Use of bhlhe40 in preparing retinoblastoma prognosis evaluation kit and therapeutic drug

Abstract

This invention discloses the application of BHLHE40 in the preparation of prognostic assessment kits and therapeutic drugs for retinoblastoma. Through a series of in vitro and in vivo experiments, this invention demonstrates that knocking down BHLHE40 expression significantly inhibits the proliferation of retinoblastoma cells, exhibiting a significant tumor-suppressive effect. Therefore, it is proposed that BHLHE40 inhibitors can be used in the preparation of therapeutic drugs for retinoblastoma, providing a new, effective, and safe treatment option for RB patients. Furthermore, BHLHE40 can serve as a drug target to guide subsequent drug development. In addition, this invention also utilizes clinical tumor tissue samples to demonstrate that high BHLHE40 expression is significantly positively correlated with high-risk pathological features such as optic nerve invasion, scleral invasion, and choroidal invasion, suggesting that BHLHE40 can serve as an effective biomarker for predicting high-risk pathological features, assisting physicians in treatment decisions.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to the application of BHLHE40 in the preparation of retinoblastoma prognostic assessment kits and therapeutic drugs. Background Technology

[0002] Retinoblastoma (RB) is the most common primary malignant intraocular tumor in infants and young children. Current mainstream treatments for RB include systemic intravenous chemotherapy, ophthalmic artery interventional chemotherapy, local treatment (laser, cryotherapy), and enucleation in severe cases. While existing treatments can control tumor progression and save lives to some extent, they still have many limitations in clinical application: systemic intravenous chemotherapy drugs lack targeting, potentially causing significant toxic side effects on the hematopoietic, digestive, and nervous systems of infants and young children during their growth and development; ophthalmic artery interventional surgery requires advanced techniques, is greatly affected by variations in the patient's ophthalmic artery, and still carries the risk of local complications; local treatment usually needs to be combined with chemotherapy, and local treatment alone is only effective for very small, localized tumors (e.g., <2 mm); while enucleation can effectively reduce the risk of tumor metastasis and save lives, it directly leads to permanent blindness, severely impacting the child's quality of life and causing heavy psychological trauma and long-term psychological burden on the child's psychological development and the entire family. Therefore, exploring new, highly effective, and low-toxicity therapeutic targets and drugs is an important direction for basic research and clinical translation of RB.

[0003] Furthermore, the current criteria for judging the prognosis of children with RB and determining the postoperative adjuvant treatment plan mainly rely on the high-risk pathological features (optic nerve invasion, scleral invasion, choroidal invasion, etc.) assessed by histopathological examination after enucleation. However, this method has the following shortcomings: (1) Delay: Pathological results can only be obtained after enucleation. For children who need eye-preservation treatment, clinicians cannot predict their risk level before treatment, making it difficult to choose a more aggressive local treatment plan; (2) Subjectivity and risk of missed detection: Pathological sections are sampled and may miss small local invasion lesions, and the interpretation results of different pathologists may also differ; (3) Inability to monitor dynamically: Pathological examination is a one-time event. In summary, there are currently no molecular markers that can predict the response rate of eye-preservation treatment. Clinicians cannot determine which children are more likely to benefit from eye-preservation treatment before treatment. There is an urgent need in this field to find new biomarkers at the molecular level that can accurately predict the prognosis at an early stage.

[0004] BHLHE40 (Basic Helix-Loop-Helix Family Member E40) is a protein-coding gene located in the 3p26.1 region of the human genome, encoding a basic helix-loop-helix (bHLH) transcription factor. This gene is involved in regulating various physiological and pathological processes, including circadian rhythms, cell differentiation, hypoxia response, and immune regulation. However, a review of relevant domestic and international patents and literature has yielded no research reports on the application of BHLHE40 in the treatment and prognostic assessment of retinoblastoma.

[0005] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of a reagent for detecting the expression level of BHLHE40 in a test sample in the preparation of a retinoblastoma prognostic assessment kit, thereby solving the problem of the lack of molecular markers in the prior art for accurately predicting the treatment response and disease progression risk of individual patients before treatment; it also provides the application of BHLHE40 inhibitors in the preparation of drugs for treating retinoblastoma, providing new targeted therapy options for RB patients to improve the effectiveness and safety of treatment.

