Use of nogo-b-gnai-yap signaling pathway biomarkers in diagnosis and treatment of nasopharyngeal carcinoma

Abstract

The application provides a use of a Nogo-B-GNAI-YAP signal pathway biomarker in diagnosis and treatment of nasopharyngeal carcinoma, and the Nogo-B-GNAI-YAP signal pathway as a biomarker is beneficial to diagnosis of the nasopharyngeal carcinoma and prognosis judgment of the nasopharyngeal carcinoma, facilitates timely provision of corresponding treatment strategies, and improves survival period of patients. Moreover, in one or more embodiments of the application, it is found that after blocking the Nogo-B-GNAI-YAP signal pathway, the metastasis of the nasopharyngeal carcinoma cell can be effectively inhibited, therefore, the Nogo-B-GNAI-YAP signal pathway blocker is expected to be used as a new nasopharyngeal carcinoma invasion and metastasis targeted treatment strategy, provides a new treatment strategy and a drug for clinical application, and makes up for the defect of limited clinical treatment drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor molecular biology, and more particularly to the use of Nogo-B-GNAI-YAP signaling pathway biomarkers in the diagnosis and treatment of nasopharyngeal carcinoma. BACKGROUND

[0002] Nasopharyngeal carcinoma (NPC) is a highly malignant tumor, which is prone to cervical lymph node and distant metastasis. In the current treatment method, radiotherapy is usually taken as the core, supplemented by chemotherapy, targeted therapy and other multidisciplinary comprehensive treatment methods, which needs to be reasonably selected according to the actual situation of the patient. At present, the clinical prognosis and treatment strategy are mainly determined by the clinical pathological stage of the patient at the time of diagnosis, that is, the prognosis and treatment strategy are determined based on the TNM evaluation system. However, the disadvantage of the TNM evaluation system is that it cannot accurately predict the risk of distant metastasis and cannot early identify the distant metastasis of nasopharyngeal carcinoma, and there are still defects in clinical application. Moreover, the treatment methods for nasopharyngeal carcinoma in the prior art are still limited, and the effect of some treatment methods is limited, which is difficult to meet the needs of clinical treatment of nasopharyngeal carcinoma.

[0003] Therefore, there is an urgent need in the clinic for a new marker or target point for more accurately predicting and diagnosing nasopharyngeal carcinoma, as well as treatment drugs and strategies based on the target point. SUMMARY

[0004] The present application aims to overcome at least one of the above-mentioned deficiencies in the prior art, and provides the use of Nogo-B-GNAI-YAP signaling pathway biomarkers in the diagnosis and treatment of nasopharyngeal carcinoma. Nogo-B-GNAI-YAP signaling pathway as a biomarker is beneficial to the diagnosis of nasopharyngeal carcinoma and the prognosis of nasopharyngeal carcinoma, which can provide corresponding treatment strategies in time and improve the survival period of patients. In addition, it can be used as a therapeutic target for nasopharyngeal carcinoma to inhibit the development of nasopharyngeal carcinoma and provide a new therapeutic target and strategy.

[0005] One object of the present application is to provide a nasopharyngeal carcinoma or a poor prognosis marker for nasopharyngeal carcinoma, which comprises specifically highly expressed Nogo-B, GNAI and / or YAP genes. In one or more embodiments of the present application, it is found that Nogo-B, GNAI and YAP are highly expressed in nasopharyngeal carcinoma cells and tissues, and the Nogo-B-GNAI-YAP signaling pathway is related to the N stage, M stage, clinical stage and poor prognosis of nasopharyngeal carcinoma patients, and is an independent predictor of OS and PFS of nasopharyngeal carcinoma patients. Therefore, Nogo-B, GNAI and YAP genes contained in Nogo-B-GNAI-YAP signaling pathway can be used as a marker for nasopharyngeal carcinoma and a poor prognosis marker for nasopharyngeal carcinoma.

[0006] Another object of the present application is to provide the use of the reagent for detecting Nogo-B, GNAI and / or YAP gene in the preparation of a product for predicting, diagnosing and / or prognosing nasopharyngeal carcinoma.

