Application of RAB32 in the preparation of products for diagnosis, treatment and prognosis of non-small cell carcinoma

Abstract

The present application discloses an application of RAB32 in the preparation of products for the diagnosis, treatment and prognosis of non-small cell lung cancer. In the present application, RAB32, a member of the small GTPase RAB family, is significantly more highly expressed in non-small cell lung cancer tissues and cell lines than in adjacent cancer tissues and normal cells, and is associated with a poor prognosis. In vitro experiments show that knocking down the highly expressed RAB32 gene in tumor cells can significantly inhibit the proliferation of lung adenocarcinoma cells. In an in vivo tumor-bearing mouse model, knocking down the RAB32 gene significantly reduced the tumor volume and inhibited its growth in the knockdown group, while also inhibiting the lymph node metastasis ability of lung adenocarcinoma cells. Therefore, the RAB32 gene can be used in the preparation of products for the diagnosis, treatment and prognosis of non-small cell lung cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of RAB32 in the preparation of products for diagnosis, treatment and prognosis of non-small cell carcinoma. Background Art

[0002] Lung cancer is the malignant tumor with the highest morbidity and mortality in my country, with approximately 85% of cases being non-small cell lung cancer (NSCLC). Tumor metastasis is the primary cause of lung cancer's high mortality rate, with lymph node metastasis being the most common and an independent risk factor for poor prognosis in NSCLC patients. Clinical data show that lymph node metastasis often occurs in patients with advanced lung cancer, with tumors often exceeding 5 cm in diameter. However, in clinical practice, some patients with T1 (≤3 cm) NSCLC have lymph node metastasis at initial diagnosis, resulting in a very poor prognosis. This clinical subtype of lung cancer (T1N2M0, or "small tumor with large lymph node metastasis"), in which lymph node metastasis occurs while the primary tumor is still at a very young stage of growth, often presents with highly aggressive, poorly differentiated, and highly malignant tumors.

[0003] Lung adenocarcinoma is a type of NSCLC. In the relevant technology, there are no public biomarkers for lung adenocarcinoma lymph node metastasis. This will affect the diagnosis, treatment intervention and prediction of lymph node metastasis of primary lung adenocarcinoma tumors (≤3CM), thereby reducing the survival rate of lung adenocarcinoma patients.

[0004] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the Invention

[0005] The technical task of the present application is to address the above deficiencies and provide an application of RAB32 in the preparation of products for the diagnosis, treatment and prognosis of non-small cell lung cancer. In the present application, RAB32, a member of the small GTPase RAB family, is significantly more highly expressed in non-small cell lung cancer tissues and cell lines than in adjacent cancer tissues and normal cells, and is associated with poor prognosis. In vitro experiments have shown that knocking down the highly expressed RAB32 gene in tumor cells can significantly inhibit the proliferation ability of lung adenocarcinoma cells. In an in vivo tumor-bearing mouse model, after knocking down the RAB32 gene, the tumor volume in the knockdown group was significantly reduced, growth was inhibited, and the lymph node metastasis ability of lung adenocarcinoma cells was inhibited. Therefore, the RAB32 gene can be used in the preparation of products for the diagnosis, treatment and prognosis of non-small cell lung cancer.

[0006] To achieve the above objectives, this application provides the following technical solutions:

[0007] According to one aspect of the present application, there is provided a use of RAB32 in preparing a product for determining the prognosis of patients with non-small cell carcinoma.

[0008] In some embodiments, the product is a kit for detecting the expression level of RAB32.

[0009] In some embodiments, the RAB32 expression level is significantly negatively correlated with the survival rate of patients with non-small cell carcinoma.

[0010] According to another aspect of the present application, a use of RAB32 in preparing a lung adenocarcinoma diagnostic product is also provided.

[0011] According to another aspect of the present application, there is also provided a use of RAB32 in preparing a diagnostic product for lung adenocarcinoma lymph node metastasis.

[0012] In some embodiments, the lung adenocarcinoma lymph node metastasis is lung adenocarcinoma mediastinal lymph node metastasis.

