An in-vitro screening / evaluation method, system and application of endometriosis drugs based on LINC02381-EGFR axis

Abstract

The application provides an in-vitro screening / evaluation method, system and application of an endometriosis drug based on a LINC02381-EGFR axis, and belongs to the technical field of medicines.The application provides an in-vitro screening / evaluation method and system of an endometriosis drug based on a LINC02381-EGFR axis, uses human ectopic endometrial cells 12Z as a carrier, uses the expression levels of LINC02381 mRNA, EGFR protein and p-PI3K / p-AKT1 and cell proliferation, migration and invasion function phenotypes as quantitative evaluation indexes, can quickly and specifically identify candidate drugs that play an anti-EMs role by interfering with the LINC02381-EGFR axis by setting a judgment threshold, has high specificity, high repeatability and high-throughput screening potential, and provides a powerful tool for the research and development of anti-EMs new drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an in vitro screening / evaluation method, system, and application of endometriosis drugs based on the LINC02381-EGFR axis. Background Technology

[0002] Endometriosis (EMs) refers to the ectopic implantation of functional endometrial tissue (glands and stroma) outside the uterine cavity, such as the ovaries, pelvic peritoneum, and rectouterine pouch. It is a common estrogen-dependent and chronic inflammatory gynecological disease. Clinically, patients often present with progressively worsening dysmenorrhea, chronic pelvic pain, dyspareunia, and menstrual abnormalities. In severe cases, it can lead to infertility, significantly impacting patients' physical and mental health and quality of life. From a pathobiological perspective, although EMs are benign, they exhibit characteristics similar to malignant tumors, including invasion, implantation, metastasis, and recurrence. Recent studies have confirmed that EMs are an independent risk factor for ovarian cancer (especially endometrioid carcinoma and clear cell carcinoma), and patients also have a significantly increased risk of developing mental health issues such as anxiety and depression. The pathogenesis of EMs is not yet fully understood, and its molecular regulatory mechanisms remain a hot topic and a challenge in research.

[0003] Clinically, treatment strategies for endometriosis (EMs) mainly include conservative drug therapy and surgical intervention. Drug therapy primarily uses hormonal medications, including oral contraceptives, progestins, and gonadotropin-releasing hormone agonists (GnRH-a), aiming to induce atrophy of ectopic lesions by suppressing ovarian function and lowering estrogen levels. However, long-term use of hormonal medications can lead to side effects such as hot flashes, night sweats, menopausal symptoms like osteoporosis, liver damage, and breakthrough bleeding, and the recurrence rate after discontinuation is high. Surgical resection of lesions can quickly relieve symptoms, but it involves surgical trauma, decreased ovarian reserve, and postoperative recurrence. Therefore, finding novel therapeutic drugs with definite efficacy, few side effects, and targeting the key molecular mechanisms of EM pathogenesis is of significant clinical importance and an urgent clinical need. Therefore, providing in vitro screening / evaluation methods for endometriosis drugs and conducting targeted drug screening provides a reliable experimental platform and technical support for the development of novel targeted therapies for endometriosis, which has significant clinical application value and scientific research significance. Summary of the Invention

[0004] To address the problems existing in the prior art, the primary objective of this invention is to provide an in vitro screening method, system, and application for endometriosis drugs based on the LINC02381-EGFR axis.

[0005] The second objective of this invention is to provide a method, system, and application for evaluating endometriosis drugs based on the LINC02381-EGFR axis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an in vitro screening method for endometriosis drugs based on the LINC02381-EGFR axis, comprising the following steps: intervening 12Z cells with the drug to be tested, and detecting the LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance and number of cells that have penetrated the membrane in 12Z cells after intervention; selecting the drug to be tested that simultaneously meets the following criteria: (1) LINC02381 mRNA transcription level decreased by ≥40% and / or EGFR protein expression level decreased by ≥15% and / or p-PI3K / p-AKT1 protein expression level decreased by ≥15%; (2) EdU positivity rate decreased by ≥30% and / or scratch healing distance decreased by ≥15% and / or number of cells that have penetrated the membrane decreased by ≥30%.

[0007] Preferably, the intervention lasts for 48 hours.

[0008] This invention provides an in vitro screening system for endometriosis drugs based on the LINC02381-EGFR axis. The in vitro screening system includes: a sample collection module, an indicator detection module, a screening module, and a result output module. The sample collection module collects 12Z cells after treatment with the drug to be tested. The indicator detection module detects the LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and number of cells that perforate the membrane in 12Z cells. The screening module analyzes the detection indicators to determine whether the drug to be tested meets the screening criteria; the screening criteria are (1) a decrease of ≥40% in LINC02381 mRNA transcription level and / or a decrease of ≥15% in EGFR protein expression level and / or a decrease of ≥15% in p-PI3K / p-AKT1 protein expression level; (2) a decrease of ≥30% in EdU positivity rate and / or a decrease of ≥15% in scratch healing distance and / or a decrease of ≥30% in the number of transmembrane cells; The result output module outputs the results.

[0009] This invention provides the application of the above-described in vitro screening method or in vitro screening system in screening or preparing drugs for endometriosis.

[0010] Preferably, the endometriosis drugs shown include traditional Chinese medicine extracts, chemically synthesized compounds, natural product monomers, peptides, or nucleic acid drugs.

[0011] This invention provides an evaluation method for endometriosis drugs based on the LINC02381-EGFR axis, comprising the following steps: intervening in 12Z cells with the drug to be evaluated, using 12Z cells with knockdown of LINC02381 as a positive reference, and evaluating the efficacy of the drug according to any one or more of the following indicators: LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and / or number of cells that have perforated the membrane.

[0012] Preferably, the intervention lasts for 48 hours.

[0013] This invention provides an in vitro evaluation system for endometriosis drugs based on the LINC02381-EGFR axis, comprising: a sample collection module, an indicator detection module, an evaluation module, and a result output module; The sample collection module collects 12Z cells after drug intervention treatment to be evaluated; The indicator detection module detects the LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and number of cells that perforate the membrane in 12Z cells. The evaluation module uses 12Z cells with knocked-down LINC02381 as a positive reference to analyze various indicators and evaluate the efficacy of the drug. The result output module outputs the results.

[0014] This invention provides the application of the above-mentioned evaluation method or in vitro evaluation system in the preparation of drugs for endometriosis.

[0015] Preferably, the endometriosis drugs shown include traditional Chinese medicine extracts, chemically synthesized compounds, natural product monomers, peptides, or nucleic acid drugs.