[0007] Based on the above, the present invention first provides the application of a reagent for detecting the expression level of BHLHE40 in a sample to be tested in the preparation of a retinoblastoma prognostic assessment kit.

[0008] Optionally, the prognostic assessment includes: when a high level of BHLHE40 expression is detected, the subject is judged to be a high-risk patient, indicating a poor prognosis; when a low level of BHLHE40 expression is detected, the subject is judged to be a low-risk patient, indicating a good prognosis.

[0009] Optionally, the sample to be tested may include: a tumor tissue sample or a blood sample.

[0010] Optionally, the methods for detecting the expression level of BHLHE40 in the sample to be tested include any one or more of the following: real-time quantitative PCR, Western blotting, flow cytometry, or immunohistochemistry.

[0011] In another aspect, the present invention provides the use of BHLHE40 inhibitors in the preparation of medicaments for the treatment of retinoblastoma.

[0012] Optionally, the BHLHE40 inhibitor is a substance that knocks out or knocks down the BHLHE40 gene, or reduces the expression level or activity of the BHLHE40 protein, or antagonizes the function of BHLHE40.

[0013] Optionally, the substance includes any one of the following: protein, small molecule compound, or gene intervention tool capable of knocking out or knocking down BHLHE40 expression.

[0014] Optionally, the gene intervention tool includes either an RNAi fragment or a gene editing tool.

[0015] Optionally, the RNAi fragment is shRNA, and the shRNA is any one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0016] In another aspect, the present invention provides a method for screening candidate drugs for the treatment of retinoblastoma, comprising: detecting the downregulation effect of candidate drugs on BHLHE40 expression or the inhibition effect of BHLHE40 activity.

[0017] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention, through experimental discovery, reveals for the first time that BHLHE40 is a core regulatory factor in glycolytic metabolism in retinoblastoma, suggesting that BHLHE40 can serve as a potential biomarker for metabolic subtyping of retinoblastoma, providing new analytical basis for this process. By detecting BHLHE40 expression levels, RB patients can be classified into those with enhanced or diminished glycolytic activity, thus achieving metabolic subtyping.

[0018] Further in vitro and in vivo experiments confirmed that knocking down BHLHE40 expression significantly inhibited the proliferation of retinoblastoma cells, demonstrating a significant tumor-suppressive effect. Therefore, this invention proposes that BHLHE40 can serve as a drug target to guide subsequent drug development, and that BHLHE40 inhibitors can be used in the preparation and application of therapeutic drugs for retinoblastoma, providing a new, effective, and safe treatment option for RB patients.

[0019] More importantly, this invention, using clinical samples, reveals for the first time the expression characteristics and clinical significance of BHLHE40 in retinoblastoma tissue. RB tumor samples confirmed a significant positive correlation between high BHLHE40 expression and high-risk pathological features such as optic nerve invasion, scleral invasion, and choroidal invasion, suggesting that BHLHE40 can serve as an independent prognostic risk factor, assisting physicians in treatment decisions. Specifically, by detecting BHLHE40 expression levels, high-risk / low-risk patients can be distinguished before treatment begins, guiding physicians to scientifically select treatment plans: eye-sparing or early enucleation, thereby reducing overtreatment and undertreatment, and enabling early, non-invasive / minimally invasive prognostic assessment without waiting until enucleation to determine the prognosis. Attached Figure Description

[0020] Figure 1The effect of changes in BHLHE40 expression on glycolysis in RB cells, including: A represents the changes in protein expression levels of key glycolytic enzymes HK2, PKM2, ENO1, and LDHA in cells of the BHLHE40 group with knockdown. B represents the changes in protein expression levels of key glycolytic enzymes HK2, PKM2, ENO1, and LDHA in cells overexpressing BHLHE40. CD represents the results of glycolysis rate measurement in cells of the BHLHE40 knockdown group; EF represents the results of glycolysis rate assay in cells overexpressing BHLHE40; Here, Basal Glycolysis represents the baseline glycolysis level, Compensatory Glycolysis represents the compensatory glycolysis level, and glycoPER represents the glycolytic proton efflux rate.

