Use of lamp5 as a target for diagnosis and treatment of bladder cancer

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a new target for bladder cancer proliferation and metastasis progression research and application thereof, more particularly to LAMP5 as a marker for bladder cancer diagnosis and prognosis, and application of LAMP5 as a bladder cancer marker in preparation of a molecular targeted drug. The present application constructs a stable cell strain with LAMP5 expression knocked down in J82 and T24T cell strains, and functional experiments show that inhibition of LAMP5 expression can inhibit the proliferation and metastasis of bladder cancer cells in vitro and ectopic tumor in vivo. It can be seen that LAMP5 expression can be inhibited by using an LAMP5 expression inhibitor, thereby providing a potential therapeutic target for bladder cancer treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a new target for bladder cancer proliferation and metastasis progression research and application thereof, more particularly to LAMP5 as a marker for bladder cancer diagnosis and prognosis, and application of LAMP5 as a bladder cancer marker in preparation of a molecular targeted drug. BACKGROUND

[0002] Bladder cancer is a malignant tumor originating from the urothelium of the bladder, and is one of the most common malignant tumors of the urinary system. According to the infiltration type of the tumor, bladder cancer can be divided into non-muscular invasive bladder cancer (NMIBC) and muscular invasive bladder cancer (MIBC). In clinical practice, 75% of patients are diagnosed with NMIBC, which belongs to early bladder cancer. Most NMIBC can be well treated by transurethral resection of bladder tumor, but the recurrence rate and progression rate are high. When NMIBC progresses, it is often accompanied by distant metastasis, and the prognosis is poor. At present, the treatment of MIBC patients mainly includes surgical resection and radiotherapy, but the overall response rate is low, and the prognosis is poor. Although the clinical treatment methods for bladder cancer are constantly improving and improving, the recurrence rate and progression rate of early bladder cancer are still high, and the prognosis of metastatic bladder cancer has not been significantly improved. Therefore, it is necessary to find sensitive and specific biomarkers for bladder cancer and diagnostic and therapeutic targets for bladder cancer.

[0003] Lysosome Associated Membrane Protein 5 (LAMP5) is the latest characterized LAMP family protein, containing 280 amino acids. Studies have shown that LAMP5 is related to the proliferation of plasmacytoid dendritic cells and is related to the immune tolerance system, which is crucial for tumor occurrence and progression. And high expression of LAMP5 is associated with poor prognosis of MLL. However, there are few studies on the expression of LAMP5 in tumors and its related functions and mechanisms. Current reports of LAMP5 in colorectal cancer and gastric cancer suggest that LAMP5 is associated with poor prognosis. However, so far, the expression of LAMP5 in bladder cancer and its related effects have not been reported. SUMMARY

[0004] The technical problem to be solved by the present application is to provide LAMP5 as a new target for bladder cancer proliferation and metastasis and a diagnostic and therapeutic marker and application thereof.

[0005] In order to solve the above technical problem, the present application provides the application of LAMP5 as a specific marker (closely related to bladder cancer proliferation and metastasis) in the preparation of a drug for diagnosing and treating bladder cancer.

[0006] The present application provides the application of LAMP5 as a specific marker in the diagnosis and treatment of bladder cancer.

[0007] As an improvement of the application: the specific marker is LAMP5 protein.

[0008] As a further improvement of the application: a kit for detecting bladder cancer is prepared.

[0009] As a further improvement of the application: the bladder cancer diagnostic kit comprises an antibody that specifically binds to LAMP5 protein.

[0010] The application also simultaneously provides the use of an inhibitor of LAMP5 gene expression in the preparation of a drug for treating bladder cancer.

[0011] As an improvement of the application: the drug comprises an inhibitor of LAMP5 gene expression and a pharmaceutically acceptable carrier.

[0012] As an improvement of the application: the inhibitor is an shRNA plasmid that inhibits LAMP5 gene expression.

[0013] As a further improvement of the application: the proliferation and metastasis of bladder cancer are inhibited.

[0014] As a further improvement of the application: LAMP5 is inhibited, which can inhibit the proliferation ability of bladder cancer cells in vitro and inhibit the in vivo proliferation ability of bladder cancer cells.

[0015] As a further improvement of the application: it can inhibit the in vitro migration and invasion ability of bladder cancer cells, and inhibit the in vivo metastasis ability of bladder cancer cells.

