Methods for studying the mechanism of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma

Abstract

This invention discloses a method for studying the mechanism of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma, including the following steps: construction and screening of lung adenocarcinoma research models, baseline detection of EGLN3 expression levels and clinical relevance analysis, verification of EGLN3 / LRPPRC protein interaction and analysis of regulatory mechanisms, troxerutin targeting screening and determination of intervention parameters, in vitro verification of troxerutin intervention in the malignant biological behavior of lung adenocarcinoma cells, in vivo verification of troxerutin intervention in lung adenocarcinoma, evaluation of intervention effect and confirmation of mechanism, and formulation of personalized consolidation intervention plan. This invention integrates the links of target verification, drug screening, in vivo and in vitro intervention, effect evaluation, and clinical translation to form a standardized and reproducible lung adenocarcinoma intervention method, filling the technical gap in B[a]P-induced lung adenocarcinoma targeted intervention, and is different from the fragmented research of existing single cell / animal experiments.

Description

Technical Field

[0001] This invention relates to the field of molecular targeted therapy for tumors, specifically to a method for studying the mechanism of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma. Background Technology

[0002] Lung cancer is the leading cause of cancer-related morbidity and mortality. Lung adenocarcinoma (LUAD) accounts for more than 55% of non-small cell lung cancer (NSCLC). It is characterized by insidious onset, rapid progression, and low early diagnosis rate. 75% of patients are diagnosed at an advanced stage, with a five-year survival rate of only 15%. Early intervention can increase the five-year survival rate to 45%. Currently, clinical targeted therapy for lung adenocarcinoma focuses on epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), Kirsten rat sarcoma virus oncogene homolog (KRAS), and C-ros proto-oncogene 1 tyrosine kinase (ROS-1). However, the mutation coverage of these targets is less than 50%, and all targeted drugs eventually develop resistance, resulting in significant limitations in clinical application.

[0003] Environmental carcinogens are a significant contributing factor to lung adenocarcinoma. Benzo[a]pyrene (B[a]P), a Group 1 carcinogen, is a core pathogenic substance among polycyclic aromatic hydrocarbons (PAHs) and is widely present in the atmosphere, drinking water, and high-temperature processed foods. Environmental B[a]P exposure concentrations are positively correlated with the incidence of lung adenocarcinoma, with approximately one-third of environment-related lung adenocarcinomas attributable to B[a]P exposure. B[a]P is activated through metabolism in vivo to produce 7,8-dihydroxy-9,10-epoxybenzo(a)pyrene (BPDE), which forms adducts with DNA, causing gene damage and inducing malignant transformation of lung epithelial cells. However, the molecular regulatory mechanism by which B[a]P mediates lung adenocarcinoma is not fully understood, and there is a lack of specific clinical interventions targeting B[a]P-induced lung adenocarcinoma.

[0004] Molecular biological studies have confirmed that hypoxia-inducible factor 3 (EGLN3) of the EGL-9 family is highly expressed in lung adenocarcinoma cells, tissues, and plasma. It is significantly negatively correlated with pathological grade, clinical stage, lymph node metastasis, and poor prognosis of lung adenocarcinoma, making it a potential diagnostic biomarker for lung adenocarcinoma. Knockdown of EGLN3 significantly inhibits the proliferation, migration, and invasion of lung adenocarcinoma cells and blocks B[a]P-induced malignant transformation of lung epithelium. The leucine-rich PPR motif protein (LRPPRC) is a core interacting protein of EGLN3. The two directly bind in the cytoplasm of lung adenocarcinoma cells, forming a positive regulatory axis, synergistically promoting the malignant phenotype of lung adenocarcinoma cells and tumorigenicity in nude mice, representing a novel potential target for targeted therapy of lung adenocarcinoma.

[0005] Small molecule drugs from traditional Chinese medicine have become a research hotspot in targeted cancer therapy due to their advantages of strong anti-tumor activity, low toxicity and side effects, and low likelihood of developing drug resistance. Troxerutin, the core flavonoid active ingredient of the traditional Chinese medicine Scutellaria baicalensis, has been proven to have anti-inflammatory, antiviral, and tumor cell proliferation-inhibiting pharmacological effects. However, whether it can target the EGLN3 / LRPPRC axis to intervene in lung adenocarcinoma, its specific mechanism of action, and standardized intervention procedures are currently unknown, with no patents or research reports available. Existing technologies have not formed a systematic approach integrating target validation, drug screening, in vivo and in vitro intervention, efficacy evaluation, and clinical translation, making it impossible to achieve precise prevention and targeted treatment of B[a]P-induced lung adenocarcinoma. Summary of the Invention

