Marker for nasopharyngeal carcinoma diagnosis and prognosis evaluation and application thereof

Abstract

The application discloses a marker for nasopharyngeal carcinoma diagnosis and prognosis evaluation and application thereof. The marker comprises LSM5 and / or RBM20. The marker LSM5 and / or RBM20 for nasopharyngeal carcinoma diagnosis and prognosis evaluation provided by the application can be accurately used for nasopharyngeal carcinoma diagnosis and prognosis evaluation, and the proliferation, invasion and metastasis of nasopharyngeal carcinoma cells can be inhibited after the marker LSM5 expression is inhibited, thereby providing a new powerful molecular biology tool for nasopharyngeal carcinoma treatment. The application has far-reaching clinical significance and important popularization and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to biomarkers for the diagnosis and prognostic evaluation of nasopharyngeal carcinoma and their applications. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) typically occurs in the posterior wall of the nasopharynx and pharyngeal recess, exhibiting distinct epidemiological, histopathological, clinical, and therapeutic characteristics. Concurrent chemoradiotherapy (CCRT) or radiotherapy is considered the primary treatment for NPC. However, many patients experience local recurrence or distant metastasis due to radiotherapy or chemotherapy resistance, ultimately leading to treatment failure. Therefore, further research into the molecular mechanisms of recurrence or metastasis is necessary to identify potential targets for maintaining or restoring treatment sensitivity and improving patient prognosis.

[0003] RNA processing is the process by which RNA transcripts are converted into mature RNA molecules. Alterations in RNA processing, such as RNA splicing and polyadenylation, are major sources of transcriptome variation in cancer and can play important oncogenic roles. Aberrant expression of RNA processing factors negatively impacts mRNA transport and editing. Furthermore, cancer treatment can induce individual splicing changes and mutations in RNA splicing factors, which can occur within a single gene or within the RNA processing factor itself, potentially affecting the splicing of downstream target genes. RNA processing factors also regulate intron removal and alternative splicing of individual genes. Changes in alternative splicing are associated with the occurrence and progression of malignant tumors. Therefore, further research into RNA processing is necessary, as it may create new opportunities for therapeutic interventions in cancer. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a biomarker for the diagnosis and / or prognostic evaluation of nasopharyngeal carcinoma.

[0005] The present invention also proposes the application of the above-mentioned markers.

[0006] The present invention also proposes a system for the diagnosis and / or prognostic evaluation of nasopharyngeal carcinoma.

[0007] The present invention also proposes a reagent for detecting the expression level of the above-mentioned biomarkers.

[0008] The present invention also proposes applications of the above-mentioned systems or reagents.

[0009] This invention also proposes an LSM5 expression inhibitor.

[0010] This invention also proposes the application of the above-mentioned LSM5 expression inhibitor.

[0011] The present invention also proposes a drug for treating nasopharyngeal carcinoma.

[0012] According to a first aspect of the invention, a biomarker for the diagnosis and / or prognostic evaluation of nasopharyngeal carcinoma is provided, said biomarker comprising LSM5 and / or RBM20.

[0013] According to some embodiments of the present invention, the prognostic evaluation of nasopharyngeal carcinoma includes evaluating the patient's survival.

[0014] According to some embodiments of the present invention, the prognostic evaluation of nasopharyngeal carcinoma includes evaluating the sensitivity of nasopharyngeal carcinoma patients to chemotherapy drugs.

[0015] According to some embodiments of the present invention, the survival status includes at least one of 1-year survival rate, 3-year survival rate, and 5-year survival rate.

[0016] According to some embodiments of the present invention, the marker is an RNA processing gene.

[0017] According to some embodiments of the present invention, the biomarker is used to calculate a prognostic risk score for nasopharyngeal carcinoma prognosis evaluation using the following formula: Risk score = 1.6243 × LSM5 expression level + (-0.9761) × RBM20 expression level.

[0018] According to a second aspect of the invention, the application of the aforementioned marker in any of the following is proposed;

[0019] (1) Establish a system for the diagnosis and / or prognostic evaluation of nasopharyngeal carcinoma; (2) Prepare products for the diagnosis and / or prognostic evaluation of nasopharyngeal carcinoma; (3) Prepare products for the treatment or adjuvant treatment of nasopharyngeal carcinoma; (4) Prepare products for the screening of nasopharyngeal carcinoma for treatment or adjuvant treatment.

[0020] According to some embodiments of the present invention, the product includes at least one of a drug, reagent, reagent kit, chip, membrane strip, or detection device.

[0021] According to some embodiments of the present invention, the treatment or adjuvant treatment of nasopharyngeal carcinoma includes inhibiting cancer cell proliferation, inhibiting cancer cell migration, invasion and / or promoting cancer cell apoptosis.