[0016] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention provides the first clearly defined, simple, and quantifiable in vitro cellular drug screening / evaluation method and system. Using human ectopic endometrial cells 12Z as a vector, and with LINC02381 knockdown as a positive control, the expression levels of LINC02381 mRNA, EGFR protein, p-PI3K / p-AKT1, and cell proliferation, migration, and invasion functional phenotypes are used as quantitative evaluation indicators. The judgment thresholds are set as follows: LINC02381 mRNA reduction ≥40% and / or EGFR protein reduction ≥15% and / or p-PI3K / p-AKT1 protein reduction ≥15%, and at least one functional phenotype achieving an EdU positivity rate reduction ≥30%, a scratch healing distance reduction ≥15%, or a perforated cell number reduction ≥30%. P adj <0.05). The reliability, specificity, and predictive accuracy of the model were comprehensively validated using serum containing the drug from *Ligustrum lucidum* and in vivo animal experiments. This demonstrates that the LINC02381-EGFR axis can rapidly and specifically identify candidate drugs that exert anti-EMs effects by intervening in the LINC02381-EGFR axis, exhibiting high specificity, high reproducibility, and high-throughput screening potential, providing a powerful tool for the development of new anti-EMs drugs. Attached Figure Description

[0017] Figure 1 Fluorescence expression of sh-EGFR after infection with different MOIs (scale bar: 500 μm).

[0018] Figure 2 The number of viable cells infected with sh-EGFR by 12Z and screened with puromycin for 48 hours.

[0019] Figure 3 Fluorescence expression of sh-LINC02381 infected with different MOIs (scale bar: 500 μm).

[0020] Figure 4 The number of viable cells 48 hours after sh-LINC02381 infection with 12Z and selection with puromycin.

[0021] Figure 5 Fluorescence expression of the three sh-EGFR sequences (scale bar: 500 μm) and knockdown status.

[0022] Figure 6 : Verify the efficiency reduction of LINC02381.

[0023] Figure 7 Effects of different concentrations of LY on 12Z survival rate and consequently p-Akt protein expression level.

[0024] Figure 8 Effects of different concentration gradients of PFH-containing serum on hEEC proliferation activity.

[0025] Figure 9 Effects of different concentration gradients of PFH-containing serum on the proliferation activity of 12Z.

[0026] Figure 10 Effects of different treatments on the expression of LINC02381, EGFR, PI3KCB and AKT1 mRNA in 12Z.

[0027] Figure 11 Effects of different treatments on the expression of EGFR, PI3K, p-PI3K, AKT1 and p-AKT1 proteins in 12Z.

[0028] Figure 12 Effects of different treatments on the proliferation activity of 12Z (scale bar: 200 μm).

[0029] Figure 13 Effects of different treatments on the invasiveness of 12Z (scale bar: 200 μm).

[0030] Figure 14 Effects of different treatments on the migration ability of 12Z (scale bar: 500 μm).

[0031] Figure 15 Effects of different treatments on the proliferation activity of 12Z (scale bar: 200 μm).

[0032] Figure 16 Effects of different treatments on the invasiveness of 12Z (scale bar: 200 μm).

[0033] Figure 17 Effects of different treatments on the migration ability of 12Z (scale bar: 500 μm).

[0034] Figure 18 Effects of different treatments on the expression of LINC02381, miR-27a-3p, EGFR, PI3KCB and AKT1 mRNA in 12Z.

[0035] Figure 19 Effects of different treatments on the expression of EGFR, PI3K, p-PI3K, AKT1 and p-AKT1 proteins in 12Z.

[0036] Figure 20 Animal model establishment and successful model verification.

[0037] Figure 21 Display of the longest diameter of lesions in each model group.

[0038] Figure 22 Endometrial condition after different intervention methods (scale bar: upper 100μm, lower 50μm).

[0039] Figure 23 Expression of EGFR, PI3K, p-PI3K, AKT1 and p-AKT1 proteins in endometrial tissue of the sham surgery group and ectopic endometrial tissue of each model group.

[0040] Figure 24 Changes in body weight of rats in different groups after different intervention methods.

[0041] Figure 25 Changes in body temperature of rats in different groups before and after treatment with different intervention methods.

[0042] Figure 26 Changes in water intake of rats in different groups before and after treatment with different intervention methods.

[0043] Figure 27 Changes in food intake of rats in different groups before and after treatment with different intervention methods.

[0044] Figure 28 Serum MMP-2 (left) and ICAM-1 (right) levels in rats of each group were measured.

[0045] Figure 29 Serum Fg (left) and T3 (right) levels in rats of each group were measured. Detailed Implementation

[0046] This invention provides an in vitro screening method for endometriosis drugs based on the LINC02381-EGFR axis, comprising the following steps: intervening 12Z cells with the drug to be tested, and detecting the LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance and number of cells that have penetrated the membrane in 12Z cells after intervention; selecting the drug to be tested that simultaneously meets the following criteria: (1) LINC02381 mRNA transcription level decreased by ≥40% and / or EGFR protein expression level decreased by ≥15% and / or p-PI3K / p-AKT1 protein expression level decreased by ≥15%; (2) EdU positivity rate decreased by ≥30% and / or scratch healing distance decreased by ≥15% and / or number of cells that have penetrated the membrane decreased by ≥30%.

[0047] This invention provides an in vitro screening system for endometriosis drugs based on the LINC02381-EGFR axis, based on the aforementioned in vitro screening method. The in vitro screening system includes: a sample collection module, an indicator detection module, a screening module, and a result output module. The sample collection module collects 12Z cells after treatment with the drug to be tested. The indicator detection module detects the LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and number of cells that have perforated the membrane in 12Z cells. The screening module analyzes the detected indicators to determine whether the drug to be tested meets the screening criteria. The screening criteria are (1) LINC02381... (1) mRNA transcription level decreased by ≥40% and / or EGFR protein expression level decreased by ≥15% and / or p-PI3K / p-AKT1 protein expression level decreased by ≥15%; (2) EdU positivity rate decreased by ≥30% and / or scratch healing distance decreased by ≥15% and / or number of permeabilized cells decreased by ≥30%; The result output module outputs the results. In this invention, the output results can be set to display "meets screening criteria" and "does not meet screening criteria", and drugs that meet the screening criteria are selected as candidate drugs for endometriosis.

[0048] This invention provides a method for evaluating endometriosis drugs based on the LINC02381-EGFR axis, comprising the following steps: intervening in 12Z cells with the drug to be evaluated, using 12Z cells with LINC02381 knockdown as a positive control, and evaluating the efficacy of the drug based on any one or more of the following indicators: LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and / or number of cells that have penetrated the membrane. The evaluation method of this invention can predict the therapeutic effect of the drug on endometriosis. The greater the reduction in LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and / or number of cells that have penetrated the membrane, the better the efficacy of the corresponding drug.