[0021] Figure 2 The effect of changes in BHLHE40 expression on the proliferation ability of RB cells, including: A represents the cell proliferation rate of the BHLHE40 knockdown group as detected by the CCK-8 assay. B indicates the cell proliferation rate of the BHLHE40 overexpressing group as detected by the CCK-8 assay. C represents photographs of tumor tissue from nude mice in the BHLHE40 knockdown (shBHLHE40) group and the control group (shCtrl, without BHLHE40 knockdown treatment); D indicates the tumor weight of nude mice in the BHLHE40 knockdown (shBHLHE40) group and the control group; E represents the proportion of Ki-67-positive cells in tumor tissues of nude mice in the BHLHE40 knockdown (shBHLHE40) group and the control group, as detected by immunofluorescence staining.

[0022] Figure 3 To analyze the association between BHLHE40 expression levels and high-risk histopathological features in patients with renal retinoblastoma (RB), and to validate the performance of BHLHE40 as a prognostic biomarker, the following analysis was conducted: A indicates the expression distribution of BHLHE40 in RB tumor tissue and normal retinal tissue detected by immunofluorescence staining; B indicates that the expression of BHLHE40 is significantly associated with high-risk pathological features in RB patients. Fisher's exact test was used to test this association (OR=24.0, 95% CI: 2.5-148.1). Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0024] As mentioned earlier, the mainstream treatments for RB currently include systemic intravenous chemotherapy, ophthalmic artery interventional chemotherapy, and enucleation. Among these, chemotherapy is currently the main method for eye preservation in intraocular RB. However, current chemotherapy regimens for RB are still mainly based on traditional cytotoxic drugs, which have problems such as large toxic side effects and high drug resistance rates, affecting long-term efficacy and the success rate of eye preservation. Although enucleation can effectively reduce the risk of tumor metastasis and save lives, it will directly lead to permanent blindness in children, seriously affecting their quality of life.

[0025] Current treatment decisions are primarily based on clinical staging and imaging assessments. However, the following situations are common in clinical practice: two children with the same clinical stage receive the same eye-preserving treatment plan, but one achieves good control while the other progresses, indicating that the latter responds poorly to eye-preserving treatment, indicating inadequate treatment and ultimately requiring enucleation, or even delaying treatment and increasing the risk of metastasis / death; or two children with the same clinical stage undergo the same enucleation surgery, but postoperative pathology reports show one with high-risk pathological features while the other is not. Clinical practice suggests that patients with high-risk pathological features generally have a poor prognosis, respond poorly to eye-preserving treatment, and are prone to relapse and progression, making early enucleation preferable to avoid delaying treatment; while those without high-risk features are more likely to respond well to eye-preserving treatment and should be given priority to avoid unnecessary enucleation, but pathological results can only be obtained after enucleation.

[0026] In summary, the existing prognostic assessment system based on clinical staging and postoperative pathology is insufficient to accurately predict the treatment response and disease progression risk of individual patients before treatment. There is an urgent need in this field to find novel biomarkers at the molecular level that can accurately determine the prognosis at an early stage, so that clinicians can determine which children are more likely to benefit from eye-preserving treatment before treatment.

[0027] To address the aforementioned technical problems, this invention has conducted extensive research and analysis. The metabolic reprogramming of tumor cells (Warburg effect, i.e., enhanced glycolysis) is widely recognized as one of the core characteristics of tumors. However, recent studies have found that even within the same type of tumor, tumor cells from different patients exhibit significant differences in metabolic patterns, a phenomenon known as "metabolic heterogeneity." Metabolic heterogeneity is closely related to tumor invasiveness, metastatic potential, and treatment resistance. Therefore, identifying and classifying tumor metabolic subtypes holds promise for providing patients with more precise treatment strategies. However, in the field of renal leukemia (RB), research on tumor metabolic heterogeneity is still in its early stages. The underlying reasons for this research gap include: (1) Difficulty in obtaining samples: RB is a rare disease, and tumor tissue can only be obtained after enucleation. It is difficult to obtain enough samples for metabolomics analysis in children undergoing eye preservation treatment; (2) Technical limitations: Traditional metabolomics analysis requires a large sample size, while RB tumors are usually small, which limits the development of high-throughput metabolic analysis; (3) Limited research ideas: Previous studies have focused on genetic changes in RB (such as RB1 gene mutations) and abnormal signaling pathways, with relatively little attention paid to metabolic reprogramming, a core feature of the tumor; (4) Lack of systematic regulatory network research: Metabolic reprogramming is a complex process involving multiple genes and pathways. Changes in a single metabolite or metabolic enzyme are difficult to reveal the regulatory mechanism of overall metabolic heterogeneity. It is necessary to start with upstream regulatory molecules such as transcription factors for systematic analysis.