[0022] According to a third aspect of the present invention, a system for the diagnosis and / or prognostic evaluation of nasopharyngeal carcinoma is provided, comprising:

[0023] The data acquisition module is used to acquire the expression levels of the aforementioned biomarkers in patients;

[0024] The data analysis module is used to input the expression levels of the biomarkers into the risk scoring model to assess the diagnosis and / or prognosis of the patient's nasopharyngeal carcinoma.

[0025] The formula for the risk scoring model includes: Risk score = 1.6243 × LSM5 expression level + (-0.9761) × RBM20 expression level.

[0026] According to some embodiments of the present invention, the application method of the system includes the following steps:

[0027] S1. Obtain the expression levels of the aforementioned biomarkers in the patient; S2. Input the expression levels of the biomarkers into the risk scoring model, calculate the patient's risk score, and analyze the patient's nasopharyngeal carcinoma diagnosis and / or prognosis.

[0028] According to some embodiments of the present invention, the analysis of the diagnosis and / or prognosis of the patient's nasopharyngeal carcinoma includes:

[0029] When the risk score is not lower than the expected risk score threshold, the patient is or is a candidate for the high-risk group; when the risk score is lower than the expected risk score threshold, the patient is or is a candidate for the low-risk group.

[0030] According to some embodiments of the present invention, the prognosis includes survival.

[0031] According to some embodiments of the present invention, the survival status includes at least one of 1-year survival rate, 3-year survival rate, and 5-year survival rate.

[0032] According to some embodiments of the present invention, the prognostic assessment includes evaluating the sensitivity of nasopharyngeal carcinoma patients to chemotherapy drugs.

[0033] According to a fourth aspect of the present invention, a reagent for detecting the expression level of the above-mentioned biomarkers is provided, the reagent comprising primers and / or probes.

[0034] In some embodiments of the present invention, the primers include primers for amplifying LSM5 and / or primers for amplifying RBM20;

[0035] The sequences of the primers used to amplify LSM5 are shown in SEQ ID NO:1 and SEQ ID NO:2;

[0036] The sequences of the primers used to amplify RBM20 are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0037] In some embodiments of the present invention, the reagent further includes PCR detection reagents.

[0038] In some embodiments of the present invention, the PCR detection reagent is a real-time fluorescence quantitative PCR detection reagent.

[0039] According to a fifth aspect of the invention, the use of the above-described system or reagent in any of the following is proposed;

[0040] 1) Prepare products for the diagnosis and / or prognostic evaluation of nasopharyngeal carcinoma; 2) Prepare products for the treatment or adjuvant therapy of nasopharyngeal carcinoma; 3) Prepare products for screening the treatment or adjuvant therapy of nasopharyngeal carcinoma.

[0041] According to some embodiments of the present invention, the product includes at least one of a drug, reagent, reagent kit, chip, membrane strip, or detection device.

[0042] According to some embodiments of the present invention, the treatment or adjuvant treatment of nasopharyngeal carcinoma includes inhibiting cancer cell proliferation, inhibiting cancer cell migration, invasion and / or promoting cancer cell apoptosis.

[0043] According to a sixth aspect of the present invention, an LSM5 expression inhibitor is provided, comprising at least one of a substance that inhibits LSM5 activity, a substance that degrades LSM5, or a substance that reduces the expression level of LSM5.

[0044] In some embodiments of the present invention, the substance that reduces LSM5 expression levels is at least one of a1)-a3):

[0045] a1) siRNA, dsRNA, miRNA, ribozyme, or shRNA targeting LSM5; a2) nucleic acid molecule encoding the siRNA, dsRNA, miRNA, ribozyme, or shRNA targeting LSM5 described in a1); a3) an expression cassette, vector, or transgenic cell line containing the nucleic acid molecule described in a2).

[0046] In some embodiments of the present invention, the siRNA includes siRNA1, siRNA2 and / or siRNA3; the sense sequence of siRNA1 is shown in SEQ ID NO:7 and the antisense sequence is shown in SEQ ID NO:8; the sense sequence of siRNA2 is shown in SEQ ID NO:9 and the antisense sequence is shown in SEQ ID NO:10; the sense sequence of siRNA3 is shown in SEQ ID NO:11 and the antisense sequence is shown in SEQ ID NO:12.

[0047] According to a seventh aspect of the present invention, the use of the above-mentioned LSM5 expression inhibitor in the preparation of products for the treatment or adjuvant treatment of nasopharyngeal carcinoma is proposed.

[0048] In some embodiments of the present invention, the product includes pharmaceuticals.

[0049] In some embodiments of the present invention, the treatment or adjuvant treatment of nasopharyngeal carcinoma includes inhibiting cancer cell proliferation, inhibiting cancer cell migration, invasion and / or promoting cancer cell apoptosis.

