Application of AML-related lncRNA biomarkers, detection kits and methods in AML diagnosis and prognosis

Abstract

This invention discloses a lncRNA biomarker associated with AML, characterized in that the lncRNA biomarker is lncRNA FOXN3-AS1, and the nucleotide sequence of lncRNA FOXN3-AS1 is shown in SEQ ID NO: 1. This invention also discloses the application of a detection kit and method in the diagnosis and prognosis of AML. The lncRNA FOXN3-AS1 of this invention, as an auxiliary diagnostic biomarker for clinical detection of AML, has the advantages of simple operation, low cost, and accurate detection, making it suitable for clinical screening. The biomarker provided by this invention can be applied to the preparation of early AML assessment products, which is beneficial for further elucidating the pathogenesis of AML and helps in the discovery of novel small molecule drug targets with potential therapeutic value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and biotechnology, and in particular relates to the application of an AML-related lncRNA biomarker, detection kit, and method in the diagnosis and prognosis of AML. Background Technology

[0002] Acute myeloid leukemia (AML) is a genetically diverse malignant disease of the hematopoietic system and the most common type of adult leukemia. Its pathogenesis is complex, involving abnormal changes in cell differentiation, proliferation, and apoptosis. Despite significant advancements in chemotherapy and hematopoietic stem cell transplantation, the survival rate of AML patients remains unsatisfactory. Overall, the current clinical prognosis of AML remains poor, with a high mortality rate; the overall 5-year survival rate is less than 50%. With the increasing aging of my country's population, the incidence of AML has been rising in recent years. AML identification methods based on fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) technologies often emphasize heterogeneity and require long testing cycles, hindering rapid identification of AML. Therefore, further elucidating the molecular mechanisms of AML development and screening for new biomarkers to provide effective diagnostic, treatment, and prognostic prediction strategies is crucial.

[0003] Recent studies have shown that non-coding RNAs are closely related to the occurrence and development of tumors and can serve as effective molecular markers for the auxiliary diagnosis, prognosis, and disease monitoring of tumors, and can provide more targeted molecules for treatment. However, in AML, the functions of a large number of non-coding RNAs remain unclear, and clinicians look forward to discovering more specific and sensitive non-coding RNAs and applying them to the clinical diagnosis and treatment of AML.

[0004] FOXN3-AS1 (Ensembl database ID ENSG00000258920) is the antisense transcript of the FOXN3 gene, belonging to the long non-coding RNA (lncRNA) class. In current research, dysregulation of lncRNA FOXN3-AS1 has only been found in non-small cell lung cancer and breast cancer (PMID: 25590602; PMID: 34703931), and further functional studies are needed to elucidate the precise role of lncRNA FOXN3-AS1 in tumorigenesis. Currently, there are no reports on the expression of lncRNA FOXN3-AS1 in AML, whether it can serve as a biomarker for AML, or methods for detecting lncRNA FOXN3-AS1 expression in AML. Summary of the Invention

[0005] The main objective of this invention is to overcome the shortcomings of existing technologies in diagnosing AML and to provide an application of AML-related lncRNA biomarkers, detection kits, and methods in the diagnosis and prognosis of AML. This invention utilizes a method based on real-time quantitative PCR to detect the expression level of human lncRNA FOXN3-AS1, using this as a biomarker for AML patients to support clinical diagnosis and prognosis assessment.

[0006] The objective of this invention and the technical problem it solves are achieved by the following technical solutions.

[0007] One aspect of the present invention provides a LncRNA marker associated with AML, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0008] Furthermore, the LncRNA marker is LncRNA FOXN3-AS1, and the primer sequences with detection specificity for LncRNA FOXN3-AS1 are shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0009] Another aspect of the present invention provides an application of a LncRNA biomarker in the preparation of AML diagnostic and prognostic kits.

[0010] Another aspect of the present invention provides an AML diagnostic and prognostic kit for detecting the relative expression level of the biomarker LncRNA FOXN3-AS1 in acute myeloid leukemia tissue. The kit includes a primer pair comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer being shown in SEQ ID NO: 2, and the nucleotide sequence of the downstream primer being shown in SEQ ID NO: 3.

[0011] Furthermore, the kit also includes qPCR amplification mix and ddH2O.

[0012] Furthermore, the qPCR amplification mix contains SYBGREEN dye.