[0006] The purpose of this invention is to provide a method for studying the mechanism of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma, in order to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for studying the mechanism of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma, comprising the following steps: Step 1: Construct lung adenocarcinoma research models, including B[a]P-induced lung epithelial malignant transformation cell model, commercially available conventionally resuscitated lung adenocarcinoma cell lines, nude mouse subcutaneous tumorigenesis model, and clinical sample screening; Step 2: Detect the mRNA and protein expression levels of EGLN3 in lung adenocarcinoma cells, animal models, and clinical samples, and complete the correlation analysis between EGLN3 and clinicopathological features and prognosis of lung adenocarcinoma; Step 3: Verify the protein-protein interaction, subcellular co-localization, and positive expression regulation mechanism of EGLN3 and LRPPRC, and confirm the oncogenic effect of the EGLN3 / LRPPRC axis; Step 4: Based on virtual screening of the natural product library, troxerutin was identified as a small molecule drug targeting the EGLN3 / LRPPRC axis, and the optimal intervention concentration of troxerutin for lung adenocarcinoma cells was detected and determined. Step 5: In vitro verification of the inhibitory effect of troxerutin on the proliferation, migration, and invasion of lung adenocarcinoma cells and malignant transformed lung epithelial cells, as well as its apoptosis-inducing effect; Step 6: In vivo verification of the inhibitory effect of troxerutin on subcutaneous tumorigenesis of lung adenocarcinoma in nude mice and its targeted regulatory effect on the EGLN3 / LRPPRC axis; Step 7: Confirm the molecular mechanism of troxerutin's intervention in lung adenocarcinoma through statistical analysis and pathway validation; Step 8: Develop personalized troxerutin consolidation intervention and follow-up plans for high-risk groups of B[a]P exposure and patients with lung adenocarcinoma.

[0008] Furthermore, the B[a]P-induced malignant transformation cell model of lung epithelium described in step one is constructed as follows: Human normal lung epithelial cells BEAS-2b are chronically exposed to benzo[a]pyrene at a concentration of 5 μg / mL and cultured for 40 generations to construct the malignant transformation cell BEAS-2b-T; the nude mouse subcutaneous tumorigenesis model is constructed using SPF-grade BALB / c-nu male nude mice, subcutaneously injected with BEAS-2b-T or A549 cells at a cell concentration of 1 × 10⁻⁶. 6 100μL / piece.

[0009] Furthermore, the detection of EGLN3 expression level in step two includes: detecting the mRNA level of EGLN3 in cells, tissues, and plasma using RT-qPCR with primer sequences F: CCTGTCTGCACGAGGCAAT and R: GCACTTCGTGTGGGTTCCTA; detecting the protein level of EGLN3 in cells and tissues using Western blot and immunohistochemistry; and analyzing the correlation between EGLN3 expression and pathological grade, clinical stage, and lymph node metastasis in lung adenocarcinoma patients using the TCGA database, and plotting ROC curves to assess diagnostic value.

[0010] Furthermore, the verification of the EGLN3-LRPPRC protein interaction in step three includes: screening EGLN3-interacting proteins using immunoprecipitation combined with high performance liquid chromatography-mass spectrometry; verifying the direct binding of the two proteins through immunoprecipitation and immunofluorescence co-localization; constructing a molecular docking model using HDOCK software to analyze hydrogen bond and salt bridge interaction sites; detecting LRPPRC expression after stable knockdown of EGLN3; and detecting EGLN3 expression after overexpression of LRPPRC to verify the positive regulatory relationship between the two proteins.

[0011] Furthermore, the optimal intervention concentration of troxerutin in step four is: 0.692 μM for A549 cells after 48 h, 0.397 μM for PC-9 cells after 48 h, and 0.610 μM for BEAS-2b-T cells after 48 h. Troxerutin can significantly downregulate the protein expression of EGLN3 and LRPPRC and completely block their protein binding.

[0012] Furthermore, the in vitro validation described in step five includes: using CCK-8 and colony formation assays to detect cell proliferation capacity, scratch healing and Transwell invasion assays to detect cell migration and invasion capacity, and Western blot detection of Bax, Bcl-2, Caspase-3, and Cleaved-Caspase-3 protein expression to assess cell apoptosis; overexpression of LRPPRC can completely reverse the intervention effect of troxerutin.

[0013] Furthermore, the in vivo verification described in step six includes: periodically measuring the long diameter a and short diameter b of tumors in nude mice, according to the formula V(mm) 3 )=ab 2 / 2 Calculate tumor volume; after intervention, take tumor tissue, observe pathological morphology by HE staining, detect the expression levels of Ki67, EGLN3 and LRPPRC by immunohistochemistry, and detect apoptosis pathway proteins by Western blot.

[0014] Furthermore, the molecular mechanism described in step seven was confirmed as follows: troxerutin induces apoptosis in lung adenocarcinoma cells and inhibits cell proliferation, migration, and invasion by targeting and inhibiting the EGLN3 / LRPPRC axis and activating the Bax / Bcl-2 / Caspase-3 signaling pathway; statistical analysis was performed using GraphpadPrism 8.0, and P < 0.05 was considered statistically significant.

