Application of genetic regulation molecular axis in gastric lesion evolution discrimination and drug screening

Abstract

The invention provides application of a genetic regulation molecular axis in gastric lesion evolution discrimination and drug screening, and belongs to the technical field of biological medicines. The specific dynamic expression track of the lnc517368 / miR-203b-3p / SMAD2 axis in gastric lesion evolution is disclosed and defined for the first time, and brand new molecular basis and objective standard are provided for stage discrimination of gastric diseases. Meanwhile, an intervention strategy taking the axis as a target is defined, and a reliable screening and evaluation system is established for developing drugs for blocking or reversing the progress of stomach lesion. And by taking a turbid-dissolving and detoxifying formula as an example, reversible intervention of a disease evolution trajectory can be realized by adjusting the shaft, so that the method not only has important scientific theoretical value, but also shows wide conversion and application prospects in the aspects of disease diagnosis, drug research and development, clinical treatment strategy formulation and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a genetic regulatory molecular axis in the differentiation of gastric lesion progression and drug screening. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors worldwide. Its development typically follows a multi-stage process, progressing from chronic superficial gastritis to precancerous lesions of gastric cancer (PLGC), characterized by intestinal metaplasia and dysplasia, and finally developing into gastric cancer. The precancerous lesion stage is considered a critical window for intervention and reversal of the cancerous progression. Currently, the diagnosis of gastric lesions mainly relies on endoscopic observation and histopathological examination. While these methods are the gold standard, they have a degree of subjectivity and limited ability to assess the risk of dynamic disease progression and provide early warning. In terms of treatment, modern medicine lacks specific drugs that can definitively reverse the pathological changes in precancerous lesions. Clinical intervention strategies primarily focus on eliminating the underlying cause (such as eradicating Helicobacter pylori) and regular monitoring.

[0003] In recent years, with the development of molecular biology, the role of epigenetic regulation in tumorigenesis has received increasing attention. Non-coding RNAs (such as long non-coding RNAs and microRNAs), as important epigenetic regulators, participate in processes such as cell proliferation, apoptosis, and differentiation through complex networks, and their abnormal expression is closely related to the progression of various tumors. Identifying specific molecular markers and regulatory networks that play a key role in the evolution of gastric lesions, and developing diagnostic tools and intervention strategies based on these, has become a research hotspot in this field. Although some individual molecules have been reported to be associated with gastritis or gastric cancer, molecular axes that can systematically characterize the dynamic patterns of the entire process from inflammation to carcinogenesis and have clinical diagnostic and intervention guidance value are still lacking. Therefore, revealing the key molecular trajectories of gastric lesion evolution and developing their applications is of great significance for the early prevention and treatment of gastric cancer. Summary of the Invention

[0004] The purpose of this invention is to provide an application of genetic regulatory molecular axis in the discrimination of gastric lesion progression and drug screening, which solves the technical problems in the prior art of lacking specific molecular trajectories that can accurately reflect the evolution of gastric lesions from chronic inflammation to precancerous lesions and gastric cancer, and lacking effective tools for disease stage discrimination and targeted drug screening based on such molecular trajectories.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of the lnc517368 / miR-203b-3p / SMAD2 axis as a target in the preparation of products for assisting in the identification of the progression stage of gastric lesions. The lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination composed of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2. The evolutionary stages include chronic superficial gastritis, precancerous lesions of the stomach, and stomach cancer; The auxiliary discrimination is based on the fact that the expression levels of lnc517368 and SMAD2 in this axis are continuously increasing, and the expression level of miR-203b-3p shows a dynamic trajectory of first decreasing, then increasing, and then decreasing again.

[0006] Preferably, the product is a kit containing reagents for detecting the expression levels of lnc517368, miR-203b-3p, and SMAD2.

[0007] This invention also provides the application of the lnc517368 / miR-203b-3p / SMAD2 axis as a target in screening drugs for the prevention and / or treatment of precancerous lesions of the stomach. The lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination composed of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2.

[0008] Preferably, the screening includes: applying candidate substances to a gastric precancerous lesion model and identifying substances that can simultaneously downregulate the expression levels of lnc517368, miR-203b-3p, and SMAD2 in the model.

[0009] This invention also provides the application of the lnc517368 / miR-203b-3p / SMAD2 axis as a target in screening drugs for the prevention and / or treatment of gastric cancer, wherein the lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination composed of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2.

[0010] Preferably, the screening includes: applying candidate substances to a gastric cancer model and identifying substances that can simultaneously downregulate the expression levels of lnc517368 and SMAD2 in the model and upregulate the expression level of miR-203b-3p.

[0011] This invention also provides the application of the lnc517368 / miR-203b-3p / SMAD2 axis as a target in constructing a model system for simulating the evolution of gastric diseases. The lnc517368 / miR-203b-3p / SMAD2 axis refers to a molecular combination composed of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2. The model system includes models capable of representing the axis expression characteristics of chronic superficial gastritis, precancerous lesions of the stomach, and stages of gastric cancer, respectively.

[0012] The present invention also provides an application of a precancerous gastric lesion cell model in screening or evaluating candidate drugs for intervention in precancerous gastric lesions, wherein the expression levels of lnc517368, miR-203b-3p and SMAD2 are significantly increased in the precancerous gastric lesion cell model.

[0013] This invention also provides the application of the lnc517368 / miR-203b-3p / SMAD2 axis as an evaluation target in the preparation of products for evaluating the potential of drugs to intervene in precancerous lesions of the stomach. The lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination composed of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2.