[0013] According to another aspect of the present application, an inhibitor is further provided, comprising shRNA targeting the RAB32 gene, wherein the nucleotide sequence of the shRNA targeting the RAB32 gene is shown as SEQ ID No. 1 or SEQ ID No. 2.

[0014] According to another aspect of the present application, there is also provided use of the inhibitor in the preparation of a drug for treating lung adenocarcinoma.

[0015] In some embodiments, the inhibitor has an inhibitory effect on the proliferation of lung adenocarcinoma cells, wherein the lung adenocarcinoma cells are A549 cell lines.

[0016] According to another aspect of the present application, there is also provided use of the inhibitor in the preparation of a drug for inhibiting lymph node metastasis of lung adenocarcinoma.

[0017] Compared with the existing technology, the advantages and positive effects of the present application are: RAB32, a member of the small GTPase RAB family in the present application, is significantly more highly expressed in non-small cell lung cancer tissues and cell lines than in adjacent cancer tissues and normal cells, and is associated with poor prognosis; in vitro experiments show that knocking down the highly expressed RAB32 gene in tumor cells can significantly inhibit the proliferation ability of lung adenocarcinoma cells; in an in vivo tumor-bearing mouse model, after knocking down the RAB32 gene, the tumor volume of the knockdown group was significantly reduced, growth was inhibited, and the lymph node metastasis ability of lung adenocarcinoma cells was inhibited. Therefore, the RAB32 gene can be used in the preparation of non-small cell cancer diagnosis, treatment and prognosis judgment products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 The Kaplan-Meier survival curve in Example 1 of the present application is shown.

[0020] Figure 2 Shown are pictures of immunohistochemical staining in Example 2 of the present application.

[0021] Figure 3 The graph shows the experimental results of CCK-8 testing tumor proliferation ability in Example 3 of the present application.

[0022] Figure 4 Shown are tumor tissue images and tumor volume curves in Example 4 of the present application.

[0023] Figure 5 The PET-CT images in Example 5 of the present application are shown.

[0024] Figure 6 Shown are pictures of the popliteal lymph nodes and plantar tumors in Example 6 of the present application. DETAILED DESCRIPTION

[0025] In order to more clearly understand the above-mentioned objects, features and advantages of the present application, the present application is further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the embodiments of the present application and the features therein can be combined with each other without conflict.

[0026] Related terms explanation:

[0027] The T1N2M0 subtype of lung adenocarcinoma is a subtype of lung adenocarcinoma with a small primary tumor (T1), metastasis to the ipsilateral mediastinal lymph nodes (N2), and no distant organ metastasis (M0).

[0028] RAB32: One of the genes in the RAB family of small GTPases.

[0029] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0030] Example 1: Analysis of RAB32 expression and survival prognosis in NSCLC patients.

[0031] Data of NSCLC patients were queried through the Kaplan-Meier Plotter online database website. Based on the median RAB32 expression level in NSCLC patients as the threshold, all patients were divided into a high expression group (RAB32-High, expression level higher than the median) and a low expression group (RAB32-Low, expression level lower than or equal to the median), containing 451 and 304 samples, respectively. Kaplan-Meier survival curves were then used to compare the overall survival (OS) of these two groups of patients. The hazard ratio (HR) and P value of the Logrank test were calculated for each gene to evaluate the impact of their expression levels on survival prognosis.

[0032] Figure 1 The Kaplan-Meier survival curve in Example 1 of the present application is shown as follows: Figure 1 As shown, the hazard ratio (HR) for the RAB32 high expression group was 1.24 (95% CI: 1.1–1.4), and the logrank test P value was 0.00033, indicating that the difference in survival between the RAB32 high expression group (RAB32-High, n=451) and the low expression group (RAB32-Low, n=304) was highly statistically significant, indicating that RAB32 expression is significantly negatively correlated with the prognosis of NSCLC patients. The survival curve of the RAB32 high expression group (RAB32-High group) is below that of the low expression group (RAB32-Low), indicating that the survival rate of patients with RAB32 high expression is significantly lower than that of patients with low expression.

[0033] Example 2: RAB32 is highly expressed in T1N2M0 lung adenocarcinoma tumor tissues.

