Application of long-chain non-coding RNA MYLK-AS1 as a biomarker in preparation of a diagnostic or prognostic kit for acute myeloid leukemia

Abstract

The application discloses application of long-chain non-coding RNA MYLK-AS1 as a biomarker in preparation of a kit for diagnosis or prognosis of acute myeloid leukemia. The application aims to provide a marker for diagnosis and / or prognosis of acute myeloid leukemia and a target point for treatment. The application discloses application of LncRNA-MYLK-AS1 as a biomarker in preparation of a kit for diagnosis and / or prognosis of acute myeloid leukemia, and the gene of the LncRNA-MYLK-AS1 is shown as SEQ ID NO. 1. The LncRNA-MYLK-AS1 provided by the application has the advantages of simple operation, accurate detection, and suitability for clinical screening, and provides more information for diagnosis and prognosis of AML, and lays a foundation for targeted treatment of AML.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of long non-coding RNA MYLK-AS1 as a biomarker in the preparation of diagnostic or prognostic kits for acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant clonal hematologic malignancy originating from hematopoietic stem cells in the bone marrow. Its core characteristics include abnormal proliferation and differentiation arrest of myeloid blast cells, which suppress normal hematopoietic function and cause symptoms such as anemia, bleeding, and infection, seriously threatening patients' lives. Adult AML is the most common type of acute leukemia, with a low survival rate and high incidence. Precise risk stratification is key to optimizing treatment decisions and improving prognosis.

[0003] Currently, AML diagnosis relies on morphological examination, cytogenetic and molecular biological testing, with bone marrow blast cells ≥20% as the core diagnostic criterion. However, morphological examination is highly subjective and has low sensitivity, which can easily lead to misdiagnosis or missed diagnosis in early-stage patients. Existing prognostic stratification relies on cytogenetic (such as specific chromosomal rearrangements) and gene mutation characteristics (such as FLT3, NPM1, DNMT3A, etc.). Due to the high heterogeneity of AML and the high relapse rate in the intermediate-risk group, existing indicators cannot fully reflect the potential for disease progression, and there is an urgent need for new and precise biomarkers. Summary of the Invention

[0004] The purpose of this invention is to provide a biomarker and therapeutic target for the diagnosis and / or prognosis of acute myeloid leukemia.

[0005] This invention provides the application of LncRNA-MYLK-AS1 as a biomarker in the preparation of kits for the diagnosis and / or prognosis of acute myeloid leukemia, the gene of LncRNA-MYLK-AS1 being shown in SEQ ID NO.1.

[0006] To further specify, if the relative expression level of LncRNA-MYLK-AS1 in the peripheral blood of patients is significantly higher than that in the peripheral blood of healthy controls, then the patients have acute myeloid leukemia or have a poor prognosis; if there is no significant difference between the relative expression level of LncRNA-MYLK-AS1 in the peripheral blood of patients and the relative expression level in the peripheral blood of healthy controls, then the patients do not have acute myeloid leukemia or have a good prognosis.

[0007] The present invention provides a kit for diagnosing and / or predicting the prognosis of acute myeloid leukemia, the kit comprising an upstream primer and a downstream primer, the sequence of the upstream primer being shown in SEQ ID NO.2 and the sequence of the downstream primer being shown in SEQ ID NO.3.

[0008] Furthermore, the kit also includes primer combinations for qPCR amplification of Mix, ddH2O, and amplification of GAPDH internal control.

[0009] The present invention provides a recombinant acute myeloid leukemia cell line, wherein the cell line knocks down LncRNA-MYLK-AS1, and the gene of LncRNA-MYLK-AS1 is shown in SEQ ID NO.1.

[0010] Further specifying, the cell line is MOLM-13 cells or KG-1 cells.

[0011] This invention provides an inhibitor of LncRNA-MYLK-AS1 expression, wherein the inhibitor is a shRNA that knocks down LncRNA-MYLK-AS1.

[0012] To further specify, the shRNA gene is a hairpin structure prepared from SEQ ID NO.6 and SEQ ID NO.7.