[0050] According to an eighth aspect of the present invention, a medicament for treating nasopharyngeal carcinoma is provided, comprising the above-mentioned LSM5 expression inhibitor.

[0051] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention provides a novel biomarker for the diagnosis and / or prognostic evaluation of nasopharyngeal carcinoma, LSM5 and / or RBM20, which can be accurately used for the diagnosis and / or prognostic evaluation of nasopharyngeal carcinoma. Simultaneously, inhibiting the expression of the biomarker LSM5 can inhibit the proliferation, invasion, and metastasis of nasopharyngeal carcinoma cells, providing a new and powerful molecular biological tool for the treatment of nasopharyngeal carcinoma. This has profound clinical significance and important prospects for widespread application. Attached Figure Description

[0052] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0053] Figure 1 The following diagrams show the identification results of differentially expressed genes (DEGs) in this embodiment of the invention. a) is a volcano plot of DEGs between tumor and normal sample groups, where orange, green, and black dots represent upregulated, downregulated, and unchanged genes (tumor / normal), respectively. b) is a heatmap showing a positive correlation between gene expression and color changes; the tree diagram on the left represents cluster analysis of different genes from different samples. c) is a Venn diagram of differentially expressed RNA-processing genes (DE-RPGs). d) is a heatmap of 10 DE-rPgs expression patterns. e) shows the expression of 10 differentially expressed RNA-processing genes in normal and nasopharyngeal carcinoma tissues (immunohistochemical results from the HPA database).

[0054] Figure 2 The above diagram shows the functional enrichment analysis results of differentially expressed RNA processing genes (DE-RPGs) in this embodiment of the invention. In the diagram, a is the GO enrichment analysis result, where black spots represent counts and color represents the adjusted P-value; b is the KEGG enrichment analysis result of DE-RPGs, where the KEGG pathway is on the vertical axis and color represents the adjusted P-value.

[0055] Figure 3 The image shows the results of dividing the NPC samples in the GSE102349 training set into high-risk and low-risk groups according to the optimal cutoff value of 2.35 in this embodiment of the invention. Among them, a, b, c, and d are all the results of dividing the NPC samples in the GSE102349 training set into high-risk and low-risk groups.

[0056] Figure 4The diagram shows the construction and identification analysis results of the prediction model in this embodiment of the invention. In diagram a, risk curves for high-risk and low-risk patients in the training set are shown. The horizontal axis of the upper and middle panels represents patient samples categorized according to risk scores. Risk scores increase from left to right. Risk scores and survival times are arranged in an appropriate order, with dashed lines representing the optimal risk score threshold and the corresponding number of patients. The lower panel represents a heatmap of model gene expression. Diagram b shows the analysis results of the survival rate of the training set. Diagram c shows the ROC curve for survival prediction in the training set, where the 3-year ROC curve overlaps with the 5-year curve.

[0057] Figure 5 To validate the risk curves for high-risk and low-risk patients in the validation group;

[0058] Figure 6 Survival analysis graph for the validation set;

[0059] Figure 7 The ROC curves for survival prediction of the internal validation set are shown, where the 3-year ROC curves overlap with the 5-year curves.

[0060] Figure 8 A plot of Cox regression analysis based on the GSE102349 dataset;

[0061] Figure 9 The graph shows the correlation detection results between prognostic genes and immune cells in this embodiment of the invention. In the graph, a is the rank value of 24 tumor-infiltrating blast cells (TIICs) in the risk group; b is the expression of 24 TIICs in the risk group; c is the univariate forest plot and TIICs; d is the correlation between DE-rpg and immune cells. "*" indicates p < 0.05, and "**" indicates p < 0.01.

[0062] Figure 10 This is a graph showing the sensitivity analysis results of the chemotherapy drugs in this embodiment of the invention.

[0063] Figure 11 This is a graph showing the sensitivity analysis results of the chemotherapy drugs in this embodiment of the invention.

[0064] Figure 12 This is a graph showing the sensitivity analysis results of the chemotherapy drugs in this embodiment of the invention.

[0065] Figure 13The figures shown are the expression verification results of RBM20 and LSM5 in the embodiments of the present invention. A represents the expression detection results of RBM20 and LSM5 in normal nasopharyngeal carcinoma tissue and nasopharyngeal carcinoma tissue; B represents the expression detection results of RBM20 and LSM5 in normal nasopharyngeal carcinoma tissue and nasopharyngeal carcinoma tissue; C represents the expression detection results of RBM20 and LSM5 in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells; D represents the expression detection results of RBM20 and LSM5 in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells; E represents the expression detection results of RBM20 and LSM5 in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells; F represents the expression detection results of RBM20 and LSM5 mRNA in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells; G represents the expression detection results of RBM20 and LSM5 mRNA in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells; ns indicates no statistically significant difference, * indicates p < 0.05, and ** indicates p < 0.01.

