Gastric cancer evaluation reagent based on expression level of TMEM43 protein and application thereof

Abstract

This invention discloses a gastric cancer assessment reagent and its application based on the expression level of TMEM43 protein, belonging to the field of precision oncology diagnosis and treatment technology. This invention is the first to discover that the TMEM43 protein is located in the endoplasmic reticulum and endoplasmic reticulum-enriched cytoplasmic regions of gastric cancer cells, and is significantly highly expressed in gastric cancer tissues and metastatic lesions. Its protein expression level is closely related to the clinical stage of gastric cancer, lymph node metastasis, peritoneal metastasis, abnormal coupling of MAMs (metastatic arteries), and patient prognosis. The TMEM43 protein regulates the structure of MAMs through interaction with MGST3, thereby driving gastric cancer EMT and invasion and metastasis, and can serve as a specific protein marker for gastric cancer metastasis and prognosis assessment. The assessment reagent provided by this invention is adaptable to various detection types such as tissue sections, cell samples, and tissue proteins. It is simple to operate, provides stable results, and offers intuitive interpretation. It can be used for early diagnosis of gastric cancer, metastasis stratification, and targeted therapy benefit screening, providing a new tool for personalized diagnosis and treatment of gastric cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a gastric cancer assessment reagent and its application based on the expression level of TMEM43 protein. Background Technology

[0002] The metastasis mechanism of gastric cancer is complex, and the lack of highly specific and sensitive protein biomarkers for clinical assessment is a key factor limiting the effectiveness of gastric cancer diagnosis and treatment. Currently, commonly used gastric cancer assessment proteins such as CEA, CA199, and CA724 have shortcomings such as low specificity, poor early detection rate, and weak association with metastasis mechanisms, failing to meet the needs of precise stratification. Although targets such as HER2 and CLDN18.2 have been used clinically, they are only applicable to a small number of patients and have limited efficacy in assessing advanced gastric cancer dominated by metastasis.

[0003] Mitochondrial-associated endoplasmic reticulum (MAMs) are crucial organelle structures regulating tumor metastasis. Abnormal coupling of these MAMs can significantly enhance the invasion and migration of gastric cancer cells, but there are currently no clinical assessment reagents targeting the regulatory proteins of MAMs. TMEM43, an endoplasmic reticulum transmembrane protein, has been shown to be associated with malignant progression in various tumors and can promote tumor proliferation and metastasis by regulating signaling pathways. However, current research has not elucidated its mechanism of action through MAMs in gastric cancer, and no gastric cancer assessment reagent based on the TMEM43 protein has been developed.

[0004] Current gastric cancer protein detection reagents mostly target secretory proteins or cell membrane receptors, resulting in insufficient coverage of organelle interaction regulatory proteins. This leads to issues such as target homogeneity, poor mechanism correlation, and limited assessment efficacy. Therefore, developing gastric cancer protein assessment reagents that target core regulatory proteins of MAMs, possess high specificity, and are directly related to metastasis mechanisms is of great significance for improving the early warning, prognosis, and personalized treatment of gastric cancer metastasis. Summary of the Invention

[0005] The purpose of this invention is to provide a gastric cancer assessment reagent based on the expression level of TMEM43 protein, its preparation method and application, in order to solve the technical defects of existing gastric cancer protein assessment markers, such as poor specificity, low early detection rate, weak association with metastasis mechanism, and lack of targeted mitochondrial-associated endoplasmic reticulum membrane (MAM) regulatory proteins, and to achieve efficient, accurate and intuitive assessment of gastric cancer invasion and metastasis risk, clinical stage, peritoneal metastasis and patient prognosis.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: A gastric cancer assessment reagent based on TMEM43 protein expression level includes a primary antibody working solution and a secondary antibody working solution. The primary antibody working solution includes a TMEM43 protein-specific antibody, and the secondary antibody working solution includes horseradish peroxidase-labeled goat anti-rabbit IgG.

[0007] Preferably, the primary antibody working solution includes TBST buffer.

[0008] Preferably, the TBST buffer comprises 10-30 mmol / L Tris-HCl, 120-180 mmol / L NaCl, and 0.05-0.2% Tween-20.

[0009] Preferably, the volume ratio of TMEM43 protein-specific antibody to TBST buffer is 1:200~1000.

[0010] Preferably, the working solution for the secondary antibody includes TBST buffer.

[0011] Preferably, the TBST buffer comprises 10-30 mmol / L Tris-HCl, 120-180 mmol / L NaCl, and 0.05-0.2% Tween-20.

[0012] Preferably, the volume ratio of horseradish peroxidase-labeled goat anti-rabbit IgG to TBST buffer is 1:1000~3000.

[0013] Preferably, the gastric cancer assessment reagent includes an antigen retrieval solution, which is a 0.01 mol / L pH 6.0 citrate antigen retrieval solution.

[0014] Preferably, the gastric cancer assessment reagent includes a blocking solution, which is a blocking solution of normal goat serum.

[0015] Preferably, the primary antibody working solution comprises 2-amino-5-methylbenzoic acid.

[0016] Preferably, the final concentration of 2-amino-5-methylbenzoic acid in the primary antibody working solution is 5~12 μmol / L.