[0013] This invention provides the use of LncRNA-MYLK-AS1, the above-mentioned cell line, or the above-mentioned expression inhibitor in the preparation of products that improve the sensitivity of chemotherapeutic drugs.

[0014] Further specifying, the chemotherapy drugs are cytarabine, daunorubicin, or homoharringtonine.

[0015] Beneficial Effects: This invention is the first to discover a correlation between LncRNA-MYLK-AS1 expression levels and AML (Acute Mycobacterium leukemia). The expression level of LncRNA-MYLK-AS1 in AML patient cells is significantly higher than that in normal cells. This invention uses the detection of LncRNA-MYLK-AS1 expression levels to determine whether a subject has AML and to assess prognosis. This allows patients to receive timely diagnosis and treatment in the early stages of the disease. The advantages of LncRNA-MYLK-AS1 as a clinical diagnostic and prognostic biomarker provided by this invention are its simplicity, accuracy, suitability for clinical screening, and provision of more information for the diagnosis and prognosis of AML, as well as laying the foundation for targeted therapy of AML. LncRNA-MYLK-AS1 has a positive regulatory effect on the malignant proliferation phenotype of AML cells, and knockdown of LncRNA-MYLK-AS1 can enhance chemotherapy sensitivity. Attached Figure Description

[0016] Figure 1 shows the Kaplan-Meier survival curves of LncRNA-MYLK-AS1 expression level and overall survival in AML patients; patients were divided into a high expression group (grey curve, n=355) and a low expression group (black curve, n=379) based on the median gene expression value. Figure 2The figure shows the expression differences of LncRNA-MYLK-AS1 in AML tumor tissues and normal control tissues based on the TNMplot database. The scatter plot represents the expression level of LncRNA-MYLK-AS1 in a single sample, and the box plot shows the expression distribution characteristics of the two groups. The left side represents normal control tissue (Normal), and the right side represents AML tumor tissue (Tumor). The vertical axis represents the gene expression level of LncRNA-MYLK-AS1, and the horizontal axis represents the group type. Figure 3 The expression difference of lncRNA-MYLK-AS1 in peripheral blood between AML patients and healthy controls was represented by the logarithmic axis. 10 The scale shows that the scatter points in the box plot represent the raw expression levels of a single sample. Figure 4 Infection efficiency of lentivirus infecting AML cells. Cell bright-field and GFP fluorescence channel images, scale bar = 100 μm; Figure 5 To construct a stable knockdown mechanism using ShRNA to measure the knockdown efficiency of LncRNA-MYLK-AS1 in AML cells after knocking down LncRNA-MYLK-AS1 in different AML cell lines, A represents the detection results of different groups in the MOLM-13 cell line, and B represents the detection results of different groups in the KG-1 cell line. Figure 6 To investigate the effect of knocking down LncRNA-MYLK-AS1 on the proliferation of AML cells; A shows the cell viability of each group at 48h, 72h, and 96h after MOLM-13 cell infection, measured using the CCK-8 assay; B shows the cell viability of each group at 48h, 72h, and 96h after KG-1 cell infection, measured using the CCK-8 assay. Figure 7 To detect the effect of LncRNA-MYLK-AS1 knockdown on apoptosis in AML cells using flow cytometry, the apoptosis level was detected using Annexin V-APC / PI double staining flow cytometry. Figure A is a scatter plot of apoptosis by flow cytometry; Figure B is a bar chart of quantitative statistical analysis of total apoptosis rate (Q2+Q3) in MOLM-13 cells; Figure C is a bar chart of quantitative statistical analysis of total apoptosis rate (Q2+Q3) in KG-1 cells; Q1 = viable cells, Q2 = late apoptotic cells, Q3 = early apoptotic cells, Q4 = necrotic cells, total apoptosis rate = Q2+Q3; Figure 8 The relative expression level of LncRNA-MYLK-AS1 in subcutaneous MOLM-13 cell xenografts in nude mice. sh-NC is the negative control transfected with sh-LncRNA-MYLK-AS1, and sh-LncRNA-MYLK-AS1 is the nude mouse xenograft group constructed by transfecting MOLM-13 cells with LncRNA-MYLK-AS1 shRNA. Figure 9 To investigate the effect of knocking down LncRNA-MYLK-AS1 combined with different chemotherapy drugs on the growth curve of subcutaneous xenografts in nude mice, tumors were induced in nude mice by subcutaneously inoculating MOLM-13 cells with sh-LncRNA-MYLK-AS1 and sh-NC as controls. Tumor volume was calculated every 2 days starting 1 week after tumor implantation in the nude mice (n=6). A represents the sh-NC+CRT group vs. the sh-LncRNA+CRT group (blank control); B represents the sh-NC+Ara-C group vs. the sh-LncRNA+Ara-C group; C represents the sh-NC+DNR group vs. the sh-LncRNA+DNR group; D represents the sh-NC+HTT group vs. the sh-LncRNA+HTT group. Figure 10 To compare the weight of subcutaneous xenografts in nude mice, samples were taken and weighed 14 days after successful treatment with the drug, with n=6; the groups were, in order, control group, cytarabine group, daunorubicin group, and homoharringtonine group. Detailed Implementation