[0013] Furthermore, the kit also includes an internal control system, which is an internal control primer designed based on the internal reference gene GAPDH sequence. The internal control primer includes an upstream primer and a downstream primer. The nucleic acid sequence of the upstream primer is shown in SEQ ID NO.4, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.5.

[0014] In another aspect, the present invention provides a method for detecting the AML biomarker LncRNAFOXN3-AS1 using an AML diagnostic and prognostic kit, the method comprising the following steps:

[0015] S1. Cell culture: Human immortalized bone marrow cell line HS-5 and human acute myeloid leukemia cell lines (MOLM-13, HL-60, KG1A) were cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics in an incubator at 37°C, 5% CO2 and 90% relative humidity, with the medium changed every 2-3 days.

[0016] S2. RNA extraction: Collect cultured cells and extract total RNA from the cells according to the instructions of the total RNA extraction kit.

[0017] S3. Reverse transcription of RNA: Perform reverse transcription to synthesize cDNA according to the instructions of the HiFiScript cDNA first strand synthesis kit.

[0018] S4. Quantitative Real-Time PCR Amplification: Using the kit described in this invention, PCR amplification is performed with the cDNA obtained in step S3 as a template, and fluorescence signals are collected.

[0019] The system composition and content for fluorescent PCR amplification are as follows:

[0020]

[0021] The reaction conditions for fluorescent PCR were: pre-denaturation at 95℃ for 10 min, followed by denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s, for a total of 38 cycles.

[0022] This invention offers significant advantages and benefits compared to existing technologies: It is the first to discover a correlation between lncRNA FOXN3-AS1 expression levels and the occurrence of AML. By detecting the expression level of lncRNA FOXN3-AS1 in a subject, it is possible to determine whether the subject has AML, allowing for appropriate preventative and treatment measures to be taken in the early stages of the disease. The superiority of lncRNA FOXN3-AS1 as an auxiliary diagnostic biomarker for AML clinical detection lies in its simplicity, low cost, and high accuracy, making it suitable for clinical screening. Compared to traditional diagnostic methods, using lncRNA to diagnose AML is more timely and sensitive, enabling patients to take appropriate preventative and treatment measures in the early stages of the disease.

[0023] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0024] Figure 1 The differential expression of lncRNA FOXN3-AS1 in AML patient samples and normal control samples was shown;

[0025] Figure 2 The ROC curves for detecting lncRNA FOXN3-AS1 in AML patient samples are shown.

[0026] Figure 3 The correlation analysis between lncRNA FOXN3-AS1 and AML patient prognosis is shown;

[0027] Figure 4 The expression of lncRNA FOXN3-AS1, lncRNA CDKN2B-AS1, lncRNA SNHG1, lncRNA FBXL19-AS1, and lncRNA ZEB2-AS1 in AML patient samples and normal control samples is shown.

[0028] Figure 5 The correlation analysis between lncRNA FOXN3-AS1, lncRNA CDKN2B-AS1, lncRNA SNHG1, lncRNA FBXL19-AS1, lncRNA ZEB2-AS1 and AML patient prognosis is shown.

[0029] Figure 6 The differential expression of lncRNA FOXN3-AS1 in normal control cells and AML cell lines was shown;

[0030] Figure 7 The differential expression of lncRNA FOXN3-AS1 in blood samples from healthy controls and AML patients was shown. Detailed Implementation

[0031] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and do not limit the present invention.

[0032] Example 1: Differences in the lncRNA FOXN3-AS1 gene between acute myeloid leukemia tissue and normal control tissue Correlation analysis of expression and prognosis

[0033] 1. The nucleotide sequence of the lncRNA FOXN3-AS1 described in this invention is shown in SEQ ID NO.1.

[0034] SEQ ID NO.1:

[0035]

[0036] 2. Using the TCGA and GTEx databases, high-throughput sequencing results from 140 AML patient samples and 70 normal control samples were analyzed to obtain differential gene expression profiles for AML. Among these, the lncRNA FOXN3-AS1 was significantly overexpressed in the acute myeloid leukemia patient group (e.g., ...). Figure 1 As shown), the area under the ROC curve (AUC) is equal to 1 (as shown). Figure 2 (As shown).

[0037] As is well known to those skilled in the art, when the area under the ROC curve is between 1.0 and 0.5, and AUC > 0.5, the closer the AUC is to 1, the better the diagnostic effect. AUC between 0.5 and 0.7 indicates lower accuracy, AUC between 0.7 and 0.9 indicates some accuracy, and AUC above 0.9 indicates high accuracy. A value greater than 0.7 indicates that the detection target can serve as a specific marker for this type of detection.

