Marker for nasopharynx cancer diagnosis and prognosis evaluation and application thereof

Abstract

The invention discloses a marker for nasopharynx cancer diagnosis and prognosis evaluation and application thereof. And the marker comprises LSM5 and / or RBM20. The nasopharyngeal carcinoma marker LSM5 and / or RBM20 for nasopharyngeal carcinoma diagnosis and / or prognosis evaluation can be accurately used for nasopharyngeal carcinoma diagnosis and / or prognosis evaluation, meanwhile, proliferation, invasion and metastasis of nasopharyngeal carcinoma cells can be inhibited after expression of the marker LSM5 is inhibited, and a new and powerful molecular biological tool is provided for nasopharyngeal carcinoma treatment. The method has profound clinical significance and important popularization and application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to markers for nasopharyngeal carcinoma diagnosis and prognosis evaluation and applications thereof. Background Art

[0002] Nasopharyngeal carcinoma (NPC) usually occurs in the posterior wall of the nasopharynx and the pharyngeal recess, with distinct epidemiological, histopathological, clinical, and therapeutic characteristics. Concurrent chemoradiotherapy (CCRT) or radiotherapy is considered the main treatment for NPC. However, many patients still experience local recurrence or distant metastasis due to radiotherapy or chemotherapy resistance, ultimately leading to treatment failure. Therefore, it is necessary to further study the molecular mechanisms of the recurrence or metastasis process to identify potential targets for maintaining or reestablishing 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 a major source of variation in the cancer transcriptome and can play an important oncogenic role. Aberrant expression of RNA processing factors can negatively affect the transport and editing of mRNA. In addition, cancer treatments can induce individual splicing changes and mutations in RNA splicing factors, which can occur within a single gene or in the RNA processing factors themselves, 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 have been associated with the development and progression of malignancies. Therefore, further investigation of RNA processing is warranted as it may create new opportunities for therapeutic intervention in cancer. Summary of the invention

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

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

[0006] The present invention also provides a system for diagnosing and / or evaluating the prognosis of nasopharyngeal carcinoma.

[0007] The present invention also provides a reagent for detecting the expression level of the above marker.

[0008] The present invention also provides the application of the above system or reagent.

[0009] The present invention also provides an LSM5 expression inhibitor.

[0010] The present invention also provides the application of the above LSM5 expression inhibitor.

[0011] The invention also provides a medicine for treating nasopharyngeal carcinoma.

[0012] According to a first aspect of the present invention, a marker for diagnosis and / or prognosis evaluation of nasopharyngeal carcinoma is provided, wherein the marker comprises LSM5 and / or RBM20.

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

[0014] According to some embodiments of the present invention, the prognosis 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 invention, the marker is an RNA processing gene.

[0017] According to some embodiments of the present invention, the marker is used to calculate a prognostic risk score by the following formula to perform prognostic evaluation of nasopharyngeal carcinoma; the calculation formula is as follows: risk score = 1.6243 × LSM5 expression level + (-0.9761) × RBM20 expression level.

[0018] According to the second aspect of the present invention, the use of the above marker in any of the following items is proposed;

[0019] (1) Establish a system for diagnosing and / or evaluating the prognosis of nasopharyngeal carcinoma; (2) Prepare products for diagnosing and / or evaluating the prognosis of nasopharyngeal carcinoma; (3) Prepare products for treating or assisting the treatment of nasopharyngeal carcinoma; (4) Prepare products for screening treatment or assisting the treatment of nasopharyngeal carcinoma.

[0020] According to some embodiments of the present invention, the product includes at least one of a drug, a reagent, a test kit, a chip, a membrane strip or a 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 diagnosing and / or evaluating prognosis of nasopharyngeal carcinoma is provided, comprising:

[0023] A data acquisition module, used to obtain the expression level of the above markers of the patient;

[0024] A data analysis module, used for inputting the expression level of the marker into a risk scoring model to evaluate the diagnosis and / or prognosis of nasopharyngeal carcinoma of the patient;

[0025] The formula of the risk score 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 comprises the following steps:

[0027] S1. Obtain the expression level of the above marker of the patient; S2. Input the expression level of the marker into the risk scoring model, calculate the risk score value of the patient, and analyze the diagnosis and / or prognosis of nasopharyngeal carcinoma of the patient.

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

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

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

[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 prognosis 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 marker is provided, wherein the reagent comprises a primer and / or a probe.