[0049] This invention provides an in vitro evaluation system for endometriosis drugs based on the LINC02381-EGFR axis, comprising: a sample collection module, an indicator detection module, an evaluation module, and a result output module. The sample collection module collects 12Z cells after treatment with the drug to be evaluated. The indicator detection module detects the LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and number of cells that have perforated the membrane in 12Z cells. The evaluation module uses 12Z cells with knocked-down LINC02381 as a positive reference to analyze each indicator and evaluate the drug's efficacy. The result output module outputs the results. In this invention, the output results can be set to rank the predicted effects of the drugs to be evaluated based on the magnitude of the decrease in the above indicators, selecting the drugs with better predicted effects as candidate drugs for endometriosis.

[0050] In this invention, the intervention time is 48 hours. As an optional implementation, the intervention method includes: introducing 12Z cells at a concentration of 5 × 10⁻⁶ cells / day. 3 Cells / well seeded in 96-well plates (for proliferation assay) or 2 × 10⁶ cells / well. 4 Cells were seeded in 24-well plates (for RT-qPCR / Western Blot) and cultured overnight. The test drug was diluted with complete culture medium to different concentrations (e.g., 0.1, 1, 10 μM), with three replicates for each concentration. A solvent control (0.1% DMSO or PBS) and a positive control were also included. The cells were incubated at 37°C for 48 h.

[0051] In this invention, the detection method for each indicator can be selected as follows: (1) RT-qPCR: Collect cells after drug incubation, extract total RNA, reverse transcribe, and perform qPCR, using β-actin as an internal control. -ΔΔCt The relative expression levels of LINC02381 and EGFR were calculated using the method.

[0052] (2) Western Blot: Collect cells after drug incubation, lyse them and detect EGFR, p-PI3K and p-AKT1 proteins.

[0053] (3) EdU proliferation: Collect cells after drug incubation, add EdU working solution (10 μM) and incubate for 2 h, fix, permeabilize, stain, and take pictures under a fluorescence microscope; count the proportion of EdU positive cells.

[0054] (4) Transwell invasion assay: Matrigel was diluted 1:8 and spread in the upper chamber of a Transwell chamber, and solidified at 37°C for 2 hours. Cells from each group were digested and resuspended in serum-free medium to a concentration of 1×10⁻⁶.5 / mL, take 100 μL and add it to the upper chamber, and add 600 μL of culture medium containing 10% FBS (or the test drug) to the lower chamber. After 48 h of culture, fix and stain with crystal violet, and count the number of transmembrane cells under a microscope.

[0055] (5) Wound healing migration assay: seed cells in a 6-well plate, administer different drug interventions after 24 h, scratch with a 200 μL pipette tip after 48 h, wash with PBS, add serum-free medium to culture the cells, take photos at 0 h and 24 h respectively, and calculate the wound healing distance.

[0056] The present invention also provides the use of the above in vitro screening method, in vitro screening system, evaluation method or in vitro evaluation system in screening or preparing drugs for endometriosis. Preferably, the endometriosis drugs include traditional Chinese medicine extracts, chemically synthesized compounds, natural product monomers, polypeptide or nucleic acid drugs.

[0057] By comprehensively utilizing network pharmacology prediction, bioinformatics analysis of GEO database and molecular biology experiments (gene knockdown, Western Blot, RT-qPCR, EdU, wound healing assay, Transwell assay, etc.), the present invention reveals for the first time that the LINC02381-EGFR axis is a key molecular pathway driving the progression of endometriosis. In Ems model cells (12Z), knockdown of LINC02381 or EGFR can significantly inhibit the proliferation, migration and invasion abilities of 12Z cells, and block the abnormal activation of the downstream PI3K / Akt signaling pathway. It is also proposed that the LINC02381-EGFR axis can be used as an ideal functional target for anti-EMs drug intervention, and the LINC02381-EGFR axis can be used for in vitro screening of endometriosis drugs or evaluating the therapeutic effect of endometriosis drugs.

[0058] The present invention uses the traditional Zhuang medicine *Polygala fallax* Hemsl. ( Polygala fallax Hemsl., PFH) as a verification tool, and uses its drug-containing serum and in vivo animal models for reverse verification. The results show that PFH-containing serum can significantly down-regulate the expression of LINC02381 and EGFR in 12Z cells, inhibit the phosphorylation of PI3K / Akt pathway, and reduce cell proliferation, migration and invasion abilities; in Ems model rats, high-dose intragastric administration of PFH can reduce the volume of ectopic lesions, alleviate pelvic adhesions, induce degeneration of ectopic endometrium, and improve the biased constitution of blood stasis, yang deficiency and qi stagnation in model rats. This reversely verifies the effectiveness of the LINC02381-EGFR axis as a drug target, and also confirms that *Polygala fallax* Hemsl. is an effective intervention agent for this axis.

[0059] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0060] Unless otherwise specified, the following embodiments are all conventional methods.

[0061] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0062] Example 1 1. Experimental Materials and Main Reagents Cell lines: Human ectopic endometrial cell line 12Z (purchased from Shanghai Jinyuan Biotechnology Co., Ltd., STR tested and approved); Human normal endometrial epithelial cells hEEC (purchased from Xiamen Yimo Biotechnology Co., Ltd.).

[0063] Lentiviral vectors: shRNA targeting LINC02381 (sh-LINC02381), shRNA targeting EGFR (sh-EGFR1, sh-EGFR2, sh-EGFR3), and negative control sh-NC were synthesized and packaged into lentiviral particles (contract number SGXM2024SIR959) by Sangon Biotech (Shanghai) Co., Ltd., with a titer of 5×10⁻⁶. 8 TU / mL, and also carries the GFP reporter gene.

[0064] Main reagents: Puromycin (Puro, purchased from Sigma); LY294002 purchased from MCE; SYBR Green qPCR mix (Takara); EGFR antibody (30847-1-AP, Proteintech), PI3K antibody (20584-1-AP, Proteintech), p-PI3K antibody (AP0427, ABclonal), AKT1 antibody (80457-1-RR, Proteintech), p-AKT1 antibody (AP0637, ABclonal), β-actin antibody (81115-1-RR, Proteintech), goat anti-rabbit secondary antibody (RGAR001, Proteintech); EdU-488 cell proliferation assay kit (Beyotime); Matrigel (Corning).

[0065] Table 1 Sequence Information

[0066] 2. Lentiviral transfection and establishment of stable knockdown cell lines 2.1 Determination of the optimal working concentration of puromycin Take 12Z cells in the logarithmic growth phase and use 1×10 4 Cells were seeded into 96-well plates and cultured for 24 hours. Different concentrations of puromycin (0, 1, 2, 3, 4, 5, 6, 8, 10 μg / mL) were added, with three replicates for each concentration. Cell death was observed under a microscope after 48 hours. Results showed that cells in the 0 μg / mL group grew well; some cells in the 1 μg / mL group died, but the surviving cells maintained acceptable morphology; and almost all cells in the ≥2 μg / mL group died. Therefore, 1 μg / mL was selected as the working concentration for selecting stably transfected cells with puromycin.