[0015] Furthermore, the personalized consolidation intervention program described in step eight is as follows: high-risk individuals exposed to B[a]P receive prophylactic intervention with troxerutin, and plasma EGLN3 levels are measured every 3 months; lung adenocarcinoma patients undergo 4 weeks of consolidation intervention, and follow-up is conducted at 1 month, 3 months, and 6 months after the intervention to monitor EGLN3 expression and the risk of tumor recurrence.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention integrates target validation, drug screening, in vitro and in vivo intervention, efficacy evaluation, and clinical translation to form a standardized and reproducible method for lung adenocarcinoma intervention, filling the technological gap in targeted intervention for B[a]P-induced lung adenocarcinoma and differing from fragmented studies based on single cell / animal experiments. It is the first to clearly demonstrate that troxerutin directly targets the EGLN3 / LRPPRC protein interaction axis, exerting its effect by inhibiting the expression and binding of these two proteins, without affecting normal lung epithelial cells. Its specificity is significantly superior to traditional chemotherapy drugs, avoiding damage to normal cells. In vitro experiments confirmed that troxerutin can significantly inhibit the proliferation, migration, and invasion of lung adenocarcinoma cells and BEAS-2b-T cells, and induce apoptosis. In vivo experiments confirmed that it can inhibit tumor growth in nude mice and block B[a]P-induced malignant transformation of lung epithelium. The intervention effect was statistically verified (P < 0.05), demonstrating stability and reliability. As a small molecule of natural flavonoid traditional Chinese medicine, troxerutin has extremely low toxicity and side effects, without the drug resistance problems of traditional targeted drugs. It has both preventive and therapeutic effects, making it suitable for long-term intervention in high-risk groups exposed to B[a]P, with broad prospects for clinical translation. Simultaneously establishing EGLN3 as a diagnostic biomarker and therapeutic target for lung adenocarcinoma enables full-process management of "early diagnosis-early intervention-early consolidation," thereby improving the early intervention rate and patient survival rate for lung adenocarcinoma. Attached Figure Description

[0017] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Please see Figure 1 This invention provides a method for studying the mechanism of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma, including the following steps: Step 1: Construction and Screening of Lung Adenocarcinoma Research Models 1. Cell model construction Using normal human lung epithelial cells BEAS-2b as the base cells, chronic exposure to B[a]P at a concentration of 5 μg / mL was performed, and the cells were cultured for 40 generations to construct the B[a]P-induced malignant transformation cell model of lung epithelial cells BEAS-2b-T. Human lung adenocarcinoma cell lines NCI-H1299, A549, PC-9, and H1975 (commercial off-the-shelf cell lines, not self-constructed) were routinely resuscitated and cultured. All cells were cultured in their respective mediums containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0020] 2. Animal Model Construction SPF-grade male BALB / c-nu nude mice (3 weeks old) were randomly divided into groups, and BEAS-2b-T cells and A549 cells (cell concentration 1×10⁻⁶) were injected subcutaneously into the axilla of the forelimbs of the nude mice, respectively. 6 A subcutaneous tumor model was constructed in nude mice (number per 100 μL). The mice were fed normally for 40 days, and tumor growth was observed regularly. The environment was sterile with a temperature of 26±2℃ and a relative humidity of 40±10%.

[0021] 3. Clinical sample screening We collected cancer tissue, adjacent tissue, and plasma samples from patients with lung adenocarcinoma, as well as plasma samples from healthy individuals. Samples with other malignant tumors, severe liver and kidney diseases, or those that had recently undergone radiotherapy, chemotherapy, or targeted therapy were excluded. Ultimately, 71 lung adenocarcinoma tissue samples, 40 plasma samples, and matched healthy control samples were included to establish a clinical sample database.

[0022] Step 2: Baseline detection of EGLN3 expression levels and clinical relevance analysis 1. Molecular level detection The expression level of EGLN3 mRNA in BEAS-2b, BEAS-2b-T, lung adenocarcinoma cells, clinical tissues, and plasma was detected by RT-qPCR. The primer sequences were: F: CCTGTCTGCACGAGGCAAT, R: GCACTTCGTGTGGGTTCCTA. The expression level of EGLN3 protein in cells and tissues was detected by Western blot and immunohistochemistry.

[0023] 2. Clinical relevance analysis Using the TCGA lung adenocarcinoma database, we analyzed the correlation between EGLN3 expression levels and patient pathological grade, clinical stage, lymph node metastasis, overall survival (OS), and disease-specific survival (DSS). We plotted ROC curves to evaluate the efficacy of EGLN3 as a diagnostic biomarker for lung adenocarcinoma and calculated the area under the curve (AUC).

[0024] 3. Knockdown efficiency verification Three EGLN3 small interfering RNAs (si-EGLN3-1, si-EGLN3-2, and si-EGLN3-3) were designed and transfected into lung adenocarcinoma cells and BEAS-2b-T cells. The knockdown efficiency was verified by RT-qPCR and Western blot, and si-EGLN3-2 was determined to be the optimal knockdown sequence.