[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 reagents further include 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 present invention, the use of the above system or reagent in any of the following items is proposed;

[0040] 1) Preparation of products for diagnosis and / or prognosis evaluation of nasopharyngeal carcinoma; 2) Preparation of products for treatment or adjuvant treatment of nasopharyngeal carcinoma; 3) Preparation of products for screening treatment or adjuvant treatment of nasopharyngeal carcinoma.

[0041] According to some embodiments of the present invention, the product includes at least one of a drug, a reagent, a test kit, a chip, a membrane strip or a 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 inhibitor of LSM5 expression is provided, comprising: at least one of a substance that inhibits the activity of LSM5, 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 the expression level of LSM5 is at least one of a1)-a3):

[0045] a1) siRNA, dsRNA, miRNA, ribozyme or shRNA targeting LSM5; a2) a 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 comprising 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 the siRNA1 is shown in SEQ ID NO:7, and the antisense sequence is shown in SEQ ID NO:8; the sense sequence of the siRNA2 is shown in SEQ ID NO:9, and the antisense sequence is shown in SEQ ID NO:10; the sense sequence of the 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, a use of the above-mentioned LSM5 expression inhibitor in the preparation of a product for the treatment or adjuvant treatment of nasopharyngeal carcinoma is proposed.

[0048] In some embodiments of the invention, the product comprises a pharmaceutical product.

[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 drug 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 new nasopharyngeal carcinoma marker LSM5 and / or RBM20 for diagnosis and / or prognosis evaluation of nasopharyngeal carcinoma, which can be accurately used for diagnosis and / or prognosis evaluation of nasopharyngeal carcinoma, and at the same time, inhibiting the expression of marker 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. It has far-reaching clinical significance and important prospects for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0053] Figure 1 : It is the identification result diagram of differentially expressed genes (DEGs) in the embodiment of the present invention, wherein a is the volcano diagram of DEGs between tumor and normal sample groups, orange, green and black dots represent up-regulated, down-regulated and unchanged genes (tumor / normal) DEGs between tumor and normal sample groups, respectively; b is a heat map, gene expression is positively correlated with color change, and the dendrogram on the left represents the clustering analysis of different genes from different samples; c is a Venn diagram of differentially expressed RNA processing genes (DE-RPGs); d is a heat map of 10 DE-RPGs expression patterns; e is the expression of 10 differential RNA processing genes in normal nasopharyngeal carcinoma tissue and nasopharyngeal carcinoma tissue (immunohistochemistry results are from the HPA database);

[0054] Figure 2 : is a functional enrichment analysis result diagram of differentially expressed RNA processing genes (DE-RPGs) in the embodiment of the present invention, wherein a is a GO enrichment analysis result diagram, black spots represent counts, and colors represent adjusted P values; b is a KEGG enrichment analysis result diagram of DE-RPGs, KEGG pathways are on the vertical axis, and colors represent adjusted P values;

[0055] Figure 3 It is a result diagram of dividing the NPC samples in the GSE102349 training set into a high-risk group and a low-risk group according to the optimal cutoff value of 2.35 in an embodiment of the present invention, wherein a, b, c, and d are all result diagrams of dividing the NPC samples in the GSE102349 training set into a high-risk group and a low-risk group;

[0056] Figure 4The figure is a result of the construction and identification analysis of the prediction model in the embodiment of the present invention, wherein a is the risk curve of the high-risk group and the low-risk group in the training set, and the horizontal axis of the upper and middle panels is the patient samples classified according to the risk score. The risk score increases from left to right; the risk score and survival time are arranged in an appropriate order, and the optimal risk score threshold and the corresponding number of patients are represented by a dotted line. The lower panel represents the heat map of the model gene expression; b is the result of the analysis of the survival rate of the training set; c is the ROC curve of the survival prediction of the training set, in which the 3-year ROC curve overlaps with the 5-year curve;

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

[0058] Figure 6 This is the survival analysis diagram of the validation set;

[0059] Figure 7 This is the ROC curve diagram for the survival prediction of the internal validation set, where the 3-year ROC curve overlaps the 5-year curve;

[0060] Figure 8 This is a Cox regression analysis diagram based on the GSE102349 dataset;

[0061] Fig. 9 : is a graph showing the correlation between prognostic genes and immune cells in an embodiment of the present invention, wherein a is the rank value of 24 tumor-infiltrating progenitor cells (TIICs) in the risk group; b is the expression of 24 TIICs in the risk group; c is a univariate forest plot with TIICS; d is the correlation between DE-rpg and immune cells, "*" indicates p < 0.05, and "**" indicates p < 0.01;