[0007] Still another object of the present application is to provide the use of Nogo-B-GNAI-YAP signal pathway inhibitor in the preparation of a medicament for preventing and treating nasopharyngeal carcinoma. In one or more embodiments of the present application, it is found that blocking Nogo-B-GNAI-YAP signal axis can inhibit the invasion and metastasis of nasopharyngeal carcinoma. Nogo-B-GNAI-YAP signal pathway inhibitor can be used as a target for treating nasopharyngeal carcinoma, and based on the target, a corresponding targeted drug for preventing and treating nasopharyngeal carcinoma can be prepared.

[0008] Further, the medicament for preventing and treating nasopharyngeal carcinoma includes a medicament for inhibiting the invasion and / or migration of nasopharyngeal carcinoma cells.

[0009] Further, the Nogo-B-GNAI-YAP signal pathway inhibitor includes an inhibitor for inhibiting the interaction between Nogo-B and GNAI.

[0010] Further, the Nogo-B-GNAI-YAP signal pathway inhibitor includes a Nogo-B inhibitor, a GNAI inhibitor and / or a YAP inhibitor.

[0011] Further, the Nogo-B inhibitor includes Nogo-B siRNA; and / or, the GNAI inhibitor includes GNAI siRNA; and / or, the YAP inhibitor includes YAP siRNA and / or Verteporfin.

[0012] Further, the Nogo-B siRNA sequence includes 5'-GAGCGTAACAGCCTACATT-3' and / or 5'-CATGTGAACTGCACGATAA-3'; and the GNAI siRNA sequence includes 5'-GAAAGATAGTGGTGTACAA-3'.

[0013] Still another object of the present application is to provide a medicament for treating nasopharyngeal carcinoma, which comprises a Nogo-B inhibitor, a GNAI inhibitor and / or a YAP inhibitor.

[0014] Further, the Nogo-B inhibitor includes Nogo-B siRNA; and / or, the GNAI inhibitor includes GNAI siRNA; and / or, the YAP inhibitor includes YAP siRNA and Verteporfin.

[0015] Further, the Nogo-B siRNA sequence comprises: 5'-GAGCGTAACAGCCTACATT-3' and / or 5'-CATGTGAACTGCACGATAA-3'; the GNAI siRNA sequence comprises: 5'-GAAAGATAGTGGTGTACAA-3'.

[0016] Further, the pharmaceutical acceptable excipient and / or carrier are further included.

[0017] Another object of the present application is to provide a vector or recombinant containing Nogo-B siRNA, GNAI siRNA and / or YAP siRNA.

[0018] Compared with the prior art, the present application has the following beneficial effects: it is found that Nogo-B-GNAI-YAP signaling pathway is closely related to the development of nasopharyngeal carcinoma, and in particular, to the poor prognosis of nasopharyngeal carcinoma. By detecting the genes contained in the Nogo-B-GNAI-YAP signaling pathway, it is beneficial to predict, diagnose and prognose nasopharyngeal carcinoma according to the relative expression state, with high accuracy, and it is beneficial to take corresponding treatment strategies in time to improve the survival period of patients. For example, early nasopharyngeal carcinoma, including distant metastasis and poor prognosis, is predicted by immunohistochemistry. Moreover, in one or more embodiments of the present application, it is found that after blocking the Nogo-B-GNAI-YAP signaling pathway, the metastasis of nasopharyngeal carcinoma cells can be effectively inhibited, and therefore, Nogo-B-GNAI-YAP signaling pathway blockers are expected to be used as a new targeted treatment strategy for nasopharyngeal carcinoma invasion and metastasis, to provide a new treatment strategy and drug for clinical application, and to make up for the limited defects of clinical treatment drugs. Based on the actual performance after blocking the Nogo-B-GNAI-YAP signaling pathway, including Verteporfin and other Nogo-B-GNAI-YAP blockers, it is beneficial to form a treatment drug for nasopharyngeal carcinoma patients with high expression of Nogo-B. Thus, a new treatment drug is provided for nasopharyngeal carcinoma patients, which is beneficial to the survival of more nasopharyngeal carcinoma patients. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Nogo-B is shown to be highly expressed in nasopharyngeal carcinoma.

[0020] Figure 2 Nogo-B high expression is shown to be related to nasopharyngeal carcinoma migration and invasion.

[0021] Figure 3 Nogo-B is shown to cause nasopharyngeal carcinoma metastasis.

[0022] Figure 4 Nogo-B is shown to promote nasopharyngeal carcinoma migration by binding to GNAI.

[0023] Figure 5 Nogo-B can be regulated by GNAI to regulate YAP.