[0028] Previous research by our research group has found that although the same anti-retinoblastoma drug shows good anti-tumor effects on different RB cells (Y79 and WERI-Rb1), there are still some differences in the intervention effect. Y79 cells are more sensitive to the drug and have a better anti-tumor effect. The research group became extremely interested in this phenomenon and launched an investigation: They performed an intersection analysis of differentially expressed genes in the transcriptomes of two groups of retinoblastoma cells (Y79 and WERI-Rb1) treated with the drug and compared them with the human transcription factor database. This revealed the differential transcription factor BHLHE40. The basal expression of BHLHE40 in the two retinoblastoma cell types was then examined, showing that BHLHE40 was relatively highly expressed in Y79 cells compared to WERI-Rb1. Further experiments revealed that BHLHE40 is a core regulator of glycolytic metabolism in retinoblastoma. Subsequent experiments confirmed that knocking down BHLHE40 expression significantly inhibited the proliferation of retinoblastoma cells, with a significant tumor-suppressive effect. More importantly, this invention used clinical samples to demonstrate that the expression level of BHLHE40 is closely related to the severity and invasiveness of retinoblastoma disease, and can serve as an effective biomarker for predicting high-risk pathological features.

[0029] Based on the above research, this invention provides the application of a reagent for detecting the expression level of BHLHE40 in a test sample in the preparation of a retinoblastoma prognostic assessment kit. The prognostic assessment includes: when a high level of BHLHE40 expression is detected, the subject is considered a high-risk patient, indicating a poor prognosis; when a low level of BHLHE40 expression is detected, the subject is considered a low-risk patient, indicating a good prognosis.

[0030] In some embodiments, the sample to be tested includes: tumor tissue sample or blood sample; in other embodiments, it may also include: body fluid sample such as urine or aqueous humor; or cytological sample such as circulating tumor cells or exfoliated cells from body fluids.

[0031] In some embodiments, the methods for detecting the expression level of BHLHE40 in the sample to be tested include any one or more of the following: real-time quantitative PCR, Western blotting, flow cytometry, or immunohistochemistry.

[0032] In another aspect, the present invention provides the use of BHLHE40 inhibitors in the preparation of medicaments for the treatment of retinoblastoma.

[0033] In some embodiments, the BHLHE40 inhibitor is a substance that knocks out or knocks down the BHLHE40 gene, reduces the expression level or activity of the BHLHE40 protein, or antagonizes BHLHE40 function. The substance includes any one of the following: proteins, small molecule compounds, or gene intervention tools capable of knocking out or knocking down BHLHE40 expression.

[0034] Furthermore, the gene intervention tool capable of knocking out or knocking down BHLHE40 expression can be any intervention tool capable of knocking out or knocking down the BHLHE40 gene at the gene level, such as RNAi fragments (such as siRNA, shRNA, miRNA) or gene editing tools (such as CRISPR-Cas9, ZFN, TALENs, etc.).

[0035] In a specific embodiment of the present invention, the BHLHE40 inhibitor is an RNAi fragment, the RNAi fragment is shRNA, and the shRNA is any one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0036] In another aspect, the present invention provides a method for screening candidate drugs for the treatment of retinoblastoma, comprising: detecting the downregulation effect of candidate drugs on BHLHE40 expression or the inhibition effect of BHLHE40 activity.

[0037] The research process of the present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0038] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Unless otherwise specified, all materials and reagents used in this invention are commercially available.

[0039] (I) Effects of BHLHE40 expression changes on RB cell glycolysis In RB cell lines Y79 (purchased from ATCC) and WERI-Rb1 (purchased from the Cell Bank of the Chinese Academy of Sciences), BHLHE40 expression was knocked down using shRNA, and BHLHE40 was overexpressed using oeBHLHE40. The effect of changes in BHLHE40 expression on glycolysis in RB cells was observed by Western blot and Seahorse metabolic analysis.

[0040] Three shRNAs were designed: shBHLHE40-1, shBHLHE40-2, and shBHLHE40-3. The specific sequences are shown in Table 1 below.