[0062] Fig.10 This is a sensitivity analysis test result diagram of the chemotherapeutic drugs in the embodiment of the present invention;

[0063] Fig.11 This is a sensitivity analysis test result diagram of the chemotherapeutic drugs in the embodiment of the present invention;

[0064] Fig.12 This is a sensitivity analysis test result diagram of the chemotherapeutic drugs in the embodiment of the present invention;

[0065] Fig.13: is the expression verification test result diagram of RBM20 and LSM5 in the embodiment of the present invention, wherein A is the expression test result diagram of RBM20 and LSM5 in normal nasopharyngeal carcinoma tissue and nasopharyngeal carcinoma tissue; B is the expression test result diagram of RBM20 and LSM5 in normal nasopharyngeal carcinoma tissue and nasopharyngeal carcinoma tissue; C is the expression test result diagram of RBM20 and LSM5 in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells; D is the expression test result diagram of RBM20 and LSM5 in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells; E is the expression test result diagram of RBM20 and LSM5 in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells; F is the expression test result diagram of RBM20 and LSM5 mRNA in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells; G is the expression test result diagram of RBM20 and LSM5 mRNA in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells; ns means no statistical difference, "*" means p < 0.05, and "**" means p < 0.01;

[0066] Fig.14 : It is a functional verification result diagram of LSM5 in nasopharyngeal carcinoma cells in the embodiment of the present invention, wherein A is a result diagram of the expression detection of LSM5 in nasopharyngeal carcinoma; B is a result diagram of the expression detection of LSM5 in nasopharyngeal carcinoma; C is a survival curve diagram of nasopharyngeal carcinoma patients with high and low LSM5 expression levels; D is a Western blot diagram for the verification of protein expression after interfering with LSM5; E is a result diagram of the protein expression detection after interfering with LSM5;

[0067] Fig.15 The figures are the functional verification result diagrams of LSM5 in nasopharyngeal carcinoma cells in the embodiments of the present invention, wherein A is a colony formation analysis diagram of cell survival and cloning ability after RNA interference with LSM5, B is a colony formation analysis diagram of cell survival and cloning ability after RNA interference with LSM5; C is an EdU analysis diagram of cell proliferation after RNA interference with LSM5; D is an EdU analysis diagram of cell proliferation after RNA interference with LSM5; E, F, and G are migration analysis diagrams of cell migration ability after RNA interference with LSM5; H and I are wound healing analysis diagrams 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 DESCRIPTION

[0068] The following will be clearly and completely described in conjunction with the embodiments of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without paying creative work all belong to the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.

[0069] Example 1 Screening of markers for diagnosis and prognosis evaluation of nasopharyngeal carcinoma

[0070] This embodiment provides a marker RNA processing factor LSM5 and RBM20 for diagnosis and prognosis evaluation of nasopharyngeal carcinoma, and the screening process is as follows:

[0071] 1. Data source selection

[0072] NPC-related transcriptome data were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) of the GSE12452 dataset (31 NPCs and 10 normal NPC epithelial tissues) were analyzed using the platform GPL570 (https: / / ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE12452). The GSE102349 dataset (88 NPC patients) was constructed and evaluated using the platform GPL11154 (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE102349).rpg from the AmiGO database (http: / / amigo.geneontology.org). By searching for the keyword “RNA processing” in the AmiGO database, 6734 genes were obtained, which 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) between NPC and normal NPC epithelial tissue. The R package “limma” based on the GSE12452 dataset was applied to identify differentially expressed genes using a threshold of p < 0.05 and |log2 fold change (FC)|> 1. Overlapping genes are genes common 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, which 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 NPC-related DEGs were identified from the GSE12452 database (e.g. Figure 1 The ab graph in Table 1 shows that 293 genes were up-regulated and 502 genes were down-regulated. Overlap analysis revealed that there were 10 common genes between NPC-related DEGs and RPGs: AHNAK2, ALYREF, FASTKD1, LSM5, PIH1D2, PNPT1, PUS7, RBM20, RBM24, and RRP15 ( Figure 1(c). Compared with the healthy control group, the expression levels of ALYREF, FASTKD1, LSM5, PNPT1, PUS7, and RRP15 in the NPC group were upregulated, while the expression levels of AHNAK2, PIH1D2, RBM20, and RBM24 were significantly decreased ( Figure 1 IHC results from the HPA database confirmed the expression of these genes ( Figure 1 By performing the following screening and verification on the above genes, the markers LSM5 and RBM20 for nasopharyngeal carcinoma diagnosis and prognosis evaluation were finally screened out.