[0014] This invention also provides the application of a specific dynamic expression trajectory of the lnc517368 / miR-203b-3p / SMAD2 axis in the preparation of decision support tools for the clinical management of gastric diseases. The specific dynamic expression trajectory refers to the continuous increase in the expression levels of lnc517368 and SMAD2 during the evolution from chronic superficial gastritis to precancerous lesions and gastric cancer, while the expression level of miR-203b-3p shows a trend of first decreasing, then increasing, and then decreasing again. The lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination consisting of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2.

[0015] The beneficial effects of this invention are: This invention elucidates the dynamic patterns and intervention strategies of the lnc517368 / miR-203b-3p / SMAD2 axis in the progression of gastric lesions, achieving the following significant beneficial effects: This study provides a novel dynamic molecular discrimination system. For the first time, it reveals and validates the spatiotemporal expression trajectory of the lnc517368 / miR-203b-3p / SMAD2 axis throughout the entire process of "chronic superficial gastritis → precancerous lesions of the stomach → gastric cancer" (lnc517368 / SMAD2 continuously increases, while miR-203b-3p exhibits an inverted U-shaped change). This axis can serve as a sensitive and specific combination of molecular markers, providing novel and quantifiable molecular evidence for the discrimination, dynamic monitoring, and risk stratification of the evolutionary stages of gastric lesions.

[0016] This enables early warning and precise staging of disease progression. Changes in the expression of this axis precede obvious morphological changes, and it can especially identify the transition point between the "compensation phase" and the "exhaustion phase" in the precancerous lesion (PLGC) stage of gastric cancer, providing a key time window and molecular decision support for clinical early intervention and blocking of carcinogenesis.

[0017] A drug screening and evaluation platform based on this axis was established. This invention clarifies the intervention strategy targeting this axis: for the PLGC stage, lnc517368, miR-203b-3p, and SMAD2 should be downregulated simultaneously; for the GC stage, lnc517368 / SMAD2 should be downregulated and miR-203b-3p should be upregulated. This provides clear screening targets and efficacy evaluation standards for developing novel drugs (including chemical drugs, biological drugs, and traditional Chinese medicine compound formulas) for the prevention or treatment of PLGC / GC, significantly improving the directionality and success rate of drug development.

[0018] This study provides a modern molecular mechanism explanation and efficacy evaluation benchmark for traditional Chinese medicine (TCM) compound therapy for partial granulocytic gastrointestinal (PLGC). Taking Huazhuo Jiedu formula as an example, it has been demonstrated that it can achieve reversible intervention in PLGC progression by specifically regulating this axis. This not only provides a solid scientific basis for the clinical application of traditional empirical formulas, but also provides objective and measurable molecular pharmacodynamic indicators for evaluating the efficacy of TCM compound therapy, thus promoting the modernization and internationalization of TCM.

[0019] In summary, this invention not only has significant scientific theoretical value, but also shows broad prospects for translational applications in disease diagnosis, drug development, and clinical treatment strategy formulation. Attached Figure Description

[0020] Figure 1 A bar chart showing the expression levels of lnc517368, miR-203b-3p, and SMAD2 genes in human gastric mucosa tissue. Where CSG represents chronic superficial gastritis, IM represents intestinal metaplasia, and GC represents gastric cancer. Data are expressed as mean ± standard deviation (x̅±s), n=3; significance was marked as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 2A bar chart showing the expression levels of lnc517368, miR-203b-3p, and SMAD2 genes in rat gastric mucosa tissue. The Control group represents the normal group, and HZJD represents the intervention group treated with the Huazhuo Jiedu formula. Data are expressed as mean ± standard deviation (x̅±s), n=3; significance was marked as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and ns indicates no statistically significant difference. Figure 3 A bar chart showing the expression levels of lnc517368, miR-203b-3p, and SMAD2 genes in MC and AGS cells, where HZJD represents the serum intervention group containing the Huazhuo Jiedu formula; data are expressed as mean ± standard deviation (x̅±s), n=3; significance was marked as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 4 Pathological images of stomach tissue from each group of rats (×100). Figure 5 The figures show the cell morphology and proliferation capacity analysis of each group, where: A is a light micrograph of cell morphology (×200); B is a line graph of cell viability detected by CCK-8 assay; C is a fluorescence image of cell proliferation detected by EdU assay (×200); and D is a bar graph of fluorescence intensity of cell proliferation detected by EdU assay. Significance was defined as follows: compared with the GES-1 group, ****P<0.0001; compared with the MC group, ****P<0.0001, **P<0.01, and ns indicates no statistically significant difference. Figure 6 The following graphs illustrate the analysis of apoptosis in each group: A is a bar chart showing the mRNA levels of apoptosis-related genes detected by RT-qPCR; B is a band chart showing the levels of apoptosis-related proteins detected by Western blot; C is a gray-scale quantitative bar chart showing the levels of apoptosis-related proteins detected by Western blot; D is a fluorescence image of apoptosis detected by TUNEL assay; E is a bar chart showing the fluorescence intensity of apoptosis detected by TUNEL assay. Significance was defined as follows: compared with the GES-1 group, ****P<0.0001, ***P<0.001, **P<0.01; compared with the MC group, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. ns indicates no statistically significant difference. Detailed Implementation

