[0047] Figure 6 The Venn diagram of prognostic genes between GPL96 and GPL97 platforms in GSE3218 and GSE10783 datasets.

[0048] Figure 7 The Venn diagram of differentially expressed genes and prognostic genes between GPL96 and GPL97 platforms in GSE3218 and GSE10783 datasets.

[0049] Figure 8Kaplan-Meier survival analysis of all cohorts with progression-free interval (PFI) and overall survival (OS) as outcome events was performed.

[0050] Fig. 9 Schematic diagram of the univariate Cox regression analysis results of clinical characteristics such as PRTFDC1, age, and tumor stage.

[0051] Fig.10 Schematic diagram of the multivariate Cox regression analysis results of PRTFDC1 in all cohorts.

[0052] Fig.11 Schematic diagram of the gene set enrichment analysis (GSEA) results related to PRTFDC1 in the TCGA cohort.

[0053] Fig.12 Schematic diagram of pathway results related to tumor occurrence, development and metastasis among the activated pathways.

[0054] Fig.13 Schematic diagram of pathway results related to tumor immune response among the inhibited pathways.

[0055] Fig.14 Schematic diagram of IHC staining results of PRTFDC1 in clinical samples.

[0056] Fig.15 Representative IHC staining images of PRTFDC1 in TGCT and normal testicular tissues.

[0057] Fig.16 Schematic diagram of the statistical analysis results of IHC scoring of PRTFDC1 in normal testicular tissue and tumors.

[0058] Fig.17 Schematic diagram of the ROC AUC results of PRTFDC1.

[0059] Fig.18 Schematic diagram of the summary results of the correlation analysis between PRTFDC1 expression and immune cell infiltration levels.

[0060] Fig.19 PRTFDC1 expression and CD8 + Scatter plot of the correlation of T cell infiltration levels.

[0061] Fig. 20 Schematic diagram of the results using the Submap algorithm to describe the relationship between the two PRTFDC1 expression subpopulations and immunotherapy response.

[0062] Fig.21Schematic diagram of the Kaplan-Meier survival analysis results of the PRTFDC1 high expression group and low expression group in two cohorts receiving CAR-T and anti-PD-L1 treatment.

[0063] Fig. 22 Schematic diagram of the top 20 potential therapeutic drug results that are highly correlated with PRTFDC1 based on NCS descending sorting.

[0064] Fig.23 Schematic diagram of the potential mechanism of action results for each compound in CMap. DETAILED DESCRIPTION

[0065] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0066] Unless otherwise specified, the reagents used in the context of the present invention are all commercially available. The experimental methods used in the present invention are all conventional methods and techniques in the art. Representative results selected from the biological experiment repetitions are presented in the context figures, and the data are displayed as mean±SD and mean±SEM as specified in the figure. All experiments were repeated at least three times. This study used R software version 4.3.3 for data processing and visualization, and some charts were completed by Sangerbox (http: / / vip.sangerbox.com / ). Correlation analysis was performed using the "spearman" method. Survival analysis used the Kaplan-Meier method to compare the survival differences between high and low expression subgroups, and the log-rank test was used to determine significance. The predictive accuracy of PRTFDC1 was evaluated by the AUC value under the RO C curve. The Wilcoxon rank sum test for independent samples was used to compare the differences between the two groups of data. All statistical tests were two-sided tests. p<0.05 was statistically significant.

[0067] Example 1

[0068] First, transcriptome data and corresponding clinical data of four testicular germ cell tumor cohorts were obtained from the Gene Expression Omnibus (GEO) database (https: / / www.ncbi.nlm.nih.gov / geo / ). These cohorts include GSE3218 (GPL96, GPL97) and GSE10783 (GPL96, GPL97). In GSE3218 (GPL96), there are 6 normal samples and 101 tumor samples; in GSE3218 (GPL97), there are 6 normal samples and 101 tumor samples; in GSE10783 (GPL96), there are 34 tumor samples; in GSE10783 (GPL97), there are also 34 tumor samples. The raw data have been background corrected, normalized, and log2 transformed using the Robust Multi-Array Average (RMA) method in the “affy” R package (version 1.76.0).