[0017] 2-Amino-5-methylbenzoic acid can significantly improve the positive staining intensity and positive cell recognition rate in immunohistochemical detection, improve the detection signal-to-noise ratio, and make the TMEM43 protein expression signal clearer and more stable in interpretation. It is speculated that its mechanism of action may be: improving the antibody binding microenvironment, maintaining the spatial conformation of the TMEM43 protein antigen epitope, thereby enhancing the binding affinity and binding stability of TMEM43-specific monoclonal antibodies to target antigens, reducing antigen-antibody binding steric hindrance, and reducing non-specific hydrophobic binding.

[0018] More preferably, the primary antibody working solution also includes 3-hydroxy-2-naphthoic acid, the final concentration of which is 1-4 μmol / L.

[0019] 3-Hydroxy-2-naphthoic acid can effectively reduce non-specific adsorption of tissue section background, improve staining specificity and uniformity, and still clearly show TMEM43 protein positive signal in low abundance samples, improve detection sensitivity and result repeatability, and further enhance the binding efficiency of TMEM43 antigen and specific antibody; it is speculated that its mechanism of action may be: optimizing the charge distribution at the antigen-antibody binding interface.

[0020] This invention also discloses a method for preparing a gastric cancer assessment reagent, comprising the following steps: Rabbit monoclonal antibody targeting the conserved antigenic epitope of human TMEM43 protein was selected as the primary antibody, and horseradish peroxidase-labeled goat anti-rabbit IgG was selected as the secondary antibody. The primary antibody was diluted with TBST buffer containing 4-6% bovine serum albumin at pH 7.3-7.5 to prepare the working solution, and the secondary antibody was diluted with TBST buffer containing 4-6% bovine serum albumin at pH 7.3-7.5 to prepare the working solution. Antigen retrieval solution, blocking solution, washing solution, colorimetric solution, counterstaining solution and mounting medium were prepared and packaged to obtain the gastric cancer assessment reagent.

[0021] This invention also discloses a method for detecting the expression level of TMEM43 protein in in vitro samples, comprising: Gastric cancer tissue was dewaxed to water using paraffin sections, and antigen retrieval was performed under high temperature and high pressure with citrate buffer to block endogenous peroxidase. After washing with TBST buffer, block with blocking buffer, incubate with primary antibody working solution at 4°C, wash again, and then incubate with secondary antibody working solution. DAB developing solution, hematoxylin counterstaining, differentiation, blue reversion, dehydration and clearing, mounting; A semi-quantitative scoring method was used, with scores based on a combination of the proportion of positive cells in the endoplasmic reticulum enriched region and the staining intensity.

[0022] This invention also discloses the application of gastric cancer assessment reagents in the preparation of gastric cancer assessment products. The gastric cancer assessment products are compatible with at least one of paraffin sections of gastric cancer tissue, fresh tissue samples, and gastric cancer cell samples.

[0023] Preferably, the evaluation reagent uses TMEM43 protein alone as a marker, or in combination with one or more of CEA, CA199, CA724, HER2, and CLDN18.2.

[0024] This invention also provides a method for preparing a gastric cancer assessment kit and a method for detecting protein expression levels, as detailed below: Step 1: Preparation of the Gastric Cancer Assessment Kit: The gastric cancer assessment kit includes a primary antibody working solution, a secondary antibody working solution, an antigen retrieval solution, a blocking solution, a washing solution, a chromogenic solution, a counterstaining solution, and a mounting medium. A rabbit monoclonal antibody targeting a conserved antigenic epitope in the endoplasmic reticulum region of human TMEM43 protein is selected as the primary antibody, and horseradish peroxidase-labeled goat anti-rabbit IgG is selected as the secondary antibody. The primary antibody is diluted with TBST buffer containing 4-6% bovine serum albumin at pH 7.3-7.5 to prepare the primary antibody working solution, and the secondary antibody is diluted with TBST buffer containing 4-6% bovine serum albumin at pH 7.3-7.5 to prepare the secondary antibody working solution.

[0025] Preferably, the antigen retrieval solution is a citrate antigen retrieval solution with a pH of 5.9-6.1 and a concentration of 0.005-0.02 mol / L.

[0026] Preferably, the blocking solution is normal goat serum blocking solution.

[0027] Preferably, the washing solution is a TBST buffer solution with a pH of 7.3 to 7.5.

[0028] Preferably, the colorimetric solution is a DAB colorimetric solution.

[0029] Preferably, the counterstaining solution is a hematoxylin counterstaining solution.

[0030] Preferably, the sealing medium is a neutral resin sealing medium.

[0031] Preferably, the TBST buffer comprises 10-30 mmol / L Tris-HCl, 120-180 mmol / L NaCl, and 0.05-0.2% Tween-20.

[0032] Preferably, the volume ratio of the primary antibody to TBST buffer is 1:200~1000.

[0033] Preferably, the volume ratio of the secondary antibody to TBST buffer is 1:1000~3000.

[0034] Preferably, the primary antibody working solution comprises 2-amino-5-methylbenzoic acid.

[0035] Preferably, the final concentration of 2-amino-5-methylbenzoic acid in the primary antibody working solution is 5~12 μmol / L.

[0036] More preferably, the primary antibody working solution also includes 3-hydroxy-2-naphthoic acid.

[0037] More preferably, the final concentration of 3-hydroxy-2-naphthoic acid in the primary antibody working solution is 1~4 μmol / L.