[0038] 3. Furthermore, a correlation analysis was conducted on the expression level of lncRNA FOXN3-AS1 in 140 AML samples from the TCGA database and patient prognosis. The 140 AML patients were grouped according to their lncRNA FOXN3-AS1 expression levels. The results showed that the overall survival of AML patients in the low lncRNA FOXN3-AS1 expression group was significantly longer than that in the high lncRNA FOXN3-AS1 expression group, and lncRNA FOXN3-AS1 was significantly negatively correlated with AML prognosis (e.g., ...). Figure 3 (As shown).

[0039] Example 2: lncRNA FOXN3-AS1 gene as a biomarker for AML diagnosis and prognosis, with efficacy comparable to current methods. Comparison with other lncRNAs in the technology

[0040] Literature (PMID: 33300059), literature (PMID: 31615767), literature (PMID: 33474753), and literature (PMID: 31186703) have reported that lncRNA CDKN2B-AS1 (Ensembl database ID: ENSG00000240498), lncRNA SNHG1 (Ensembl database ID: ENSG00000255717), lncRNA FBXL19-AS1 (Ensembl database ID: ENSG00000260852), and lncRNA ZEB2-AS1 (Ensembl database ID: ENSG00000238057) can be used as biomarkers for the diagnosis and prognosis of AML. The inventors of this application compare the lncRNA FOXN3-AS1 of this application with the aforementioned lncRNAs in terms of its diagnostic and prognostic effects on AML.

[0041] 1. Using the GEPIA2 (Gene Expression Profiling Interactive Analysis 2) database, high-throughput sequencing results were obtained from 106 AML patient samples and 70 normal control samples. The expression levels of lncRNAs FOXN3-AS1, CDKN2B-AS1, SNHG1, FBXL19-AS1, and ZEB2-AS1 in AML patient and normal control samples were analyzed to assess the diagnostic efficacy of these genes for AML. Compared with normal control tissues, lncRNAs FOXN3-AS1, FBXL19-AS1, and ZEB2-AS1 were significantly overexpressed in AML samples, while the expression levels of lncRNAs CDKN2B-AS1 and SNHG1 showed no statistically significant difference in AML samples (e.g., ...). Figure 4 (As shown). Therefore, the lncRNA FOXN3-AS1 gene is superior to the previously reported lncRNAs CDKN2B-AS1 and SNHG1 in terms of diagnostic efficacy for AML.

[0042] 2. Furthermore, a correlation analysis was conducted on the expression levels of lncRNA FOXN3-AS1, lncRNA CDKN2B-AS1, lncRNA SNHG1, lncRNA FBXL19-AS1, and lncRNA ZEB2-AS1 in 106 AML patients from the GEPIA2 database and their prognosis. The 106 AML patients were grouped according to the expression levels of these four lncRNAs. It was found that the overall survival of AML patients in the low-expression lncRNA FOXN3-AS1 group was significantly longer than that in the high-expression lncRNA FOXN3-AS1 group, and lncRNA FOXN3-AS1 was significantly negatively correlated with AML patient prognosis. However, the expression levels of lncRNA CDKN2B-AS1, lncRNA SNHG1, lncRNA FBXL19-AS1, and lncRNA ZEB2-AS1 were significantly lower than those of lncRNA CDKN2B-AS1, lncRNA SNHG1, lncRNA FBXL19-AS1, and lncRNA ZEB2-AS1 were significantly lower than those of lncRNA CDKN2B-AS1, lncRNA SNHG1, lncRNA FBXL19-AS1, and lncRNA ZEB2-AS1. ZEB2-AS1 was not significantly associated with the prognosis of AML patients (e.g. Figure 5 (As shown). Therefore, the lncRNA FOXN3-AS1 gene in this application is superior to the known lncRNAs CDKN2B-AS1, SNHG1, FBXL19-AS1 and ZEB2-AS1 in predicting the prognosis of AML.

[0043] Example 3: lncRNA FOXN3-AS1 gene in normal control cells and acute myeloid leukemia cell lines Differential expression

[0044] 1. Cell culture: Human immortalized bone marrow cell line HS-5 and human acute myeloid leukemia cell lines (MOLM-13, HL-60, KG1A) were cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics in an incubator at 37°C, 5% CO2, and 90% relative humidity, with the medium changed every 2-3 days.