[0021] This invention provides the application of the lnc517368 / miR-203b-3p / SMAD2 axis as a target in the preparation of products used to assist in the identification of the progression stages of gastric lesions. The lnc517368 / miR-203b-3p / SMAD2 axis refers to a molecular combination composed of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2. In this invention, the "product" includes, but is not limited to, diagnostic reagents, detection devices, computer software, or systems that can be used to assist in clinical judgment. Preferably, the product is a kit. The kit may contain reagents for detecting the expression levels of lnc517368, miR-203b-3p, and SMAD2, such as primers, probes, antibodies, fluorescent labels, enzyme-labeled substrates, and other conventional molecular detection materials. The progression stages include chronic superficial gastritis, precancerous lesions of the stomach, and gastric cancer. Precancerous lesions of the stomach can be further subdivided into pathological stages such as mild intestinal metaplasia and severe intestinal metaplasia. The auxiliary discrimination is based on the sustained increase in the expression levels of lnc517368 and SMAD2 along this axis, and the dynamic trajectory of miR-203b-3p expression level showing an initial decrease followed by an increase and then a decrease. This dynamic trajectory can be detected and analyzed using conventional molecular biology or pathological methods such as real-time quantitative PCR, gene chips, high-throughput sequencing, and immunohistochemistry. In this invention, the "auxiliary discrimination" refers to providing clinicians with reference information on the disease progression stage by detecting the expression pattern along this axis, assisting them in diagnosis, staging, or prognostic assessment.

[0022] This invention also provides the application of the lnc517368 / miR-203b-3p / SMAD2 axis as a target in screening drugs for the prevention and / or treatment of precancerous lesions of the stomach. The screening method includes: applying candidate substances to a precancerous lesion model of the stomach and identifying substances that can simultaneously downregulate the expression levels of lnc517368, miR-203b-3p, and SMAD2 in the model. The candidate substances can be chemically synthesized drugs, natural products, traditional Chinese medicine compound formulas, biological agents, gene therapy vectors, etc. The precancerous lesion model of the stomach can be an animal model (such as an MNNG-induced PLGC rat model) or a cell model (such as an MNNG-induced MC cell model). The expression levels can be detected using conventional techniques such as RT-qPCR, Western blot, immunofluorescence, and flow cytometry. "Simultaneous downregulation" means that in this model, after treatment with the candidate substances, the expression levels of lnc517368, miR-203b-3p, and SMAD2 are all significantly lower than those in the model control group.

[0023] This invention also provides the application of the lnc517368 / miR-203b-3p / SMAD2 axis as a target in screening drugs for the prevention and / or treatment of gastric cancer. The screening method includes: treating candidate substances in a gastric cancer model and identifying substances capable of simultaneously downregulating the expression levels of lnc517368 and SMAD2 in the model and upregulating the expression level of miR-203b-3p. The gastric cancer model can be an animal model (such as an MNNG-induced gastric cancer rat model) or a cell model (such as the AGS gastric cancer cell line). The expression levels can be detected using conventional molecular biology methods. The phrase "simultaneously downregulating lnc517368 and SMAD2 and upregulating miR-203b-3p" means that in the model, after treatment with the candidate substance, the expression of lnc517368 and SMAD2 decreases, while the expression of miR-203b-3p increases, indicating that the substance has the potential to reverse the abnormal expression of this axis.

[0024] This invention also provides the application of the lnc517368 / miR-203b-3p / SMAD2 axis as a target in constructing a model system for simulating the evolution of gastric diseases. The model system includes models capable of characterizing the expression features of this axis in chronic superficial gastritis, precancerous lesions of the stomach, and gastric cancer stages, respectively. The models can be in vitro cell models (such as GES-1, MC, AGS cell lines), in vivo animal models (such as MNNG-induced rat PLGC / GC models), or computational models (such as machine learning models based on the expression data of this axis). The "expression features" refer to the expression pattern of this axis in different disease stages, such as persistently elevated lnc517368 and SMAD2, and an inverted U-shaped change in miR-203b-3p. This model system can be used in disease mechanism research, drug screening, and efficacy evaluation.

[0025] This invention also provides the application of a precancerous gastric lesion cell model in screening or evaluating candidate drugs for intervention in precancerous gastric lesions. In this precancerous gastric lesion cell model, the expression levels of lnc517368, miR-203b-3p, and SMAD2 are significantly increased. This cell model can be obtained by inducing human gastric mucosal epithelial cells GES-1 with MNNG, exhibiting a PLGC phenotype. In this model, the efficacy of candidate drugs can be evaluated by detecting changes in the expression levels of this axis, cell proliferation, apoptosis, migration, and other phenotypic indicators. This model has the advantages of simple operation, good reproducibility, and low cost, making it suitable for high-throughput drug screening.

[0026] This invention also provides the application of the lnc517368 / miR-203b-3p / SMAD2 axis as an evaluation target in the preparation of products for assessing the intervention potential of drugs against precancerous lesions of the gastric stomach. The products may include reagent kits, detection systems, evaluation software, etc. The evaluation method includes: detecting changes in the expression levels of this axis after drug treatment; if the drug can simultaneously downregulate the expression of lnc517368, miR-203b-3p, and SMAD2, it indicates that it has the potential to intervene in PLGC. This evaluation result can serve as a reference in the early stages of drug development, assisting in decisions regarding whether to proceed with further research.