[0066] Figure 14 The figures shown are functional verification results of LSM5 in nasopharyngeal carcinoma cells according to embodiments of the present invention. A represents the expression detection results of LSM5 in nasopharyngeal carcinoma; B represents the expression detection results of LSM5 in nasopharyngeal carcinoma; C represents the survival curves of nasopharyngeal carcinoma patients with high and low LSM5 expression levels; D represents the Western blot diagram verifying protein expression after LSM5 interference; and E represents the protein expression detection results after LSM5 interference.

[0067] Figure 15 The figures shown are the functional verification results of LSM5 in nasopharyngeal carcinoma cells in the embodiments of the present invention. Among them, A is the colony formation analysis of cell survival and clonal ability after RNA interference with LSM5; B is the colony formation analysis of cell survival and clonal ability after RNA interference with LSM5; C is the EdU analysis of cell proliferation after RNA interference with LSM5; D is the EdU analysis of cell proliferation after RNA interference with LSM5; E, F, and G are migration analysis of cell migration ability after RNA interference with LSM5; H and I are wound healing analysis of cell migration ability after RNA interference with LSM5. ns indicates no statistically significant difference, * indicates p < 0.05, and ** indicates p < 0.01. Detailed Implementation

[0068] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0069] Example 1: Screening of biomarkers for the diagnosis and prognostic evaluation of nasopharyngeal carcinoma

[0070] This embodiment provides RNA processing factors LSM5 and RBM20 as biomarkers for the diagnosis and prognostic evaluation of nasopharyngeal carcinoma. The screening process is as follows:

[0071] 1. Selection of data source

[0072] NPC-related transcriptome data were downloaded from the Gene Expression Comprehensive Database (GEO). Differentially expressed genes (DEGs) in the GSE12452 dataset (31 nasopharyngeal carcinoma and 10 normal nasopharyngeal epithelial tissues) were analyzed using the GPL570 platform (https: / / ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE12452). The GSE102349 dataset (88 nasopharyngeal carcinoma patients) was constructed and evaluated using the GPL11154 platform (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE102349).rpg from the AmiGO database (http: / / amigo.geneontology.org). 6734 genes were obtained by searching the AmiGO database for the keyword "RNA processing," and were subsequently defined as RNA processing genes (RPGs) for this study.

[0073] 2. Identification of differentially expressed RNA processing genes

[0074] Differential expression analysis revealed differentially expressed genes (DEGs) in nasopharyngeal carcinoma and normal nasopharyngeal epithelial tissue. The R package "limma," based on the GSE12452 dataset, was used to identify differentially expressed genes using thresholds p < 0.05 and |log² change (FC)| > 1. Overlapping genes are genes shared between two or more groups of elements. Jvenn (http: / / jvenn.toulouse.inra.fr / app / example.html) was used to identify common genes in the DEGs and RPGs lists; these common genes were differentially expressed RNA processing genes (DE-RPGs) associated with NPC.

[0075] Table 1

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] The results are as follows Figure 1 As shown in Table 1, a total of 795 DEGs related to NPCs were identified from the GSE12452 database (such as...). Figure 1 (See Figure 1). Of these, 293 genes were upregulated and 502 genes were downregulated. Overlap analysis revealed 10 common genes between NPC-related DEGs and RPGs: AHNAK2, ALYREF, FASTKD1, LSM5, PIH1D2, PNPT1, PUS7, RBM20, RBM24, and RRP15. Figure 1(As shown in Figure c). Compared with the healthy control group, the expression levels of ALYREF, FASTKD1, LSM5, PNPT1, PUS7, and RRP15 were all upregulated in the nasopharyngeal carcinoma group, while the expression levels of AHNAK2, PIH1D2, RBM20, and RBM24 were significantly decreased. Figure 1 As shown in Figure d). IHC results from the HPA database confirmed the expression of these genes ( Figure 1 (As shown in Figure e). Through the following screening and verification of the above genes, the biomarkers LSM5 and RBM20 for the diagnosis and prognostic evaluation of nasopharyngeal carcinoma were finally obtained.

[0087] 3. Functional enrichment analysis of GO and KEGG

[0088] Cluster Profiler was used to perform functional enrichment analysis of GO and KEGG to identify common functions and pathways of DE-RPG associated with NPC. The significance threshold was p < 0.05.

[0089] Table 2

[0090]

[0091]

[0092] Table 3 shows the KEGG analysis of NPC-related DE-RPGs.