[0041] Table 1: BHLHE40 shRNA sequence

[0042] The results are as follows Figure 1 As shown, Western blot analysis revealed that shRNA successfully downregulated BHLHE40 expression levels in Y79 and WERI-Rb1 cells; while oeBHLHE40 successfully upregulated BHLHE40 expression levels in both cells. Further observation showed that knocking down BHLHE40 downregulated the expression levels of key glycolytic enzymes HK2, PKM2, ENO1, and LDHA; while overexpression of BHLHE40 upregulated the expression levels of these key glycolytic enzymes. Figure 1 (A and B).

[0043] In addition, Seahorse metabolic analysis was performed, with RB cell lines Y79 and WERI-Rb1 mixed at 1×10⁻⁶ cells per well. 5 Cells were seeded in Seahorse XF96 plates to detect the glycolysis rate. Results showed that knockdown of BHLHE40 decreased the glycolysis rate of RB cells, manifested as a reduction in basal glycolysis levels and a weakened glycolytic reserve. Figure 1(C and D); Conversely, overexpression of BHLHE40 upregulated the glycolysis rate in RB cells, manifested as an upregulation of basal glycolysis levels and enhanced glycolytic reserve capacity (C and D). Figure 1 (E and F).

[0044] The above results indicate that BHLHE40 is a core regulator of glycolytic metabolism in retinoblastoma.

[0045] (ii) Knockdown of BHLHE40 inhibits the in vitro proliferation ability of RB cells. 1. Cell experiments BHLHE40 expression was knocked down using shBHLHE40-3 in the Y79 RB cell line and knocked down using shBHLHE40-2 in the WERI-Rb1 cell line. BHLHE40 was overexpressed using oeBHLHE40 in both Y79 and WERI-Rb1 cells. The effect of changes in BHLHE40 expression on the proliferation capacity of RB cells was observed by CCK-8 assay.

[0046] The results showed that knocking down BHLHE40 significantly reduced the proliferation capacity of Y79 cells and WERI-Rb1 cells, with cell viability decreasing by approximately 50% and 30% respectively on day 3 of treatment compared to the control group. Figure 2 (A). Conversely, overexpression of BHLHE40 significantly enhanced the proliferation capacity of WERI-Rb1 cells (A). Figure 2 B).

[0047] 2. Animal experiments To further evaluate the effect of BHLHE40 expression level on the in vivo proliferation of RB cells, Y79 cells stably transfected with shCtrl and shBHLHE40-3 were inoculated into the subretinal space of nude mice (4 weeks old, female) via microinjection. After tumor formation, the animals were sacrificed and the eyeballs were enucleated for photography and weighing.

[0048] The results showed that the tumor volume and weight in the shBHLHE40 group of nude mice were significantly lower than those in the shCtrl group. Figure 2 (C and D).

[0049] Furthermore, immunofluorescence staining showed that the proportion of Ki-67-positive cells in tumor tissue of the shBHLHE40 group was significantly lower than that of the control group. Figure 2 E).

[0050] The above results indicate that knocking down BHLHE40 expression can significantly inhibit the proliferation of retinoblastoma cells, and the tumor-suppressing effect is significant.

[0051] (III) Association analysis between BHLHE40 and high-risk pathological features in RB patients To explore the value and clinical significance of BHLHE40 in the prognostic assessment of RB, this invention collected tumor tissue samples from 24 RB patients, of which 14 were HRF positive and 10 were HRF negative (HRF stands for High-Risk Features), as well as 6 normal retinal tissue samples (from normal organ donors).

[0052] Immunofluorescence staining results showed that the expression level of BHLHE40 in all RB tumor tissues was significantly higher than that in normal retinal tissues, but heterogeneity still existed within the tumors. Based on staining intensity, the expression level of BHLHE40 in 14 tumor tissues was interpreted as high, and the expression level of BHLHE40 in the remaining 10 tumor tissues was interpreted as low. Figure 3 A).

[0053] Further analysis showed that high BHLHE40 expression was significantly positively correlated with high-risk pathological features in RB patients (including optic nerve involvement, extensive choroidal involvement, anterior segment infiltration, and scleral involvement) (Fisher's exact test, OR = 24.0, 95% CI: 2.5-148.1) (see [link to relevant documentation]). Figure 3 (See Table B and Table 2). Diagnostic efficacy analysis showed that the BHLHE40 had a sensitivity of 85.7%, a specificity of 80.0%, and a Youden index of 0.657 in predicting high-risk pathological features in patients with renal retardation (RB). (See Table 3).