[0069] Based on the same platform, the above dataset was used to remove the batch effect using the ComBat function in the “sva” R package (version 3.50.0). The corrected results are as follows: Figure 1 As shown, the two merged cohorts GSE3218-10783 (GPL96) and GSE3218-10783 (GPL97) (see Figure 2 ). In the two merged cohorts, the “limma” R package (version 3.58.1) was used to identify differentially expressed genes between tumor samples and normal samples, and the screening was based on the criteria of |log2 FC|>1.5, adjPval<0.05. In order to intuitively display the results of differentially expressed genes, a volcano plot was used for visualization. The results are shown in Figure 2. Figure 3 shown.

[0070] A total of 1306 differentially expressed genes were screened in the GSE3218-10783 (GPL96) cohort, of which 457 genes were upregulated and 849 genes were downregulated. A total of 764 differentially expressed genes were screened in the GSE3218-10783 (GPL97) cohort, of which 129 genes were upregulated and 635 genes were downregulated. In order to achieve comprehensive differentially expressed gene screening, the differentially expressed genes screened from the merged cohorts of the two different platforms were integrated to obtain 2015 differentially expressed genes, including 571 genes upregulated and 1444 genes downregulated (see Figure 4 ). The expression levels of these differentially expressed genes showed obvious differences between TGCT and normal testicular tissues.

[0071] In order to further explore the functions of these differentially expressed genes in TGCT, the KEGG functional enrichment analysis of the up-regulated and down-regulated differentially expressed genes was performed using the "cluster Profiler" R package (version 4.10.0). The results showed that the up-regulated differentially expressed genes were mainly enriched in pathways such as Phagosome, Focal adhesion, cell adhesion molecules, ECM-receptor interaction, and antigen processing and presentation; while the down-regulated differentially expressed genes were mainly enriched in pathways such as Cell cycle, Glycolysis / Gluconeogenesis, Central carbon metabolism in cancer, and Purine metabolism (see Figure 5 ). The above results indicate that these genes may be involved in the occurrence and development of tumors and the biological processes of cancer cell proliferation, migration and invasion.

[0072] In order to screen out genes related to the prognosis of TGCT patients, the “survival” R package (version 3.5.8) was used to perform univariate Cox regression analysis on TCGA and 4 GEO cohorts. Genes with Hazard ratio (HR) > 1or < 1 and p value < 0.05 were considered to be genes related to prognosis. 3499 prognostic genes were screened in the TCGA cohort. In order to comprehensively screen prognostic genes, the prognostic genes screened from the same data sets of two different platforms were merged. Specifically, GSE3218 screened 2900 prognostic genes, while GSE10783 screened 3699 prognostic genes (see Figure 6 ).according to Figure 7 The Venn diagram in Figure 3 shows that among the differentially expressed genes in the merged cohort GSE3218-10783, the genes associated with PFI in the TCGA cohort, the genes associated with OS in the GSE3218 cohort, and the genes associated with OS in the GSE10783 cohort, a differentially expressed gene closely associated with prognosis was identified, namely PRTFDC1.

[0073] To further verify the effect of PRTFDC1 on the prognosis of TGCT, the optimal cutoff value of PRTFDC1 expression was determined using the "survminer" R package (version 0.4.9), and the patients were divided into high expression group and low expression group, and Kaplan-Meier curve analysis and log-rank test were performed. In the survival analysis of all cohorts, it was shown that the prognosis of the PRTFDC1 high expression group was significantly worse than that of the low expression group (see Figure 8Univariate and multivariate Cox regression analysis showed that PRTFDC1 was significant in predicting PFI and OS, and was an independent prognostic factor for TGCT patients in the TGCA and GSE3218 cohorts after adjusting for other clinical characteristics (see Figure 9-10 ). Based on the above results, PRTFDC1 can be regarded as an effective prognostic marker for TGCT patients.