[0016] The technical solution adopted by the present application is as follows: through bioinformatics analysis of RNASeq V2 and clinical data of bladder cancer in TCGA database, LAMP5 is screened out as a high expression gene in bladder cancer, and LAMP5 is also highly expressed in advanced bladder cancer (stage III+IV). Further, the expression of LAMP5 in human bladder cancer tissue and adjacent normal tissue is detected by immunohistochemical staining (IHC) technology, and the expression level of LAMP5 in human bladder epithelial normal cells and bladder cancer cell lines is detected by Western blot.

[0017] In vitro ATP experiment and soft agar colony formation experiment (soft agar assay) prove that LAMP5 can promote the in vitro proliferation ability of bladder cancer cells, and Transwell experiment in vitro proves that LAMP5 can promote the migration and invasion of bladder cancer cells in vitro. Further, the subcutaneous tumor transplantation experiment in nude mice proves that LAMP5 can significantly promote the growth of bladder cancer cell transplantation tumor in vivo, and the lung metastasis model experiment in nude mice finds that LAMP5 can significantly promote the formation of lung metastasis foci in nude mice.

[0018] It should be noted that: some bladder cancer molecular markers known at present are NMP22, BTA and FGFR3, etc., but these target molecules and the LAMP5 of the application are essentially different in genetic sources and property characteristics.

[0019] The application has the following beneficial effects:

[0020] 1. Bioinformatics analysis of TCGA database and clinical bladder cancer tissues showed that LAMP5 expression was significantly up-regulated, and LAMP5 expression was negatively correlated with the overall survival (OS) of bladder cancer patients. Meanwhile, LAMP5 was highly expressed in advanced bladder cancer (stage III+IV). Therefore, LAMP5 can assist in the diagnosis of bladder cancer and serve as a prognostic indicator for bladder cancer.

[0021] 2. The application constructed a stable cell line with knockdown of LAMP5 expression in J82 and T24T cell lines, and functional experiments showed that inhibition of LAMP5 expression can inhibit the proliferation and metastasis of bladder cancer cells in vitro and ectopic tumor in vivo. Therefore, LAMP5 expression inhibitors can inhibit LAMP5, thereby providing a potential therapeutic target for the treatment of bladder cancer. BRIEF DESCRIPTION OF DRAWINGS

[0022] The specific embodiments of the application will be further described in detail below with reference to the accompanying drawings.

[0023] Figure 1-1 Bioinformatics analysis of LAMP5 expression in human bladder cancer tissues and adjacent tissues (A) in TCGA database, and the relationship with the overall survival of bladder cancer patients (B).

[0024] Figure 1-2 Immunohistochemical staining (IHC) technology was used to analyze the expression of LAMP5 in human bladder cancer tissues and adjacent tissues;

[0025] Figure 1-2 In the middle:

[0026] A. Immunohistochemical staining was used to detect the protein expression of LAMP5 in 19 pairs of matched bladder cancer clinical tissues;

[0027] B. Image-Pro Plus software was used to analyze the relative quantification results of LAMP5 protein expression levels in 19 pairs of clinical bladder cancer tissues and their corresponding adjacent tissues.

[0028] Figure 2-1 Western blot technology was used to detect the expression of LAMP5 protein in human bladder cancer cell lines and normal bladder urothelial cells;

[0029] Note: SV-HUC-1 is normal bladder epithelial cell, RT4, RT112, J82, UMUC3, T24, T24T, TCCSUP are bladder cancer cell lines, the order of bladder cancer cell lines is according to the clinical stage of cell source.

[0030] Figure 2-2 To construct LAMP5 knockdown cell lines by RNA interference technology, the results of LAMP5 knockdown effect identification; GAPDH is used as an internal reference.

[0031] A corresponds to J82 cells, B corresponds to T24T cells.

[0032] Figure 3 To detect the effect of LAMP5 on the growth and proliferation of bladder cancer cells by ATP experiment and soft agar colony formation experiment.

[0033] Figure 3 In:

[0034] A and B detect the effect of LAMP5 knockdown on the proliferation of bladder cancer cells J82 and T24T by ATP experiment.

[0035] C detects the effect of LAMP5 knockdown on the proliferation of bladder cancer cells J82 and T24T by soft agar experiment.

[0036] D is the soft agar colony formation experiment cell colony number statistics: more than 32 cells are counted as a colony, the results are expressed as colonies per 10 4 cells, the number of cell colonies in each image is counted and compared.