[0025] Step 3: Verification of EGLN3 / LRPPRC protein interaction and analysis of regulatory mechanisms 1. Screening of interacting proteins We used co-immunoprecipitation (CO-IP) combined with high performance liquid chromatography-mass spectrometry to screen for interacting proteins of EGLN3. After removing cytoskeletal proteins, LRPPRC was identified as the core candidate interacting protein.

[0026] 2. Combined verification CO-IP experiments were conducted using Flag-EGLN3 and HA-LRPPRC as bait proteins to verify their binding. Immunofluorescence colocalization experiments were used to observe the colocalization of EGLN3 and LRPPRC in the cytoplasm of lung adenocarcinoma cells. An EGLN3 / LRPPRC molecular docking model was constructed using HDOCK software to analyze hydrogen bond and salt bridge interaction sites.

[0027] 3. Expression regulation verification A stable BEAS-2b cell line with EGLN3 knockdown (sh-EGLN3) was constructed, and the expression level of LRPPRC was detected. A commercially available lung adenocarcinoma cell line overexpressing LRPPRC was constructed, and the expression level of EGLN3 was detected to verify the positive regulatory relationship between the two.

[0028] 4. Functional recovery verification After knocking down EGLN3, LRPPRC was overexpressed. The rescue effect of LRPPRC on the suppression of malignant phenotypes caused by EGLN3 knockdown was detected by CCK-8, colony formation, scratch healing, Transwell invasion and nude mouse tumorigenesis experiments.

[0029] Step 4: Choxerutin Targeted Screening and Intervention Parameter Determination 1. Virtual Filtering Based on the Taoshu natural product library (19,543 small molecules), virtual screening was performed using Schrödinger and AutodockVina software. Using binding energy as the evaluation index, troxerutin was determined to be the optimal small molecule drug targeting the EGLN3 / LRPPRC axis, with a binding energy of -66.18 kcal / mol.

[0030] 2. IC50 value detection The half-maximal inhibitory concentration (IC50) of troxerutin against lung adenocarcinoma cells and BEAS-2b-T cells was determined using the CCK-8 assay: 48h IC50 = 0.692 μM for A549 cells, 0.397 μM for PC-9 cells, and 0.610 μM for BEAS-2b-T cells. The 48h IC50 was thus determined to be the optimal intervention concentration.

[0031] 3. Targeted effect verification Western blot was used to detect the expression levels of EGLN3 and LRPPRC proteins after troxerutin intervention; CO-IP assay was used to detect the inhibitory effect of troxerutin on the binding of EGLN3 and LRPPRC proteins.

[0032] Step 5: In vitro validation of troxerutin's intervention on the malignant biological behavior of lung adenocarcinoma cells. 1. Proliferation capacity detection Experimental groups: control group (NC group), troxerutin intervention group (TRO group), and rescue group (TRO and oeLRPPRC group); cell proliferation activity was detected at 0h, 24h, 48h, 72h, and 96h using the CCK-8 assay, and cell colony formation assay was used to detect cell colony formation ability.

[0033] 2. Detection of migration and invasion capabilities The scratch healing assay was used to detect cell migration ability, the Transwell invasion assay (Matrigel coating) was used to detect cell invasion ability, and Image-J software was used to quantitatively analyze migration distance and the number of cells that penetrated the membrane.

[0034] 3. Apoptosis induction detection Western blot analysis was performed on proteins involved in the apoptosis pathway: pro-apoptotic protein Bax, apoptosis inhibitory protein Bcl-2, cysteine ​​protease 3 (Caspase-3), and activating Caspase-3 (Cleaved-Caspase-3). The ratios of Bax / Bcl-2 and Cleaved-Caspase-3 / Caspase-3 were calculated.

[0035] 4. Verification through rescue experiments After overexpressing LRPPRC, troxerutin was administered, and the above proliferation, migration, invasion, and apoptosis experiments were repeated to verify that the intervention effect of troxerutin depends on the EGLN3 / LRPPRC axis.

[0036] Step Six: In vivo validation of troxerutin intervention in lung adenocarcinoma 1. Nude mouse grouping and intervention The successfully constructed BEAS-2b-T and A549 nude mouse tumorigenesis models were randomly divided into a control group, a troxerutin intervention group, and a rescue group (overexpressing LRPPRC and troxerutin); the long diameter (a) and short diameter (b) of the tumor were measured periodically according to the formula V(mm) 3 )=ab 2 / 2 Calculate the tumor volume and plot the growth curve.

[0037] 2. Histopathological examination Nude mice were euthanized by cervical dislocation after intervention. Tumor tissue was dissected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin (HE) staining was used to observe the pathological morphology of the tumor, and immunohistochemistry was used to detect the expression levels of Ki67 (proliferation marker), EGLN3, and LRPPRC.