[0038] Step 2: Detecting protein expression levels: Take paraffin sections of gastric cancer tissue, dewax to water using standard procedures, and perform antigen retrieval using citrate buffer under high temperature and high pressure for 1-3 minutes, followed by natural cooling to room temperature; add 2-4% H2O2 solution and incubate at room temperature in the dark for 5-15 minutes to block endogenous peroxidase; wash sections 2-4 times with TBST buffer, 3-8 minutes each time; add normal goat serum blocking solution and block at 36-38℃ for 20-40 minutes; discard the blocking solution, add primary antibody working solution, and place in a humidified chamber at 0-5℃. Incubate for 10-15 hours; wash 2-4 times with TBST buffer, 3-10 min each time, add secondary antibody working solution, and incubate at 36-38℃ for 20-40 min; after thorough washing with TBST buffer, add DAB chromogenic solution and develop at room temperature for 3-5 min, then rinse with tap water to stop the chromogenic process; counterstain with hematoxylin for 0.5-2 min, differentiate with hydrochloric acid and alcohol, return to blue with ammonia, perform gradient dehydration and clearing, and mount with neutral resin; observe under a microscope, and the appearance of brownish-yellow granules in the endoplasmic reticulum region is used to determine positive expression of TMEM43 protein.

[0039] Preferably, a semi-quantitative scoring method is used, with a comprehensive score based on the proportion of positive cells in the cytoplasmic endoplasmic reticulum enriched area and the staining intensity. The positive cell proportion score is as follows: ≤5% = 0 points, 6%-25% = 1 point, 26%-50% = 2 points, 51%-75% = 3 points, and >75% = 4 points. The staining intensity score is as follows: no staining = 0 points, pale yellow = 1 point, brownish yellow = 2 points, and brownish brown = 3 points. The final total score is the sum of the proportion score and the intensity score. A total score ≥3 points is considered as high expression of TMEM43 protein, indicating that gastric cancer tissue has a high risk of invasion and metastasis and a poor prognosis.

[0040] This invention, employing a technical solution that uses TMEM43 protein as a specific molecular marker, combined with an optimized antibody system and synergistic components, offers the following advantages: It establishes for the first time a gastric cancer protein assessment system targeting the core protein regulated by MAMs. TMEM43 protein is specifically and highly expressed in gastric cancer tissues and metastases, and is highly correlated with lymph node metastasis, peritoneal metastasis, clinical stage, and poor prognosis, demonstrating strong detection specificity and a direct link to the metastasis mechanism. The selected antibody targets conserved epitopes with strong binding affinity; combined with synergistic components, it significantly enhances staining intensity and positive recognition rate, reduces background interference, and is compatible with various sample types, including paraffin sections and cell samples. The system is simple to operate, provides intuitive interpretation, and yields stable results. It can be used alone or in combination with existing clinical markers for the full-cycle diagnosis and treatment assessment of gastric cancer, effectively overcoming the shortcomings of traditional protein markers, such as poor specificity and weak metastasis correlation, and possesses outstanding clinical application value and translational prospects. Therefore, this invention is a gastric cancer assessment reagent and application technology based on TMEM43 protein expression levels that is highly targeted, performs excellently, has wide applicability, and combines high specificity with clinical practicality. Attached Figure Description

[0041] Figure 1 This diagram illustrates the difference in TMEM43 expression between gastric cancer tissue and adjacent normal tissue as shown by proteomics analysis.

[0042] Figure 2 This diagram illustrates the difference in TMEM43 expression between gastric cancer and normal tissues.

[0043] Figure 3 This is a schematic diagram showing the results of multiplex immunohistochemical fluorescence staining.

[0044] Figure 4 This is a schematic diagram illustrating the expression location of TMEM43.

[0045] Figure 5 This diagram illustrates the differential expression of TMEM43 in different cellular components.

[0046] Figure 6 Volcano plot of differentially expressed genes in the transcriptome of HGC27 cells with different TMEM43 expression levels.

[0047] Figure 7 A schematic diagram of HGC27 cells with different TMEM43 expression levels (MAMs).

[0048] Figure 8 A schematic diagram showing the changes in the number of MAMs and the morphology of the endoplasmic reticulum after 8 hours of treatment with Thapsigargin.

[0049] Figure 9 This is a schematic diagram of tumor tissue MAMs expressing different TMEM43.

[0050] Figure 10 This is a schematic diagram of MAMs in cancerous tissue and adjacent normal tissue of a gastric cancer patient.

[0051] Figure 11 This is a schematic diagram showing the protein expression levels of TMEM43 and MGST3 in gastric cancer and adjacent tissues.

[0052] Figure 12 This is a graph showing the correlation between co-expression of TMEM43 and MGST3 proteins and prognosis. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0055] Example 1: This example provides a method for preparing a gastric cancer assessment kit and a method for detecting protein expression levels, as detailed below.

