A kit for detecting expression amount of fnip1 and an acute myeloid leukemia diagnosis product

Abstract

This invention belongs to the field of medical testing technology, specifically relating to a kit for detecting FNIP1 expression and a diagnostic product for acute myeloid leukemia (AML). The kit includes a specific primer pair for detecting FNIP1 expression, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2. The kit is used to detect FNIP1 expression in biological samples, and subsequently for the diagnosis and prognosis of AML. The expression level of FNIP1 in AML patient biological samples is associated with FAB subtype distribution, risk, and prognosis. Multivariate Cox regression analysis shows that FNIP1 expression is an independent risk factor for overall survival; compared with the high FNIP1 expression group, patients with low FNIP1 expression have lower overall survival and poorer prognosis, indicating accurate prognosis for AML.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to a kit for detecting FNIP1 expression and a diagnostic product for acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is highly heterogeneous, characterized by uncontrolled proliferation, differentiation arrest, accumulation of immature myeloid progenitor cells, and suppression of normal hematopoiesis, ultimately leading to clinical manifestations such as anemia, bleeding, and infection. AML can occur at any age and is a common type of leukemia. Most AML patients experience a rapid and severe course of disease with a high mortality rate.

[0003] Currently, chemotherapy is the main treatment for AML. Although chemotherapy regimens are constantly being improved, problems such as drug resistance, relapse, and residual disease still exist. Moreover, the overall survival rate of AML has not improved significantly. Except for M3 AML, up to 50% of patients with other types of AML experience relapse or death, and the 5-year survival rate is only 40%-60%. Even with hematopoietic stem cell transplantation, only 30%-40% of adult patients and 60%-70% of pediatric patients can achieve long-term disease-free survival.

[0004] Current technologies primarily rely on chromosomal karyotype, age, medical history, initial white blood cell count, immunophenotype, and early treatment response to determine prognosis. While these methods can broadly differentiate between extremely good and extremely poor outcomes, such as M3 type and complex karyotypes, they cannot provide fine-grained stratification for AML patients with normal karyotypes. This results in some low-risk patients receiving excessive treatment while others receive insufficient chemotherapy, leading to inaccurate prognostic assessments and a lack of products that accurately detect AML. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a kit for detecting FNIP1 expression and a diagnostic product for acute myeloid leukemia, which can solve the technical problem of inaccurate prognosis in AML.

[0006] This invention is achieved by adopting the following technical solution: A kit for detecting FNIP1 expression levels, the kit comprising a specific primer pair for detecting FNIP1 expression levels, the nucleotide sequences of the primer pair being shown in SEQ ID NO.1 and SEQ ID NO.2; The kit is used to detect the expression level of FNIP1 in biological samples, and then to diagnose and predict the prognosis of acute myeloid leukemia.

[0007] Furthermore, the biological sample is selected from peripheral blood, bone marrow, or bone marrow biopsy tissue.

[0008] Furthermore, the kit also includes an internal reference gene detection reagent and / or an FNIP1 protein detection antibody; the internal reference gene is GAPDH.

[0009] Furthermore, the method for detecting FNIP1 expression level using the kit is any one of real-time quantitative PCR, reverse transcription PCR, RNA sequencing, Western blotting, or enzyme-linked immunosorbent assay.

[0010] Furthermore, the method for detecting FNIP1 expression level using the kit is any one of real-time quantitative PCR, reverse transcription PCR, RNA sequencing, Western blotting, or enzyme-linked immunosorbent assay.

[0011] A product for the diagnosis and prognosis of acute myeloid leukemia, the product comprising a gene chip containing the specific primer pair for detecting FNIP1 expression.

[0012] Furthermore, the product also includes reagents for detecting FNIP1 gene expression levels using methods such as Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry, protein chip assay, RT-PCR, and DNA blotting.

[0013] Furthermore, the enzyme-linked immunosorbent assay (ELISA) includes a sandwich assay.

[0014] Furthermore, it also includes a protein chip containing an FNIP1 protein detection antibody.

[0015] Furthermore, the method for prognostic assessment includes: detecting the expression level of FNIP1 in biological samples of patients diagnosed with acute myeloid leukemia; compared with the low FNIP1 expression group, patients with high FNIP1 expression have lower overall survival, shorter relapse-free survival, and poorer prognosis.