[0087] 3. GO and KEGG functional enrichment analysis

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

[0089] Table 2

[0090]

[0091]

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

[0093]

[0094] The results are as follows Figure 2 , as shown in Table 2-3, it can be seen that GO functional annotation and enrichment analysis and KEGG pathway annotation and enrichment analysis elucidated the potential molecular mechanisms of NPC-related DE-RPGs in NPC. 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 a, in the BP category, these genes were mainly related to RNA processing activities, including splicing, catabolism, metabolism, processing, and translocation. In addition, terms related to the cell cycle were significantly enriched. In the MF category, terms related to RNA binding were significantly enriched. KEGG pathway analysis showed that NPC-related DE-RPGs were 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 prediction 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 NPC-related DE-rpgs, 88 NPC patients were randomly divided into GSE102349 training set (n=44) and 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 evaluate the association of the identified DE-RPGs with disease-free survival (DFS) of NPC patients to identify NPC-related DE-RPGs with prognostic value. The significance threshold was p < 0.05.

[0099] The R package "survminer" was used to perform univariate and multivariate COX analysis on NPC-related DE-RPGs with a threshold P value of <0.05 to screen genes associated with the NPC prognostic risk model. The risk value of each patient was obtained by simulating gene expression and the risk coefficient (coef) obtained from multivariate Cox regression. Subsequently, the patients were divided into high-risk and low-risk groups according to the optimal threshold of the risk score. The formula used for the risk score is as follows.

[0100] Risk score = risk factor × gene 1 + risk factor × gene 2 + risk factor × gene n.

[0101] Kaplan-Meier survival analysis was used to visualize the results of prognostic survival analysis between the two groups. The ROC curve drawn using the “pROC” R package was used to evaluate the validity of the risk model. Finally, the independent prognostic value of the risk signature was verified by univariate and multivariate Cox analysis based on the risk score and clinicopathological factors.

[0102] Table 4 Correlation analysis between 10 DE-RPGs and patient survival.

[0103]

[0104] Table 5 Regression coefficients of LSM5 and RBM20

[0105]

[0106] The results are shown in Table 4. It can be seen from the table that LSM5 (p=0.022) and RBM20 (p=0.035) are significantly correlated with the DFS of NPC patients.

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

[0108] In order to evaluate the prognostic value of the risk model, NPC patients were scored by the expression of the two model genes and the risk coefficient obtained by multivariate COX regression to obtain the risk value of each patient. The patients were divided into high-risk group and low-risk group according to the optimal threshold of the risk score. The risk score calculation formula is:

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

[0110] The above risk score formula was used to calculate the risk score of NPC patients in the GSE102349 training set. The NPC samples in the GSE102349 training set were divided into high-risk group and low-risk group according to the optimal cutoff value of 2.35 (e.g. Figure 3 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 in the hazard curves and disease progression status (disease-free and progressive) of patients in the training set, it is shown that patients with progressive disease have higher hazard scores. Figure 4 b and Figure 6 It can be seen that the DFS of the low-risk group was significantly higher than that of the high-risk group (p = 0.00025). Figure 4 c and Figure 7 It can be seen that the area under the ROC curve (AUC) for one, three, and five years was 0.836, 0.652, and 0.652, respectively. In addition, in the high-risk group, the expression level of LSM5 was relatively high, while the longer DFS rate in the low-risk group was associated with high expression of RBM20.

[0112] Cox regression analysis based on the GSE102349 dataset showed that the risk score can affect the DFS of NPC patients independently of the stage characteristics (e.g. Figure 8 shown).

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

[0114] Example 2 Correlation analysis between prognostic markers and immune cells

[0115] Using the one-sample GSEA method, 24 immune-related gene sets were obtained for each sample in the GSE102349 dataset. The samples were 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, and the significance threshold was set at p < 0.05.