[0024] Figure 6 GNAI is associated with poor prognosis of NPC.

[0025] Figure 7 Knocking down GNAI can inhibit the migration of NPC cells.

[0026] Figure 8 Nogo-B-GNAI-YAP axis is associated with poor prognosis of NPC.

[0027] Figure 9 Blocking Nogo-B-GNAI-YAP axis can inhibit the metastasis of NPC. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed description is illustrative only, and is intended to provide further description in addition to the foregoing description of the exemplary embodiments according to this application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0029] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to this application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components and / or combinations thereof.

[0030] The application will be further described with reference to the following examples without however, limiting the application thereto. The following examples are intended to be illustrative only and are not intended to limit the application. The test samples and test procedures used in the following examples include the following (if the specific conditions of the experiment are not noted in the examples, the general conditions are usually used, or the conditions recommended by the reagent company are used; the reagents, consumables, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified).

[0031] Example 1

[0032] I. Nogo-B is highly expressed in NPC and is associated with poor prognosis

[0033] GESADatabase found that Nogo-B is highly expressed in high-stage Figure 1 A), Western Blot and Real-time PCR results show that Nogo-B is highly expressed in NPC cells and tissues Figure 1BC). Subsequently, the mRNA expression level of Nogo-B was detected in 12 nasopharyngeal carcinoma tissues and 9 chronic nasopharyngitis tissues, and it was found that the expression of Nogo-B in most nasopharyngeal carcinoma tissues was higher than that in nasopharyngeal inflammation tissues (BC). Figure 1 D), further immunohistochemical analysis revealed that Nogo-B was generally highly expressed in nasopharyngeal carcinoma. Figure 1 E) Nogo-B expression was significantly correlated with clinical characteristics (N stage, M stage, clinical stage, survival time, and progression-free survival). High Nogo-B expression can shorten overall survival (OS) and progression-free survival (PFS). Cox proportional hazards model analysis revealed that Nogo-B is a major independent predictor of 3-year OS and PFS in nasopharyngeal carcinoma patients. Figure 1 As shown in F, high expression of Nogo-B was associated with shortened overall survival (OS) and progression-free survival (PFS) in nasopharyngeal carcinoma patients, suggesting that Nogo-B is associated with poor prognosis in nasopharyngeal carcinoma patients.

[0034] II. Nogo-B is associated with nasopharyngeal carcinoma invasion and metastasis.

[0035] 1. We overexpressed Nogo-B in nasopharyngeal carcinoma cells (TW03, HK1) and verified it using Western blotting and PCR. Figure 2 Stable cell lines overexpressing Nogo-B were established (TW03 in A and HK1 in B, verified by Western blotting and real-time PCR), indicating successful cell line construction. In vitro functional experiments revealed an increase in the number of nasopharyngeal carcinoma cells crossing the transwell chambers after Nogo-B overexpression, suggesting that Nogo-B promotes the migration and invasion of nasopharyngeal carcinoma cells. Figure 2 As shown in CD, compared with the vector cells, the nasopharyngeal carcinoma cell lines that stably overexpress Nogo-B (TW03 in C and HK1 in D) showed an increase in the number of migrating and invading cells. Figure 2 EF established stable cell lines with Nogo-B knockdown (TW03 in E and HK1 in F), and verified them by Western blotting and real-time PCR. Figure 2 Compared with the vector cells, the nasopharyngeal carcinoma cell lines with knocked-down Nogo-B (TW03 in G and HK1 in H) showed a reduction in the number of migrating and invading cells.

[0036] 2. An in vivo model of lung metastasis in nude mice was established. Results showed that high expression of Nogo-B exacerbated lung metastasis in nude mice. Figure 3 As shown. Figure 3 A represents the lung images of each group of mice. Figure 3 B showed that the incidence of lung metastasis in the Nogo-B group of nude mice was significantly higher than that in the vector control group. Figure 3In C, H&E staining results confirmed the presence of lung xenografts, and the lung xenograft size in the Nogo-B group was the largest among all groups. Figure 3 D Immunohistochemical results confirmed the high expression of Nogo-B in the Nogo-B group

[0037] The above results suggest that Nogo-B promotes the migration, invasion, and metastasis of nasopharyngeal carcinoma cells.