[0056] Step 1: Preparation of the Gastric Cancer Assessment Kit: The gastric cancer assessment kit includes primary antibody working solution, secondary antibody working solution, antigen retrieval solution, blocking solution, washing solution, chromogenic solution, counterstaining solution, and mounting medium. A rabbit monoclonal antibody targeting the conserved epitope of human TMEM43 protein is selected as the primary antibody, and horseradish peroxidase-labeled goat anti-rabbit IgG is used as the secondary antibody. The primary antibody is diluted with TBST buffer (pH 7.4) containing 5% bovine serum albumin to prepare the primary antibody working solution, and the secondary antibody is diluted with TBST buffer (pH 7.4) containing 5% bovine serum albumin to prepare the secondary antibody working solution. The antigen retrieval solution is 0.01... The antigen retrieval solution was prepared using citrate solution at pH 6.0 with a concentration of mol / L. The blocking solution was normal goat serum blocking solution. The washing solution was TBST buffer at pH 7.4. The chromogenic solution was DAB chromogenic solution. The counterstaining solution was hematoxylin counterstaining solution. The mounting medium was neutral resin mounting medium. All commercially available qualified reagents were used. The TBST buffer consisted of 20 mmol / L Tris-HCl, 150 mmol / L NaCl and 0.1% Tween-20. The volume ratio of primary antibody to TBST buffer was 1:500, and the volume ratio of secondary antibody to TBST buffer was 1:2000.

[0057] Step 2: Detecting protein expression levels: Paraffin sections of gastric cancer tissue were routinely dewaxed to water, and subjected to high-temperature, high-pressure antigen retrieval using citrate buffer for 2 minutes, followed by natural cooling to room temperature. 3% H2O2 solution was added, and the sections were incubated at room temperature in the dark for 10 minutes to block endogenous peroxidase. The sections were washed three times with TBST buffer, 5 minutes each time. Normal goat serum blocking solution was added, and the sections were blocked at 37°C for 30 minutes. The blocking solution was discarded, and primary antibody working solution was added. The sections were incubated in a humidified chamber at 4°C for 12 hours. The sections were washed three times with TBST buffer, 5 minutes each time, and secondary antibody working solution was added. The sections were incubated at 37°C. Incubate for 30 min; after thorough washing with TBST buffer, add DAB chromogenic solution and incubate at room temperature for 4 min, then rinse with tap water to stop the incubation; counterstain with hematoxylin for 1 min, differentiate with hydrochloric acid alcohol, return to blue with ammonia, undergo gradient dehydration and clearing, and mount with neutral resin; observe under a microscope, and the appearance of brownish-yellow granules in the endoplasmic reticulum region is used to determine positive expression of TMEM43 protein; a semi-quantitative scoring method is used, and the ratio of positive cells in the endoplasmic reticulum enrichment region and the staining intensity are used to score the total score. A total score ≥3 points is used to determine high expression of TMEM43 protein, indicating that gastric cancer tissue has a high risk of invasion and metastasis and a poor prognostic tendency.

[0058] Example 2: The only difference between this example and Example 1 is in the preparation steps of the gastric cancer assessment kit. The primary antibody working solution of Example 2 also includes 2-amino-5-methylbenzoic acid, and the final concentration of 2-amino-5-methylbenzoic acid in the primary antibody working solution is 8 μmol / L.

[0059] Example 3: The only difference between this example and Example 2 is that in the preparation steps of the gastric cancer assessment kit, the final concentration of 2-amino-5-methylbenzoic acid in the primary antibody working solution in Example 3 is adjusted from 8 μmol / L to 10 μmol / L.

[0060] Example 4: The only difference between this example and Example 3 is in the preparation steps of the gastric cancer assessment kit. The primary antibody working solution of Example 4 also contains 3-hydroxy-2-naphthoic acid, and the final concentration of 3-hydroxy-2-naphthoic acid in the primary antibody working solution is 2 μmol / L.

[0061] Example 5: The only difference between this example and Example 4 is that in the preparation steps of the gastric cancer assessment kit, the final concentration of 3-hydroxy-2-naphthoic acid in the primary antibody working solution in Example 5 is adjusted from 2.0 μmol / L to 3 μmol / L.

[0062] Comparative Example 1: The only difference between this comparative example and Example 2 is that in the preparation steps of the gastric cancer assessment kit, the final concentration of 2-amino-5-methylbenzoic acid in the primary antibody working solution of Comparative Example 1 was adjusted from 8 μmol / L to 20 μmol / L.

[0063] Comparative Example 2: The only difference between this comparative example and Example 3 is that in the preparation steps of the gastric cancer assessment kit, 2-amino-5-methylbenzoic acid was replaced with benzoic acid, and the final concentration of benzoic acid in the primary antibody working solution was 10 μmol / L.

[0064] Comparative Example 3: The only difference between this comparative example and Example 5 is that in the preparation steps of the gastric cancer assessment kit, 3-hydroxy-2-naphthoic acid was changed to 2-naphthoic acid, and the final concentration of 2-naphthoic acid in the primary antibody working solution was 3 μmol / L.

[0065] Experimental Case 1: Expression characteristics and clinical relevance analysis of TMEM43 protein in gastric cancer tissue.

[0066] Test samples: cancer tissue and adjacent normal tissue samples from 112 patients with gastric adenocarcinoma.