[0017] Example 1. 1. To clarify the relationship between the expression level of lncRNA-MYLK-AS1 and the prognosis of patients with acute myeloid leukemia (AML), the KM plotter database (https: / / kmplot.com / analysis / ) was used to analyze the relationship between the expression level of MYLK-AS1 in AML and patient prognosis. Kaplan-Meier survival analysis was performed, and log-rank test was used to compare survival differences between groups.

[0018] The results are as follows Figure 1 As shown, based on the expression level of LncRNA-MYLK-AS1, the 734 included patients were divided into a high-expression group (355 cases) and a low-expression group (379 cases). Survival curve trends showed that the survival probability of patients in the high-expression group was lower than that in the low-expression group throughout the follow-up period, suggesting that high expression of LncRNA-MYLK-AS1 may be associated with worse survival outcomes. Statistical analysis showed that the survival difference between the two groups was statistically significant, as determined by the log-rank test. p =0.068, risk ratio HR =1.18 (95%) CI (0.99-1.41) Statistical analysis showed that the survival difference between the two groups of patients was obtained by log-rank test. p =0.068, not reached p The risk ratio is below the traditional statistical significance criterion of <0.05, but it is within the critical range of statistical significance, showing a tendency towards marginal significance; at the same time, the risk ratio... HR=1.18 (95%) CI The range of MYLK-AS1 expression (0.99-1.41) suggests that high MYLK-AS1 expression has a potential trend of increasing the risk of poor prognosis in patients by 18%. The upper limit of its 95% confidence interval is greater than 1 and the lower limit is close to the null line 1, which further supports the existence of this poor prognostic trend. Combined with the consistent results of subsequent clinical peripheral blood tests, cell function experiments and in vivo animal experiments, it can be confirmed that MYLK-AS1 has clinical value as a potential diagnostic and prognostic molecular marker for AML.

[0019] 2. To investigate the expression changes of lncRNA-MYLK-AS1 in tumorigenesis, the TNMplot database (https: / / tnmplot.comanalysis / ) was used to analyze the expression differences of lncRNA-MYLK-AS1 in AML and normal tissues. Researchers performed RNA-Seq screening analysis on normal and tumor tissues based on TNMplot. Further analysis of the expression differences of lncRNA-MYLK-AS1 in AML tumor tissues and normal tissues (…) Figure 2 The analysis results showed that the expression level of LncRNA-MYLK-AS1 in AML tumor tissues was significantly higher than that in normal tissues. Quantitative statistical results (Table 1) showed that the mean relative expression level of LncRNA-MYLK-AS1 in AML tumor tissues was 9.15 times that in normal tissues, and the median expression level reached 9.50 times, with statistically significant differences between groups. p =9.55×10 -62 <0.0001). The above results indicate that LncRNA-MYLK-AS1 is specifically upregulated in AML tumor tissues and has the potential to serve as a prognostic molecular marker for AML.

[0020] Table 1. Differences in MYLK-AS1 expression between AML and normal tissues.