[0116] In addition, in order to correlate the differential immune cell profiles of NPC patients with DFS rates, a univariate Cox regression analysis was performed. Subsequently, Pearson's correlation analysis was used to reveal the correlation between immune cells and the markers (prognostic genes) prepared in Example 1. |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 Fig. 9 As shown, from Fig. 9 As can be seen in a, ssGSEA was used to detect the abundance of 24 immune cells in the high-risk group and the low-risk group; Fig. 9 As can be seen in b, the Wilcoxon rank sum test showed that the percentages of 21 immune cells were statistically different between the two risk groups.

[0120] In addition, from Fig. 9 c, as can be seen in Table 6, CD8 T cells, cytotoxic cells, Tem, Tcm, mast cells, eosinophils, T cells, pDCs, and DCs are significantly associated with the DFS rate of patients with NPC. The main roles of DCs in anti-tumor immune responses include phagocytosis of dead tumor cells, capture and presentation of tumor-associated antigens, and activation of various T cells, thereby jointly stimulating a series of immune responses to kill tumor cells. In tumors, a reduction in DC counts leads to weaker antigen processing, which alters the proliferation and differentiation of T cells, negatively affects their tumor killing effects, and induces immune tolerance.

[0121] from Fig. 9 As can be seen in d, the Pearson correlation of differential immune cells with RBM20 and LSM5, in the low-risk group, DCs were significantly negatively correlated with RBM20, CD8+ T cells, T cells, DCs, eosinophils, and Tem, while pDCs were negatively correlated with LSM5. However, in the high-risk group, there was no significant correlation between immune cells and prognostic genes.

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

[0123] This example verifies the guiding significance of the prediction model constructed in Example 2 for the use of chemotherapeutic drugs. The specific method is as follows: The IC50 of nasopharyngeal carcinoma samples for common chemotherapeutic drugs was calculated using the R language "pRRophetic" package, and the differences in IC50 of 138 chemotherapeutic drugs between high-risk and low-risk groups were compared using the rank sum test. Then, the R language ggplot2 was used to draw a box plot to visualize the results. The rank sum test results showed that 55 of the 138 chemotherapeutic drugs had significant differences. The result with a p value of 0.05 showed that the difference was statistically significant. The lower the half-inhibitory concentration value, the higher the sensitivity to the chemotherapeutic drug.

[0124] Table 7

[0125]

[0126]

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

[0128] Example 4 Application of RBM20 and / or LSM5 in the preparation of a preparation for diagnosing nasopharyngeal carcinoma

[0129] This example provides the use of RBM20 and / or LSM5 in the preparation of a preparation for diagnosing nasopharyngeal carcinoma, and 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, we 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 fluorescence quantitative PCR detection of mRNA expression of RBM20 and LSM5 molecules

[0136] Real-time fluorescence quantitative PCR was performed in normal human nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells to detect the expression of RBM20 and LSM5 molecules. The specific experimental process 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 Conway Century in Beijing, China.

[0138] (2) mRNA reverse transcription: mRNA reverse transcription was performed using an mRNA reverse transcription kit (purchased from Conway Century, Beijing, 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 sequence for detecting the internal reference gene is:

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

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

[0149] The total reaction system was prepared as follows: 10 μL of 2×SYBR Green MIX, 1 μL of each of the upstream and downstream primers (10 mM), 2 μL of cDNA template, and the mixture was made up to 20 μL with enzyme-free water;

[0150] The reaction procedure was: 95℃5min pre-denaturation, 95℃30s denaturation, 60℃5s annealing, 70℃10s extension, 40 cycles in total, and then melting curve analysis: the temperature was 70℃-95℃ to collect fluorescence signals. After the reaction was completed, the amplification curve and melting curve of qRT-PCR were confirmed, and the expression intensity of each gene was standardized according to the CT value (threshold cycle values) and the internal reference gene (GAPDH) and then 2 -△△ct Computation of gene expression.

[0151] 3. Immunohistochemistry (IHC) analysis of protein expression of RBM20 and LSM5 in tumor tissues of NPC patients

[0152] Seventy paraffin-embedded tissue specimens were stained using the IHCSP method. Positive staining results were shown as brown-yellow granules. Immunohistochemical staining was quantified using mean optical density (MOD) obtained using Image J image analysis software. The PV-9000 Universal Two-Step Detection Kit was purchased from Zhongshan Jinqiao, Beijing, China.

[0153] Test results such as Fig.13 As shown, from Fig.13 As can be seen from the AB graph, compared with normal NPC tissue (n=30), LSM5 expression was increased and RBM20 expression was decreased in NPC tissue (n=40).