[0074] Example 2

[0075] To gain a deeper understanding of the biological characteristics of PRTFDC1, Gene Set Enrichment Analysis (GSEA) was performed based on the correlation between PRTFDC1 and other genes. The top 15 activated and inhibited pathways were displayed in bubble plots according to the Normalized Enrichment Scores (NES) (see Fig.11 ). It is worth noting that during the analysis, some pathways related to tumor development were also observed to be activated (see Fig.12 ), such as focal adhesion, ECM receptor interaction, pathways in cancer, TGF-beta signaling pathway, WNT signaling pathway. In contrast, some immune-related pathways are inhibited (see Fig.13 Based on the above results, it is speculated that PRTFDC1 may promote the malignant progression of TGCT by activating pathways involved in tumor cell proliferation and growth while inhibiting processes related to antigen presentation and immune cell activation.

[0076] To verify the high expression of PRTFDC1 in testicular germ cell tumor tissues, 5 cases of B-cell lymphoma were excluded from the microarray chip of 48 human testicular germ cell tumor tissues, and the expression level and subcellular localization of PRTFDC1 protein in 35 seminoma tissues and 8 normal testicular tissues were detected by immunohistochemical staining (the antibody used was anti-PRTFDC1 antibody (ProteinTech Group, Inc, 11986-1-AP) (see Fig.14 The results showed that PRTFDC1 was not only highly expressed in seminoma tissues, but also mainly localized in the nucleus and cytoplasm, while it was weakly expressed or negative in normal testicular tissues (see Fig.15 ). The above 43 tissue samples were scored by immunohistochemistry, with the percentage of positive cells scored as 0 (<5%), 1 (5–25%), 2 (25–50%), 3 (50–75%), and 4 (>75%); the staining intensity score was 0 (negative), 1 (weak), 2 (moder ate), and 3 (strong). The final IHC score was obtained by adding the positive cell percentage score and the staining intensity score. The results showed that the score of the tumor group was significantly higher than that of the normal group (see Fig.16 Subsequently, the “pROC” R package (version 1.18.5) was used to perform receiver operating characteristic (ROC) curve analysis to evaluate the prediction accuracy of PFTFDC1, where the area under the ROC curve (AUC) greater than 0.7 was considered to have good predictive performance. The results showed that the area under the ROC curve (AUC) = 0.775, indicating that the immunohistochemical score had good predictive performance (see Fig.17 ).

[0077] In order to explore the biological characteristics of PFTFDC1, the Spearman correlation method was used to calculate the correlation coefficient between PFTFDC1 and other genes, and genes with |correlation|>0.4 and false discovery rate (FDR)<0.05 were selected and sorted in descending order. The sorted gene list was further placed in the clusterProfiler package to perform GSEA to study whether genes with strong correlation were clustered in meaningful functional pathways.

[0078] Example 3

[0079] The above results clearly show that the expression of PRTFDC1 is associated with pathways related to the inhibition of tumor immunity. To further study this result, the TCGA-TGCT dataset was used to systematically analyze the relationship between PRTFDC1 and the TGCT immune microenvironment using the TIMER, EPIC, MCPcounter, xCell, and CIBERSORT algorithms, and the Spearman correlation between PRTFDC1 and the infiltration of major immune cells was evaluated (see Fig.18 The results showed that the expression level of PRTFDC1 was significantly higher than that of CD8 + The immune infiltration score of T cells was significantly negatively correlated with Fig.19 These results suggest that high expression of PRTFDC1 may inhibit CD8 + The infiltration of T cells in TGCT leads to poor prognosis. This finding provides a new perspective for understanding the potential role of PRTFDC1 in tumor immune escape and suggests that PRTFDC1 may be an important regulatory factor affecting the immune microenvironment of TGCT.