[0067] Methods: Cancerous tissues and paired adjacent normal tissues from 112 patients with gastric adenocarcinoma were selected as test samples. Label-free quantitative proteomics and multiplex fluorescence immunohistochemistry were used to detect the expression level and specific location of TMEM43 protein in different tissues. Simultaneously, transcriptomic data and clinicopathological data from 875 gastric cancer patients with complete clinical staging, lymph node metastasis, peritoneal metastasis, and survival follow-up information were collected. SPSS statistical software was used to analyze the correlation between TMEM43 expression level and clinicopathological features. Kaplan-Meier survival curves were plotted, and statistical differences were analyzed using the Log-rank test. Furthermore, gastric cancer tissues from different clinical stages were fixed, dehydrated, embedded, and ultrathinly sectioned. Transmission electron microscopy was used to observe and quantitatively count the number of mitochondrial-endoplasmic reticulum structural couplings (MAMs) in each group of samples. This comprehensively analyzed the expression characteristics of TMEM43 in gastric cancer tissues and its correlation with clinical progression, prognosis, and MAM abnormalities.

[0068] Proteomic analysis of gastric cancer tissue and adjacent normal tissue showed differences in TMEM43 expression, such as... Figure 1 As shown, the expression differences of TMEM43 in gastric cancer and normal tissues are as follows: Figure 2 As shown, the results of multiplex immunohistochemical fluorescence staining are as follows: Figure 3As shown, proteomics and multiplex immunofluorescence results consistently confirmed that the expression level of TMEM43 protein in gastric cancer tissues and metastatic lesions such as peritoneum and liver was significantly higher than that in adjacent normal tissues. Furthermore, its expression was further increased in samples with lymph node and peritoneal metastases. Statistical analysis showed that high TMEM43 expression was significantly positively correlated with malignant pathological features such as stage III / IV gastric cancer, positive lymph node metastasis, and positive peritoneal metastasis. Survival analysis results indicated that the overall survival of patients with high TMEM43 expression was significantly shorter than that of patients with low expression, and the risk of recurrence and death was significantly increased. Simultaneously, transmission electron microscopy results showed that the number of MAMs in tumor tissues of stage III and IV gastric cancer patients was significantly higher than that of stage I and II patients, and the number of MAMs was positively correlated with the TMEM43 expression level. This fully demonstrates that TMEM43 is a molecular marker specifically highly expressed in gastric cancer, and its abnormal expression is closely related to gastric cancer invasion and metastasis, poor prognosis, and abnormal coupling with MAMs, possessing important clinical value for gastric cancer metastasis stratification and prognostic assessment.

[0069] Experimental Example 2: Expression and subcellular localization analysis of TMEM43 protein in gastric cancer cell lines.

[0070] Test samples: human gastric cancer cell lines BGC823, GCIY, HGC27, NUGC3, AGS, and SNU668.

[0071] Methods: Six commonly used human gastric cancer cell lines (BGC823, GCIY, HGC27, NUGC3, AGS, and SNU668) were selected as test samples. The basal expression level of TMEM43 protein in each cell line was detected and quantitatively compared using Western blot. Cell smear immunofluorescence colocalization assays were performed, using Calnexin as an endoplasmic reticulum (ER)-specific marker. The colocalization of TMEM43 with ER markers was observed and the correlation coefficient was calculated. ER component separation kits were used to extract total cellular proteins, cytoplasmic proteins, ER proteins, and mitochondrial proteins stepwise. Western blot was then used to verify the distribution of TMEM43 in each subcellular component. Transcriptome sequencing was performed on TMEM43-knockdown HGC27 cells and negative control cells. Bioinformatics methods were used to analyze differentially expressed genes, focusing on changes in ER stress-related pathway genes. The expression profile, subcellular localization, and related biological functions of TMEM43 in gastric cancer cells were systematically clarified.

[0072] Detecting the expression location of TMEM43 as follows Figure 4 As shown, the expression differences of TMEM43 in different cellular components were detected as follows: Figure 5 As shown, the differential gene volcano plot of the HGC27 cell transcriptome with different TMEM43 expression levels is as follows: Figure 6As shown, Western blot results indicated that TMEM43 was expressed to varying degrees in all six gastric cancer cell lines, with the highest expression levels in HGC27 and NUGC3 cells, and relatively low expression levels in AGS and SNU668 cells. Immunofluorescence co-localization and subcellular component separation experiments clearly confirmed that TMEM43 protein is mainly located in the endoplasmic reticulum (ER) of gastric cancer cells, highly co-localized with the ER marker Calnexin, and almost not distributed in other subcellular structures such as the cytoplasm and mitochondria. Transcriptome sequencing analysis showed that knockdown of TMEM43 significantly upregulated the expression levels of key ER stress genes such as IRE1a and GADD153, suggesting that TMEM43 is involved in maintaining the structural and functional homeostasis of the ER in gastric cancer cells, and its absence triggers a significant ER stress response. This experiment is the first to accurately determine the ER localization characteristics of TMEM43 in gastric cancer cells, providing a solid subcellular structural basis for subsequent research on its molecular mechanism of regulating gastric cancer metastasis through MAMs.

[0073] Experimental Example 3: Functional verification of TMEM43 protein regulating the formation of MAMs in gastric cancer cells.

[0074] Test samples: TMEM43 knockdown group, negative control group HGC27 cells; mouse subcutaneous xenograft tissue.