[0021] Example 2. Differential expression analysis of LncRNA-MYLK-AS1 in peripheral blood of AML relapse patients and healthy controls LncRNA-MYLK-AS1 gene sequence: SEQ ID NO: .

[0022] This study included 14 patients with recurrent AML admitted to the Department of Hematology of our hospital as the research subjects, and recruited 14 healthy individuals as healthy controls. Peripheral blood samples were collected from all research subjects, and the expression level of LncRNA-MYLK-AS1 was detected by RT-qPCR technology.

[0023] Total RNA extraction: (1) Clinical peripheral blood samples need to be pretreated with human peripheral blood lymphocyte separation solution. The entire process of sample, reagent and experimental environment must be carried out at 20±2℃ (reagents need to be warmed up. 20℃ in summer, 23℃ in winter). Strict aseptic operation procedures must be followed. After centrifugation at 500g for 25min, density gradient centrifugation was performed to separate white membrane cells (PBMCs). After washing twice with sterile PBS, RNA extraction solution was added for the next step of RNA extraction.

[0024] (2) Tumor tissue samples need to be washed with PBS 1-3 times, and then 1 ml of RNA extraction solution and the corresponding number of sterile and enzyme-free grinding beads are added. The samples are then ground into homogenate on a grinder for later use.

[0025] (3) After collecting the cells, centrifuge at 3000 rpm for 5 min, wash once with sterile PBS, then add 1 ml of RNA extraction solution, pipette to mix evenly, lyse the cells, and let stand at room temperature for 5 min.

[0026] (4) After the above different pretreatments, add an appropriate amount of chloroform substitute to each sample, shake vigorously for 15 seconds, emulsify thoroughly, and let stand at room temperature for 3 minutes.

[0027] (5) Centrifuge at 12000 rpm for 15 min at 4°C, take the supernatant aqueous phase and transfer it into a pre-cooled 1.5 ml EP tube. Use a 200 μl pipette to aspirate twice (do not aspirate the lower layer; it is better to omit it than 400 μl, but remember the volume to prepare for the addition of isopropanol).

[0028] (6) Add an equal volume of isopropanol, mix thoroughly, and let stand at 4°C for 10 minutes.

[0029] (7) Centrifuge at 12000rpm for 10min at 4°C (the white precipitate is RNA), discard the supernatant, add 1ml of DEPC water to prepare 75% ethanol to wash, and let it air dry at room temperature until there is no ethanol in the tube.

[0030] (8) Add 25-50 μl of DEPC water to dissolve the RNA precipitate, and then proceed with the next step of RNA reverse transcription, or aliquot the RNA and store it in a -80℃ freezer for later use.

[0031] RNA reverse transcription: (1) RNA concentration measurement: First, use 1 μl of DEPC water to measure the Blank value. Then, take 1 μl of RNA extract from each sample and add it to a trace spectrophotometer. Measure the absorption peaks and ratios at 230 nm, 260 nm, and 280 nm respectively, and calculate the concentration and purity of RNA in each sample.

[0032] (2) Prepare the following reaction mixture in a nuclease-free microcentrifuge tube (the reaction mixture is prepared on ice).

[0033] Table 2

[0034] (3) After briefly centrifuging the above reaction mixture, reverse transcription was carried out under the following conditions.

[0035] Table 3

[0036] qPCR: (1) The qPCR primer sequence for LncRNA-MYLK-AS1 was designed on the NCBI website: forward,5'-CGTTGGCCAAGGTTTCGTTA-3' (SEQ ID NO.2); reverse,5'-AGGCAGATAACCTGGCTTCCA-3' (SEQ ID NO. 3).

[0037] GAPDH primers - forward, 5'-TCAAGGCTGAGAACGGGAAG-3' (SEQ ID NO.4); reverse, 5'-TCGCCCCACTTGATTTTGGA-3' (SEQ ID NO.5).

[0038] (2) Prepare qPCR reaction solution according to the following components (the reaction solution is prepared on ice).

[0039] Table 4

[0040] (3) Two-step qPCR procedure qPCR amplification program: Holding stage: pre-denaturation 95℃ for 30s; Cycling Stage: PCR reaction, Number of Cycles: 40; 95℃ for 5s, 60℃ for 30s.