[0034] Immunohistochemical analysis was performed on normal lung tissue, T1N0M0 lung adenocarcinoma (no lymph node metastasis) tumor tissue, and T1N2M0 lung adenocarcinoma tumor tissue (n = 20 per group). All tissue samples were obtained from patients diagnosed postoperatively by pathology at Tianjin Medical University General Hospital. Staging was determined according to the 8th edition of the AJCC TNM staging system (American Joint Committee on Cancer, 8th edition). The immunohistochemical method was as follows:

[0035] (1) Preparation of paraffin sections: Fix lung tissue or tumor tissue with 4% formaldehyde solution. Wash the sample three times in phosphate buffer for 5 minutes each time. Treat with 70%, 80%, and 90% ethanol solutions for 30 minutes each, followed by 95% and 100% ethanol solutions (twice each, 20 minutes each), and then treat with a 1:1 mixture of 100% ethanol and xylene for 15 minutes, and then treat with xylene alone until transparent. Pour the melted paraffin into the prepared container and quickly transfer the paraffin-soaked tissue to the container. After cooling, a paraffin block is obtained. Fix the paraffin block on the microtome and slice it into standard 3mm thickness. Flatten the slices in warm water and quickly pick them up with a slide. Place the slices on a 45°C drying machine to dry.

[0036] (2) Immunohistochemical staining: The sections were placed in an oven at 60°C for 2 hours, then treated with a series of xylene solutions and the ethanol concentration was gradually reduced to 70% for dewaxing, and finally washed with PBS. The endogenous peroxidase activity was blocked by treating with 3% hydrogen peroxide solution for 20 minutes, and then washed with PBS. The samples were placed in a boiling EDTA buffer solution (1x) and heated for 20 minutes. After completion, they were naturally cooled to room temperature and finally washed with PBS. Blocked with 5% bovine serum albumin (BSA) and 0.5% Triton X-100 and incubated at room temperature for 1 hour. The primary antibody solution was added and incubated in a humidified chamber overnight. After the temperature returned to room temperature, the sections were washed with PBS and the secondary antibody solution corresponding to the primary antibody was added, incubated at room temperature for 1 hour, and then washed again with PBS. DAB color development solution was added for color development. After observing the appearance of a brown-yellow positive reaction, the color development was stopped with double distilled water. Stained with hematoxylin for about 1 minute and rinsed with tap water. After treating with differentiation solution for 30 seconds, rinse with tap water to continue differentiation. Dehydrate using ethanol solutions of varying concentrations, and finally treat with xylene. Once sections are dry, seal with neutral gum.

[0037] Figure 2 The immunohistochemical staining pictures in Example 2 of the present application are shown as follows: Figure 2 As shown in the figure, RAB32 was significantly overexpressed in T1N2M0 tumor tissues compared with normal lung tissues and T1N0M0 lung adenocarcinoma (no lymph node metastasis) tumor tissues (darker brown staining in immunohistochemistry indicates high expression).

[0038] Example 3: Knockdown of RAB32 expression inhibits the proliferation of lung adenocarcinoma cells.

[0039] 1. Construction of stably transfected cell lines: Lentiviral-mediated shRNA specifically targeting the RAB32 gene (shRAB32) and the nonspecific control shCtrl lentiviral particles were purchased from Shanghai GeneGene Co., Ltd.

[0040] The nucleotide sequence of the shRNA targeting the RAB32 gene is 5′-CCTTGAGAGCAGAGAACAAAT-3′ (SEQ ID No. 1) or 5′-AGATTCTTGTAAACCACCAAA-3′ (SEQ ID No. 2).

[0041] The nucleotide sequence of the nonspecific control shCtrl is 5′-TTCTCCGAACGTGTCACGT-3′ (SEQ ID No. 5).

[0042] A stable cell line (shRAB32) with stably silenced RAB32 gene and a control cell line (shCtrl) were constructed according to the instructions. The process is as follows:

[0043] 1) Determine the MOI of lentivirus infection: Select A549 cells for lentiviral transfection. When cells are in good condition, digest the cells and prepare a single-cell suspension. Culture in an incubator. When the cell density reaches 20%-30%, replace the culture medium, add different titers of virus and the corresponding infection enhancer solution, and observe the GFP fluorescence intensity under a microscope.