[0038] 3. In vivo pathway validation Proteins were extracted from tumor tissue, and Western blot was used to detect the expression of Bax / Bcl-2 / Caspase-3 pathway proteins to confirm the in vivo mechanism of action.

[0039] Step 7: Evaluation of Intervention Effectiveness and Validation of Mechanism 1. Statistical Analysis Data analysis was performed using GraphpadPrism 8.0 software. Quantitative data were expressed as mean ± standard deviation (x±s). Student's test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0040] 2. Confirmation of the mechanism of action It was confirmed that troxerutin induces apoptosis in lung adenocarcinoma cells and inhibits their proliferation, migration, and invasion by targeting and inhibiting the EGLN3 / LRPPRC axis, downregulating the expression of EGLN3 and LRPPRC, blocking their binding, and thereby activating the Bax / Bcl-2 / Caspase-3 apoptosis signaling pathway.

[0041] 3. Diagnostic and prognostic value assessment By integrating clinical samples and TCGA data, EGLN3 was confirmed as an independent prognostic factor and potential diagnostic biomarker for lung adenocarcinoma. The diagnostic AUC of plasma EGLN3 was 0.7078, and the AUC of tissue EGLN3 was 0.6497.

[0042] Step 8: Development of Personalized Consolidation Intervention Plan 1. Prevention plan for high-risk groups For high-risk groups with occupational and environmental exposure to B[a]P, troxerutin was used for preventive intervention, and plasma EGLN3 levels were tested regularly, with a follow-up examination every 3 months to assess the risk of lung adenocarcinoma.

[0043] 2. Clinical patient consolidation program For lung adenocarcinoma patients who received the intervention, a 4-week consolidation intervention cycle was established to maintain troxerutin-targeted intervention. Tumor markers and EGLN3 expression were detected every 2 weeks. Follow-up was conducted at 1 month, 3 months and 6 months after the intervention to monitor the risk of recurrence.

[0044] Example 1: Construction of a lung adenocarcinoma research model 1. Construction of BEAS-2b-T malignant transformation cell model Normal human lung epithelial cells BEAS-2b were cultured in DMEM complete medium and divided into a control group (BEAS-2b-N, containing 0.1‰ DMSO) and a virus-treated group (BEAS-2b-T, containing 5 μg / mL B[a]P). After cell adhesion, the cells were chronically exposed to the virus and passaged every 3 days for 40 generations. ELISA was used to detect BPDE-DNA adducts. The results showed that the adduct content in BEAS-2b-T cells was significantly higher than that in BEAS-2b-N (P < 0.0001). HE staining showed irregular cell morphology and obvious mitotic figures, confirming the successful construction of the malignant transformation model.

[0045] 2. Culture of lung adenocarcinoma cells NCI-H1299, A549, PC-9, and H1975 cells (commercial off-the-shelf cell lines, not self-constructed) were cultured in 1640 complete medium at 37°C in a 5% CO2 incubator. When the cells reached 80% confluence, they were trypsinized and passaged. Cells in the logarithmic growth phase were used for subsequent experiments.

[0046] 3. Construction of a nude mouse tumor model Twenty SPF-grade BALB / c-nu nude mice were selected and divided into four groups: BEAS-2b-N, BEAS-2b-T, A549, and SK-MES-1, with five mice in each group. Each mouse was subcutaneously injected with 1×10⁻⁶ ppm of the drug. 6 After 20 days of cell inoculation, tumors were palpable in the BEAS-2b-T and A549 groups. After 30 days, the tumors were dissected, and HE staining and immunohistochemistry confirmed that the pathological type was lung adenocarcinoma, and the model was successfully constructed.

[0047] 4. Clinical sample screening Tissue samples from 71 patients with lung adenocarcinoma and plasma samples from 40 patients were included. Patients with other malignant tumors, severe liver and kidney diseases, or a history of radiotherapy and chemotherapy were excluded. All samples were pathologically confirmed. Plasma samples were stored at -80°C, and tissue samples were paraffin-embedded for later use.

[0048] Example 2: EGLN3 Expression Detection and Clinical Relevance Analysis 1. RT-qPCR detection Total RNA was extracted from cells, tissues, and plasma, and reverse transcribed into cDNA using TAKARSYBR® Premix ExTaq. TM qPCR was performed using reagent II under the following conditions: 95℃ for 30s, 95℃ for 3s, 60℃ for 30s, for 40 cycles. The results showed that EGLN3 expression in BEAS-2b-T cells, lung adenocarcinoma cells, cancer tissue, and patient plasma was significantly higher than that in the control group (P < 0.05).