[0037] Figure 4 To detect the growth and formation of tumors in vivo promoted by LAMP5 in nude mice subcutaneous tumor experiment.

[0038] Figure 4 In:

[0039] A is to take out the tumor and take a photo after the nude mice are killed according to the regulations of animal ethics, and the comparison chart of tumor in the experimental group and the control group.

[0040] B is to weigh the tumor tissue taken out, and compare the weight of the control group and the experimental group to find that the weight of the tumor in the experimental group is also significantly reduced.

[0041] C detects the expression level of Ki67 in bladder cancer tumor tissue by immunohistochemical staining.

[0042] D calculates and analyzes the ratio of IOD / Area using Image-Pro Plus 6.0 software, and the relative quantitative results of Ki67 protein expression level.

[0043] Figure 5 Statistical analysis of LAMP5 mRNA levels in 124 cases of stage I and II, 256 cases of stage III and IV bladder cancer patients in TCGA database by bioinformatics.

[0044] Figure 6 Transwell experiment to detect the effect of LAMP5 on the migration and invasion ability of bladder cancer cells.

[0045] Figure 6 In the middle:

[0046] A, C, through Transwell experiment to detect the effect of LAMP5 on the migration and invasion ability of J82 and T24T cells;

[0047] B, D, count the number of cells passing through the chamber three times independently, and statistically analyze the difference in the number of cells between LAMP5 knockdown and its control group.

[0048] Figure 7 To verify the effect of LAMP5 on the migration and invasion ability of bladder cancer cells T24T in vivo in a nude mouse lung metastasis model;

[0049] Figure 7 In the middle:

[0050] A, the nude mouse lung metastasis model was established by tail vein injection, and the lung tissue of the nude mouse was photographed after the model was successfully established;

[0051] B, the nude mouse lung metastasis model was established by tail vein injection, and the number of metastatic nodules on the surface of the lung of the nude mouse was counted after the model was successfully established, and a columnar diagram of the difference in the number of lung surface nodules was made. DETAILED DESCRIPTION

[0052] The application will be further described below in conjunction with specific examples, but the scope of protection of the application is not limited to this:

[0053] Example 1, LAMP5 is significantly overexpressed in bladder cancer clinical tissue samples, and is negatively correlated with the overall survival of bladder cancer patients

[0054] 1. TCGA database analysis

[0055] In order to clarify the possible role of LAMP5 in bladder cancer and its relationship with bladder cancer patients, the present application analyzes the expression of LAMP5 in 19 pairs of bladder cancer clinical tissues (A) and the relationship between LAMP5 and the overall survival (OS) of bladder cancer patients (B) by TCGA database. Figure 1-1 Figure 1-1 ​B) According to the median of LAMP5 expression in bladder cancer tissues, the bladder cancer tissues were divided into two groups, the results showed that the patients with low expression of LAMP5 had longer OS time and higher survival rate, on the contrary, the patients with high expression of LAMP5 had shorter OS time and lower survival rate, and the difference between the two groups was significant (P<0.05).

[0056] 2. Tissue samples

[0057] In the present application, 19 pairs of clinical bladder cancer pathological tissue samples (bladder cancer tissues, cancer adjacent tissues) were used and approved by the Ethics Committee of Wenzhou Medical University.

[0058] 3. Immunohistochemistry

[0059] The conventional immunohistochemical method was referred to, and the specific steps were as follows:

[0060] 3.1 Baking slices: the prepared slices were placed on a special slide rack, and then baked in a 65℃ oven for 2-3h;

[0061] 3.2 Dewaxing and hydration: the tissue slices were sequentially placed in xylene I for 10min, xylene II for 10min, and 1 / 2 xylene+1 / 2 alcohol mixture for 2min for dewaxing; then subjected to hydration by different gradient ethanol: 100% ethanol I→100% ethanol II→95% ethanol→90% ethanol→80% ethanol→70% ethanol→50% ethanol, each for 90s, and finally washed in a beaker containing double distilled water for 10min, during which the water was changed for 3 times;

[0062] 3.3 Antigen repair: a certain amount of 1×antigen repair solution (pH 6.0, 0.01M) was added to the slide box, and then heated to boiling in a microwave oven. Then the tissue slices were placed in the slide box, and the box was wrapped with plastic wrap to prevent overheating and volatilization overflow, the temperature of the microwave oven was adjusted to 30%, and the repair was performed for 7min / time, a total of 4 times. After repair, it was cooled at room temperature;

