Application of RAB32 in preparation of non-small cell carcinoma diagnosis, treatment and prognosis product

Abstract

The invention discloses application of RAB32 in preparation of a product for diagnosis, treatment and prognosis of non-small cell carcinoma. According to the application, a small GTP enzyme RAB family member RAB32 is remarkably and highly expressed in non-small cell lung cancer tissues and cell lines compared with para-carcinoma tissues and normal cells, and is related to poor prognosis; an in-vitro experiment shows that the multiplication capacity of lung adenocarcinoma cells can be remarkably inhibited after the high-expression RAB32 gene is knocked down in tumor cells; in an in-vivo tumor-bearing mouse model, after the RAB32 gene is knocked down, the tumor volume of a knock-down group is obviously reduced, the growth of the knocked-down group is inhibited, and meanwhile, the lymph node metastasis capability of lung adenocarcinoma cells is inhibited, so that the RAB32 gene can be applied to preparation of non-small cell carcinoma diagnosis, treatment and prognosis products.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies. Specifically, it relates to the application of RAB32 in the preparation of products for the diagnosis, treatment, and prognosis judgment of non-small cell carcinoma. Background Art

[0002] Lung cancer is the malignant tumor with the highest incidence and mortality in China, and about 85% of it is non-small cell lung cancer (NSCLC). Tumor metastasis is the main reason for the high fatality rate of lung cancer. Among them, lymph node metastasis is 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, and the tumor diameter is mostly greater than 5 cm. However, in clinical practice, it is found that some T1 (≤3CM) NSCLC patients already have lymph node metastasis at the initial diagnosis, and their survival prognosis is very poor. This clinical subtype of lung cancer (T1N2M0, that is, "small tumor with large lymph node metastasis") with lymph node metastasis at a very small growth stage of the primary tumor mostly shows strong tumor invasiveness, poor differentiation, and high malignancy.

[0003] Lung adenocarcinoma is a type of NSCLC. In the related technologies, there is no publicly disclosed biomarker for lymph node metastasis of lung adenocarcinoma, which will affect the diagnosis, treatment intervention, and predictive judgment of lymph node metastasis in primary tumors (≤3CM) of lung adenocarcinoma, thereby reducing the survival probability of lung adenocarcinoma patients.

[0004] It should be noted that the information disclosed in the above background art section is only used to strengthen the understanding of the background of the present disclosure. Therefore, it may include information that does not constitute the prior art known to those of ordinary skill in the art. Summary of the Invention

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

[0006] To achieve the above object, this application provides the following technical solutions: According to one aspect of the present application, there is provided an application of RAB32 in the preparation of a product for predicting the prognosis of patients with non-small cell carcinoma.

[0007] In some embodiments, the product is a kit for detecting the expression level of RAB32.

[0008] In some embodiments, the expression level of RAB32 is significantly negatively correlated with the survival rate of patients with non-small cell carcinoma.

[0009] According to another aspect of the present application, there is also provided an application of RAB32 in the preparation of a product for diagnosing lung adenocarcinoma.

[0010] According to another aspect of the present application, there is also provided an application of RAB32 in the preparation of a product for diagnosing lymph node metastasis of lung adenocarcinoma.

[0011] In some embodiments, the lymph node metastasis of lung adenocarcinoma is mediastinal lymph node metastasis of lung adenocarcinoma.

[0012] According to another aspect of the present application, there is also provided an inhibitor, the inhibitor includes shRNA targeting the RAB32 gene, and the nucleotide sequence of the shRNA targeting the RAB32 gene is as shown in SEQ ID No.1 or SEQ ID No.2.

[0013] According to another aspect of the present application, there is also provided an application of the above-mentioned inhibitor in the preparation of a drug for treating lung adenocarcinoma.

[0014] In some embodiments, the inhibitor has an inhibitory effect on the proliferation of lung adenocarcinoma cells, wherein the lung adenocarcinoma cells are A549 cell line.