[0049] 2. Western blot detection Protein was extracted using RIPA lysis buffer, quantified by BCA, subjected to SDS-PAGE electrophoresis, transferred to a membrane, blocked with 5% skim milk powder, incubated with primary antibody (EGLN3 antibody 1:1000) at 4°C for 14 h, incubated with secondary antibody (goat anti-rabbit IgG 1:10000) at room temperature for 2 h, and then visualized by ECL. The results showed that EGLN3 protein was highly expressed in lung adenocarcinoma cells and BEAS-2b-T cells.

[0050] 3. Clinical relevance analysis Analysis of the TCGA database showed that patients in the high EGLN3 expression group had significantly shorter overall survival (OS) and daily minimum survival (DSS) than those in the low expression group (P < 0.01). Univariate and multivariate Cox regression confirmed that EGLN3 is an independent prognostic factor for lung adenocarcinoma. ROC curves showed that plasma EGLN3 had a diagnostic AUC of 0.7078 and tissue AUC of 0.6497, indicating diagnostic value.

[0051] 4. Knockdown efficiency verification Transfecting si-EGLN3-1, si-EGLN3-2, and si-EGLN3-3 into A549, PC-9, and BEAS-2b-T cells, 48 ​​hours later, showed that si-EGLN3-2 had the highest knockdown efficiency (P < 0.001), and was determined to be the optimal sequence.

[0052] Example 3: EGLN3 / LRPPRC Interaction Verification and Regulation Mechanism 1. CO-IP combined with mass spectrometry screening BEAS-2b-T cells were lysed, and EGLN3 antibody was added for immunoprecipitation. Mass spectrometry analysis identified 52 EGLN3-binding proteins, with the highest LRPPRC score (11.88), which was identified as the core interacting protein.

[0053] 2. Combined verification CO-IP experiment: Flag-EGLN3 group can precipitate LRPPRC, and HA-LRPPRC group can precipitate EGLN3, confirming that the two directly bind; Immunofluorescence co-localization shows that EGLN3 (green) and LRPPRC (red) are co-localized in the cytoplasm of lung adenocarcinoma cells, with significant yellow fluorescence signal; Molecular docking shows that the two bind through hydrogen bonds (Asn155 / Glu692, etc.) and salt bridges (Lys172 / Glu834, etc.), with a docking score of -213.16.

[0054] 3. Expression regulation verification After stable knockdown of EGLN3, LRPPRC protein expression was significantly downregulated (P < 0.001); after overexpression of LRPPRC, EGLN3 protein expression was significantly upregulated (P < 0.01), confirming the positive regulation between the two.

[0055] 4. Functional rescue experiment Knockdown of EGLN3 significantly inhibited cell proliferation, migration, and invasion (P < 0.001), while overexpression of LRPPRC completely restored the above phenotypes (P < 0.05), confirming that EGLN3 regulates the malignant phenotype of lung adenocarcinoma cells through LRPPRC.

[0056] Example 4: Choxerutin Targeted Screening and Intervention Parameter Determination 1. Virtual Filtering Based on virtual screening of the Taoshu natural product library, troxerutin ranked first with a binding energy of -66.18 kcal / mol. It can form hydrogen bonds and hydrophobic interactions with the EGLN3 / LRPPRC interaction site, and was identified as a targeted drug.

[0057] 2. IC50 testing A troxerutin concentration gradient of 1 μM, 0.8 μM, 0.6 μM, 0.4 μM, 0.2 μM, and 0 μM was set, and intervention was performed for 12 h, 24 h, and 48 h. Cell viability was detected by CCK-8 assay. The results showed that the IC50 was optimal at 48 h: A549 = 0.692 μM, PC-9 = 0.397 μM, and BEAS-2b-T = 0.610 μM.

[0058] 3. Targeted effect verification After 48 hours of troxerutin intervention, Western blot showed that the expression of EGLN3 and LRPPRC proteins was significantly downregulated (P < 0.05); CO-IP showed that troxerutin completely blocked the binding of EGLN3 and LRPPRC, confirming the targeted inhibitory effect.

[0059] Example 5: In vitro intervention of troxerutin in lung adenocarcinoma cells 1. Proliferation inhibition Cell cultures of the NC group, TRO group, and TRO+oeLRPPRC group were analyzed using CCK-8 assays. The results showed that the cell proliferation activity of the TRO group was significantly lower than that of the NC group (P < 0.0001), and the number of colonies formed was reduced by more than 70%. Cell proliferation was restored after overexpression of LRPPRC (P < 0.001).

[0060] 2. Inhibition of migration and invasion Scratch healing assay showed that the migration distance of TRO group cells after 24 hours was only 30% of that of NC group (P < 0.001); Transwell invasion assay showed that the number of cells that penetrated the membrane decreased by more than 80% (P < 0.0001); migration and invasion ability was restored after overexpression of LRPPRC.

[0061] 3. Apoptosis induction Western blot analysis showed that Bax expression was upregulated and Bcl-2 was downregulated in the TRO group, with the Bax / Bcl-2 ratio increasing by 2.3-fold (P < 0.001); the Cleaved-Caspase-3 / Caspase-3 ratio increased by 1.8-fold (P < 0.01), confirming apoptosis induction; and the apoptosis effect was reversed after overexpression of LRPPRC.