[0027] This invention also provides the application of a specific dynamic expression trajectory along the lnc517368 / miR-203b-3p / SMAD2 axis in the development of decision support tools for the clinical management of gastric diseases. The specific dynamic expression trajectory refers to the sustained increase in the expression levels of lnc517368 and SMAD2 during the progression from chronic superficial gastritis to precancerous lesions and gastric cancer, while the expression level of miR-203b-3p exhibits a trend of first decreasing, then increasing, and then decreasing again. The decision support tools include, but are not limited to, clinical diagnostic assistance systems, prognostic assessment models, treatment recommendation generation software, and mobile medical applications. This tool can integrate patient test data and, combined with the dynamic trajectory of this axis, provide doctors with auxiliary suggestions regarding disease staging, risk stratification, and treatment timing.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example To explore molecular targets that can be used to assist in identifying disease stages and screening interventional drugs, this embodiment provides the "lnc517368 / miR-203b-3p / SMAD2 axis" as a research object. The dynamic expression patterns of this axis during the CSG→PLGC→GC evolution process are systematically analyzed, and its feasibility as a diagnostic marker and interventional target is verified. In the verification process, the Huazhuo Jiedu formula is used as an example intervention method. This formula, created by Professor Wang Yangang under the guidance of the theory of turbidity and toxins by Master Li Diangui, is an empirical formula for PLGC. Its composition is: Artemisia capillaris 15g, Scutellaria baicalensis 12g, Coptis chinensis 12g, Hedyotis diffusa 15g, Lobelia chinensis 15g, Scutellaria barbata 15g, Isatis indigotica 15g, Gynostemma pentaphyllum 15g, Sophora flavescens 10g, Pogostemon cablin 9g, Eupatorium fortunei 9g. The medicinal materials were uniformly purchased and identified by the pharmacy of the Third Affiliated Hospital of Beijing University of Chinese Medicine.

[0030] Against this backdrop, this embodiment systematically elucidates the expression characteristics and modifiability of the lnc517368 / miR-203b-3p / SMAD2 axis in the progression of gastric lesions through multi-level validation including clinical sample analysis, animal model construction, and cell experiments. Specific experiments and results are as follows: Clinical samples: chronic superficial gastritis, mild intestinal metaplasia, severe intestinal metaplasia, gastric cancer Patient diagnostic criteria: The diagnostic criteria for chronic superficial gastritis (CSA) refer to the "Expert Consensus on the Diagnosis and Treatment of Chronic Non-Atrophic Gastritis with Integrated Traditional Chinese and Western Medicine (2025)". The diagnostic criteria for chronic atrophic gastritis (CAG) and intestinal metaplasia (IM) refer to the "Expert Consensus on the Diagnosis and Treatment of Chronic Atrophic Gastritis with Integrated Traditional Chinese and Western Medicine (2025)" and the "Guidelines for the Diagnosis and Treatment of Chronic Gastritis in China (2022, Shanghai)". The diagnostic criteria for gastric cancer (GC) refer to the WHO 2019 "Diagnostic Criteria for Digestive System Tumors" and the "Consensus on Endoscopic Diagnosis and Treatment of Early Gastric Cancer in China (2023, Taiyuan)".

[0031] Endoscopic diagnostic criteria: 1. Under CSG endoscopy, mucosal erythema, bleeding points or plaques can be seen, and the mucosa is rough with or without edema, congestion and exudation.

[0032] 2. Under CAG endoscopy, the mucosa appears red and white, with white predominating. The color is grayish, the folds are flattened or even disappear, and some mucosal blood vessels are exposed. It may also be accompanied by granular or nodular mucosa.

[0033] 3. IM appears as an uneven mucosa or gray spots under endoscopy.

[0034] 4. Early GC: It may manifest as mild mucosal redness, discoloration, erosion, granular unevenness or small elevations / depressions, with indistinct borders.

[0035] 5. Advanced stage GC (Borrmann classification): Type I (polypoid): Obvious polypoid or umbrella-shaped mass protruding into the gastric lumen. Type II (localized ulcer): Well-defined discoid ulcer with raised, embankment-like edges. Type III (infiltrative ulcer): Indistinctly defined ulcer with extensive infiltration of the surrounding mucosa. Type IV (diffuse infiltrative): The mucosal surface may not be significantly damaged, but the gastric wall is diffusely thickened and rigid, and gastric lumen distension is restricted ("linoleic stomach").

[0036] Pathological and histological diagnostic criteria: 1. CSG: The pathological features of the gastric mucosa are mainly lymphatic and plasma cell infiltration, without atrophy of the gastric mucosal glands or intestinal metaplasia.

[0037] 2. CAG: The degree of atrophy is determined based on the extent of reduction in the intrinsic glands. Mild atrophy is defined as a reduction of less than 1 / 3 of the intrinsic glands; moderate atrophy is defined as a reduction of 1 / 3 to 2 / 3 of the intrinsic glands; and severe atrophy is defined as a reduction of more than 2 / 3 of the intrinsic glands.

[0038] 3.IM: Mild: Intestinal metaplasia area accounts for less than 1 / 3 of the total area of ​​glands and surface epithelium; Moderate: Intestinal metaplasia area accounts for 1 / 3 to 2 / 3 of the total area of ​​glands and surface epithelium; Severe: Intestinal metaplasia area exceeds 2 / 3 of the total area of ​​glands and surface epithelium.

[0039] 4. Genoscopic globulin carcinoma (GC) is mainly classified into adenocarcinoma (tubular, papillary, mucinous adenocarcinoma, low-adhesion carcinoma, etc.), signet ring cell carcinoma, and adenosquamous carcinoma. The Lauren classification (intestinal type, diffuse type, mixed type) is also recommended as a supplementary description. Tumor differentiation is histologically graded as high, intermediate, or low differentiation.

[0040] Reagents: 2×Universal Blue SYBR Green qPCR Master Mix, purchased from Wuhan Saiwei Biotechnology Co., Ltd., catalog number G3326; TransZol Up Plus RNA Kit, purchased from Beijing TransGen Biotech Co., Ltd., catalog number S40524.