[0015] According to another aspect of the present application, there is also provided an application of the above-mentioned inhibitor in the preparation of a drug for inhibiting lymph node metastasis of lung adenocarcinoma.

[0016] Compared with the prior art, the advantages and positive effects of the present application are as follows: in the present application, the small GTPase RAB family member RAB32 is significantly highly expressed in non-small cell lung cancer tissues and cell lines compared with 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 the in vivo tumor-bearing mouse model, after knocking down the RAB32 gene, the tumor volume in the knockdown group is significantly reduced, the growth is inhibited, and at the same time, the lymph node metastasis ability of lung adenocarcinoma cells is inhibited. Therefore, the RAB32 gene can be applied in the preparation of products for diagnosing, treating and predicting the prognosis of non-small cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the accompanying drawings required for use in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0018] Figure 1 Show the Kaplan-Meier survival curve in Embodiment 1 of the present application.

[0019] Figure 2 Show the immunohistochemical staining pictures in Embodiment 2 of the present application.

[0020] Figure 3 Show the experimental result curve of CCK-8 test for tumor proliferation ability in Embodiment 3 of the present application.

[0021] Figure 4 Show the tumor tissue pictures and tumor volume curve in Embodiment 4 of the present application.

[0022] Figure 5 Show the PET-CT photos in Embodiment 5 of the present application.

[0023] Figure 6 Show the popliteal lymph node and plantar tumor pictures in Embodiment 6 of the present application. Detailed implementation manners

[0024] In order to more clearly understand the above objects, features, and advantages of the present application, the following further describes the present application in conjunction with the accompanying drawings and embodiments. It should be noted that, without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other.

[0025] Explanation of related terms: The lung adenocarcinoma subtype of T1N2M0 is a lung adenocarcinoma subtype with a relatively small primary tumor (T1), having metastasized to the ipsilateral mediastinal lymph nodes (N2), and no distant organ metastasis (M0).

[0026] RAB32: One of the genes in the RAB family of small GTPases.

[0027] The following further describes the present application in conjunction with the accompanying drawings and specific embodiments.

[0028] Embodiment 1: Analysis of the expression of RAB32 and the survival prognosis of NSCLC patients.

[0029] Data of NSCLC patients were queried through the online database website of Kaplan-Meier Plotter. According to the median expression level of RAB32 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), which included 451 and 304 samples respectively. Subsequently, the Kaplan-Meier survival curve was used to compare the overall survival (OS) of these two groups of patients, and the hazard ratio (HR) and P value of the Logrank test of each gene were calculated to evaluate the impact of their expression levels on survival prognosis.

[0030] Figure 1 The Kaplan-Meier survival curve in Example 1 of the present application is shown, as Figure 1 shown, the hazard ratio (HR) of the RAB32 high-expression group was 1.24 (95% CI: 1.1–1.4), and the P value of the logrank test was 0.00033, indicating that there was a highly statistically significant difference in the survival rates between the RAB32 gene high-expression group (RAB32-High, n = 451) and the low-expression group (RAB32-Low, n = 304), suggesting a significant negative correlation between RAB32 expression and the prognosis of NSCLC patients. According to the survival curve of the RAB32 gene high-expression group (RAB32-High group) being below that of the low-expression group (RAB32-Low), it indicates that the survival rate of patients with high RAB32 gene expression was significantly lower than that of patients with low expression.

[0031] Example 2: RAB32 was highly expressed in the tumor tissues of lung adenocarcinoma with T1N2M0.