[0075] Test methods: HGC27 gastric cancer cells from the TMEM43 knockdown group (shTMEM43) and the negative control group (shCtrl), as well as the corresponding mouse subcutaneous xenograft tissue, were used as test samples. All samples were fixed with 2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide, dehydrated with graded ethanol, embedded in epoxy resin, sectioned at 70 nm, and stained with uranium acetate-lead citrate. After treatment, the samples were observed and photographed using cryo-transmission electron microscopy. No less than 10 fields of view were randomly selected. The number of MAMs was determined according to the standard that the distance between mitochondria and endoplasmic reticulum was less than 30 nm and the contact length was greater than 100 nm. At the same time, a Thapsigargin endoplasmic reticulum stress inducer treatment group and a TMEM43 overexpression recovery experimental group were set up to observe the changes in the number of MAMs under the same detection standards. The regulatory role of TMEM43 on the formation and homeostasis of gastric cancer MAMs was systematically verified from two levels: in vitro cells and in vivo animal models.

[0076] HGC27 cells with different TMEM43 expression levels, such as MAMs Figure 7 As shown, the changes in the number of MAMs and the morphology of the endoplasmic reticulum after 8 hours of Thapsigargin treatment are as follows: Figure 8 As shown, tumor tissue MAMs expressing different TMEM43 expression levels are as follows: Figure 9As shown, cryo-transmission electron microscopy and quantitative statistical results revealed that after TMEM43 knockdown, the number of MAMs in HGC27 gastric cancer cells was significantly reduced compared to the control group, and the structural coupling between mitochondria and endoplasmic reticulum was significantly disrupted. Similarly, in mouse subcutaneous xenograft tissue, the number of MAMs in the shTMEM43 group was significantly lower than that in the shCtrl group, and the in vitro and in vivo experimental results were completely consistent. After endoplasmic reticulum stress induced by Thapsigargin, the number of MAMs in the control group did not change significantly, while the number of MAMs in the TMEM43 knockdown group was still significantly lower and accompanied by typical stress morphology such as endoplasmic reticulum swelling. At the same time, TMEM43 overexpression could significantly reverse the MAM reduction phenotype caused by knockdown, which fully confirms that TMEM43 is the core key molecule regulating the abnormal coupling of MAMs in gastric cancer cells. It participates in the regulation of organelle interactions in gastric cancer cells by maintaining the structural stability of MAMs, revealing an important mechanism by which TMEM43 drives gastric cancer invasion and metastasis at the organelle structure level.

[0077] Experiment 4: Correlation analysis of TMEM43 / MGST3 protein co-expression with malignant progression and prognosis of gastric cancer Test samples: 4 pairs of gastric cancer and adjacent tissue samples; 433 gastric cancer tissue samples with complete clinical information.

[0078] Methods: Four pairs of gastric cancer and their paired adjacent normal tissues, along with 433 gastric cancer tissue samples with complete clinicopathological information and survival data, were selected as test samples. Western blot was used to detect the expression levels of TMEM43 and MGST3 proteins in tissues and cells, and the correlation between their expression was analyzed. Exogenous and endogenous co-immunoprecipitation experiments were used in 293T cells and HGC27 cells to verify the protein interaction between TMEM43 and MGST3. Simultaneously, changes in MGST3 protein levels were detected in gastric cancer cell models with TMEM43 knockdown and overexpression to clarify the regulatory role of TMEM43 on MGST3. Based on bioinformatics analysis, the 433 patients were divided into three groups: high TMEM43 / MGST3 expression, high TMEM43 / MGST3 expression, and low TMEM43 / MGST3 expression. Kaplan-Meier survival analysis and Log-rank test were used to explore the association between the synergistic expression of these two proteins and the malignant progression of gastric cancer and patient prognosis.

[0079] MAMs in cancerous tissue and adjacent normal tissue of gastric cancer patients, such as Figure 10 As shown, the protein expression levels of TMEM43 and MGST3 in gastric cancer and adjacent tissues are as follows: Figure 11 As shown in the figure, the correlation between TMEM43 and MGST3 protein co-expression and prognosis is as follows: Figure 12As shown, Western blot results revealed that both TMEM43 and MGST3 exhibited high expression trends in gastric cancer tissues, and their expression levels were significantly positively correlated. Immunoprecipitation experiments confirmed a direct and stable endogenous protein interaction between TMEM43 and MGST3. MGST3 is located in the outer mitochondrial membrane, while TMEM43 is located in the endoplasmic reticulum. Together, they constitute the core linker complex of MAMs. Cellular experiments further demonstrated that TMEM43 can positively stabilize MGST3 protein expression by inhibiting its ubiquitination and degradation. Survival analysis showed that patients with high synergistic expression of TMEM43 and MGST3 had a higher risk of lymph node metastasis and peritoneal metastasis, shorter overall survival, and significantly worse prognosis than patients with high expression of either TMEM43 or MGST3. This indicates that the TMEM43 / MGST3 axis is a key functional axis regulating abnormal MAMs and invasion and metastasis in gastric cancer. The combined assessment of the two markers has better efficacy in metastasis risk stratification and prognostic judgment than a single marker, providing a novel and highly specific combined marker for precision diagnosis and treatment of gastric cancer.

[0080] Experimental Example 5: Gastric Cancer Assessment Kit for Immunohistochemical Staining Intensity of TMEM43 Protein.

[0081] Test samples: Gastric cancer assessment kits prepared in Examples 1-5 and Comparative Examples 1-3.