[0041] Instruments: Real-time quantitative PCR instrument, ultra-micro spectrophotometer (Shanghai Thermo Fisher Scientific Instruments Co., Ltd.), clean bench (Suzhou Purification Equipment Co., Ltd.), carbon dioxide constant temperature cell culture incubator (Shanghai Lishen Scientific Instruments Co., Ltd.), medical centrifuge (Qingdao Haite Biomedical Co., Ltd.).

[0042] Statistical methods: Experimental data were analyzed using GraphPad Prism 10.0. Quantitative data were expressed as mean ± standard deviation (x̅±s). One-way ANOVA was used to compare the significance of differences between group means. A p-value < 0.05 was considered statistically significant.

[0043] Gastric mucosal tissue samples were collected from 12 patients meeting the diagnostic criteria in the Department of Gastroenterology, Third Affiliated Hospital of Beijing University of Chinese Medicine, including 3 cases each of CSA, mild IM, severe IM, and GC. Total RNA was extracted from the gastric mucosal tissue, and the RNA concentration and OD260 / OD280 ratio were measured using a spectrophotometer. cDNA was synthesized by reverse transcription. Amplification was performed using 2 μL of cDNA as a template. The reaction program was set as follows: 95℃ pre-denaturation for 30 s, followed by 40 cycles of: 95℃ denaturation for 15 s, 60℃ annealing for 10 s, and 72℃ extension for 30 s. The melting curve was set to the instrument default. CT values ​​were obtained after amplification, and the relative expression levels of the target gene in each group were calculated using the 2^-ΔΔCt method. Primers required for the experiment were designed and synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are shown in Table 1.

[0044] Table 1 Primer Sequences

[0045] RT-qPCR results showed that, compared with the CSG group, the expression level of lnc517368 in the gastric mucosa tissue of the mild IM group was increased ( P <0.05), miR-203b-3p expression level decreased ( P <0.05), SMAD2 expression level increased ( P <0.0001); lnc517368 expression remained elevated in the severe IM group ( P <0.001), miR-203b-3p expression increased ( P <0.01), SMAD2 expression increased ( P <0.05); in the gastric cancer group, lnc517368 and SMAD2 further increased to the highest level ( P <0.0001), miR-203b-3p decreased to the lowest level ( P <0.01). Compared with the mild IM group, the expression level of lnc517368 in the gastric mucosa tissue of the severe IM group was increased ( P <0.05), miR-203b-3p expression level increased ( P <0.001), SMAD2 expression levels are downregulated ( P <0.001), therefore, miR-203b-3p expression levels are higher in the pathological stage, and miR-203b-3p negatively regulates SMAD2, but SMAD2 expression levels are still upregulated compared to the CSG group. Compared to the severe IM group, the GC group showed increased lnc517368 expression in gastric mucosa tissue ( P <0.0001), miR-203b-3p expression decreased ( P <0.0001), SMAD2 expression is elevated ( P <0.0001). See Figure 1 。

[0046] PLGC Rat Model (April, June, August) Gastric Cancer Rat Model: Animals: Male SD rats, SPF grade, 6 weeks old, weighing 220 - 250 g, purchased from Beijing Huafukang Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019 - 0008.

[0047] Reagents: 1 - Methyl - 3 - nitro - 1 - nitrosoguanidine (MNNG), purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number R030453. Drugs: Sodium salicylate, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: F2124136.

[0048] Construction of PLGC and GC Animal Models: After 1 week of adaptive feeding of 60 male SD rats at 6 weeks old, 10 rats were randomly selected as the normal group. Rats in the normal group had a regular diet and free access to water. The remaining 50 rats were used for the construction of PLGC and GC animal models. The methods for establishing PLGC and GC animal models refer to the literature and previous foundations, and the combined - factor modeling method was used: 1 - Methyl - 3 - nitro - 1 - nitrosoguanidine (MNNG) at a concentration of 200 μg / mL was freely available for drinking daily, stored in the dark, and freshly prepared before use; Fasting was carried out every other day, that is, fed for 1 day and fasted for 1 day, without restricting water intake; On the fasting day, 2% sodium salicylate (10 mL / kg / d) was administered by gavage, freshly prepared before use. At the 12th week of modeling, every 4 weeks, 2 rats were randomly sacrificed to observe the pathological conditions of the gastric mucosa to determine whether the PLGC modeling was successful. The criteria for successful PLGC modeling were that the pathological examination of the gastric mucosa of 2 rats showed reduced intrinsic glands, intestinal metaplasia, and dysplasia. The criteria for successful GC modeling were the appearance of a mass with a diameter of more than 1 cm.

[0049] The daily raw drug dose of Huazhuo Jiedu Decoction for adults is 142 g, and the daily dose of traditional Chinese medicine formula granules is 37 g. Referring to the equivalent dose ratio table calculated based on the body surface area of humans and animals, the equivalent dose of rats was calculated to be 6.25 times the human clinical dose. Rats were given 3.85 g / kg / d of Huazhuo Jiedu formula granules by gavage daily. Both the normal group and the model group were given an equal volume of distilled water by gavage daily, and each group continued for 12 weeks.