[0093]

[0094] The results are as follows Figure 2 As shown in Tables 2-3, GO functional annotation and enrichment analysis and KEGG pathway annotation and enrichment analysis elucidated the potential molecular mechanisms of nasopharyngeal carcinoma-related DE-RPGs in nasopharyngeal carcinoma. GO analysis identified 55 enriched biological process (BP) terms and 16 molecular function (MF) terms (as shown in Table 2). The top five terms in the BP and MF categories are as follows... Figure 2 As shown in Figure a, in the BP category, these genes are mainly associated with RNA processing activities, including splicing, catabolism, metabolism, processing, and translocation. Furthermore, terms related to the cell cycle are significantly enriched. In the MF category, terms related to RNA binding are significantly enriched. KEGG pathway analysis indicates that DE-RPGs associated with NPCs are involved in RNA degradation and spliceosome pathways. Figure 2 (b, as shown in Table 3).

[0095] 4. Screening of RNA processing factors with prognostic value and construction and identification of predictive models.

[0096] The GSE102349 dataset was used to screen RNA processing factors with prognostic value. Specifically, the method is as follows:

[0097] After matching the expression profiles of DE-rpg related to NPC, 88 NPC patients were randomly divided into the GSE102349 training set (n=44) and the GSE102349 test set (n=44) in a 1:1 ratio.

[0098] Univariate Cox regression analysis was performed using the GSE102349 training set (n=44) to assess the association between identified DE-RPGs and disease-free survival (DFS) in NPC patients, in order to identify NPC-related DE-RPGs with prognostic value. The significance threshold was p<0.05.

[0099] Univariate and multivariate Cox regression analyses were performed on NPC-related DE-RPGs using the R package "survminer," with a p-value threshold of <0.05, to screen for genes relevant to the NPC prognostic risk model. Risk values ​​for each patient were obtained by simulating gene expression and using risk coefficients (coef) derived from multivariate Cox regression. Subsequently, patients were divided into high-risk and low-risk groups based on the optimal threshold for the risk score. The formula used for the risk score is as follows.

[0100] Risk score = Risk coefficient × Gene 1 + Risk coefficient × Gene 2 + Risk coefficient × Gene n.

[0101] Kaplan-Meier survival analysis was used to visualize the results of the prognostic survival analysis between the two groups. ROC curves plotted using the "pROC" R package were used to evaluate the validity of the risk model. Finally, univariate and multivariate Cox analyses based on risk scores and clinicopathological factors were used to validate the independent prognosis of risk characteristics.

[0102] Table 4. Correlation analysis of 10 DE-RPGs with patient survival.

[0103]

[0104] Table 5 Regression coefficients of LSM5 and RBM20

[0105]

[0106] The results are shown in Table 4. As can be seen from the table, LSM5 (p = 0.022) and RBM20 (p = 0.035) were significantly associated with DFS in NPC patients.

[0107] The regression coefficients of LSM5 and RBM20 were calculated (as shown in Table 5) to be used in the formula for constructing the prognostic risk score of RNA processing factors.

[0108] To assess the prognostic value of the risk model, NPC patients were scored using the expression levels of two model genes and risk coefficients obtained from multivariate Cox regression. Each patient's risk score was then assigned to a high-risk or low-risk group based on the optimal threshold for the risk score. The risk score calculation formula is as follows:

[0109] Risk score = 1.6243 × LSM5 expression level + (-0.9761) × RBM20 expression level.

[0110] The above risk scoring formula was used to calculate the risk scores of NPC patients in the GSE102349 training set. Based on the optimal cutoff value of 2.35, the NPC samples in the GSE102349 training set were divided into high-risk and low-risk groups (e.g., ...). Figure 3 (As shown).

[0111] The prediction analysis results in the GSE102349 training set are as follows: Figure 4-7 As shown, from Figure 4 a and Figure 5 As can be seen from the risk curves and disease progression states (disease-free and progressive) of patients in the training set, patients with progressive disease have higher risk scores. From... Figure 4 b and Figure 6 As can be seen, the DFS in the low-risk group was significantly higher than that in the high-risk group (p = 0.00025). Figure 4 c and Figure 7 As can be seen, the areas under the ROC curves (AUC) for one, three, and five years were 0.836, 0.652, and 0.652, respectively. Furthermore, in the high-risk group, LSM5 expression levels were relatively high, while the longer DFS rate in the low-risk group was associated with high RBM20 expression.

[0112] Cox regression analysis based on the GSE102349 dataset showed that risk score can influence DFS in nasopharyngeal carcinoma patients independently of stage characteristics (e.g., Figure 8 (As shown).

[0113] The results showed that LSM5 and RBM20 were independent prognostic factors for nasopharyngeal carcinoma.

[0114] Example 2: Correlation Analysis of Prognostic Biomarkers and Immune Cells

[0115] Using the single-sample GSEA method, 24 immune-related gene sets were obtained for each sample in the GSE102349 dataset. Samples were then divided into high-risk and low-risk groups based on the rich immune-related information. The Wilcoxon rank-sum test was used to compare the differences in tumor-infiltrating immune cells between these risk groups, with a significance threshold set at p < 0.05.