[0067] 2.2 Determination of the optimal MOI value for lentiviral infection (1) Optimal MOI for sh-EGFR: 12Z cells were inoculated at 5×10 4 Cells were seeded into 6-well plates. When the cell density reached 30-50%, complete medium containing polybrene (5 μg / mL) and sh-EGFR lentiviral solutions (sh-EGFR1, sh-EGFR2, sh-EGFR3) with different MOIs (1, 5, 10, 20, 30, 50) were added. After 4 hours, the medium was replenished to the normal volume. GFP fluorescence expression was observed after 48 hours. After 72 hours, the medium was replaced with complete medium containing 1 μg / mL puromycin for drug screening for 48 hours. The number of viable cells was detected by CCK-8 assay. The results for sh-EGFR1 are shown below. Figures 1-2 As shown. Figure 1 Fluorescence expression of sh-EGFR1 after infection with different MOIs (scale bar: 500 μm). Figure 2 The number of viable cells after 48 hours of sh-EGFR1 infection with 12Z and selection with puromycin.

[0068] The results showed that at MOI=30 and 50, the proportion of GFP-positive cells observed under the microscope was >80%, and there was no significant difference in the number of viable cells. P adj >0.05), considering the potential toxicity of high MOI to cells, MOI=30 was selected as the optimal multiplicity of infection. The optimal multiplicity of infection for sh-EGFR1, sh-EGFR2, and sh-EGFR3 was MOI=30.

[0069] Exploring the optimal MOI using sh-LINC02381: The operation is the same as sh-EGFR1, but set MOI=1, 5, 10, 20, 30, 50, 70, 100. The results are as follows... Figures 3-4 As shown. Figure 3 Fluorescence expression after infection with sh-LINC02381 at different MOIs (scale bar: 500 μm). Figure 4 The number of viable cells after 48 hours of puromycin selection following infection with sh-LINC02381 by 12Z.

[0070] The results showed that the GFP positivity rate observed under a microscope was >80% at MOI=50, 70, and 100, and the CCK-8 results showed no significant difference in the number of viable cells at MOI=50, 70, and 100. MOI=50 was chosen to balance transfection efficiency and cell viability.

[0071] 2.3 Optimal shRNA sequence screening 12Z cells were infected with sh-EGFR1, sh-EGFR2, and sh-EGFR3 sequences at MOI=30. After 24 h, the medium was replaced with fresh complete medium, and after 72 h, the medium was replaced with medium containing 1 μg / mL puromycin for selection. After 48 h, the protein was collected and EGFR expression was detected by Western blotting.

[0072] Fluorescence expression of the three sh-EGFR sequences (scale bar: 500 μm) and knockdown status are as follows: Figure 5 As shown, P adj <0.05、 P adj <0.01、 P adj <0.001 and P adj <0.0001. The results showed that sh-EGFR2 knockdown was the most efficient, with EGFR protein expression reduced by more than 50% compared to sh-NC. P adj <0.0001). Therefore, sh-EGFR2 was chosen for subsequent experiments.

[0073] 2.4 Establishment and validation of stable knockdown cell lines According to the determined MOI value and puromycin concentration, formal transfection was performed: 12Z cells were seeded in 6-well plates (5 × 10⁶ cells / well). 4Cells / well), the next day, add virus solution containing polybrene (sh-EGFR2, MOI=30; sh-LINC02381, MOI=50), replace with fresh medium after 24 hours, and replace with medium containing 1 μg / mL puromycin after 72 hours. After 48 hours of screening, stable cell lines are obtained.

[0074] Cells from the sh-LINC02381 and sh-NC groups were collected, total RNA was extracted, and LINC02381 expression was detected by RT-qPCR. Results are as follows: Figure 6 As shown, the results indicate that the LINC02381 mRNA level in the sh-LINC02381 group decreased by approximately 70% compared to the sh-NC group. P adj <0.001).

[0075] Cells from the sh-EGFR2 and sh-NC groups were collected, total protein was extracted, and EGFR protein expression was detected by Western blot. The results showed that the knockdown efficiency was over 50%. P adj <0.0001) (Results are the same as above) Figure 5 This indicates that the stable knockdown cell line was successfully constructed.

[0076] 3. Establishment and validation of core model indicators 3.1 Determination of the optimal concentration of LY294002 (a broad-spectrum PI3K inhibitor), a downstream pathway activity control drug To verify the model's ability to detect downstream PI3K / Akt pathway activity, the CCK-8 assay was used to detect the cell viability of 12Z cells treated with different concentrations of LY294002 (0, 1, 5, 10, 15, 20, 30, 40, 50 μM) for 24 h. Furthermore, Western blotting was used to detect p-AKT1 expression after 6 h and 24 h of treatment with 0 μM, 1 μM, and 5 μM LY294002. The results are as follows: Figure 7 As shown in the figure, (A) the effect of different concentrations of LY on the survival rate of 12Z; (B) the expression level of 12Z p-Akt protein. The results showed that the survival rate was >80% at 1 μM and 5 μM concentrations, and treatment with 1 μM for 6 h significantly inhibited p-AKT1 (… P adj <0.05). Therefore, 1 μM LY294002 was selected as the control concentration for subsequent downstream pathway activity assays. (Note: LY294002 is only used to verify the reliability of the downstream p-PI3K / p-AKT1 assay system and is not used to set the decision threshold for the LINC02381-EGFR axis.) 3.2 Model Detection Indicators and Threshold Settings Based on the results of the above knockdown experiment (sh-LINC02381), the core detection indicators and judgment criteria of the model are determined as follows: 3.2.1 Determination of positive reference Using 12Z cells with stable knockdown of LINC02381 (sh-LINC02381) as a positive control, the results showed that compared with the sh-NC group, the LINC02381 mRNA level in the sh-LINC02381 group was reduced by ≥80%. Figure 6 EGFR protein decreased by approximately ≥20% Figure 19 ), p-PI3K / p-AKT1 protein decreased by approximately ≥20% ( Figure 19 EdU positivity rate decreased by ≥30% Figure 15 ), the number of transmembrane cells decreased by ≥30% ( Figure 16 ), scratch healing distance reduced by ≥15% ( Figure 17 The above data defines the maximum effect achievable by completely blocking the LINC02381-EGFR axis.

[0077] 3.2.2 Setting the Judgment Threshold Judgment criteria: Molecular indicators (at least one of the following must be met): ① ≥40% reduction in LINC02381 mRNA (equivalent to approximately 50% of the knockdown effect); ② ≥15% reduction in EGFR protein (equivalent to approximately 75% of the knockdown effect); ③ ≥15% reduction in p-PI3K or p-AKT1 protein (equivalent to approximately 75% of the knockdown effect).