[0050] Gastric mucosal tissue samples were collected from rats in the normal group, PLGC group, and GC group at 4, 6, and 8 months of modeling. The expression levels of lnc517368, miR-203b-3p, and Smad2 in the gastric mucosal tissue of each group were detected by RT-qPCR, using the same method as for clinical samples. RT-qPCR results showed that, compared with the control group, the expression levels of lnc517368 (…) in the gastric mucosa of rats in the 4, 6, 8, and GC groups were significantly higher. P <0.0001) and Smad2 ( P <0.05, P <0.01, P <0.05, P <0.001) expression was significantly increased and increased month by month; miR-203b-3p showed a pattern of first decreasing, then increasing, and then decreasing again. P <0.05, P <0.01, P <0.001), consistent with the expression levels of lnc517368, miR-203b-3p, and SMAD2 in the gastric mucosa at various pathological stages of the clinical Correa cascade reaction. Compared with the PLGC group in June, intervention with HZJD (Huazhuo Jiedu Fang) downregulated the expression of lnc517368, miR-203b-3p, and SMAD2. P <0.001, P <0.01, P <0.05). See Figure 2 .

[0051] Precancerous lesion cells (MC) and gastric cancer cell lines (AGS): Experimental cells: Human gastric mucosal epithelial cells (GES-1) were purchased from Wuhan Pronosei Biotechnology Co., Ltd., catalog number: CL-0563. Human gastric adenocarcinoma cells (AGS) were purchased from Wuhan Pronosei Biotechnology Co., Ltd., catalog number: CL-0022.

[0052] Reagents: 1640 culture medium, purchased from Suzhou Mossex Instruments Co., Ltd., catalog number 6124537; fetal bovine serum, purchased from Pansera, catalog number ST210819ES; penicillin-streptomycin solution, purchased from Gibco, catalog number 15140122; PBS buffer, purchased from Beijing Lanjieke Technology Co., Ltd., catalog number 28224968CJ; trypsin digestion solution, purchased from Beijing Solarbio Technology Co., Ltd., catalog number T1300.

[0053] Culture and MC induction of GES-1 and AGS: (1) After thawing the frozen GES-1 cells in warm water at 37°C, add 1640 medium, centrifuge, and collect the cell pellet. Then add 1640 complete medium containing 10% fetal bovine serum, 100 IU / mL streptomycin, and 100 μg / mL penicillin to prepare a cell suspension. Seed the suspension in culture dishes and place them in a 37°C, 5% CO2 incubator. Change the medium every 2 days. When the cell monolayer adheres to the dish and covers more than 80% of the bottom, trypsin is added for digestion. After observing that the cells become round and partially detach, add medium to stop digestion, collect all cells, centrifuge at 1000 rpm / min for 5 min, discard the supernatant, add 1640 complete medium, pipette to form a cell suspension, seed the suspension in culture dishes, and place them in a 37°C, 5% CO2 incubator to culture the cells into the logarithmic growth phase. The AGS cell culture method is the same as that for GES-1 cells.

[0054] (2) Based on literature and previous research, an MNNG-induced PLGC cell model (MNNG-induced cell, MC) was used. 2 × 10⁶ cells were collected. 5 GES-1 cells in the logarithmic growth phase were seeded onto culture plates, and 1640 complete medium was added. The plates were incubated overnight at 37°C with 5% CO2. Then, 20 μM / L MNNG medium was added to the medium, and the cells were cultured for 24 hours. By day 7, a large number of cells had died and detached. Culture was continued until day 11 to obtain MC cells. Following the above GES-1 cell passage method, once the monolayer reached 80% confluence, the cells were cryopreserved for subsequent experiments.

[0055] Preparation of drug-containing serum: Twenty 8-week-old male SD rats were acclimatized for one week and then randomly divided into a blank serum group and a serum containing Huazhuo Jiedu Fang (a traditional Chinese medicine formula) drug. The serum containing Huazhuo Jiedu Fang drug was administered 3.85 g / kg / day of Huazhuo Jiedu Fang granules via gavage, equivalent to the dosage calculated based on body surface area for humans and rats. The blank serum group was administered an equal volume of distilled water via gavage. This gavage treatment was continued for 7 days. One hour after the last administration, the rats were anesthetized, and blood was collected from the abdominal aorta. After standing at room temperature for 2 hours, the blood was centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. The supernatant was then incubated in a 56℃ water bath for 30 minutes to inactivate complement, and filtered through a 0.22 μm sterile filter to obtain the blank serum and the serum containing Huazhuo Jiedu Fang drug, which were stored at -80℃ for later use.

[0056] MC and AGS cells were added at 100 μL per well (2~4 × 10⁻⁶). 4Cells were seeded in 96-well plates and pre-cultured for 24 h. Cells were then treated with different concentrations of drug-containing serum (0, 0.25%, 0.5%, 1%, 2%, 4%, 8%, 16%) for 24 h. The culture medium was then changed, and 10 μL of CCK-8 solution was added to each well. Cells were incubated in the dark for 1 h. The absorbance at 450 nm was measured using a microplate reader. The optimal concentration of drug-containing serum was determined by calculating cell viability.

[0057] Cell groups: GES-1 group, MC group, AGS group, MC+HZJD group, and AGS+HZJD group. GES-1 group was treated with blank serum, MC and AGS groups were treated with blank serum or AGS cells respectively, and MC+HZJD group and AGS+HZJD group were treated with drug-containing serum or AGS cells respectively.

[0058] Once the monolayer reaches 80% coverage, wash the cells once with PBS, add TransZol Up, and repeatedly pipette to fully lyse the cells. Transfer the cell lysis buffer to a centrifuge tube, add RNA Extraction Agent, and repeatedly pipette and vortex at room temperature for 5 minutes. Subsequent steps are the same as for clinical samples.