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

[0155] Normal human nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells in the logarithmic growth phase were collected and the protein concentration was determined using a BCA kit (Lanco, Hangzhou, China). A total of 80 μg of protein in each group was loaded into an 8% polyacrylamide gel and separated by SDS-PAGE. The proteins were then transferred to a PVDF cell membrane. The primary antibodies were incubated overnight at 4°C (RBM20, 1:500, bs-9606R, Bioss, China; LSM5, 1:500, ab184568, Abcam, UK). Signals were visualized using a dual-color infrared laser imaging system (Odyssey CLx, LI-COR, New Jersey, USA). Endogenous actin (1:1000, 66009-1-Ig, China) and GAPDH (1:20000, 10494-1-AP, China) were used for protein expression normalization.

[0156] Test results such as Fig.13 As shown, from Fig.13As can be seen from the CG figure, in the NPC cell line, the protein level and mRNA level of LSM5 were increased, while the expression level of RBM20 was still low.

[0157] Example 5 Application of LSM5 in the preparation of a product for nasopharyngeal carcinoma prognosis

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

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

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

[0161] Example 6 An expression inhibitor of LSM5

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

[0163] Table 8

[0164]

[0165] Example 7 Application of LSM5 expression inhibitor in the preparation of products for inhibiting nasopharyngeal carcinoma

[0166] This example provides the use of the LSM5 expression inhibitor prepared in Example 6 in the preparation of a tumor-suppressing product. Specifically, CNE2 and 5-8F cells were used for verification experiments. 5-8F and CNE2 cells were cultured and a blank control group was established. The small interfering RNA (siRNA1-LSM5, siRNA2-LSM5 and siRNA3-LSM5) targeting LSM5 prepared in Example 6 was used as the experimental group. The siRNA-NC group was a negative control. The siRNA targeting LSM5 is from Jiman Biotechnology (Shanghai) Co., Ltd., and the sequence is shown in Table 8. The specific verification process is as follows:

[0167] (1) Cell transfection

[0168] Cells were seeded into 6-well plates 24 hours before transfection (approximately 1×10 5 cells / well) to ensure that the cells were grown at 60-70% confluency during transfection. In RPMI-1640 medium without antibiotics, pcDNA3.1-RBM20-3xFlag vector or LSM5 expression inhibitor siRNA-LSM5 (siRNA1-LSM5, siRNA2-LSM5 or siRNA3-LSM5) were respectively transfected with 3000 transfection reagent. For pcDNA3.1-RBM20-3xFlag transfection, mix the vector DNA with the transfection reagent at a mass ratio of 1:2 and incubate for 5 minutes. For siRNA-LSM5 transfection, mix the siRNA with the transfection reagent at a mass ratio of 1:2 and incubate for 5 minutes. Add the above mixture to the cell culture dish and shake gently 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 after transfection, Western Blot (WB) and quantitative reverse transcription PCR (RT-qPCR) were performed to detect the transfection efficiency and LSM5 gene expression level. The LSM5 expression detection method was consistent with that in Example 5. The detection results are shown in FIG. Fig.14 As shown in Figures D and E.

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

[0172] The single cell suspension was inoculated into a 6-well plate at a rate of 300 cells per well and stained with crystal violet after 24 h of culture. Subsequently, the cells were inoculated into a 6-well plate at a rate of 1×10 4 Cells were cultured for 24 h. Then they were co-cultured with 50 μM EdU solution for 2 h and then fixed with 4% paraformaldehyde for 20 min. 500 μL of click reaction solution was added to each well and incubated at room temperature for 30 min. After DAPI staining, images were taken using a fluorescence microscope (Carl Zeiss Axio Observer3m). EdU cell proliferation imaging detection kit was purchased from Wuhan Electronic Science Biotechnology Co., Ltd., China.

[0173] (4) Scratch healing experiment

[0174] The steps of the scratch experiment are as follows: 1) Incubate the treated cells with a healing degree close to 100% in advance with a culture medium containing 2% FBS for 12 hours; 2) Use a 200μL pipette tip to make a "cross" scratch in each well of a 6-well plate, and wash the cells 5 times with PBS until there are almost no suspended cells under a microscope; 3) Remove PBS and add 2mL of culture 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 keep records; 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 Fig.15 As shown in the figure, clonogenicity and EdU detection showed that LSM5 knockdown inhibited the viability and proliferation of 5-8F and CNE2 cells (as shown in Fig.15 Wound healing and invasion experiments showed that downregulation of LSM5 reduced the migration and invasion abilities of NPC cells (as shown in Figure AF). Fig.15 HI).