[0078] Functional indicators (at least one of which is significantly inhibited) P adj <0.05): ① EdU positive cell proportion decreased by ≥30%; ② Transwell cell number decreased by ≥30%; ③ Scratch healing distance decreased by ≥15%.

[0079] Comprehensive judgment: If a drug meets at least one molecular indicator and at least one functional indicator, it is judged to be an anti-EMs candidate drug.

[0080] 4. Stability and repeatability verification Preparation of serum containing the herb *Heliotropium indicum*: Ten female SD rats (weighing 200±20g) were administered a decoction of *Heliotropium indicum* by gavage. The dosage was 0.54g crude drug / day (equivalent to a clinical dose of 30g crude drug / day / 70kg human), calculated based on the human and animal body surface area. This was administered once daily for 7 consecutive days. One hour after the last administration, blood was collected from the abdominal aorta. After standing for 2 hours, the blood was centrifuged at 3000 rpm and 4℃ for 15 min. The supernatant was collected, filtered through a 0.22μm filter for sterilization, inactivated in a 56℃ water bath for 30 min, aliquoted, and stored at -80℃. The control group serum was prepared by gavage with an equal volume of physiological saline using the same method.

[0081] Three batches of independently cultured 12Z cells (each batch spaced > 1 month apart) were treated with serum containing *Ligustrum lucidum* for 48 h. The following parameters were then measured: LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and number of cells that permeated the cell membrane. Results showed that the reduction in LINC02381 mRNA in the three batches was 45%, 43%, and 40%, respectively, with a coefficient of variation (CV) of 5.9%; the reduction in the number of cells that permeated the cell membrane was 63%, 52%, and 50%, respectively, with a CV of 12.7%. These results indicate that the model has good stability and reproducibility.

[0082] Example 2 The effectiveness of the method / system / model was validated using serum containing the drug-containing substance *Liriope muscari*. 1. Preparation of serum containing *Phyllostachys edulis* (PFH-containing serum): Ten female SD rats (weighing 200±20g) were administered *Phyllostachys edulis* decoction by gavage at a dose equivalent to 0.54g crude drug / day (equivalent to a clinical dose of 30g crude drug / day / 70kg human) calculated based on human and animal body surface area, once daily for 7 consecutive days. One hour after the last administration, blood was collected from the abdominal aorta, allowed to stand for 2 hours, centrifuged at 3000rpm and 4℃ for 15min, and the supernatant was collected. The supernatant was then filtered through a 0.22μm filter membrane for sterilization, inactivated in a 56℃ water bath for 30min, aliquoted, and stored at -80℃. The control group serum was prepared by gavage with an equal volume of physiological saline using the same method.

[0083] 2. Model Response Detection to Yellow Water Lily 2.1 Concentration Screening PFH-containing serum was diluted to 10%, 50%, and 100% (v / v) in DMEM basal medium and used to treat 12Z cells for 48 hours. Cell growth curves were observed using the Incucyte live cell dynamics analysis system. The effects of different concentration gradients of PFH-containing serum on hEEC proliferation activity are shown below. Figure 8 As shown; the effects of serum containing different concentration gradients of PFH on the proliferation activity of 12Z are as follows. Figure 9As shown in the figure. The results showed that a 10% concentration could inhibit the growth of approximately 50% of 12Z cells, while a 100% concentration caused cells to almost stop growing, and the 10% concentration had no significant toxicity to normal hEEC cells. Therefore, 10% PFH-containing serum was chosen as the validation concentration.

[0084] 12Z cells were divided into a control group (10% control serum), a PFH group (10% PFH-containing serum), an LY group (1 μM LY294002), and a combination group (10% PFH + 1 μM LY). Molecular markers and functional phenotypes were detected 24 h after treatment.

[0085] 2.2 Molecular index detection ①RT-qPCR detection of mRNA expression: Total RNA was extracted from cells in each group, and cDNA was synthesized using a reverse transcription kit according to the TRIzol method. qPCR was performed using the SYBR Green method, with primer sequences shown in the table below. β-actin was used as an internal control. -ΔΔCt The relative expression level is calculated using this method.

[0086] Table 2 Primer Sequences

[0087] The results are as follows Figure 10 As shown: Compared with the control group, the PFH group showed a significant decrease in LINC02381 mRNA levels of approximately 43% ( P adj <0.0001), the combined group reduced by approximately 46% ( P adj <0.0001); EGFR mRNA showed a decreasing trend in the PFH group, but the result was not statistically significant. P adj >0.05); PI3KCB and AKT1 mRNA were significantly reduced in both the PFH group and the combined group ( P adj <0.05).

[0088] ②Western Blot detection of protein expression: Cells from each group were collected, RIPA lysis buffer (containing protease and phosphatase inhibitors) was added, and the cells were lysed on ice for 30 min. After centrifugation at 12,000 rpm for 15 min, the supernatant was collected. Protein concentration was determined by BCA method. Equal amounts of protein were subjected to SDS-PAGE electrophoresis, transferred to a membrane, and blocked. Primary antibodies (EGFR 1:1000, PI3K 1:1000, p-PI3K 1:500, AKT1 1:1000, p-AKT1 1:500, β-actin 1:5000) were added sequentially, and the membrane was incubated overnight at 4°C. After washing, secondary antibody (1:5000) was added and incubated at room temperature for 1 h. The membrane was then developed by ECL, and the gray values ​​were analyzed by ImageJ.

[0089] The results are as follows Figure 11 As shown: Compared with the control group, EGFR protein expression in the PFH group decreased by approximately 43% ( P adj <0.05), p-PI3K decreased by about 40% ( P adj <0.01), p-AKT1 decreased by approximately 40% ( P adj <0.05); the combined group showed a more significant decrease in the above indicators (all <0.05); P adj <0.001). No significant changes were observed in the expression of total PI3K and total AKT1 proteins.

[0090] 2.3 Functional Phenotypic Testing ①EdU proliferation assay: Cells from each group were seeded in 6-well plates. After 24 hours of drug intervention, EdU working solution (10 μM) was added and incubated for 2 hours. Cells were then fixed, permeabilized, stained, and photographed using a fluorescence microscope. The proportion of EdU-positive cells was counted.

[0091] The results are as follows Figure 12 As shown: The EdU positivity rate in the PFH group decreased by approximately 22% compared to the control group. P adj <0.0001), the combined group showed a more significant decrease of approximately 30% ( P adj <0.0001) ②Transwell invasion assay: Matrigel was diluted 1:8 and spread in the upper chamber of a Transwell chamber, then solidified at 37°C for 2 hours. Cells from each group were digested and resuspended in serum-free medium to a concentration of 1×10⁻⁶. 5 / mL, take 100μL and add it to the upper chamber, and add 600μL of culture medium containing 10% FBS (or the drug to be tested) to the lower chamber. After culturing for 48h, fix, stain with crystal violet, and count the number of cells that have penetrated the membrane under a microscope.