[0062] Example 6: In vivo intervention of troxerutin in tumor formation in nude mice 1. Tumor growth inhibition Nude mice were divided into sh-NC group, sh-EGLN3 group, sh-EGLN3+oeLRPPRC group, and troxerutin intervention group. The tumor volume in the troxerutin intervention group was significantly smaller than that in the control group (P<0.0001), and the tumor weight was reduced by more than 65%.

[0063] 2. Histopathology and Immunohistochemistry HE staining showed that the tumor cell density and mitotic figures were reduced in the intervention group; immunohistochemistry showed that the Ki67 positivity rate was reduced by 70% (P<0.0001), and the expression of EGLN3 and LRPPRC was significantly downregulated.

[0064] 3. In vivo pathway validation Western blot analysis of tumor tissue showed significantly elevated Bax / Bcl-2 and Cleaved-Caspase-3 / Caspase-3 ratios, confirming activation of the apoptosis pathway in vivo.

[0065] Example 7: Effect Evaluation and Consolidation Plan 1. Statistical evaluation All data were analyzed using GraphpadPrism 8.0, and P < 0.05 was considered statistically significant. In vitro experiments were repeated 3 times, and in vivo experiments were performed with 5 animals per group, so the results were reproducible.

[0066] 2. Mechanism Validation The core mechanism by which troxerutin inhibits the EGLN3 / LRPPRC axis, activates the Bax / Bcl-2 / Caspase-3 pathway, and induces apoptosis and inhibits malignant phenotypes was clarified.

[0067] 3. Consolidate the intervention plan High-risk groups exposed to B[a]P: Oral administration of troxerutin, with plasma EGLN3 measured every 3 months; Lung adenocarcinoma patients: 4-week consolidation intervention, followed up at 1, 3, and 6 months after the intervention to monitor EGLN3 levels and tumor recurrence.

[0068] In summary, this invention integrates target validation, drug screening, in vitro and in vivo intervention, efficacy evaluation, and clinical translation to form a standardized and reproducible method for lung adenocarcinoma intervention, filling the technological gap in targeted intervention for B[a]P-induced lung adenocarcinoma and differing from existing fragmented studies based on single cell / animal experiments. It is the first to clearly demonstrate that troxerutin directly targets the EGLN3 / LRPPRC protein interaction axis, exerting its effect by inhibiting the expression and binding of these two proteins, without affecting normal lung epithelial cells. Its specificity is significantly superior to traditional chemotherapy drugs, avoiding damage to normal cells. In vitro experiments confirmed that troxerutin can significantly inhibit the proliferation, migration, and invasion of lung adenocarcinoma cells and BEAS-2b-T cells, and induce apoptosis. In vivo experiments confirmed that it can inhibit tumor growth in nude mice and block B[a]P-induced malignant transformation of lung epithelium. The intervention effect was statistically verified (P < 0.05), demonstrating stability and reliability. Troxerutin, a small molecule natural flavonoid traditional Chinese medicine, has extremely low toxicity and side effects, and does not have the drug resistance problems of traditional targeted drugs. It has both preventive and therapeutic effects, making it suitable for long-term intervention in high-risk groups exposed to B[a]P, and has broad prospects for clinical translation. Simultaneously establishing EGLN3 as a diagnostic biomarker and therapeutic target for lung adenocarcinoma enables full-process management of "early diagnosis-early intervention-early consolidation," improving the early intervention rate and patient survival rate of lung adenocarcinoma.

[0069] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for mechanism research of targeting EGLN3 / LRPPRC by troxerutin in lung adenocarcinoma, characterized in that: Includes the following steps: Step 1: Construct lung adenocarcinoma research models, including B[a]P-induced lung epithelial malignant transformation cell model, commercially available conventionally resuscitated lung adenocarcinoma cell lines, nude mouse subcutaneous tumorigenesis model, and clinical sample screening; Step 2: Detect the mRNA and protein expression levels of EGLN3 in lung adenocarcinoma cells, animal models, and clinical samples, and complete the correlation analysis between EGLN3 and clinicopathological features and prognosis of lung adenocarcinoma; Step 3: Verify the protein-protein interaction, subcellular co-localization, and positive expression regulation mechanism of EGLN3 and LRPPRC, and confirm the oncogenic effect of the EGLN3 / LRPPRC axis; Step 4: Based on virtual screening of the natural product library, troxerutin was identified as a small molecule drug targeting the EGLN3 / LRPPRC axis, and the optimal intervention concentration of troxerutin for lung adenocarcinoma cells was detected and determined. Step 5: In vitro verification of the inhibitory effect of troxerutin on the proliferation, migration, and invasion of lung adenocarcinoma cells and malignant transformed lung epithelial cells, as well as its apoptosis-inducing effect; Step 6: In vivo verification of the inhibitory effect of troxerutin on subcutaneous tumorigenesis of lung adenocarcinoma in nude mice and its targeted regulatory effect on the EGLN3 / LRPPRC axis; Step 7: Confirm the molecular mechanism of troxerutin's intervention in lung adenocarcinoma through statistical analysis and pathway validation; Step 8: Develop personalized troxerutin consolidation intervention and follow-up plans for high-risk groups of B[a]P exposure and patients with lung adenocarcinoma.