[0032] Immunohistochemical analysis was performed on normal human lung tissues, tumor tissues of lung adenocarcinoma with T1N0M0 (without lymph node metastasis), and tumor tissues of lung adenocarcinoma with T1N2M0 (20 cases in each group). All tissue samples were from patients diagnosed by postoperative pathology in Tianjin Medical University General Hospital, and the staging was determined according to the 8th edition of the AJCC TNM staging standard (American Joint Committee on Cancer, 8th edition). The immunohistochemical method is as follows: (1)Preparation of paraffin sections: Fix lung tissue or tumor tissue with 4% formaldehyde solution. Wash the samples 3 times in phosphate buffer, 5 minutes each time. Treat with 70%, 80%, and 90% ethanol solutions for 30 minutes respectively, then use 95% and 100% ethanol solutions (twice each, 20 minutes each time), then treat with a 1:1 mixture of 100% ethanol and xylene for 15 minutes, and then use xylene alone until transparent. Pour the melted paraffin into a prepared container, and quickly transfer the paraffin-impregnated tissue into the container. After cooling, paraffin blocks can be obtained. Fix the paraffin block on a microtome and cut sections at a standard thickness of 3 mm. Place the sections in warm water to flatten them, quickly pick them up with a glass slide, and place the sections on a baking machine at 45 °C to dry.

[0033] (2)Immunohistochemical staining: Heat the sections in an oven at 60 °C for 2 hours, then dewax them with a series of xylene solutions and gradually reduce the ethanol concentration to 70% for dewaxing, and finally wash with PBS. Block endogenous peroxidase activity by treating with 3% hydrogen peroxide solution for 20 minutes, and wash with PBS. Place the samples in a boiling EDTA buffer solution (1x) and heat-treat for 20 minutes. After completion, cool naturally to room temperature, and finally wash with PBS. Block with 5% bovine serum albumin (BSA) and 0.5% Triton X-100, and incubate at room temperature for 1 hour. Add the primary antibody solution and incubate overnight in a wet box. After the temperature returns to room temperature, wash with PBS and add the secondary antibody solution corresponding to the primary antibody, incubate at room temperature for 1 hour, and then wash with PBS again. Add DAB chromogenic solution for color development. After observing the appearance of a brownish-yellow positive reaction, stop the color development with double-distilled water. Stain with hematoxylin for about 1 minute and rinse with tap water. Treat with a differentiating solution for 30 seconds and then rinse with tap water for differentiation. Gradually dehydrate with different concentrations of ethanol solutions, and finally treat with xylene. After the sections are dry, seal them with neutral gum to finish.

[0034] Figure 2 The immunohistochemical staining pictures in Example 2 of the present application are shown, as Figure 2 shown. Compared with normal lung tissue and T1N0M0 lung adenocarcinoma (not metastasized to lymph nodes) tumor tissue, RAB32 was significantly highly expressed in T1N2M0 tumor tissue (deeper brown staining in immunohistochemistry represents high expression).

[0035] Example 3: Knockdown of RAB32 expression inhibits the proliferation of lung adenocarcinoma cells.

[0036] 1. Construction of stable transfected cell lines: Lentiviral particles of shRNA (shRAB32) specifically targeting the RAB32 gene and non-specific control shCtrl were both purchased from GeneChem Co., Ltd. in Shanghai.

[0037] 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).

[0038] The nucleotide sequence of the non-specific control shCtrl is 5′-TTCTCCGAACGTGTCACGT-3′ (SEQ ID No.5).

[0039] According to the instructions, stable cell lines with stably silenced RAB32 gene (shRAB32) and control cell lines (shCtrl) were constructed as follows: 1) Determine the MOI of lentivirus infection on cells: Select the A549 cell line as the lentivirus transfection cell line. When the cell state is good, digest the cells and prepare a single-cell suspension, and culture it in an incubator. When the cell density reaches 20% - 30%, change the medium, add lentiviruses with different titers and corresponding infection enhancer solutions, and observe the GFP fluorescence intensity under the microscope.

[0040] 2) Determine the puromycin screening concentration.