[0059] RT-qPCR results showed that, compared with the GES-1 group, the expression levels of lnc517368, miR-203b-3p, and SMAD2 were increased in the MC group. P <0.01, P <0.0001, P <0.001); the expression of lnc517368 and SMAD2 was elevated in the AGS group ( P <0.0001), miR-203b-3p expression decreased ( P <0.01). Compared with the MC group, the AGS group showed increased expression of lnc517368 and SMAD2 ( P <0.05), miR-203b-3p expression decreased ( P <0.0001); in the MC+HZJD group, the expression of lnc517368, miR-203b-3p, and SMAD2 was all decreased ( P <0.05, P <0.01, P <0.01). Compared with the AGS group, the expression of lnc517368 and SMAD2 was decreased in the AGS+HZJD group. P <0.01, P <0.001), miR-203b-3p expression increased ( P <0.05). See Figure 3 .

[0060] Clinical tissue samples, PLGC rat models, and gastric precancerous lesion cells and gastric cancer cell lines (MC, AGS) were used to systematically reveal the dynamic expression characteristics of the lnc517368 / miR-203b-3p / SMAD2 axis in the progression of chronic superficial gastritis → intestinal metaplasia (mild → severe) → gastric cancer and its intervention mechanism with traditional Chinese medicine. 1. Clinical samples: miR-203b-3p showed a trend of "first decreasing, then increasing, then decreasing again", while lnc517368 / SMAD2 remained elevated. In the mild IM stage, miR-203b-3p expression was slightly low, while in the severe IM stage, miR-203b-3p expression was increased, which may be a compensatory protection. In gastric cancer tissue, miR-203b-3p decreased to the lowest level, while lnc517368 / SMAD2 reached the peak, indicating that the compensatory mechanism failed and the tumor suppressor molecules were exhausted.

[0061] 2. Animal model: PLGC rats exhibited a "first decrease, then increase, then decrease" pattern of miR-203b-3p, with HZJD staged intervention. In PLGC rats (April → June → August), miR-203b-3p showed a trend of first decreasing (April), then increasing (June), and then decreasing again (August), while lnc517368 / Smad2 remained elevated. During the GC phase, miR-203b-3p reached its lowest point, while lnc517368 / Smad2 reached its peak. After HZJD intervention, during the PLGC phase (June), the body's microenvironment was restored, miR-203b-3p was downregulated, and overcompensation was avoided. This blocked the malignant transformation pathway.

[0062] 3. Cell Model: HZJD bidirectionally regulates miR-203b-3p, inhibiting the activity of gastric cancer cells. MC cells (PLGC phenotype): miR-203b-3p was highly expressed, and HZJD downregulated its expression; AGS cells (gastric cancer phenotype): miR-203b-3p was lowly expressed, and HZJD upregulated its expression; lnc517368 / SMAD2 was highly expressed in both cell types, and HZJD significantly inhibited its expression.

[0063] Summary of dynamic features miR-203b-3p exhibits an inverted U-shaped curve: an initial compensatory increase (protective) → a peak followed by a rapid decline (functional exhaustion). lnc517368 and SMAD2 show an ascending curve: once initiated, they continuously rise, driving a positive feedback loop of inflammation-carcinogenesis. The rat model's timeline perfectly replicates the clinical curve, demonstrating the reproducibility and intervention window of this curve's evolution.

[0064] The time and space window of intervention by traditional Chinese medicine: April–June (PLGC peak window): HZJD inhibits the overexpression of miR-203b-3p, preventing "compensatory exhaustion," while suppressing the upward slope of lnc517368 / SMAD2, thus delaying pathological progression.

[0065] In August and after tumor formation (cancerous window): HZJD reversed the decline of miR-203b-3p, restored its anti-cancer activity, and continuously reduced lnc517368 / SMAD2, achieving "late-stage braking".

[0066] In summary, the spatiotemporal dynamic curve of lnc517368 / miR-203b-3p / SMAD2 axis for the first time depicts the complete molecular trajectory of "compensation → attack-defense conversion → loss of control" in the process of "chronic superficial gastritis → precancerous lesions of gastric cancer". Huazhuo Jiedu Formula can achieve reversible intervention in the progression of PLGC by specifically adjusting this curve at each stage.

[0067] Validation of the intervention effect based on the lnc517368 / miR-203b-3p / SMAD2 axis: 1. Animal experiments HE staining of rats in the Control group showed a thicker mucosal layer, intact surface epithelial structure, well-preserved gastric pits, and regularly arranged glands; the mucosal glands secreted neutral mucin, thus staining purplish-red with AB / PAS; Ki-67 positive cells were locally present in the gland necks within the intrinsic epithelium. HE staining of the Model group showed defects in the surface epithelial structure, a significant reduction in the number of intrinsic glands, and cystic dilation and disordered arrangement of the glands; AB / PAS staining showed the mucosal glands stained blue, indicating acidic mucus and intestinal metaplasia in the rat gastric mucosa; Ki-67 positive cells were confined to the area below the glandular pits, representing complete intestinal metaplasia. HE staining of the HZJD group showed thickened mucosa, with a relatively intact surface epithelial structure but still some damage; the number of glands was relatively normal, their arrangement was orderly, their shape was relatively regular, and their size was relatively normal; AB / PAS staining showed purplish-red, but the number was less than in the Control group; inflammation was still present, manifested by an increase in Ki-67 positive cells and an enlarged proliferative zone. See Figure 4 .