[0063] 3.4 Endogenous peroxidase removal: after cooling to room temperature, 1×TBS was soaked for 5min / time, a total of three times, and then the tissue was circled with a histological pen; 3% H2O2 was added dropwise on the slide, and placed in a wet box, and incubated at room temperature for 30min;

[0064] 3.5 Blocking: the H2O2 on the tissue slide was shaken off, and then 1×TBS was soaked for 5min / time, a total of three times; 5% BSA was added dropwise on the slide, and placed in a wet box, and incubated at room temperature for 30min;

[0065] 3.6 Primary antibody incubation: the BSA on the tissue slide was shaken off, and then the prepared primary antibody was directly added without washing, and incubated at 4℃ overnight;

[0066] 3.7 Second antibody incubation: The primary antibody was recovered, and 1xTBS was added for 5 minutes each time for a total of three times. The biotinylated secondary antibody corresponding to the primary antibody was added, and the incubation was performed at 37°C for 1 hour in a constant temperature incubator.

[0067] Note: The primary antibody was purchased from Invitrogen with the item number 14-9778-80, and the secondary antibody was from the BOSTER ready-to-use SABC-POD kit (SA1022).

[0068] 3.8 SABC incubation: The secondary antibody was removed, and 1xTBS was added for 5 minutes each time for a total of three times. SABC was then added, and the incubation was performed at 37°C for 1 hour in a constant temperature incubator.

[0069] 3.9 DAB color development: The SABC was removed, and 1xTBS was added for 5 minutes each time for a total of three times. The freshly prepared DAB color development solution was added, and the slide was observed under a microscope. The color development was stopped with distilled water, and the slide was finally washed with distilled water for 5 minutes.

[0070] 3.10 Hematoxylin counterstaining: Hematoxylin was added, and the slide was stained in the dark for 2-3 minutes. The staining was stopped with distilled water, and the slide was finally washed with distilled water for 10 minutes.

[0071] 3.11 Decolorization and transparency: The slide was placed in 50% ethanol, 70% ethanol, 80% ethanol, 95% ethanol, 100% I ethanol, and 100% II ethanol for 90 seconds each. The slide was then placed in a 1 / 2 xylene + 1 / 2 alcohol mixture for 2 minutes, xylene I for 5 minutes, and xylene II for 5 minutes, and was then dried in a fume hood.

[0072] 3.12 Mounting, microscopic examination, and analysis: After drying, the tissue was mounted with neutral resin. After mounting, the slide was dried in a fume hood, and photographs were taken under a microscope for analysis. It was found that LAMP5 was highly expressed in bladder cancer tissue, but was lowly expressed or not expressed in paracancerous tissue, and the difference was statistically significant (p<0.05). Figure 1-2

[0073] Example 2: LAMP5 is up-regulated in bladder cancer cell lines, so a stable cell line with knocked-down LAMP5 is constructed

[0074] 1. Western blotting

[0075] Reference: Western blotting was performed according to the following procedure:

[0076] ​1.1 Collect cells: when the density of cultured cells reaches about 85%, terminate the culture. Discard the old culture medium, add pre-cooled PBS to wash away the old culture medium, discard the PBS, add an appropriate amount (about 100 μL) of BB (Boiling Buffer cell lysis solution) according to the amount of cells, quickly scrape the cells on ice with a cell scraper, and collect them in a 1.5 mL EP tube, and place it in a metal bath at 100°C for 5 min;

[0077] 1.2 Ultrasonic: set the ultrasonic amplitude according to the sample amount, and the ultrasonic time is 2 min (1 s / time, interval 1 s);

[0078] 1.3 Measure the volume and concentration of protein: place it in a centrifuge at 13000 rpm for 5 min, use a pipette to aspirate the supernatant and measure the volume, and then transfer the supernatant to a new 1.5 mL EP tube; use a Nanodrop spectrophotometer to measure the protein sample concentration, measure each sample 2-3 times, and take the average value;

[0079] 1.4 Volumetric: input the measured protein sample volume, concentration in the volumetric electronic template, calculate the volume of BB and 6xSB needed to be added, and dilute the sample to the same concentration;

[0080] 1.5 SDS-PAGE electrophoresis: place the prepared gel plate in the electrophoresis tank, add the newly prepared 1xRunning Buffer to the inner slot of the gel plate, gently pull out the comb and remove the residual gel in the hole with a pipette; add the protein marker and the treated protein sample to the loading hole in order, pour the recovered 1xRunning Buffer into the outer slot of the gel plate, and finally set the voltage (150V) to start; stop when the indicator bromophenol blue runs to the bottom of the gel;