[0016] Furthermore, the diagnostic method includes: detecting the expression level of FNIP1 in the biological sample to be tested; if the expression level of FNIP1 in the sample to be tested is significantly increased compared with the expression level of FNIP1 in the healthy control sample, it suggests that the individual to be tested has acute myeloid leukemia or has a high risk of developing acute myeloid leukemia.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a kit for detecting FNIP1 expression levels. The kit includes a specific primer pair for detecting FNIP1 expression levels, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2. The kit is used to detect FNIP1 expression levels in biological samples, and subsequently for the diagnosis and prognosis of acute myeloid leukemia (AML). FNIP1 expression levels in AML patient biological samples are associated with FAB subtype distribution, risk, and prognosis. Multivariate Cox regression analysis shows that FNIP1 expression level is an independent risk factor for overall survival. Compared with the high FNIP1 expression group, patients with low FNIP1 expression have lower overall survival and poorer prognosis, indicating accurate prognosis for AML. Attached Figure Description

[0018] Figure 1 Survival analysis of FNIP1 in AML; P<0.05.

[0019] Figure 2 Pan-cancer analysis of FNIP1 in AML from the GEPIA database.

[0020] Figure 3 To detect the mRNA expression of FNIP1 in AML patients by RT-qPCR, *P<0.05.

[0021] Figure 4 To analyze the difference in N-WASP expression between AML samples and normal controls; where TCGA represents AML samples and GTEx represents normal control samples.

[0022] Figure 5 For pan-cancer analysis related to N-WASP.

[0023] Figure 6 Kaplan-Meier survival analysis was performed on AML patients with low N-WASP expression (35 patients) and high N-WASP expression (103 patients); where A represents overall survival (OS), P < 0.05; and B represents event-free survival (EFS), P > 0.05. Survival curves were generated using the logrank test, and the survival difference between the low-expression and high-expression groups was analyzed.

[0024] Figure 7 Correlation analysis of N-WASP with its co-expressed gene FNIP1.

[0025] Figure 8 To investigate the effect of FNIP1 on cell proliferation in THP-1 cells, cell proliferation was detected using CCK8 assay.

[0026] Figure 9To investigate the effect of FNIP1 on apoptosis in THP-1 cells, the apoptosis rate was detected by flow cytometry. *P<0.05, **P<0.01.

[0027] Figure 10 ROC curve for FNIP1 in diagnosing acute myeloid leukemia. Detailed Implementation

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods. Specific explanations are needed as follows:

[0029] Acute myeloid leukemia, abbreviated as AML.

[0030] Example 1: Application of a kit for detecting FNIP1 expression levels in the prognosis of acute myeloid encephalopathy. To understand the importance of FNPI1 in the prognosis of AML patients, the GEPIA database was used, http: / / gepia.cancer-pku.cn / . Survival analysis of FNPI1 in AML was performed.

[0031] like Figure 1 As shown, Kaplan-Meier survival analysis revealed that patients in the high-expression group had a significantly better survival trend than those in the low-expression group, with a significantly longer median survival time. Patients in the low-expression group had a poorer prognosis, with their survival rate declining rapidly in the early stages of follow-up. The Log-rank test confirmed that the difference in overall survival between the two groups was statistically significant.

[0032] The above results indicate that low FNIP1 expression is closely associated with poor prognosis in AML patients. Specifically, AML patients with low FNIP1 expression have a significantly lower overall survival than those with high FNIP1 expression. Therefore, FNIP1 can serve as an independent prognostic biomarker for AML. By detecting the expression level of FNIP1 in biological samples (such as peripheral blood or bone marrow) from AML patients, high-risk and low-risk patients can be effectively distinguished; that is, compared with the high FNIP1 expression group, patients with low FNIP1 expression have lower overall survival and poorer prognosis. This confirms the practical application value of kits for detecting FNIP1 expression in the preparation of prognostic kits for acute myeloid leukemia. Therefore, FNIP1 is a gene associated with AML prognosis.

[0033] Using the GEPIA database, a pan-cancer analysis of the FNIP1 gene in 30 human cancers revealed that aberrant expression of all genes exhibits both tumor-suppressive and tumor-promoting properties. Figure 2 As shown.