[0044] 2) Determine the puromycin selection concentration.

[0045] 3) Lentiviral infection of cells to construct a cell line with stable RAB32 gene silencing: Observe GFP fluorescence intensity, perform qPCR and WB to detect RAB32 expression, and select cells with the best knockdown effect. The screening criteria are: qPCR detection shows that RAB32 mRNA expression levels are reduced by ≥70% compared with the shCtrl group; WB results are analyzed by grayscale value (ImageJ software) and RAB32 protein expression is downregulated by ≥60% relative to the internal reference GAPDH. GFP fluorescence is also used to observe infection efficiency. Cells with stable and obvious interference effects are selected for subsequent experiments. RAB32 primer sequences used in qPCR are:

[0046] Forward primer: 5′-CAGGTGGACCAATTCTGCAAA-3′ (SEQ ID No. 3); Reverse primer: 5′-GGCAGCTTCCTCTATGTTTATGT-3′ (SEQ ID No. 4); The primer sequence for the internal reference gene GAPDH is as follows:

[0047] Upstream primer (forward): 5′-GGAGCGAGATCCCTCCAAAAT-3′ (SEQ ID No. 6); downstream primer (reverse): 5′-GGCTGTTGTCATACTTCTCATGG-3′ (SEQ ID No. 7).

[0048] 2. CCK-8 Cell Proliferation Assay: When the cell density reaches approximately 70% to 90%, wash with PBS, digest with trypsin, and prepare a single-cell suspension. Count the cells in the single-cell suspension. Seed 5,000 cells per well in 100 μl of culture medium in a 96-well plate and continue culturing in an incubator. After 2 hours of cell culture and adherence, add 10 μl of CCK-8 solution to each well and measure the absorbance of the cells at 450 nm. Subsequently, measure the absorbance of the cells at 450 nm 24, 48, 72, and 96 hours after adherence.

[0049] Figure 3 The results of the CCK-8 test for tumor proliferation ability in Example 3 of the present application are shown as follows: Figure 3 As shown in Figure 3, the proliferation ability of A549 cells was significantly decreased after knockdown of RAB32 compared with the control group.

[0050] Example 4: Knockdown of RAB32 expression inhibits tumor growth.

[0051] 1. Transfected cell lines:

[0052] The method for constructing a stably transfected cell line is the same as that in Example 3 and will not be described again here.

[0053] 2. Subcutaneous tumor formation in nude mice:

[0054] 1) Experimental Animals: Twenty-four healthy female Balb / c nude mice (aged 4-6 weeks) were selected. Animals, feed, and bedding were provided by Beijing Huafukang Biotechnology Co., Ltd. All nude mice were housed in a specific pathogen-free (SPF) laboratory animal facility provided by the Lung Cancer Research Institute of Tianjin Medical University General Hospital. The ambient temperature was maintained between 22°C and 26°C, the relative humidity was maintained between 50% and 60%, and a 12-hour daylight cycle was implemented. Before the experiment, nude mice were randomly divided into experimental (sh-RAB32) and control (shCtrl) groups, with six mice per cage. A one-week acclimatization period was performed before the experiment began.

[0055] 2) Cell preparation: On the day of tumor cell inoculation, digest and collect the cells and prepare a cell suspension at a concentration of 2×104 cells / μL.

[0056] 3) Subcutaneous inoculation: Use a pre-chilled pipette tip to take 100 μL of the prepared cell suspension and mix it with Matrigel at a 1:1 ratio. Use an insulin syringe to draw up the mixture. Insert the needle into the nude mouse cortex and inject. After the injection is complete, slowly withdraw the needle and press the injection site for a few seconds to prevent fluid leakage.

[0057] 4) Observation of tumor formation: Starting from the first week after inoculation, observe tumor growth and measure tumor size every three days.