[0041] 3) Construct a cell line with stably silenced RAB32 gene by lentivirus infection of cells: Observe the GFP fluorescence intensity, and perform qPCR and WB to detect the expression level of RAB32, and select the cells with the best knockdown effect. Among them, the screening criteria are as follows: qPCR detection shows that the expression level of RAB32 mRNA decreases by ≥70% compared with the shCtrl group; the WB results are analyzed by gray value (ImageJ software), and the expression of RAB32 protein is downregulated by ≥60% relative to the internal reference GAPDH. At the same time, refer to the GFP fluorescence to observe the infection efficiency, and select the cells with stable and obvious interference effects for subsequent experiments. The primer sequences of RAB32 used in qPCR are as follows: Forward primer: 5′-CAGGTGGACCAATTCTGCAAA-3′ (SEQ ID No.3); Reverse primer: 5′-GGCAGCTTCCTCTATGTTTATGT-3′ (SEQ ID No.4); The primer sequences of the internal reference gene GAPDH are as follows: Forward primer: 5′-GGAGCGAGATCCCTCCAAAAT-3′ (SEQ ID No.6); Reverse primer: 5′-GGCTGTTGTCATACTTCTCATGG-3′ (SEQ ID No.7).

[0042] 2. CCK-8 cell proliferation assay: When the cell growth density is about 70% - 90%, add PBS for washing, digest with trypsin and prepare a single-cell suspension; perform cell counting on the above single-cell suspension. Inoculate 100 μL of medium with 5000 cells per well into a 96-well plate and continue culturing in an incubator; after the cells adhere for 2 h, add 10 μL of CCK-8 solution to each well and measure the absorbance of the cells at 450 nm; then measure the absorbance of the cells at 450 nm at 24 h, 48 h, 72 h, and 96 h after adhesion respectively.

[0043] Figure 3 The curve showing the experimental results of the CCK-8 assay for tumor proliferation ability in Example 3 of the present application is as Figure 3 shown. Compared with the control group, the proliferation ability of A549 cells decreased significantly after knocking down RAB32.

[0044] Example 4: Knocking down RAB32 expression inhibits tumor growth.

[0045] 1. Transfected cell line: The method for constructing a stable transfected cell line is the same as that in Example 3 and will not be elaborated here.

[0046] 2. Subcutaneous tumor formation in nude mice: 1) Experimental animals: Select 24 healthy female Balb / c nude mice (aged 4 - 6 weeks). The animals, feed, and bedding are from Beijing Huafukang Biotechnology Co., Ltd. All experimental nude mice are raised in a specific pathogen-free (SPF) level experimental animal facility provided by the Lung Cancer Research Institute of Tianjin Medical University General Hospital, maintaining the environmental temperature between 22°C and 26°C, the relative humidity at 50% - 60%, and implementing a 12-hour day-night alternating light regime. Before the experiment, the nude mice are randomly grouped into 6 per cage, divided into an experimental group (sh-RAB32) and a control group (shCtrl), and a one-week environmental adaptation period is carried out before the start of the experiment.

[0047] 2) Cell preparation: Digest and collect the cells on the day of inoculating the tumor cells to prepare a cell suspension with a concentration of 2×104 cells / μL.

[0048] 3) Subcutaneous inoculation: Use a pre-cooled pipette tip to take 100 μL of the prepared cell suspension and mix it evenly with Matrigel matrix gel in an equal volume ratio of 1:1. Aspirate the mixture with an insulin syringe, insert the syringe into the cortex of the nude mouse and then inject. After injection, slowly withdraw the needle and press the injection site for a few seconds to avoid liquid leakage. 4) Observe tumor formation: Starting from the first week after inoculation, observe the tumor growth and measure the tumor size every three days.

[0049] A549-shRAB32 (shRNA targeting RAB32 transfected into A549 cell line by 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 sacrificed on the 48th day to dissect the tumors. Figure 4 Show the tumor tissue pictures and tumor volume curves in Example 4 of the present application, as Figure 4 shown. The tumor growth rate of the control group (sh-control) cells was significantly stronger than that of the RAB32 knockdown group (experimental group, sh-RAB32).

[0050] Example 5: Knockdown of RAB32 reduces the ability of lymph node metastasis.

[0051] 1. Transfected cell line: The method for constructing a stably transfected cell line is the same as that in Example 3 and will not be elaborated here.