[0080] After clarifying the potential association between PRTFDC1 and the immune microenvironment in TGCT, the potential utility of PRTFDC1 as an immunotherapy biomarker was further investigated. To explore the relationship between PRTFDC1 expression level and immunotherapy response, SubMap analysis revealed that the expression profiles of patients with low PRTFDC1 expression during anti-PD-1 treatment were significantly similar (see Fig. 20 (p<0.01), which indicates that patients with low PRTFDC1 expression may be more sensitive to anti-PD-1 treatment. Subsequently, two cohorts (GSE100797 and IMvigor210) were further analyzed, and survival analysis was performed on patients who received Chimeric Antigen ReceptorT-Cell Immunothera py (CAR-T) and anti-PD-L1 immunotherapy. The results showed that the prognosis of patients in the PRTFDC1 low expression group was significantly better, suggesting that patients in the low expression group benefited more from CAR-T and anti-PD-L1 immunotherapy (see Fig.21 ). This result further supports the potential of PRTFDC1 expression level as a predictor of immunotherapy response and emphasizes the importance of PRTFDC1 in personalized treatment strategies.

[0081] Connectivity Map (CMap) is a tool that predicts the activation or inhibition of compounds based on gene expression signatures. Based on the expression levels of key genes, the CMap tool was used to screen potential therapeutic drugs to further explore which drug targets may be helpful in treating TGCT and their mechanisms of action. By analyzing the TCGA-TGCT dataset, the top 20 potential TGCT therapeutic drugs that are highly correlated with PRTFDC1 were screened out based on the normalized connectivity score (NCS), including puromycin, simeprevir, flumethasone-pivalate, colchicine, avicin-g, otilonium, etc. (see Fig. 22 ). Further mechanism analysis revealed the possible mechanism of action of these 20 potential drugs, which may work by inhibiting acetylcholine receptors, HCV, protein synthesis, tubulin, etc. (see Fig.23 ).

[0082] It is clear from the above that the present invention successfully screened and identified a biomarker PRTFDC1 with important clinical value by combining bioinformatics analysis and immunohistochemistry. The results show that PRTFDC1 is closely related to the prognosis of TGCT and can effectively predict the patient's response to immunotherapy.

[0083] In order to further explore the role of up-regulated and down-regulated differentially expressed genes in TGCT in the occurrence and progression of tumors, the present invention respectively carried out functional enrichment analysis on these genes. The results showed that the up-regulated differentially expressed genes were mainly enriched in the following pathways: Phagosome, Focal adhesion, cell adhesion molecules, ECM-receptor interaction, antigen processing and presentation, etc. Studies have shown that malignant cells use cell adhesion molecules to adhere to the extracellular matrix to promote tumor growth and metastasis, and cell adhesion to the extracellular matrix (ECM) also plays an important role in the drug resistance of cancer cells. On the other hand, down-regulated differentially expressed genes were mainly enriched in the following pathways: Cellcycle, Glycolysis / Gluconeogenesis, Central carbon metabolism in cancer, Purinemetaboli sm pathways. Many studies have shown that abnormal regulation of tumor cells in metabolism and proliferation can affect tumor growth and development. The present invention comprehensively analyzed the independent cohorts of PFI and OS of TGCT and found that the overall prognosis of the PRTFDC1 high expression group was worse than that of the low expression group. This observation suggests that PRTFDC1 may be a pro-oncogenic factor leading to poor prognosis. However, there are relatively few studies on PRTFDC1. Interestingly, the role of PRTFDC1 in other types of cancer is as a tumor suppressor. For example, in studies of oral squamous cell carcinoma, PRTFDC1 was found to be a potential tumor suppressor gene. In addition, in studies of ovarian cancer, silencing of PRTFDC1 may be involved in the development of ovarian cancer. These studies suggest that PRTFDC1 may have different functions in different types of cancer, acting as both a tumor promoter and a tumor suppressor.

[0084] In order to study the potential mechanism of PRTFDC1 in cancer, the present invention performed GSEA analysis on PRTFDC1-related genes. The results showed that it activated pathways related to tumor occurrence and development, while inhibiting pathways related to immune cell function. These findings further suggest that PRTFDC1 may promote the occurrence and progression of tumors by affecting the immune microenvironment. In addition, through immunohistochemical staining, the present invention confirmed that PRTFDC1 is highly expressed in TGCT tissues and lowly expressed in normal tissues.