[0116] In addition, univariate Cox regression analysis was performed to correlate the differential immune cell profile of nasopharyngeal carcinoma patients with the disease-free survival (DFS) rate. Subsequently, Pearson's correlation analysis was used to reveal the correlation between immune cells and the biomarkers (prognostic genes) prepared in Example 1. A correlation coefficient (cor) > 0.3 and p < 0.05 were established as significance thresholds.

[0117] Table 6

[0118]

[0119] The results of the correlation between prognostic genes and immune cells are as follows: Figure 9 As shown, from Figure 9 As shown in a, the abundance of 24 immune cells in the high-risk and low-risk groups was detected using ssGSEA; from Figure 9 As shown in b, the Wilcoxon rank-sum test revealed a statistically significant difference in the percentage of 21 immune cells between the two risk groups.

[0120] In addition, from Figure 9 As shown in Table 6, CD8 T cells, cytotoxic cells, Tem, Tcm, mast cells, eosinophils, T cells, pDCs, and DCs are significantly associated with disease-free survival (DFS) in nasopharyngeal carcinoma patients. The main roles of DCs in antitumor immune responses include phagocytosis of dead tumor cells, capture and presentation of tumor-associated antigens, and activation of various T cells, thereby collectively stimulating a series of immune responses to kill tumor cells. In tumors, a decrease in DC count leads to weaker antigen processing, thereby altering T cell proliferation and differentiation, negatively impacting their tumor-killing efficacy, and inducing immune tolerance.

[0121] from Figure 9 As shown in Figure d, Pearson correlations between differentially expressed immune cells and RBM20 and LSM5 were observed. In the low-risk group, dendritic cells (DCs) showed a significant negative correlation with RBM20, CD8+ T cells, T cells, DCs, eosinophils, and Tem, while pDCs showed a negative correlation with LSM5. However, in the high-risk group, no significant correlation was found between immune cells and prognostic genes.

[0122] Example 3: Application of the predictive model in guiding the use of chemotherapy drugs

[0123] This embodiment verifies the guiding significance of the predictive model constructed in Example 2 for the use of chemotherapy drugs. The specific method is as follows: Using the R language package "pRRophetic", the IC50 of nasopharyngeal carcinoma samples for common chemotherapy drugs was calculated. The rank-sum test was used to compare the differences in IC50 of 138 chemotherapy drugs between high- and low-risk groups. Then, box plots were created using the R language ggplot2 to visualize the results. The rank-sum test results showed that 55 of the 138 chemotherapy drugs showed significant differences. A p-value < 0.05 indicates statistical significance. A lower half-inhibitory concentration (WIC) value indicates higher sensitivity to chemotherapy drugs.

[0124] Table 7

[0125]

[0126]

[0127] The results are as follows Figure 10-12 As shown in Table 7, there is a statistically significant difference in sensitivity between the high-risk and low-risk groups for the 55 drugs, which is reflected in the half-maximal inhibitory concentration (WMC) values. It can be inferred that patients in the low-risk group are more sensitive to these 55 drugs. The correlation between risk scores and 10 common chemotherapy drugs is shown in Table 7. Figure 10 As shown. The differences between the two risk groups for the remaining 45 drugs are as follows: Figure 11-12 As shown.

[0128] Example 4: Application of RBM20 and / or LSM5 in the preparation of formulations for nasopharyngeal carcinoma diagnosis

[0129] This embodiment provides the application of RBM20 and / or LSM5 in the preparation of agents for nasopharyngeal carcinoma diagnosis. The specific verification steps are as follows:

[0130] 1. Sample selection

[0131] (1) Cell selection

[0132] Human nasopharyngeal carcinoma cells 5-8F, CNE2, S18, 6-10B, CNE1, S26, and human nasopharyngeal epithelial cells NP69 were purchased from BeNa Cell Culture Company, Hebei Province, China. The culture media for human nasopharyngeal carcinoma cells and NP69 cells were RPMI-1640 supplemented with 10% fetal bovine serum and K-SFM supplemented with 10% fetal bovine serum, respectively.

[0133] (2) Tumor tissue collection

[0134] From January 2019 to December 2020, this invention collected paraffin-embedded specimens of 40 nasopharyngeal carcinoma (NPC) cases and 30 normal nasopharyngeal tissues from Xiangya Hospital of Central South University.

[0135] 2. Real-time quantitative PCR detection of RBM20 and LSM5 mRNA expression.

[0136] The expression of RBM20 and LSM5 molecules was detected by real-time quantitative PCR in normal human nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells. The specific experimental procedure is as follows:

[0137] (1) RNA was extracted from normal human nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells using an RNA extraction kit purchased from Beijing Conway Century, China.

[0138] (2) mRNA reverse transcription: mRNA reverse transcription was performed using an mRNA reverse transcription kit (purchased from Beijing Conway Century, China (No.: CW2569)).