[0081] 1.6 Transfer membrane: soak the sponge and filter paper in pre-cooled 1xTransfer Buffer in advance and remove the bubbles; then activate the PVDF membrane in methanol for 30 s, and after activation, transfer it to 1xTransfer Buffer to remove methanol repeatedly; place it in the transfer membrane box from negative to positive in the order of sponge, filter paper, gel, PVDF membrane, filter paper, and sponge; finally set the voltage to 25V and the time according to the experimental needs;

[0082] 1.7 Milk blocking: after the transfer is completed, take out the PVDF membrane and mark it, add 1xTBS to the shaker and wash for 5 min, then add skimmed milk to the shaker and block at room temperature for 1 h;

[0083] 1.8 Incubate the primary antibody: recover the milk, wash the membrane with 1xTBS for 5 minutes each time, a total of 3 times, place the membrane in the antibody incubation box and label it, add the corresponding primary antibody, incubate overnight on a 4°C shaker;

[0084] 1.9 Incubate the secondary antibody: recover the primary antibody, wash the membrane with 1xTBST for 5 minutes each time, a total of 3 times; shake off the residual liquid in the antibody box, add the corresponding secondary antibody to the primary antibody, incubate for 3 hours on a 4°C shaker;

[0085] 1.10 Development: recover the secondary antibody, wash the membrane with 1xTBST for 5 minutes each time, a total of 3 times, and then wash it with 1xTBS for 10 minutes; dilute the developing solution with 1xTBS, according to the strength of the antibody, place the PVDF membrane in the developing solution for a few seconds, and scan the membrane with a Typhoon FLA 7000 scanner to obtain the image.

[0086] 1.11 Result processing: analyze the target band and find that LAMP5 is up-regulated in human bladder cancer cell lines compared with normal bladder cells SV-HUC-1, and the expression amount in bladder cancer J82 and T24T cells is relatively high. Figure 2-1 ). GAPDH was used as an internal reference.

[0087] 2. Construct a stable cell line with LAMP5 knockdown

[0088] Select bladder cancer cell lines J82 and T24T with relatively high LAMP5 expression, Beijing Qikang Biotechnology Co., Ltd. constructs PTSB-SH-copGFP-2A-PURO-LAMP5 knockdown plasmid (containing one control plasmid Nonsense and four knockdown plasmids); according to the slow virus transfection system method, transfect Nonsense control plasmid and LAMP5-shRNA-1, LAMP5-shRNA-2, LAMP5-shRNA-3, LAMP5-shRNA-4 knockdown plasmid into J82, T24T cells respectively, to establish stable transfection cells J82-LAMP5-shRNA, T24T-LAMP5-shRNA and its control J82-Nonsense, T24T-Nonsense, and verify the knockdown efficiency by Western Blot experiment. The results show that T24T and J82 cells successfully knock down LAMP5 protein expression, and GAPDH was used as an internal reference Figure 2-2 ).

[0089] Note: LAMP5-shRNA-1, LAMP5-shRNA-2, LAMP5-shRNA-3, LAMP5-shRNA-4 knockdown plasmid is clearly informed in the shRNA Data Instruction Manual of Qikang Biotechnology.

[0090] Example 3, LAMP5 significantly promotes the in vitro proliferation ability of bladder cancer cells

[0091] 1. ATP experiment was used to detect the change of growth activity of tumor cells J82-shLAMP5#1, J82-shLAMP5#2, T24T-shLAMP5#1, T24T-shLAMP5#2 compared with control J82-Nonsense, T24T-Nonsense cells, as shown in Figure 3 A-B.

[0092] The specific steps are as follows: 0.25% trypsin was used to digest cells in logarithmic growth phase, and a single cell suspension was prepared by blowing with medium. After counting, the corresponding cell suspension volume was taken to add corresponding cell number to the 96-well plate. After the cells adhered, the corresponding cells were taken out from the incubator, and the state was observed under a microscope. ATP detection reagent was taken out from -20℃ and dissolved at room temperature. The old culture medium in the 96-well plate was shaken off, 25μl of PBS was added to each well, and then 25μl of ATP detection reagent was added to each well, avoiding light operation. Avoid light, shake on the oscillator for 3min, and stand at room temperature for 10min. The cell lysate in the 96-well plate was transferred to a light-proof plate, and 40μl was added to each well. The machine was detected.