[0176] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A marker for diagnosis and / or prognosis evaluation of nasopharyngeal carcinoma, characterized in that: The markers include LSM5 and / or RBM20.

2. Use of the marker according to claim 1 in any of the following: (1) Establish a system for nasopharyngeal carcinoma diagnosis and / or prognostic evaluation; (2) Preparation of products for diagnosis and / or prognosis evaluation of nasopharyngeal carcinoma; (3) Preparation of products for the treatment or adjuvant treatment of nasopharyngeal carcinoma; (4) Preparation and screening of products for treatment or adjuvant treatment of nasopharyngeal carcinoma; Preferably, the product comprises at least one of a drug, a reagent, a test kit, a chip, a membrane strip or a detection device.

3. The use according to claim 2, characterized in that: The treatment or adjuvant treatment of nasopharyngeal carcinoma includes inhibiting cancer cell proliferation, inhibiting cancer cell migration and invasion and / or promoting cancer cell apoptosis.

4. A system for nasopharyngeal carcinoma diagnosis and / or prognosis evaluation, characterized in that: include: A data acquisition module, used to obtain the expression level of the marker according to claim 1 of the patient; A data analysis module, for inputting the expression level of the marker according to claim 1 into a risk scoring model to evaluate the diagnosis and / or prognosis of nasopharyngeal carcinoma of the patient; The formula of the risk score model includes: risk score=1.6243×LSM5 expression level+(-0.9761)×RBM20 expression level.

5. The system according to claim 4, characterized in that The application method of the system comprises the following steps: S1. obtaining the expression level of the marker according to claim 1 of the patient; S2. Inputting the expression level of the marker according to claim 1 into the risk scoring model, calculating the risk score value of the patient, and analyzing the diagnosis and / or prognosis of nasopharyngeal carcinoma of the patient.

6. A reagent for detecting the expression level of the marker according to claim 1, characterized in that: The reagents include primers and / or probes; Preferably, the primers include primers for amplifying LSM5 and / or primers for amplifying RBM20; The sequences of the primers used to amplify LSM5 are shown in SEQ ID NO: 1 and SEQ ID NO: 2; The sequences of the primers used to amplify RBM20 are shown in SEQ ID NO:3 and SEQ ID NO:

4.

7. Use of the system according to any one of claims 4 to 5 or the reagent according to claim 6 in any of the following; 1) Preparation of products for diagnosis and / or prognosis evaluation of nasopharyngeal carcinoma; 2) Preparation of products for the treatment or adjuvant treatment of nasopharyngeal carcinoma; 3) Prepare and screen products for treatment or adjuvant treatment of nasopharyngeal carcinoma; Preferably, the product comprises at least one of a drug, a reagent, a kit, a chip, a membrane strip or a detection device; Preferably, the treatment or adjuvant treatment of nasopharyngeal carcinoma includes inhibiting cancer cell proliferation, inhibiting cancer cell migration, invasion and / or promoting cancer cell apoptosis.

8. A LSM5 expression inhibitor, characterized in that include: 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; Preferably, the substance that reduces the expression level of LSM5 is at least one of a1)-a3): a1) siRNA, dsRNA, miRNA, ribozyme or shRNA targeting LSM5; a2) a nucleic acid molecule encoding the siRNA, dsRNA, miRNA, ribozyme or shRNA targeting LSM5 described in a1); a3) an expression cassette, a vector or a transgenic cell line comprising the nucleic acid molecule described in a2); More preferably, the siRNA includes siRNA1, siRNA2 and / or siRNA3; The sense sequence of the siRNA1 is shown in SEQ ID NO:7, and the antisense sequence is shown in SEQ ID NO:8; The sense sequence of the siRNA2 is shown in SEQ ID NO:9, and the antisense sequence is shown in SEQ ID NO:10; The sense sequence of the siRNA3 is shown in SEQ ID NO:11, and the antisense sequence is shown in SEQ ID NO:

12.

9. Use of the LSM5 expression inhibitor according to claim 8 in the preparation of a product for treating or assisting in the treatment of nasopharyngeal carcinoma.

10. A drug for treating nasopharyngeal carcinoma, characterized in that: Contains the LSM5 expression inhibitor as claimed in claim 8.

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