[0139] (3) Real-time quantitative PCR reaction

[0140] The primers for real-time quantitative PCR detection of LSM5 are:

[0141] Forward primer: 5'-TGTCCGATTCCTTCACCTCC-3' (SEQ ID NO:1);

[0142] Reverse primer: 5'-CATTTGTCCACAAGCTCTCACC-3' (SEQ ID NO:2).

[0143] The primers for real-time quantitative PCR detection of RBM20 are:

[0144] Forward primer: 5'-CCTCCCTTGAGCTCTCTCGC-3' (SEQ ID NO:3);

[0145] Reverse primer: 5'-AGCTTGGCGGCATTTTGGAT-3' (SEQ ID NO:4).

[0146] The internal reference gene is GAPDH, and the primer sequences for detecting the internal reference gene are:

[0147] Forward primer: 5'-GAAAGCCTGCCGGTGACTAA-3' (SEQ ID NO:5);

[0148] Reverse primer: 5'-GCCCAATACGACCAAATCAGAG-3' (SEQ ID NO:6).

[0149] The total reaction volume was prepared as follows: 10 μL of 2×SYBR Green MIX, 1 μL each of upstream and downstream primers (10 mM), 2 μL of cDNA template, and enzyme-free water to a final volume of 20 μL.

[0150] The reaction program was as follows: 95℃ for 5 min pre-denaturation, followed by 95℃ for 30 s denaturation, 60℃ for 5 s annealing, and 70℃ for 10 s extension, for a total of 40 cycles. Melting curve analysis was then performed: fluorescence signals were collected at temperatures ranging from 70℃ to 95℃. After the reaction, the amplification and melting curves of qRT-PCR were confirmed. The expression intensities of each gene were standardized according to CT values ​​(threshold cycle values) and the internal reference gene (GAPDH). -△△ct Calculate gene expression.

[0151] 3. Immunohistochemical (IHC) analysis of RBM20 and LSM5 protein expression in nasopharyngeal carcinoma tumor tissues.

[0152] Seventy paraffin-embedded tissue specimens were stained using the IHCSP method. Positive staining results appeared as brownish-yellow granules. Immunohistochemical staining was quantified using mean optical density (MOD) obtained through ImageJ image analysis software. The PV-9000 universal two-step detection kit was purchased from Zhongshan Jinqiao Clinic in Beijing, China.

[0153] Test results as follows Figure 13 As shown, from Figure 13 As can be seen from the AB diagram, compared with normal nasopharyngeal carcinoma tissue (n=30), LSM5 expression was increased and RBM20 expression was decreased in nasopharyngeal carcinoma tissue (n=40).

[0154] 4. Western blot (WB) analysis of RBM20 and LSM5 protein expression in nasopharyngeal carcinoma cell lines.

[0155] Normal human nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells in logarithmic growth phase were collected, and protein concentrations were determined using a BCA assay kit (Lanco, Hangzhou, China). 80 μg of protein from each group was loaded into an 8% polyacrylamide gel and separated by SDS-PAGE. These proteins were then transferred to PVDF cell membranes. Primary antibodies were incubated overnight at 4°C (RBM20, 1:500, bs-9606R, Bioss, China; LSM5, 1:500, ab184568, Abcam, UK). Signal visualization was performed using a dual-color infrared laser imaging system (Odyssey CLx, LI-COR, NJ, USA). Protein expression normalization was performed using endogenous actin (1:1000, 66009-1-Ig, China) and GAPDH (1:20000, 10494-1-AP, China).

[0156] Test results as follows Figure 13 As shown, from Figure 13As can be seen from the CG image, in the nasopharyngeal carcinoma cell line, the protein and mRNA levels of LSM5 are elevated, while the expression level of RBM20 remains low.

[0157] Example 5: Application of LSM5 in the preparation of products for nasopharyngeal carcinoma prognosis

[0158] This embodiment provides a scheme for verifying the application of LSM5 in the preparation of products for nasopharyngeal carcinoma prognosis. The specific verification steps are as follows:

[0159] Forty patients with nasopharyngeal carcinoma from Xiangya Hospital of Central South University were followed up every 3-6 months via outpatient visit or telephone to collect survival and mortality data. The follow-up cutoff points were death, follow-up failure, or the end of the follow-up period (December 2023). The follow-up rate for the 40 nasopharyngeal carcinoma patients was 100%.

[0160] Prognostic analysis results as follows Figure 14 As shown, from Figure 14 The AC plot shows that the median survival time was 7.75 months for the 16 patients with high LSM5 expression, while the median survival time was 21 months for the 14 patients with low LSM5 expression. The chi-square value of the Log-Rank test was 15.93. These results indicate that LSM5 can be effectively used for the prognosis of nasopharyngeal carcinoma.