[0092] The results are as follows Figure 13 As shown: the number of transmembrane cells in the PFH group was reduced by approximately 55% compared to the control group. P adj <0.001), the combined group reduced by approximately 76% ( P adj <0.0001).

[0093] ③ Scratch migration assay: Cells from each group were seeded in 6-well plates. After 24 hours, different drugs were administered. After 48 hours, the cells were scratched with a 200 μL pipette tip, washed with PBS, and cultured in serum-free medium. Photos were taken at 0 hours and 24 hours, and the scratch healing distance was calculated.

[0094] The results are as follows Figure 14 As shown: the scratch healing distance in the PFH group was reduced by approximately 35% compared to the control group. P adj <0.05%, the combined group reduced by approximately 39% ( P adj <0.01).

[0095] Among the above indicators, LINC02381 mRNA, EGFR protein, and p-PI3K / p-AKT1 all reached the molecular indicator threshold; among the functional indicators, scratch healing distance and permeabilized cell number reached the threshold, meeting the positive judgment criteria. *Echinochloa crus-galli* exhibits significant anti-EMs activity, and this also verifies that the method / system / model can accurately identify effective anti-EMs drugs.

[0096] Example 3 The specificity of the method / system / model was further validated using gene knockdown cell lines. 1. Effects of knocking down LINC02381 or EGFR on 12Z cell phenotype 12Z cells (constructed according to the method in Example 1) were stably transfected with sh-LINC02381 and sh-EGFR2, respectively, along with the corresponding negative control sh-NC. Cells were divided into six treatment groups: sh-NC, sh-NC+PFH, sh-LINC, sh-LINC+PFH, sh-EGFR, and sh-EGFR+PFH. All groups were treated with either 10% PFH-containing serum or control serum. Specific treatments are as follows: Table 3 Treatment methods for each group

[0097] Note: CON is the serum of the control group; the preparation method of 10% PFH-containing serum is the same as in Example 2.

[0098] Molecular index and functional phenotype detection were performed on each group 24 hours after treatment, using the same detection methods as in Example 2.

[0099] EdU proliferation experiment such as Figure 15 As shown: Compared with the sh-NC group, the EdU positivity rate was significantly lower in the sh-LINC group, sh-EGFR group, and all PFH-added groups. P adj <0.05, with the sh-EGFR+PFH group showing the most significant decrease ( P adj <0.0001).

[0100] Transwell invasion experiments such as Figure 16 As shown: Compared with the sh-NC group, the number of transmembrane cells decreased by approximately 40% in the sh-LINC and sh-EGFR groups.P adj <0.01), further reduced after adding PFH ( P adj <0.0001).

[0101] Scratch migration experiment such as Figure 17 As shown: Compared with the sh-NC group, the scratch healing distance in the sh-LINC and sh-EGFR groups was reduced by approximately 18-25%. P adj <0.05), the effect is more significant after adding PFH.

[0102] RT-qPCR results are as follows Figure 18 As shown: LINC02381 expression was significantly decreased in the sh-LINC group ( P adj <0.0001), EGFR mRNA showed no significant change, but miR-27a-3p expression increased ( P adj <0.05); EGFR protein decreased in the sh-EGFR group ( P adj <0.0001), LINC02381 mRNA showed a feedback increase ( P adj <0.0001), while LINC02381 expression was reversed after combining with PFH ( P adj <0.0001).

[0103] Western Blot results are as follows Figure 19 As shown, the expression of EGFR, PI3K, p-PI3K, AKT1, and p-AKT1 proteins decreased in both the sh-LINC and sh-EGFR groups, with the decrease being more significant after the combination with PFH.

[0104] The above results further confirm that the LINC02381-EGFR axis is a key target of anti-EMs drugs such as PFH, and that intervention effects targeting this axis can be specifically detected.

[0105] Example 4 In vivo efficacy validation in animals (in vivo evidence of the predictive power of methods / systems / models): To verify the consistency between drug screening results and in vivo efficacy, an SD rat model of endometriosis (autologous transplantation) was established, consisting of 6 groups: sham-operated group, model group, LY group (40 mg / kg LY294002, intraperitoneal injection, once weekly), low-dose PFH group (2.7 g crude drug / kg / day, gavage), medium-dose PFH group (5.4 g crude drug / kg / day), and high-dose PFH group (10.8 g crude drug / kg / day), with 6 rats in each group. The drugs were administered continuously for 28 days. The preparation method for PFH-containing serum was the same as in Example 2. Molecular marker detection and functional phenotypic detection methods were also the same as in Example 2.

[0106] 1. Animal model establishment (autologous transplantation method) Eight-week-old female SD rats, weighing 197.9±6.2g, were used. The estrous cycle was confirmed via vaginal smear. Twenty-four hours prior to modeling, 0.02mg / 100g of estradiol benzoate was subcutaneously injected into the posterior neck. After anesthesia, a midline abdominal incision was made, one uterus was located, ligated, and a 1cm segment was harvested. This segment was longitudinally dissected and cut into two tissue blocks approximately 5mm × 5mm. The endometrial surface was sutured tightly against the abdominal wall muscles. Postoperatively, penicillin was administered intramuscularly for 3 days, followed by estradiol injections every 3 days for a total of 5 times. Twenty-eight days postoperatively, laparotomy was performed to observe the graft growth as vesicular with visible surface blood vessels. HE staining confirmed the growth of ectopic endometrial tissue, indicating successful modeling.

[0107] like Figure 20 As shown in the figure, (A) endometrial tissue implanted into the abdominal wall during modeling (indicated by the green arrow); (B) after 28 days of modeling, the endometrial tissue implanted into the abdominal wall grows in a vesicular pattern with visible blood vessels on the surface (indicated by the blue arrow); (C) HE-stained pathological sections of the uterus and ectopic lesions of rats in the sham-operated group and the modeling group. Green arrows represent columnar epithelial cells, red arrows represent stem cell-like cells, and yellow arrows represent inflammatory cells (scale bar: upper 100μm, lower 50μm).

[0108] 2. Experimental Results 2.1 The following table shows the statistical statistics of ectopic lesion volume: Table 4. Volume of ectopic lesions in each group

[0109] Note: Due to severe adhesions, the size of the lesion in model group 2 could not be accurately measured.