[0052] 2. Digest and collect cells on the day of tumor cell inoculation to prepare a cell suspension with a concentration of 2×10 4 cells / μL.

[0053] 3. Take 30 μL of the prepared cell suspension and mix it evenly with Matrigel matrix gel in an equal volume ratio of 1:1. Use an insulin syringe to inject the tumor cell suspension into the footpad.

[0054] 4. After 30 days, anesthetize the mice with isoflurane, and then inject 18F-FDG (240 μU / mouse) via the tail vein. After 20 min, place the mice in an InliView-3000B PET-SPECT-CT triple-modal imaging system for small animal in vivo imaging to analyze the lymphatic metastasis of the mice.

[0055] Figure 5 Show the PET-CT pictures in Example 5 of the present application, as Figure 5 shown. Compared with the control group, the uptake of 18F-FDG by tumors at the footpad and popliteal fossa sites in the experimental group (shRAB32) was significantly less, indicating that the proliferation ability of tumors at the primary site of the footpad was weakened, and the invasiveness of lymphatic metastasis to the popliteal lymph nodes was significantly reduced.

[0056] Example 6: Knockdown of RAB32 weakens the ability of lymph node metastasis.

[0057] 1. Transfected cell line: The method for constructing a stably transfected cell line is the same as that in Example 3 and will not be elaborated here.

[0058] 2. Cell preparation: Digest and collect the cells on the day of inoculation, and prepare them into a cell suspension with a concentration of 2×10 4 cells / μL.

[0059] 3. Subcutaneous inoculation: Take 30 μL of the prepared cell suspension with a pre-cooled pipette tip and mix it with Matrigel matrix gel in an equal volume ratio of 1:1. Aspirate the mixture with an insulin syringe. Use the insulin syringe to inject the tumor cell suspension into the raised dermal papilla of the footpad, then slowly withdraw the needle. After injection, slowly withdraw the needle and press the injection site for a few seconds to avoid liquid leakage.

[0060] 4. Observation of tumor formation: Regularly monitor the body weight of the mice, the growth of footpad tumors, and the lymph node metastasis from about one week after inoculation.

[0061] Figure 6 Pictures of popliteal lymph nodes and footpad tumors in Example 6 of the present application are shown. As Figure 6 shown, compared with the control group, the volume of lymph nodes in the experimental group was significantly reduced, and no lymph nodes were found in 1 mouse in the experimental group. That is to say, the ability of lymph node metastasis in the experimental group was weaker than that in the control group.

[0062] Through the above specific implementation manners, those skilled in the art of the present application can easily implement the present application. However, it should be understood that the present application is not limited to the above specific implementation manners. Based on the disclosed implementation manners, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. Use of RAB32 in the preparation of a product for predicting the prognosis of patients with non-small cell carcinoma.

2. The application according to claim 1, characterized in that, The product is a kit for detecting the expression level of RAB32.

3. The application according to claim 2, characterized in that, The expression level of RAB32 is significantly negatively correlated with the survival rate of patients with non-small cell carcinoma.

4. Use of RAB32 in the preparation of a product for diagnosing lung adenocarcinoma.

5. Use of RAB32 in the preparation of a product for diagnosing lymph node metastasis of lung adenocarcinoma.

6. The application according to claim 5, characterized in that, The lymph node metastasis of lung adenocarcinoma is mediastinal lymph node metastasis of lung adenocarcinoma.

7. An inhibitor, characterized in that, The inhibitor includes 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.

8. Use of the inhibitor according to claim 7 in the preparation of a drug for treating lung adenocarcinoma.

9. The application according to claim 8, wherein The inhibitor has an inhibitory effect on the proliferation of lung adenocarcinoma cells, wherein the lung adenocarcinoma cells are A549 cell line.

10. Use of the inhibitor according to claim 7 in the preparation of a drug for inhibiting lymph node metastasis of lung adenocarcinoma.

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