[0054] Table 2: Association analysis between BHLHE40 expression level and high-risk histopathological features in RB patients

[0055] Note: High-risk pathological features include optic nerve invasion, extensive choroidal invasion, anterior segment infiltration, and scleral invasion; OR value represents the strength of the association between high BHLHE40 expression and high-risk pathological features.

[0056] Table 3: Predictive power of BHLHE40

[0057] In addition, this invention also analyzed the correlation between patients' chemotherapy history, gender, age, and monocular / bicular expression and BHLHE40 expression. The results showed that there was no significant correlation between BHLHE40 expression level and whether the patient had received chemotherapy treatment before eye removal (p > 0.05), nor was there a significant correlation between BHLHE40 expression level and patient gender, age, or monocular / bicular expression.

[0058] The above results indicate that the expression level of BHLHE40 is closely related to the severity and aggressiveness of RB disease, and can serve as an effective biomarker for predicting high-risk pathological features. It can provide molecular evidence to assist clinicians in developing postoperative adjuvant therapy plans and determining whether more aggressive intervention is needed, reducing overtreatment and undertreatment, and enabling early, non-invasive / minimally invasive prognostic assessment. Specifically, by detecting the expression level of BHLHE40 in patients, high-risk / low-risk patients can be effectively distinguished: when the BHLHE40 expression level is high, the subject is considered a high-risk patient, indicating a poor prognosis, poor response to eye-preserving treatment, and a high risk of recurrence and progression; early enucleation is preferred to avoid delaying treatment. When the BHLHE40 expression level is low, the subject is considered a low-risk patient, indicating a good prognosis, and eye-preserving treatment is more likely to be effective; eye-preserving treatment can be prioritized to avoid unnecessary enucleation.

[0059] In summary, based on extensive research and experimental verification, this invention proposes that BHLHE40 inhibitors can be used in the preparation and application of therapeutic drugs for retinoblastoma (RB), providing a new, effective, and safe treatment option for RB patients. Furthermore, BHLHE40 can serve as a drug target to guide subsequent drug development. In addition, this invention has demonstrated using clinical tumor tissue samples that high BHLHE40 expression is significantly positively correlated with high-risk pathological features such as optic nerve invasion, scleral invasion, and choroidal invasion, suggesting that BHLHE40 can serve as a prognostic biomarker for RB, assisting physicians in making treatment decisions.

[0060] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Application of reagents for detecting BHLHE40 expression levels in test samples in the preparation of retinoblastoma prognostic assessment kits.

2. The application as described in claim 1, characterized in that, The prognostic assessment includes: when the BHLHE40 expression level is detected to be high, the subject is judged to be a high-risk patient, indicating a poor prognosis; when the BHLHE40 expression level is detected to be low, the subject is judged to be a low-risk patient, indicating a good prognosis.

3. The application as described in claim 1, characterized in that, The samples to be tested include: tumor tissue samples or blood samples.

4. The application as described in claim 1, characterized in that, Methods for detecting the expression level of BHLHE40 in the sample to be tested include any one or more of the following: real-time quantitative PCR, Western blotting, flow cytometry, or immunohistochemistry.

5. Application of BHLHE40 inhibitors in the preparation of drugs for the treatment of retinoblastoma.

6. The application as described in claim 5, characterized in that, The BHLHE40 inhibitor is a substance that knocks out or knocks down the BHLHE40 gene, or reduces the expression level or activity of the BHLHE40 protein, or antagonizes the function of BHLHE40.

7. The application as described in claim 6, characterized in that, The substance includes any one of the following: protein, small molecule compound, or gene intervention tool capable of knocking out or knocking down BHLHE40 expression.

8. The application as described in claim 7, characterized in that, The gene intervention tool includes either an RNAi fragment or a gene editing tool.

9. The application as described in claim 8, characterized in that, The RNAi fragment is shRNA, and the shRNA is any one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:

3.

10. A method for screening candidate drugs for the treatment of retinoblastoma, characterized in that, include: The study aimed to detect the downregulation effect of candidate drugs on BHLHE40 expression or the inhibition effect on BHLHE40 activity.

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