[0034] Newly diagnosed AML patients from the Department of Pediatrics and the Department of Adult Hematology at the Affiliated Hospital of Zunyi Medical University were selected, and relevant clinical data, including clinical manifestations and hematological indicators, were collected. This included residual bone marrow samples from 30 newly diagnosed AML patients and residual bone marrow samples from 20 patients of the same age and period with non-hematologic malignancies and non-severe infections, or bone marrow samples from volunteers, as a control group. Informed consent was obtained from patients, their families, and volunteers, and this study was reviewed by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University (ethics number: KLLY-2020-188). RT-qPCR was used to verify the mRNA expression of FNIP1 in AML patients, revealing low expression of FNIP1 in AML patients. Figure 3 As shown; the primers for RT-qPCR are as follows:

[0035] FNIP1-F, 5'-GCATAGCACGGTCTGCATCT-3', SEQ ID NO.1; FNIP1-R, 5'-ACTTGTTGTTTGGCTGCGTC-3', SEQ ID NO.2; GAPDH-F, 5'-GGAGCGAGATCCCTCCAAAAT-3', SEQ ID NO.3; GAPDH-R, 5'-GGCTGTTGTCATACTTCTCATGG-3', SEQ ID NO. 4.

[0036] Experiment 1: Mining FNIP1 N-WASP is expressed at low levels in AML patients and normal controls. To explore the transcriptional expression profile of the N-WASP gene in AML, we first analyzed the mRNA expression levels of N-WASP in the GEPIA database, including the TCGA and GTEx projects.

[0037] The results showed that N-WASP was expressed at low levels in AML patients compared to normal controls. Figure 4 As shown. Furthermore, by downloading the expression levels of N-WASP in 31 human cancers from the GEPIA database and generating a pan-cancer analysis using GraphPad Prism software, abnormal expression of all N-WASP members could be observed, such as... Figure 5 As shown, the N-WASP gene therefore plays a role in AML progression.

[0038] To understand the importance of N-WASP in the prognosis of AML patients, these patients were further divided into two groups based on the 25th percentile level of gene expression: a low N-WASP expression group and a high N-WASP expression group. Kaplan-Meier survival curves were generated for overall survival (OS) and event-free survival (EFS) for patients with low and high expression.

[0039] The results showed that patients with lower N-WASP expression had lower overall survival (OS) compared to patients with higher N-WASP expression, but there was no statistically significant difference in emergency survival (EFS) between the low-N-WASP expression group and the high-N-WASP expression group. Figure 6 As shown in the figure. Therefore, N-WASP was identified as a gene associated with AML prognosis.

[0040] Next, co-expression network analysis was performed on RNA sequencing data from 138 AML patients in the database. Based on the Pearson correlation between N-WASP and genes (Pearson |R|>0.3), a total of 475 N-WASP co-expressing genes were identified, of which 416 genes were positively correlated with N-WASP expression and 59 genes were negatively correlated. Furthermore, heatmap analysis was performed on some significantly co-expressed genes, with FNIP1 showing a Pearson R of 0.6646 with N-WASP. GO functional enrichment analysis was also performed on N-WASP co-expression regulatory genes to further explore the biological pathways involved by N-WASP in AML. GO functional analysis revealed involvement in processes including protein ubiquitination, transcriptional regulation, gene expression regulation, and participation in apoptosis and cell cycle. Simultaneously, KEGG pathway analysis of N-WASP co-expression regulatory genes showed that 264 co-expressed genes were significantly enriched in oncogenic pathways, such as the mTOR signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, JAK-STAT signaling pathway, and AMPK signaling pathway.

[0041] Further correlation analysis of N-WASP and its co-expressed gene FNPI1 using the cBiportal database showed a positive correlation between N-WASP and FNPI1. Figure 7 As shown.

[0042] Experiment 2: Functionality of FNIP1 in AML After successfully constructing THP-1 cells with stable FNIP1 overexpression, their proliferation and apoptosis rates were detected by CCK8 assay and flow cytometry, respectively.

[0043] The results showed that in THP-1 cells, the OD450 value of the Over-FNIP1 group was significantly lower than that of the Over-Ctrl group, such as... Figure 8 As shown; the apoptosis rate was significantly higher in the group than in the Over-Ctrl group, as shown in the figure. Figure 9 As shown.