2. The method for studying the mechanism of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma according to claim 1, characterized in that: The B[a]P-induced malignant transformation cell model of lung epithelium described in step one was constructed as follows: Normal human lung epithelial cells BEAS-2b were chronically exposed to benzo[a]pyrene at a concentration of 5 μg / mL and cultured for 40 generations to construct the malignant transformation cell line BEAS-2b-T. The subcutaneous tumorigenesis model in nude mice was constructed by subcutaneously injecting BEAS-2b-T or A549 cells into SPF-grade BALB / c-nu male nude mice at a cell concentration of 1 × 10⁻⁶ cells. 6 100μL / piece.

3. The method for studying the mechanism of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma according to claim 1, characterized in that: Step two, the detection of EGLN3 expression level, includes: using RT-qPCR to detect the mRNA level of EGLN3 in cells, tissues, and plasma, with primer sequences F: CCTGTCTGCACGAGGCAAT and R: GCACTTCGTGTGGGTTCCTA; using Western blot and immunohistochemistry to detect the protein level of EGLN3 in cells and tissues; and combining the TCGA database to analyze the correlation between EGLN3 expression and pathological grade, clinical stage, and lymph node metastasis in lung adenocarcinoma patients, and plotting ROC curves to assess diagnostic value.

4. The method of claim 1, wherein the mechanism of study of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma is characterized by: Step 3, the verification of the interaction between EGLN3 and LRPPRC proteins, includes: screening EGLN3 interacting proteins using immunoprecipitation combined with high performance liquid chromatography-mass spectrometry; verifying the direct binding of the two proteins through immunoprecipitation and immunofluorescence co-localization; constructing a molecular docking model using HDOCK software to analyze hydrogen bond and salt bridge interaction sites; detecting LRPPRC expression after stable knockdown of EGLN3; and detecting EGLN3 expression after overexpression of LRPPRC to verify the positive regulatory relationship between the two proteins.

5. The method of claim 1, wherein the mechanism of study of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma is characterized by: The optimal intervention concentration of troxerutin in step four is: IC50 of 0.692 μM for A549 cells after 48 h, IC50 of 0.397 μM for PC-9 cells after 48 h, and IC50 of 0.610 μM for BEAS-2b-T cells after 48 h. Troxerutin can significantly downregulate the protein expression of EGLN3 and LRPPRC and completely block their protein binding.

6. The method of claim 1, wherein the mechanism of study of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma is characterized by: The in vitro validation described in step five includes: using CCK-8 and colony formation assays to detect cell proliferation capacity, scratch healing and Transwell invasion assays to detect cell migration and invasion capacity, and Western blot detection of Bax, Bcl-2, Caspase-3, and Cleaved-Caspase-3 protein expression to assess cell apoptosis; overexpression of LRPPRC can completely reverse the intervention effect of troxerutin.

7. The method of claim 1, wherein the mechanism of study of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma is characterized by: Step six, the in vivo validation, includes: periodically measuring the long diameter (a) and short diameter (b) of tumors in nude mice, according to the formula V(mm). 3 )=ab 2 / 2 Calculate tumor volume; after intervention, take tumor tissue, observe pathological morphology by HE staining, detect the expression levels of Ki67, EGLN3 and LRPPRC by immunohistochemistry, and detect apoptosis pathway proteins by Western blot.

8. The method of claim 1, wherein the mechanism of study of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma is characterized by: The molecular mechanism described in step seven was confirmed as follows: troxerutin induces apoptosis of lung adenocarcinoma cells and inhibits cell proliferation, migration and invasion by targeting and inhibiting the EGLN3 / LRPPRC axis and activating the Bax / Bcl-2 / Caspase-3 signaling pathway. Statistical analysis was performed using GraphpadPrism 8.0, and P < 0.05 was considered statistically significant.

9. The method of claim 1, wherein the mechanism of study of troxerutin targeting EGLN3 / LRPPRC in lung adenocarcinoma is characterized by: The personalized consolidation intervention program described in step eight is as follows: high-risk individuals exposed to B[a]P receive prophylactic intervention with troxerutin and plasma EGLN3 levels are measured every 3 months; lung adenocarcinoma patients receive a 4-week consolidation intervention and are followed up at 1 month, 3 months, and 6 months after the intervention to monitor EGLN3 expression and the risk of tumor recurrence.

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