[0058] A549-shRAB32 (shRNA targeting RAB32 was transfected into A549 cell line via lentivirus) cells were inoculated subcutaneously on the back of nude mice. Tumor formation was detected on the 7th day. The long and short diameters of the tumor were measured every 3 days to calculate the volume and growth rate. The nude mice were killed and the tumors were removed on the 48th day. Figure 4 The tumor tissue image and tumor volume curve in Example 4 of this application are shown as follows: Figure 4 As shown, the tumor growth rate of the control group (sh-control) was significantly stronger than that of the RAB32 knockdown group (experimental group, sh-RAB32).

[0059] Example 5: Knockdown of RAB32 reduces lymph node metastasis ability.

[0060] 1. Transfected cell lines:

[0061] The method for constructing a stably transfected cell line is the same as that in Example 3 and will not be described again here.

[0062] 2. On the day of tumor cell inoculation, digest and collect the cells and prepare a cell suspension with a concentration of 2×10 4 cells / μL.

[0063] 3. Take 30 μL of the prepared cell suspension and mix it with Matrigel at a ratio of 1:1. Inject the tumor cell suspension into the footpad using an insulin syringe.

[0064] 4. After 30 days, the mice were anesthetized by inhalation of ethylflurane and injected with 18F-FDG (240 μU / mouse) through the tail vein. 20 minutes later, the mice were placed in the InliView-3000B PET-SPECT-CT trimodality imaging system for in vivo imaging and analysis of lymph node metastasis.

[0065] Figure 5 The PET-CT images in Example 5 of the present application are shown as follows: Figure 5 As shown in the data, compared with the control group, the uptake of 18F-FDG in the footpad and popliteal tumors of the experimental group (shRAB32) was significantly lower, indicating that the proliferation ability of the tumor in the primary site of the footpad was weakened and the invasiveness of metastasis to the popliteal lymph nodes through lymphatic vessels was significantly reduced.

[0066] Example 6: Knockdown of RAB32 reduces lymph node metastasis ability.

[0067] 1. Transfected cell lines:

[0068] The method for constructing a stably transfected cell line is the same as that in Example 3 and will not be described again here.

[0069] 2. Cell preparation: Digest and collect cells on the day of inoculation and prepare a cell suspension with a concentration of 2×10 4 cells / μL.

[0070] 3. Subcutaneous Inoculation: Use a pre-chilled pipette tip to dispense 30 μL of the prepared cell suspension and mix it with Matrigel at a 1:1 ratio. Aspirate the mixture using an insulin syringe. Inject the tumor cell suspension into the raised skin bump on the footpad using the insulin syringe and slowly withdraw the needle. After the injection is complete, slowly withdraw the needle and apply pressure to the injection site for several seconds to prevent fluid leakage.

[0071] 4. Observe tumor formation: Regularly monitor mouse body weight, footpad tumor growth, and lymph node metastasis starting approximately one week after inoculation.

[0072] Figure 6 Showing the popliteal lymph node and footpad tumor pictures in Example 6 of this application, Figure 6 As shown, compared with the control group, the lymph node volume of the experimental group was significantly reduced, and no lymph nodes were found in one mouse in the experimental group, that is, the lymph node metastasis ability of the experimental group was weaker than that of the control group.

[0073] Through the above specific embodiments, those skilled in the art can easily implement the present application. However, it should be understood that the present application is not limited to the above specific embodiments. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to implement different technical solutions.

Claims

1. Use of a reagent for detecting RAB32 protein levels in the preparation of a diagnostic product for lung adenocarcinoma lymph node metastasis, characterized in that: The lung adenocarcinoma lymph node metastasis refers to lung adenocarcinoma lymph node metastasis in which the primary tumor is ≤3 cm, has metastasized to the ipsilateral mediastinal lymph nodes, and has no distant organ metastasis.

2. Use of an inhibitor in the preparation of a drug for inhibiting lymph node metastasis of lung adenocarcinoma, characterized in that: The inhibitor is shRNA targeting the RAB32 gene, and the nucleotide sequence of the shRNA targeting the RAB32 gene is shown as SEQ ID No. 1 or SEQ ID No. 2.

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