[0068] 2. Cell experiments 2.1 The formula for clearing turbidity and detoxifying inhibits the proliferation of MC cells. In the normal group, GES-1 cells grew in a single layer, with mostly round or oval nuclei and a spindle shape. In the model group, MC cells grew in multiple layers, with tightly packed cells exhibiting irregular morphology and pseudopodia formation. In the HZJD group, cells were mostly spindle-shaped or fusiform, with a few polygonal cells and fewer suspended cells. In the FA group, cells were irregularly arranged, with triangular, polygonal, and other irregular shapes visible, and some vacuoles appearing in the cytoplasm. (See...) Figure 5 A.

[0069] The CCK-8 and EdU experiments assessed cell viability in each group and the effect of the Huazhuo Jiedu formula on cell proliferation. Compared with GES-1 cells, MC cells showed significantly enhanced proliferation capacity. P <0.01), while the Huazhuo Jiedu formula could downregulate the proliferation of MC cells (P<0.05), especially at 48h and 72h. See Figure 5 BD.

[0070] 2.2 The formula for clearing turbidity and detoxifying promotes apoptosis in MC cells. Compared with the GES-1 group, the MC group showed decreased expression levels of caspase 3, caspase 9, and Bax mRNA, and increased expression levels of Bcl-2 and PARP mRNA. P <0.01). After intervention with the Huazhuo Jiedu formula, the expression levels of caspase 3, caspase 9, and Bax mRNA increased ( P <0.05), Bcl-2 and PARP mRNA levels decreased ( P <0.01). See Figure 6 A.

[0071] Compared with the GES-1 group, the MC group showed decreased expression levels of cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9, and Bax proteins, and increased expression levels of Bcl-2 and PARP proteins. P <0.01). In the HZJD group, the expression of cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9, and Bax proteins was upregulated, while the expression of Bcl-2 and PARP proteins was downregulated. P <0.05). See Figure 6 BC.

[0072] TUNEL results showed that, compared with the GES-1 group, the apoptosis rate in the MC group was significantly reduced. P <0.01%. Compared with the MC group, the apoptosis rate in the HZJD group was significantly increased ( P <0.01). See Figure 6 DE.

[0073] As demonstrated by the above embodiments, this invention provides a specific dynamic expression trajectory of the lnc517368 / miR-203b-3p / SMAD2 axis in the evolution of gastric lesions. This trajectory has been validated in clinical tissue samples, animal models at different time points, and cell models, exhibiting clear and reproducible characteristics. Furthermore, this invention provides a method for constructing a precancerous gastric lesion cell model based on this molecular axis, as well as the resulting model, and verifies the feasibility of using this axis as a target for stage-specific drug screening and efficacy evaluation.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of lnc517368 / miR-203b-3p / SMAD2 axis as a target in the preparation of products for assisting in the differentiation of the progression stage of gastric lesions, characterized in that, The lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination consisting of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2. The evolutionary stages include chronic superficial gastritis, precancerous lesions of the stomach, and stomach cancer; The auxiliary discrimination is based on the fact that the expression levels of lnc517368 and SMAD2 in this axis are continuously increasing, and the expression level of miR-203b-3p shows a dynamic trajectory of first decreasing, then increasing, and then decreasing again.

2. The application according to claim 1, characterized in that, The product is a kit containing reagents for detecting the expression levels of lnc517368, miR-203b-3p, and SMAD2.

3. The application of lnc517368 / miR-203b-3p / SMAD2 axis as a target in screening drugs for the prevention and / or treatment of precancerous lesions of the stomach, characterized in that, The lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination consisting of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2.

4. The application according to claim 3, characterized in that, The screening process includes: applying candidate substances to a gastric precancerous lesion model and identifying substances that can simultaneously downregulate the expression levels of lnc517368, miR-203b-3p, and SMAD2 in the model.

5. The application of lnc517368 / miR-203b-3p / SMAD2 axis as a target in screening drugs for the prevention and / or treatment of gastric cancer, characterized in that, The lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination consisting of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2.

6. The application according to claim 5, characterized in that, The screening process includes: applying candidate substances to a gastric cancer model and identifying substances that can simultaneously downregulate the expression levels of lnc517368 and SMAD2 in the model and upregulate the expression level of miR-203b-3p.

7. The application of lnc517368 / miR-203b-3p / SMAD2 axis as a target in constructing a model system for simulating the evolution of gastric diseases, characterized in that... The lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination consisting of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2. The model system includes models capable of representing the axis expression characteristics of chronic superficial gastritis, precancerous lesions of the stomach, and stages of gastric cancer, respectively.

8. The application of a precancerous gastric lesion cell model in screening or evaluating candidate drugs for intervening in precancerous gastric lesions, characterized in that, The expression levels of lnc517368, miR-203b-3p, and SMAD2 were significantly increased in the gastric precancerous lesion cell model. The application of 9.lnc517368 / miR-203b-3p / SMAD2 axis as an evaluation target in the preparation of products for assessing the potential of drugs to intervene in precancerous lesions of the stomach, characterized in that, The lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination consisting of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2. The application of a specific dynamic expression trajectory of the 10.lnc517368 / miR-203b-3p / SMAD2 axis in the development of a decision support tool for the clinical management of gastric diseases, characterized in that... The specific dynamic expression trajectory refers to the continuous increase in the expression levels of lnc517368 and SMAD2 during the evolution from chronic superficial gastritis to precancerous lesions and gastric cancer, while the expression level of miR-203b-3p shows a trend of first decreasing, then increasing, and then decreasing again. The lnc517368 / miR-203b-3p / SMAD2 axis refers to the molecular combination consisting of the long non-coding RNA lnc517368, the microRNA miR-203b-3p, and the signal transduction molecule SMAD2.

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