[0093] Note: Cell plating will plate three plates at the same time. After 24h of culture, the ATP value of the corresponding cell plate is measured as day 1 ATP value, after 72h of culture, the ATP value of the corresponding cell plate is measured as day 3 ATP value, and after 120h of culture, the ATP value of the corresponding cell plate is measured as day 5 ATP value.

[0094] The results are: knockdown of LAMP5 significantly inhibits the cell growth activity of bladder cancer cells, indicating that LAMP5 promotes the in vitro proliferation ability of bladder cancer cells.

[0095] 2. Soft agar colony formation experiment was used to evaluate the anchorage-independent malignant proliferation ability of tumor cells J82-shLAMP5#1, J82-shLAMP5#2, T24T-shLAMP5#1, T24T-shLAMP5#2 compared with control J82-Nonsense, T24T-Nonsense cells, as shown in Figure 3 C-D.

[0096] The specific steps are as follows: preheated medium and agar (1.25%) were added to a 15 mL centrifuge tube in a ratio of 3:2, mixed well, and then added to a six-well plate as soon as possible (3 mL / well). Care should be taken not to produce bubbles when plating. After plating, it was allowed to solidify at room temperature for about 2h, and the medium system was as follows:

[0097] Reagent Amount 2 x Basal Medium Eagle 4 mL FBS 1 mL PBS 1 mL Double antibody 0.1 mL Glutamine 0.1 mL 7.5% NaHCO3 0.1 mL

[0098] The prepared cells were counted and 1 x 10 4 The cells were counted and added to the prepared medium. The medium mixed with the cell suspension was added to the corresponding proportion of agar, and then mixed well with a pipette gun. Immediately after adding the corresponding underlayer glue, attention should be paid to avoid air bubbles during plating. After plating, it was allowed to solidify at room temperature for about 2 hours. Finally, the 6-well plate was sealed with sealing film, and then placed in a 37°C 5% carbon dioxide cell incubator for continuous culture. After about 7 days of culture, the growth state of the clones was observed. After 3-4 weeks of culture, the cells were counted and photographed under a microscope with a 5x lens. The colony formation rate was calculated.

[0099] The results showed that knocking down LAMP5 significantly inhibited the anchorage-independent growth ability of bladder cancer cells, indicating that LAMP5 can promote the malignant growth of bladder cancer cells.

[0100] Example 4, LAMP5 significantly promotes the formation of ectopic transplanted tumors in nude mice

[0101] The right side of the back of 3 groups of 4-5 week old BALA / c nude mice were injected with 2 x 10 6 After 4-5 weeks of subcutaneous tumor formation, the nude mice were anesthetized and the tumors were dissected for photography and weighing according to the Wenzhou Medical University Animal Experiment Management Method. The results directly showed that knocking down LAMP5 significantly inhibited the size and weight of subcutaneous tumors in nude mice Figure 4 A-B).

[0102] To further explore the role of LAMP5 in the proliferation of bladder cancer cells in vivo, the subcutaneous tumor tissue was cut off after the nude mice were sacrificed for immunohistochemical staining, and the expression level of Ki67 (a cell proliferation-related marker) was detected. The results are shown in Figure 4 C-D: Compared with the control group (LAMP5-Nonsense), the expression of Ki67 decreased after knocking down LAMP5, indicating that the proliferation of tumor cells was significantly inhibited when the expression of LAMP5 was knocked down.

[0103] In summary, the results showed that knocking down LAMP5 significantly inhibited the in vivo proliferation of bladder cancer cells, indicating that LAMP5 has a role in promoting the proliferation of bladder cancer cells in vitro and in vivo, fully demonstrating that LAMP5 plays an important role in the growth and proliferation of bladder cancer cells.

[0104] Example 5, Compared with stage I and II bladder cancer patients, the mRNA level of LAMP5 was significantly higher in stage III and IV bladder cancer patients

[0105] In order to clarify the possible role of LAMP5 in bladder cancer metastasis, the present application uses TCGA database to analyze the LAMP5 mRNA level of 124 cases of stage I and II, 256 cases of stage III and IV bladder cancer patients, and the results are shown in Figure 5 The mRNA level of LAMP5 in stage III and IV bladder cancer patients is significantly higher than that in stage I and II bladder cancer patients, and the above results show that the mRNA level of LAMP5 is positively correlated with bladder cancer metastasis. Therefore, it is speculated that LAMP5 promotes the metastasis of bladder cancer.