[0038] III. Nogo-B has a direct interaction with GNAI

[0039] To further investigate the relationship between Nogo-B and GNAI in nasopharyngeal carcinoma and their role in the invasion and metastasis of nasopharyngeal carcinoma, the inventors simultaneously overexpressed Nogo-B and GNAI in nasopharyngeal carcinoma cell lines and performed immunofluorescence and Co-IP experiments. It was found that Nogo-B and GNAI co-localized ( Figure 4 A) and directly bound ( Figure 4 B). To investigate the effect of Nogo-B binding to GNAI on the invasion and metastasis of nasopharyngeal carcinoma cells, the inventors simultaneously overexpressed Nogo-B and GNAI in nasopharyngeal carcinoma cells and performed in vitro Transwell migration experiments. It was found that the migration ability of the cells was significantly enhanced, as evidenced by a significant increase in the number of cells penetrating the Transwell chamber. Moreover, the addition of GNAI siRNA to the Nogo-B-overexpressing cells or the addition of Nogo-B siRNA to the GNAI-overexpressing cells significantly reduced the migration ability of the cells ( Figure 4 C)

[0040] IV. Nogo-B can regulate YAP through GNAI

[0041] Nogo-B induces EMT in nasopharyngeal carcinoma cells by YAP nuclear entry. RNA-seq was performed on nasopharyngeal carcinoma cells overexpressing Nogo-B, and the results are shown in Figure 5 : Overexpression of Nogo-B can regulate the Hippo pathway ( Figure 5 A). Western Blot results showed that YAP phosphorylation was enhanced after Nogo-B knockdown ( Figure 5 B). Immunofluorescence also found that YAP nuclear export increased after Nogo-B knockdown ( Figure 5 C), YAP nuclear entry increased after GNAI overexpression ( Figure 5 D), and overexpression of GNAI reversed the YAP nuclear export caused by Nogo-B knockdown, suggesting that Nogo-B can regulate YAP through GNAI.

[0042] V. GNAI is associated with poor prognosis of nasopharyngeal carcinoma

[0043] We performed immunohistochemical staining on 91 nasopharyngeal carcinoma tissue samples and combined it with clinical follow-up data. The results showed that nasopharyngeal carcinoma patients with high GNAI expression had shorter overall survival (OS) and progression-free survival (PFS). Figure 6 As shown in A and 6B, GNAI is associated with poor prognosis in patients with nasopharyngeal carcinoma.

[0044] VI. Knocking down GNAI can inhibit the migration of nasopharyngeal carcinoma cells.

[0045] GNAI was knocked down in nasopharyngeal carcinoma cells (TW03). In vitro functional experiments showed that knocking down GNAI reduced the number of nasopharyngeal carcinoma cells crossing the transwell chambers, such as... Figure 7 As shown, GNAI can regulate the migration of nasopharyngeal carcinoma cells.

[0046] VII. The Nogo-B-GNAI-YAP signal axis is associated with poor prognosis in nasopharyngeal carcinoma.

[0047] Immunohistochemical staining of nasopharyngeal carcinoma tissue, combined with clinical data, revealed that high expression of the Nogo-B-GNAI-YAP signaling axis can shorten overall survival (OS) and progression-free survival (PFS). Figure 8 This indicates that it is associated with poor prognosis in nasopharyngeal carcinoma.

[0048] 8. Blocking the Nogo-B-GNAI-YAP signaling axis can inhibit nasopharyngeal carcinoma metastasis.

[0049] Experiments were conducted using a nude mouse model of nasopharyngeal carcinoma lung metastasis. After administration of the YAP inhibitor Verteporfin (ip 100 mg / kg), the increase in lung metastasis induced by Nogo-B overexpression was reversed, with no statistically significant difference compared to the control group. Figure 9 This suggests that blocking the Nogo-B-GNAI-YAP signaling axis can inhibit nasopharyngeal carcinoma metastasis and may become a therapeutic target for nasopharyngeal carcinoma.

[0050] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. Use of GNAI siRNA in the preparation of a drug for treating nasopharyngeal carcinoma, characterized in that, The sequence of the GNAI siRNA is 5'-GAAAGATAGTGGTGTACAA-3'.

2. Use of a vector or recombinant in the preparation of a medicament for the treatment of nasopharyngeal carcinoma, characterized in that, The sequence of the GNAI siRNA is 5'-GAAAGATAGTGGTGTACAA-3'. The sequence of the GNAI siRNA is 5'-GAAAGATAGTGGTGTACAA-3'.

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