[0110] The longest diameter of the lesions in each model group is shown as follows: Figure 21 As shown in the figure. The results show that the average volume of the model group is 39.66 mm. 3 The longest diameter of the lesion was 6.07 mm; the average volume of the LY group was 15.08 mm. 3 (reduced by 62.0%) P adj<0.01), the longest diameter of the lesion was 3.39 mm; 26.47 mm in the low-dose PFH group. 3 (reduced by 33.3%) P adj <0.05), the longest diameter of the lesion was 4.15 mm; 21.63 mm in the medium-dose PFH group. 3 (reduced by 45.5%) P adj <0.01), the longest diameter of the lesion was 3.71 mm; in the high-dose PFH group it was 10.92 mm. 3 (reduced by 72.5%) P adj <0.001), the longest diameter of the lesion was 2.57 mm.

[0111] 2.2 The degree of adhesion is scored as shown in the table below: Table 5 Adhesion Scoring Criteria

[0112] Table 6 Adhesion Degree Scoring

[0113] The results showed that the model group had an average score of 2.17 (extensive thick adhesions); the LY group had a score of 1.0 (thin and easily separated); and the high-dose PFH group had a score of 0.67 (almost no adhesions). P adj <0.001).

[0114] 2.3 HE pathological sections as follows Figure 22 As shown in the figure (scale bar: top 100μm, bottom 50μm. Green arrows represent normal columnar epithelial cells, blue arrows represent significant degenerative changes in the endometrium, with epithelial cell damage, peeling, and disordered arrangement), the results showed that columnar epithelial cells and abundant blood vessels were visible in the model group; in the high-dose PFH group, glandular epithelial cells were disordered, damaged, and peeled, stromal cells were loosely arranged, and significant degenerative changes were observed.

[0115] 2.4 Results of molecular marker expression are as follows Figure 23 As shown, the results indicated that in ectopic lesion tissue, the levels of EGFR, PI3K, p-PI3K, AKT1, and p-AKT1 proteins in the model group were significantly higher than those in the Sham group. P adj <0.05); the above proteins were significantly reduced in both the high-dose PFH group and the LY group ( P adj <0.05).

[0116] 2.5 Results of improvement in physical condition as follows Figures 24-27 As shown, the results indicated that both the LY group and the low-dose PFH group experienced weight gain (P adj <0.05. The body temperature of the model group decreased after modeling ( P adj <0.05%, body temperature rebounded after intervention in the medium-dose PFH group ( P adj <0.05. The model group showed a significant reduction in water and food intake ( P adj <0.001), the high-dose PFH group recovered ( P adj <0.05).

[0117] 2.6. Serum marker results as follows Figures 28-29 As shown, the results indicate that ICAM-1, MMP-2, and Fg levels were significantly elevated in the model group ( P adj <0.0001), T3 decreased significantly ( P adj <0.01); PFH and LY groups all reduced ICAM-1, MMP-2, and Fg ( P adj (<0.01-0.0001), T3 shows an upward trend.

[0118] The above in vivo data are highly consistent with the model screening results (in vitro), further demonstrating that the method / system / model based on the LINC02381-EGFR axis has reliable predictive capabilities.

[0119] Example 5 An in vitro screening method for endometriosis drugs based on the LINC02381-EGFR axis: (1) Steps: Cell plating: 12Z cells were plated at a density of 5 × 10⁻⁶. 3 Cells / well seeded in 96-well plates (for proliferation assay) or 2 × 10⁶ cells / well. 4 Cells were seeded in 24-well plates (for RT-qPCR / Western Blot) and cultured overnight.

[0120] Drug administration: The test drug was diluted with complete culture medium to different concentrations (e.g., 0.1, 1, 10 μM), with 3 replicates for each concentration. A solvent control (0.1% DMSO or PBS) and a positive control (12Z cells knocked down with LINC02381) were also included. The cells were incubated at 37°C for 24 h.

[0121] (2) Testing: RT-qPCR: Cells were collected, total RNA was extracted, reverse transcribed, and qPCR was performed using primers listed in Table 2, with β-actin as an internal control. -ΔΔCtThe relative expression levels of LINC02381 and EGFR were calculated using the method.

[0122] Western Blot: Another batch of cells was taken, lysed, and the EGFR, p-PI3K, and p-AKT1 proteins were detected.

[0123] EdU proliferation: Take another independently cultured 96-well plate, add EdU working solution (10 μM) and incubate for 2 hours after 24 hours of drug intervention, fix, permeabilize, stain, and photograph under a fluorescence microscope. Count the proportion of EdU-positive cells.

[0124] Transwell invasion assay: A separate 96-well plate was used for independent cell culture. Matrigel was diluted 1:8 and placed in the upper chamber of a Transwell chamber, where it was incubated at 37°C for 2 hours. Cells from each group were digested and resuspended in serum-free medium to a concentration of 1×10⁶ cells / well. 5 / mL, take 100μL and add it to the upper chamber, and add 600μL of culture medium containing 10% FBS (or the drug to be tested) to the lower chamber. After culturing for 48h, fix, stain with crystal violet, and count the number of cells that have penetrated the membrane under a microscope.

[0125] Scratch migration assay: Cells from each group were seeded in 6-well plates. After 24 hours, different drugs were administered. After 48 hours, scratches were made with a 200 μL pipette tip. Cells were washed with PBS and cultured in serum-free medium. Photos were taken at 0 hours and 24 hours, and the scratch healing distance was calculated.

[0126] Criteria: If a drug meets at least one molecular marker and at least one functional marker, it is considered a candidate drug for anti-EMs. Molecular markers (meeting at least one): ① LINC02381 mRNA reduction ≥40%; ② EGFR protein reduction ≥15%; ③ p-PI3K or p-AKT1 protein reduction ≥15%. Functional markers (at least one showing significant inhibition) P adj <0.05): ① EdU positive cell proportion decreased by ≥30%; ② Transwell cell number decreased by ≥30%; ③ Scratch healing distance decreased by ≥15%.

[0127] Example 6 An in vitro screening system for endometriosis drugs based on the LINC02381-EGFR axis includes: a sample collection module, an indicator detection module, a screening module, and a result output module.

[0128] Sample collection module: Collect 12Z cells after treatment with the drug to be tested; the drug treatment method is the same as step (1) in Example 5.

[0129] Indicator detection module: The LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance and number of cells that have perforated the membrane in 12Z cells were detected; the detection method is the same as step (2) in Example 5.

[0130] Screening module: Analyze the detection indicators to determine whether the drug to be tested meets the screening criteria; the screening criteria are (1) a decrease of ≥40% in LINC02381 mRNA and / or a decrease of ≥15% in EGFR protein and / or a decrease of ≥15% in p-PI3K / p-AKT1 protein; (2) a decrease of ≥30% in EdU positivity rate and / or a decrease of ≥15% in scratch healing distance and / or a decrease of ≥30% in the number of transmembrane cells.

[0131] The results output module outputs the results; the output results are set to display "meets screening criteria" and "does not meet screening criteria", and drugs that meet the screening criteria are selected as candidate drugs for endometriosis.