[0045] 2. RNA extraction: Collect cultured cells and perform total RNA extraction according to the instructions of the total RNA extraction kit (purchased from Shanghai Feijie Biotechnology Co., Ltd.).

[0046] 3. Reverse transcription: Perform reverse transcription to synthesize cDNA according to the instructions of the HiFiScript cDNA First Strand Synthesis Kit (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.).

[0047] 4. Quantitative PCR amplification test.

[0048] The reaction system is as follows:

[0049]

[0050] The reaction procedure was as follows: pre-denaturation at 95℃ for 10 min, followed by denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s, for a total of 38 cycles.

[0051] The ABI 7500 real-time PCR instrument was programmed with a selected melting curve, and fluorescence signals from the samples were continuously collected during the ramp-up process to obtain the melting curve. Real-Time PCR was performed using 2... -△△Ct Relative quantitative analysis was performed using this method. The primer sequences for FOXN3-AS1 and GAPDH are shown in Table 1.

[0052] Table 1. Specific amplification primer sequences and related information

[0053]

[0054] Figure 6 The differential expression of lncRNA FOXN3-AS1 in normal control cells and AML cell lines is shown. Figure 6 As shown, compared with the human immortalized bone marrow cell line (HS-5), the expression of lncRNA FOXN3-AS1 gene was upregulated in human acute myeloid leukemia cell lines (MOLM-13, HL-60, KG1A), and the difference was statistically significant (P<0.05).

[0055] Example 4: Differential expression of lncRNA FOXN3-AS1 gene in blood samples from healthy controls and AML patients

[0056] 1. Sample collection: 40 blood samples were collected from AML patients and 40 from healthy controls.

[0057] 2. Total RNA extraction from samples: Total RNA was extracted from samples using the Thermo Fisher Blood RNA Isolation Kit, following the instructions in the kit's manual.

[0058] 3. Same as in Example 3, perform reverse transcription and quantitative PCR amplification tests.

[0059] Figure 7 The differential expression of lncRNA FOXN3-AS1 in blood samples from healthy controls and AML patients is shown. Figure 7 As shown, compared with blood samples from healthy controls, the expression of lncRNA FOXN3-AS1 was upregulated in blood samples from AML patients, and the difference was statistically significant (P<0.05).

[0060] This invention obtained high-throughput sequencing (RNA-Seq) data and clinical information from 140 AML patient samples through the National Cancer Institute (NCI) TCGA database (https: / / portal.gdc.cancer.gov / ); and RNA-Seq data from 70 normal control samples through the GTEx database (https: / / gtexportal.org / home / datasets). Analysis showed that lncRNA FOXN3-AS1 was significantly highly expressed in AML patient samples compared to normal control samples, with an area under the ROC curve (AUC) of 1, suggesting that lncRNA FOXN3-AS1 can serve as a diagnostic biomarker for AML.

[0061] Subsequently, 140 AML patients were grouped according to the expression level of lncRNA FOXN3-AS1. It was found that the overall survival of AML patients in the low expression group of lncRNA FOXN3-AS1 was significantly longer than that in the high expression group of lncRNA FOXN3-AS1, suggesting that lncRNA FOXN3-AS1 can be used as a prognostic biomarker for AML.

[0062] In vitro experiments confirmed that, compared with control human immortalized bone marrow cells (HS-5), the expression level of lncRNA FOXN3-AS1 was significantly increased in three human acute myeloid leukemia cell lines (MOLM-13, HL-60, and KG1A), consistent with the results of TCGA and GTEx database analysis.

[0063] Furthermore, by collecting peripheral blood samples from 40 AML patients and 40 healthy controls, analysis revealed that lncRNA FOXN3-AS1 was significantly more expressed in peripheral blood samples from AML patients compared to peripheral blood samples from healthy controls.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of a reagent for detecting the relative expression level of the biomarker LncRNA FOXN3-AS1 in tissues in the preparation of an acute myeloid leukemia kit, characterized in that, The reagent includes a primer pair, which includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

3.

2. The application according to claim 1, characterized in that, The reagents also include qPCR amplification Mix and ddH2O.

3. The application according to claim 2, characterized in that, The qPCR amplification mix contains SYBR Green dye.

4. The application according to claim 1, characterized in that, The kit also includes an internal standard system, which is an internal standard primer designed based on the internal reference gene GAPDH sequence. The internal standard primer includes an upstream primer and a downstream primer. The nucleic acid sequence of the upstream primer is shown in SEQ ID NO.4, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.5.

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