[0082] Test method: Paraffin samples of gastric cancer tissue from the same gastric cancer patient were divided into 8 groups, corresponding to Example 1, Example 2, Example 3, Example 4, Example 5, Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively. The experiments were conducted according to the preparation method of the primary antibody working solution and the complete immunohistochemical detection process of the corresponding examples and comparative examples of the present invention. The antigen retrieval, incubation conditions, color development time, counterstaining and mounting steps were kept completely consistent. The average optical density (IOD) of TMEM43 protein positive staining in each group was detected and statistically analyzed.

[0083] The results of the immunohistochemical staining intensity test of TMEM43 protein using the gastric cancer assessment kit are shown in Table 1.

[0084] Table 1. Results of TMEM43 protein immunohistochemical staining intensity test using the gastric cancer assessment kit.

[0085] Example 1 presents the standard basic detection scheme of this invention, with stable and reliable detection results that meet the requirements of routine TMEM43 protein immunohistochemical detection. Example 2 introduces 2-amino-5-methylbenzoic acid (2-amino-5-methylbenzoic acid) based on Example 1, resulting in a significant increase in positive protein staining intensity compared to Example 1, indicating that 2-amino-5-methylbenzoic acid can effectively enhance antigen-antibody binding and optimize TMEM43 protein staining performance. Example 3 adjusts the concentration of 2-amino-5-methylbenzoic acid based on Example 2, further improving staining intensity compared to Example 2, demonstrating that within a suitable range, 2-amino-5-methylbenzoic acid exhibits superior synergistic effects with concentration adjustments. Example 4, based on the system containing the optimal amount of 2-amino-5-methylbenzoic acid in Example 3, additionally introduces 3-hydroxy-2-naphthoic acid for compound use, further improving staining intensity compared to Example 3, demonstrating a significant synergistic effect between 2-amino-5-methylbenzoic acid and 3-hydroxy-2-naphthoic acid. Example 5 optimizes the concentration of 3-hydroxy-2-naphthoic acid based on Example 4, achieving a better staining intensity than Example 4, further demonstrating that an appropriate concentration of 3-hydroxy-2-naphthoic acid can sustainably improve the staining effect of the overall detection system. Comparative Example 1 used an excessively high concentration of 2-amino-5-methylbenzoic acid, which resulted in significant binding inhibition, a marked decrease in staining intensity, and a substantial reduction in detection performance compared to Examples 2 and 3. This demonstrates that 2-amino-5-methylbenzoic acid has an optimal range of action, and excessive use can have adverse effects. In Comparative Example 2, 2-amino-5-methylbenzoic acid was replaced with benzoic acid, which has a similar structure. The staining synergistic effect was significantly lower than that of Example 3, failing to reach the synergistic level of 2-amino-5-methylbenzoic acid, demonstrating that 2-amino-5-methylbenzoic acid has unique structural specificity and technical advantages. In Comparative Example 3, 3-hydroxy-2-naphthoic acid was replaced with 2-naphthoic acid, which has a similar structure. The synergistic effect completely disappeared, and the staining effect was significantly worse than that of Example 5. This demonstrates that 3-hydroxy-2-naphthoic acid also possesses unique structural specificity and outstanding innovative technical effects.

[0086] Experimental Example 6: Gastric Cancer Assessment Kit for Detecting the Percentage of TMEM43 Protein Immunohistochemically Positive Cells.

[0087] Test samples: Gastric cancer assessment kits prepared in Examples 1-5 and Comparative Examples 1-3.

[0088] Test method: Paraffin samples of the same gastric cancer tissue as in Experiment 5 were divided into 8 groups corresponding to Examples 1-5 and Comparative Examples 1-3, with 3 biological replicates in each group. The primary antibody preparation scheme and immunohistochemical detection procedure were completely consistent with those of each example and comparative example. All experimental conditions were kept uniform. The non-specific background staining level and overall signal-to-noise ratio of each group were statistically analyzed by image grayscale analysis.

[0089] The results of the gastric cancer assessment kit for the percentage of TMEM43 protein-positive cells by immunohistochemistry are shown in Table 2.

[0090] Table 2. Results of TMEM43 protein immunohistochemical positive cell percentage assay using the gastric cancer assessment kit.