[0044] Experiment 3 analyzed the correlation between FNIP1 mRNA expression levels and general clinical characteristics of patients. FNIP1 mRNA expression was related to patient subtype; however, there were no statistically significant differences with patient gender, age, degree of anemia, WBC count, PB blasts, PLT, LDH, etc., as shown in Table 1.

[0045] Table 1. Relationship between FNIP1 expression levels and general clinical characteristics in AML patients. Note: # represents t-test; ## represents Mann-Whitney U test; & represents chi-square test (Pearson chi-square test); && represents chi-square test (continuity correction); &&& represents chi-square test (Fisher's test).

[0046] Compared with the control group, the expression level of FNIP1 in AML M2 and M5 types was significantly reduced and the difference was statistically significant; the expression level of FNIP1 in M3 and M4 types was not significantly different from that in the control group; there was no statistically significant difference among different FAB subtypes, as shown in Table 2.

[0047] Table 2. Relationship between FNIP1 expression level and FAB classification in AML patients. Note: " / " indicates that the item does not exist. * The value represents a significant difference compared to the control.

[0048] Compared with the control group, the expression levels of FNIP1 mRNA in both the non-high-risk and high-risk groups were significantly reduced, with statistically significant differences; there were no statistically significant differences among different risk groups. Spearman correlation analysis of the AML group compared with the control group showed that the correlation coefficient between the expression level of this molecule and the risk level of the AML group was less than 0, as shown in Table 3.

[0049] Table 3 Relationship between FNPI1 expression level and risk in AML patients Note: *Compared with the control group; Non-HR: not high risk; HR: high risk; " / " indicates that the item was not present.

[0050] Cox regression analysis: The independent risk factor affecting overall survival (OS) in AML patients was the expression level of FNIP1 mRNA, as shown in Table 4.

[0051] Table 4. Clinical characteristics of AML patients and Cox regression survival analysis Experiment 4: Analysis of the diagnostic value of FNIP1 in AML patients The data comes from the GDC database, https: / / portal.gdc.cancer.gov / ; 138 screened acute myeloid leukemia (AML) samples were included, excluding those with a history of other malignant tumors. The ROC curve of FNIP1 was significantly higher than the reference diagonal, with an area under the curve (AUC) of 0.720, suggesting that FNIP1 expression has good diagnostic efficacy for AML. Figure 10 As shown, FNIP1 can serve as a biomarker for auxiliary diagnosis.

[0052] Database analysis of FNIP1 expression in AML patients and its correlation with clinical characteristics revealed abnormal FNIP1 expression in AML, which is a biomarker for poor prognosis. This study provides direction for research on the role and mechanism of FNIP1 in inducing apoptosis and inhibiting cell proliferation in AML, and provides a certain theoretical basis for researching new therapeutic targets for AML.

[0053] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A kit for detecting FNIP1 expression levels, characterized in that, The kit includes specific primer pairs for detecting FNIP1 expression levels, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2; The kit is used to detect the expression level of FNIP1 in biological samples, and then to diagnose and predict the prognosis of acute myeloid leukemia.

2. The kit for detecting FNIP1 expression levels according to claim 1, characterized in that, The biological samples were selected from peripheral blood, bone marrow, or bone marrow biopsy tissue.

3. The kit for detecting FNIP1 expression levels according to claim 1, characterized in that, The kit also includes an internal reference gene detection reagent and / or an FNIP1 protein detection antibody; the internal reference gene is GAPDH.

4. The kit for detecting FNIP1 expression levels according to claim 1, characterized in that, The method for detecting FNIP1 expression level using the kit is any one of real-time quantitative PCR, reverse transcription PCR, RNA sequencing, Western blotting, or enzyme-linked immunosorbent assay.

5. A product for the diagnosis and prognosis of acute myeloid leukemia, characterized in that, The product includes a gene chip containing the specific primer pair for detecting FNIP1 expression as described in claim 1.

6. The product according to claim 5, characterized in that, The product also includes reagents for detecting FNIP1 gene expression levels using methods such as Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry, protein chip assay, RT-PCR, and DNA blotting.

7. The product according to claim 6, characterized in that, The enzyme-linked immunosorbent assay (ELISA) includes a sandwich assay.

8. The product according to claim 5, characterized in that, It also includes a protein chip containing an FNIP1 protein detection antibody.

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