[0106] Example 6, LAMP5 significantly promotes the in vitro migration and invasion ability of bladder cancer cells

[0107] The effect of LAMP5 knockdown on the migration and invasion of bladder cancer cells was investigated by Transwell / Invasion experiment method. J82 / T24T (shLAMP5#1, shLAMP5#2) and its control cells were used for the experiment. The specific operation is as follows:

[0108] In the Transwell experiment, J82 / T24T (shLAMP5#1, shLAMP5#2) and its control cells were resuspended in 0.1% FBS medium and then seeded in the upper chamber of the migration and invasion chamber. The lower chamber used full culture medium. After 24h, 4% paraformaldehyde was used for fixation for 30 min, 100% methanol was used for permeation for 30 min, and Giemsa staining solution was used for staining in the dark for 30 min. Images were collected (Fig. Figure 6 A, 6C), and the number of migrated and infiltrated cells was quantitatively analyzed, as shown in Figure 6 B, 6D, and the difference was statistically significant.

[0109] The results show that knockdown of LAMP5 significantly inhibits the in vitro migration and invasion ability of bladder cancer cells, indicating that LAMP5 plays an important role in the metastasis of bladder cancer cells.

[0110] Example 7, LAMP5 significantly promotes the formation of lung metastasis in nude mice

[0111] 1. Animal feeding

[0112] Twenty-one 4-week-old BALB / c nude mice were purchased and normally fed in the SPF level animal laboratory. All animal experiments were in accordance with the requirements of the Wenzhou Medical University Animal Committee;

[0113] 2. Expansion of target cells T24T (Nonsense), T24T (LAMP5 shRNA#1), T24T (LAMP5 shRNA#2);

[0114] 3. Digest the target cells in logarithmic growth phase, centrifuge, discard the culture medium, wash once with PBS, and discard the PBS;

[0115] 4. Resuspend the cell pellet with 1 mL PBS, and take 1 μL of the cell suspension in 1 mL PBS for dilution counting;

[0116] 5. Calculate the required cell amount: inject 100 μL of the cell suspension into the tail vein of each nude mouse, and each 100 μL of the cell suspension contains 2.5 × 10 6 cells;

[0117] 6. Animal room operation: first, tag each nude mouse for marking, and randomly divide 21 nude mice into 3 groups with 7 mice in each group; inject 100 μL of the prepared corresponding cell suspension into the tail vein of each nude mouse, and feed them in separate cages;

[0118] 7. Normally feed the nude mice for about 10 weeks, anesthetize the nude mice with 0.5% sodium pentobarbital, sacrifice them, dissect the whole lung tissue of the nude mice, and fix it in Bouin's fixative (saturated picric acid: formalin: glacial acetic acid 15:5:1) for 24-36 h, then take photos to count the number of metastatic nodules, as shown in Figs. A-7B. Figure 7

[0119] The results show that knocking down LAMP5 can inhibit the formation of lung metastatic nodules in nude mice, indicating that LAMP5 promotes the metastasis of bladder cancer cells in vivo.

[0120] Finally, it should be noted that the above only lists several specific embodiments of the present application. Obviously, the present application is not limited to the above embodiments, and there can be many variations. All variations that can be directly derived or inferred from the disclosure of the present application by those of ordinary skill in the art should be considered within the scope of the present application.​

Claims

1. The application of LAMP5 as a specific biomarker in the preparation of reagent kits for the diagnosis and treatment of bladder cancer, characterized by: The specific biomarker is the LAMP5 protein.

2. The application according to claim 1, characterized in that: The kit includes an antibody that specifically binds to the LAMP5 protein.

3. The application of LAMP5 gene expression inhibitors in the preparation of drugs for treating bladder cancer, characterized by: The inhibitor is an shRNA plasmid that inhibits LAMP5 gene expression.

4. The application according to claim 3, characterized in that: Inhibits bladder cancer proliferation and metastasis.

5. The application according to claim 4, characterized in that: Inhibiting LAMP5 can suppress the proliferation of bladder cancer cells in vitro and in vivo.

6. The application according to claim 5, characterized in that: It can inhibit the in vitro migration and invasion of bladder cancer cells, and also inhibit the in vivo metastasis of bladder cancer cells.

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