[0132] Example 7 An evaluation method for endometriosis drugs based on the LINC02381-EGFR axis: (1) Steps: Cell plating: 12Z cells were plated at a density of 5 × 10⁻⁶. 3 Cells / well seeded in 96-well plates (for proliferation assay) or 2 × 10⁶ cells / well. 4 Cells were seeded in 24-well plates (for RT-qPCR / Western Blot) and cultured overnight.

[0133] Drug administration: The test drug was diluted with complete culture medium to different concentrations (e.g., 0.1, 1, 10 μM), with 3 replicates for each concentration. A solvent control (0.1% DMSO or PBS) and a positive control (12Z cells knocked down with LINC02381) were also included. The cells were incubated at 37°C for 24 h.

[0134] (2) Testing: RT-qPCR: Cells were collected, total RNA was extracted, reverse transcribed, and qPCR was performed using primers listed in Table 2, with β-actin as an internal control. -ΔΔCt The relative expression levels of LINC02381 and EGFR were calculated using the method.

[0135] Western Blot: Another batch of cells was taken, lysed, and the EGFR, p-PI3K, and p-AKT1 proteins were detected.

[0136] EdU proliferation: Take another independently cultured 96-well plate, add EdU working solution (10 μM) and incubate for 2 hours after 24 hours of drug intervention, fix, permeabilize, stain, and photograph under a fluorescence microscope. Count the proportion of EdU-positive cells.

[0137] Transwell invasion assay: A separate 96-well plate was used for independent cell culture. Matrigel was diluted 1:8 and placed in the upper chamber of a Transwell chamber, where it was incubated at 37°C for 2 hours. Cells from each group were digested and resuspended in serum-free medium to a concentration of 1×10⁶ cells / well. 5 / mL, take 100μL and add it to the upper chamber, and add 600μL of culture medium containing 10% FBS (or the drug to be tested) to the lower chamber. After culturing for 48h, fix, stain with crystal violet, and count the number of cells that have penetrated the membrane under a microscope.

[0138] Scratch migration assay: Cells from each group were seeded in 6-well plates. After 24 hours, different drugs were administered. After 48 hours, scratches were made with a 200 μL pipette tip. Cells were washed with PBS and cultured in serum-free medium. Photos were taken at 0 hours and 24 hours, and the scratch healing distance was calculated.

[0139] Predicting the therapeutic effect of the drug to be evaluated on endometriosis: The greater the reduction in the LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and / or number of perforated cells, the better the efficacy of the corresponding drug.

[0140] Example 8 An in vitro evaluation system for endometriosis drugs based on the LINC02381-EGFR axis includes: a sample collection module, an indicator detection module, an evaluation module, and a result output module.

[0141] Sample collection module: Collect 12Z cells after drug intervention treatment to be evaluated; the drug intervention treatment method is the same as step (1) in Example 7.

[0142] Indicator detection module: The LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance and number of cells that have perforated the membrane in 12Z cells were detected; the detection method is the same as step (2) in Example 7.

[0143] Evaluation module: Using 12Z cells with knockdown of LINC02381 as a positive control, the efficacy of the drug was evaluated by analyzing various indicators; the mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance and number of cells that perforated the membrane were compared between the drug to be evaluated and the positive control.

[0144] The results output module outputs the results; the output results are set to compare and rank the predicted effects of the drugs to be evaluated based on the decrease of the above indicators. The greater the decrease, the better the drug treatment effect.

[0145] 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. An in vitro screening method for endometriosis drugs based on the LINC02381-EGFR axis, characterized in that, Includes the following steps: 12Z cells were treated with the test drug. After intervention, the LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and number of cells that perforated the membrane were measured in 12Z cells. Test drugs that simultaneously met the following criteria were selected: (1) The LINC02381 mRNA transcription level decreased by ≥40% and / or the EGFR protein expression level decreased by ≥15% and / or the p-PI3K / p-AKT1 protein expression level decreased by ≥15%; (2) The EdU positivity rate decreased by ≥30% and / or the scratch healing distance decreased by ≥15% and / or the number of transmembrane cells decreased by ≥30%.

2. The in vitro screening method according to claim 1, characterized in that, The intervention lasted for 48 hours.

3. An in vitro screening system for endometriosis drugs based on the LINC02381-EGFR axis, characterized in that, The in vitro screening system includes: a sample collection module, an indicator detection module, a screening module, and a result output module; The sample collection module collects 12Z cells after treatment with the drug to be tested. The indicator detection module detects the LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and number of cells that perforate the membrane in 12Z cells. The screening module analyzes the detection indicators to determine whether the drug to be tested meets the screening criteria; the screening criteria are (1) a decrease of ≥40% in LINC02381 mRNA transcription level and / or a decrease of ≥15% in EGFR protein expression level and / or a decrease of ≥15% in p-PI3K / p-AKT1 protein expression level; (2) a decrease of ≥30% in EdU positivity rate and / or a decrease of ≥15% in scratch healing distance and / or a decrease of ≥30% in the number of transmembrane cells; The result output module outputs the results.

4. The use of the in vitro screening method according to any one of claims 1 to 2 or the in vitro screening system according to claim 3 in screening or preparing drugs for endometriosis.

5. The application according to claim 4, characterized in that, The drugs shown for endometriosis include extracts of traditional Chinese medicine, chemically synthesized compounds, natural product monomers, peptides, or nucleic acid drugs.

6. A method for evaluating endometriosis drugs based on the LINC02381-EGFR axis, characterized in that, Includes the following steps: 12Z cells were treated with the drug to be evaluated, with 12Z cells knocked down by LINC02381 as a positive control. The efficacy of the drug was evaluated based on any one or more of the following indicators: LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance and / or number of cells that permeate the membrane.

7. The evaluation method according to claim 6, characterized in that, The intervention lasted for 48 hours.

8. An in vitro evaluation system for endometriosis drugs based on the LINC02381-EGFR axis, characterized in that, include: Sample collection module, indicator detection module, evaluation module, and result output module; The sample collection module collects 12Z cells after drug intervention treatment to be evaluated; The indicator detection module detects the LINC02381 mRNA transcription level, EGFR protein expression level, p-PI3K / p-AKT1 protein expression level, EdU positivity rate, scratch healing distance, and number of cells that perforate the membrane in 12Z cells. The evaluation module uses 12Z cells with knocked-down LINC02381 as a positive reference to analyze various indicators and evaluate the efficacy of the drug. The result output module outputs the results.

9. The use of the evaluation method according to any one of claims 6 to 7 or the in vitro evaluation system according to claim 8 in the preparation of drugs for endometriosis.

10. The application according to claim 9, characterized in that, The drugs shown for endometriosis include extracts of traditional Chinese medicine, chemically synthesized compounds, natural product monomers, peptides, or nucleic acid drugs.

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