[0085] The tumor immune microenvironment includes components such as tumor cells, immune cells, cytokines and immune checkpoint molecules. The interactions between these components have an important impact on the occurrence, progression, metastasis and treatment response of cancer. In GSEA, the present invention found that PRTFDC1 may inhibit immune-related pathways. + T cells can recognize tumor-associated antigens, selectively kill tumor cells, and induce anti-tumor responses. Increased infiltration of T cells is usually associated with a good prognosis. The results of multiple algorithm analysis showed that the expression level of PRTFDC1 was related to CD8 + T cells showed a significant negative correlation, indicating that high expression of PRTFDC1 may inhibit CD8 + T cell infiltration leads to a poor prognosis in TGCT.

[0086] Previous studies have confirmed that anti-PD-1 and PD-L1 treatments activate the immune system by inhibiting immune checkpoints, thereby killing cancer cells. CAR-T cell therapy recognizes tumor cells in the body and releases a large number of effector factors through immune action to efficiently kill tumor cells. Submap analysis results showed that the PRTFDC1 low expression group responded to anti-PD-1 treatment, while the high expression group had no significant response. In addition, the present invention performed a survival analysis in two immunotherapy cohorts receiving CAR-T and anti-PD-L1 immunotherapy. The results showed that the prognosis of patients in the PRTFDC1 low expression group was significantly better, suggesting that patients in the low expression group are most likely to benefit more from CAR-T and anti-PD-L1 immunotherapy. Low PRTFDC1 expression, upregulation of immune checkpoints, and CD8 + Patients with increased T cell infiltration have a better prognosis and are most likely to benefit from immunotherapy. Finally, the present invention studied potential drug targets for TGCT patients, discovered a variety of potential TG CT therapeutic drugs related to PRTFDC1, and further explored their mechanisms of action.

[0087] In general, the present invention clarifies the key role of PRTFDC1 in TGCT, provides new ideas for the acquisition of relevant target points and the treatment of related diseases in subsequent research, and helps to provide a clear direction for subsequent drug development, clinical treatment, etc., and has extremely high social value and market application prospects.

[0088] The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting the expression level of PRTFDC1 in the preparation of a product for auxiliary diagnosis and / or prognosis evaluation of testicular germ cell tumors.

2. The use according to claim 1, characterized in that: The reagent for detecting the expression level of PRTFDC1 includes primers for detecting the expression level of PRTFDC1 gene and / or a reagent for detecting the content of PRTFDC1 protein.

3. The use according to claim 2, characterized in that: The primers for detecting the expression level of the PRTFDC1 gene are selected from one or more of the following primer pairs: Primer pair 1: the upstream sequence is shown in SEQ ID NO: 1, and the downstream sequence is shown in SEQ ID NO: 2; Primer pair 2: the upstream sequence is shown in SEQ ID NO: 3, and the downstream sequence is shown in SEQ ID NO:

4.

4. The use according to claim 2, characterized in that: The reagent for detecting the PRTFDC1 protein content is selected from anti-PRTFDC1 antibodies.

5. Use of a PRTFDC1 inhibitor in the preparation of a medicament for preventing and / or treating testicular germ cell tumors.

6. The use according to claim 5, characterized in that: The PRTFDC1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the PRTFDC1 gene.

7. A kit for auxiliary diagnosis and / or prognosis assessment of testicular germ cell tumors, characterized in that: Reagents for detecting PRTFDC1 expression levels are included.

8. The kit according to claim 7, characterized in that The reagent for detecting the expression level of PRTFDC1 includes primers for detecting the expression level of PRTFDC1 gene and / or a reagent for detecting the content of PRTFDC1 protein.

9. Application of PRTFDC1 inhibitors in the preparation of drugs for improving the immunotherapeutic activity of testicular germ cell tumors.

10. The use according to claim 9, characterized in that: The PRTFDC1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the PRTFDC1 gene.

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