[0091] Example 1 presents the standard basic detection scheme of this invention, with stable and reliable detection results that meet the requirements of routine TMEM43 protein immunohistochemical detection. Example 2 introduces 2-amino-5-methylbenzoic acid (2-Methylbenzoic acid) based on Example 1, resulting in a significantly increased proportion of positive tumor cell recognition compared to Example 1. This indicates that 2-amino-5-methylbenzoic acid can effectively enhance the effective binding efficiency of antigen and antibody, improving the recognition ability of TMEM43 positive cells. Example 3 adjusts the concentration of 2-amino-5-methylbenzoic acid based on Example 2, further increasing the proportion of positive cells compared to Example 2. This demonstrates that within a suitable range, 2-amino-5-methylbenzoic acid exhibits superior synergistic effects with concentration adjustments. Example 4, based on the system containing the optimal amount of 2-amino-5-methylbenzoic acid in Example 3, additionally introduces 3-hydroxy-2-naphthoic acid for compound use. Compared to Example 3, the proportion of positive cell recognition continues to increase, demonstrating a significant synergistic effect between 2-amino-5-methylbenzoic acid and 3-hydroxy-2-naphthoic acid. Example 5 optimizes the concentration of 3-hydroxy-2-naphthoic acid based on Example 4, achieving a better positive cell recognition rate than in Example 4, further demonstrating that an appropriate concentration of 3-hydroxy-2-naphthoic acid can sustainably improve the positive recognition effect of the overall detection system. Comparative Example 1 used an excessively high concentration of 2-amino-5-methylbenzoic acid, resulting in a significantly lower proportion of positive cells compared to Examples 2 and 3. Effective binding was significantly inhibited, and detection performance was greatly reduced, demonstrating that 2-amino-5-methylbenzoic acid has an optimal range of action, and excessive use will have adverse effects. In Comparative Example 2, 2-amino-5-methylbenzoic acid was replaced with benzoic acid, which has a similar structure. The positive recognition enhancement effect was significantly lower than that of Example 3, and could not reach the enhancement level of 2-amino-5-methylbenzoic acid, demonstrating that 2-amino-5-methylbenzoic acid has a unique structural specificity advantage. In Comparative Example 3, 3-hydroxy-2-naphthoic acid was replaced with 2-naphthoic acid, which has a similar structure. The synergistic effect completely disappeared, and the positive cell recognition effect was significantly worse than that of Example 5, demonstrating that 3-hydroxy-2-naphthoic acid also has unique structural specificity and outstanding innovative technical effects.

[0092] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0093] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A gastric cancer evaluation reagent based on the expression level of TMEM43 protein, characterized by: The gastric cancer assessment reagent includes a primary antibody working solution and a secondary antibody working solution. The primary antibody working solution includes a TMEM43 protein-specific antibody, and the secondary antibody working solution includes horseradish peroxidase-labeled goat anti-rabbit IgG.

2. The gastric cancer evaluation reagent based on the expression level of TMEM43 protein according to claim 1, characterized by: The primary antibody working solution includes TBST buffer, which comprises 10-30 mmol / L Tris-HCl, 120-180 mmol / L NaCl and 0.05-0.2% Tween-20, and the volume ratio of the TMEM43 protein-specific antibody to the TBST buffer is 1:200-1000.

3. The gastric cancer assessment reagent based on TMEM43 protein expression level according to claim 1, characterized in that: The secondary antibody working solution includes TBST buffer, which comprises 10-30 mmol / L Tris-HCl, 120-180 mmol / L NaCl, and 0.05-0.2% Tween-20. The volume ratio of horseradish peroxidase-labeled goat anti-rabbit IgG to TBST buffer is 1:1000-3000.

4. A gastric cancer assessment reagent based on TMEM43 protein expression level according to claim 1, wherein the kit includes an antigen retrieval solution, wherein the antigen retrieval solution is a 0.005~0.02mol / L citrate antigen retrieval solution with pH 5.9~6.

1.

5. The gastric cancer assessment reagent based on TMEM43 protein expression level according to claim 1, characterized in that: The gastric cancer assessment reagent includes a blocking solution, which is a blocking solution made from normal goat serum.

6. The gastric cancer assessment reagent based on TMEM43 protein expression level according to claim 1, characterized in that: The primary antibody working solution includes 2-amino-5-methylbenzoic acid, and the final concentration of 2-amino-5-methylbenzoic acid in the primary antibody working solution is 5~12 μmol / L.

7. A method for preparing a gastric cancer assessment reagent according to any one of claims 1 to 6, characterized in that: Includes the following steps, Rabbit monoclonal antibody targeting the conserved antigenic epitope of human TMEM43 protein was selected as the primary antibody, and horseradish peroxidase-labeled goat anti-rabbit IgG was selected as the secondary antibody. The primary antibody was diluted with TBST buffer containing 4-6% bovine serum albumin at pH 7.3-7.5 to prepare the working solution, and the secondary antibody was diluted with TBST buffer containing 4-6% bovine serum albumin at pH 7.3-7.5 to prepare the working solution. Antigen retrieval solution, blocking solution, washing solution, colorimetric solution, counterstaining solution and mounting medium were prepared and packaged to obtain the gastric cancer assessment reagent.

8. A method for detecting the expression level of TMEM43 protein in ex vivo samples, characterized in that: Immunohistochemical detection using the evaluation reagents described in any one of claims 1 to 6 includes: Gastric cancer tissue was dewaxed to water using paraffin sections, and antigen retrieval was performed under high temperature and high pressure with citrate buffer to block endogenous peroxidase. After washing with TBST buffer, block with blocking buffer, incubate with primary antibody working solution at 4°C, wash again, and then incubate with secondary antibody working solution. DAB developing solution, hematoxylin counterstaining, differentiation, blue reversion, dehydration and clearing, mounting; A semi-quantitative scoring method was used, with scores based on a combination of the proportion of positive cells in the endoplasmic reticulum enriched region and the staining intensity.

9. The use of the gastric cancer assessment reagent according to any one of claims 1 to 6 in the preparation of gastric cancer assessment products, characterized in that: The gastric cancer assessment product is compatible with at least one of the following samples: paraffin sections of gastric cancer tissue, fresh tissue samples, and gastric cancer cell samples.

10. The application of the gastric cancer assessment reagent according to claim 9 in the preparation of gastric cancer assessment products, characterized in that: The evaluation reagent is used alone as a marker of TMEM43 protein, or in combination with one or more of CEA, CA199, CA724, HER2, and CLDN18.2.

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