[0161] Example 6: An LSM5 expression inhibitor

[0162] This embodiment provides an LSM5 expression inhibitor, including siRNA1-LSM5, siRNA2-LSM5, and siRNA2-LSM5, which were synthesized by Jiman Biotechnology (Shanghai) Co., Ltd., and the specific sequences are shown in Table 8 below.

[0163] Table 8

[0164]

[0165] Example 7: Application of LSM5 expression inhibitors in the preparation of products that inhibit nasopharyngeal carcinoma

[0166] This embodiment demonstrates the application of the LSM5 expression inhibitor prepared in Example 6 in the preparation of a tumor-inhibiting product. Specifically, CNE2 and 5-8F cells were used for validation experiments. 5-8F and CNE2 cells were cultured, and a blank control group was established. The small interfering RNAs (siRNA1-LSM5, siRNA2-LSM5, and siRNA3-LSM5) targeting LSM5 prepared in Example 6 were used as experimental groups. The siRNA-NC group served as a negative control. The LSM5-targeting siRNAs were obtained from Jiman Biotechnology (Shanghai) Co., Ltd., and their sequences are shown in Table 8. The specific validation process is as follows:

[0167] (1) Cell transfection

[0168] Cells were seeded into 6-well plates (approximately 1 × 10⁶ cells) 24 hours before transfection. 5 (cells / well), ensuring cell growth at 60-70% confluency at transfection. In antibiotic-free RPMI-1640 medium, pcDNA3.1-RBM20-3xFlag vector or LSM5 expression inhibitor siRNA-LSM5 (siRNA1-LSM5, siRNA2-LSM5, or siRNA3-LSM5) were respectively added to... Mix 3000 units of transfection reagent. For pcDNA3.1-RBM20-3xFlag transfection, mix the vector DNA with the transfection reagent at a 1:2 mass ratio and incubate for 5 minutes. For siRNA-LSM5 transfection, mix the siRNA with the transfection reagent at a 1:2 mass ratio and incubate for 5 minutes. Add the above mixture to cell culture dishes and gently shake to ensure even distribution. Continue cell culture for 24 hours for subsequent analysis. The control group uses empty vector pcDNA3.1 or non-targeting siRNA.

[0169] (2) LSM5 expression detection.

[0170] Within 24 hours post-transfection, Western blotting (WB) and quantitative reverse transcription PCR (RT-qPCR) were performed to detect transfection efficiency and LSM5 gene expression levels. The LSM5 expression detection method was the same as in Example 5, and the results are as follows: Figure 14 The figures D and E are shown in the diagram.

[0171] (3) Colony formation test and 5-ethyl-20-deoxyuridine (EdU) test

[0172] Single-cell suspensions were seeded into 6-well plates at a rate of 300 cells per well and cultured for 24 hours before crystal violet staining. Subsequently, cells were seeded into 6-well plates at a rate of 1 × 10⁶ cells per well. 4 Cells were cultured for 24 h. Then, they were co-cultured with 50 μM EdU solution for 2 h, followed by fixation with 4% paraformaldehyde for 20 min. 500 μL of click reaction solution was added to each well, and the cells were incubated at room temperature for 30 min. After DAPI staining, images were captured using a fluorescence microscope (Carl Zeiss Axio Observer 3m). The EdU cell proliferation imaging kit was purchased from Wuhan Electronic Science and Biotechnology Co., Ltd., China.

[0173] (4) Scratch healing test

[0174] The scratch assay steps are as follows: 1) Pre-culture cells with a healing rate close to 100% in medium containing 2% FBS for 12 hours; 2) Use a 200μL pipette tip to make a cross-shaped scratch in each well of a 6-well plate, wash the cells 5 times with PBS until there are almost no suspended cells under the microscope; 3) Remove the PBS and add 2mL of medium containing 2% serum to each well; 4) Observe the healing of the scratches under an inverted microscope at the same time every day, take pictures, and record the results; 5) Use Image-ProPlus image analysis software to analyze the scratch distance and obtain the average width of the scratch.

[0175] The results are as follows Figure 15 As shown in the figure, clonogenesis and EdU assays indicate that LSM5 knockdown inhibits the viability and proliferation of 5-8F and CNE2 cells (e.g., Figure 15 (As shown in AF). Wound healing and invasion experiments showed that downregulation of LSM5 reduced the migration and invasion ability of nasopharyngeal carcinoma cells (e.g., Figure 15 (As shown in HI).

[0176] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. The application of a reagent for detecting LSM5 expression levels in the preparation of products for prognostic evaluation of nasopharyngeal carcinoma, characterized in that, The reagents include primers and / or probes; The primers include primers for amplifying LSM5; The sequences of the primers used to amplify LSM5 are shown in SEQ ID NO:1 and SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The product includes a reagent kit.

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