[0041] Fluorescence signals from samples were continuously collected during the ascent process to obtain amplification and melting curves. Further statistical analysis using the ΔΔCT method was performed to obtain gene expression levels. Time PCR uses 2 △△Ct The method is used for relative quantitative analysis.

[0042] Test results as follows Figure 3As shown, the relative expression level of LncRNA-MYLK-AS1 in the peripheral blood of patients with relapsed AML was 24.30 (11.03~75.64), which was significantly higher than that in the healthy control group (0.99 (0.92~1.19)) and the difference was statistically significant. p <0.0001). The above results confirm that LncRNA-MYLK-AS1 is specifically highly expressed in the peripheral blood of patients with recurrent AML, which is consistent with the results of previous tumor tissue expression analysis in public databases. This suggests that LncRNA-MYLK-AS1 may be involved in the disease development of AML, and its easy detection and significant abundance differences in peripheral blood make it a potential molecular marker for non-invasive auxiliary diagnosis of AML.

[0043] Example 3. Reagent Kit 1. Primer combination: forward,5'-CGTTGGCCAAGGTTTCGTTA-3' (SEQ ID NO.2); reverse,5'-AGGCAGATAACCTGGCTTCCA-3' (SEQ ID NO.3); GAPDH primers——forward, 5'-TCAAGGCTGAGAACGGGAAG-3' (SEQ ID NO.4); reverse, 5'-TCGCCCCACTTGATTTTGGA-3' (SEQ ID NO.5); 2.5× PrimeScipt Master Mix reagent; 3. Healthy blood was used as a negative control.

[0044] Example 4. Construction of lentivirus-mediated stable knockdown of LncRNA-MYLK-AS1 in MOLM-13 and KG-1 cell lines 1. Design and synthesize oligonucleotide single strands for constructing shRNA, the sequences of which are as follows: Chain of Justice: 5'-TCCCCTCTTAAATGCTATACTACTTCCTGTCAGATAGTATAGCATTTAAGAGGTTTTTGAA-3' (SEQ ID NO.6) antisense chain: 5'-AGGGGAGAATTTACGATATGATGAAGGACAGTCTATCATATCGTAAATTCTCCAAAAACTT-3' (SEQ ID NO.7) 2. (1) Cell culture: Collect MOLM-13 and KG-1 cell suspensions, with a cell density of approximately 1×10⁻⁶ cells / mL.6 The cells / ml were mixed evenly by pipetting and mixing with RPMI 1640 medium (containing fetal bovine serum + antibiotics).

[0045] (2) Infecting cells: Add (MOLM-13 and KG-1 MOI: 10 and 20) / 1ml of viral supernatant (containing pSIH-LncRNA-MYLK-AS1 shRNA or pSIH-control shRNA) to the above MOLM-13 and KG-1 cell suspensions, and add polybrene to a final concentration of 5μg / ml. Shake gently, transfect by centrifugation, and then put back into the culture plate for continued culture at 37℃.

[0046] (3) Change medium: 24 hours after infection, collect the cells into a sterile centrifuge tube, centrifuge at 300g for 5 minutes, remove the supernatant, replace with complete culture medium, mix gently and put back into the culture plate to continue culturing at 37℃.

[0047] (4) 72 hours post-infection, the proportion of GFP-positive cells was observed using a fluorescence microscope to assess the lentiviral infection efficiency. Figure 4 The results showed that the GFP positivity rate of both MOLM-13 and KG-1 cells was over 80%, and there was no significant difference in infection efficiency between the two groups of cells, indicating that the lentiviral infection system was stable and reliable.

[0048] (5) Screening for positive cells: After the MOLM-13 and KG-1 cells from step ③ are dispersed, they are re-seeded in 10cm cell culture dishes. When the cell confluence is about 80%, puromycin is added. If the cells contain the pSIH vector, they can survive. Dead cells are screened and discarded. When the cells grow to a suitable density, puromycin is added again for drug screening. When the cells no longer die, MOLM-13 and KG-1 cells that stably express the pSIH vector (containing LncRNA-MYLK-AS1shRNA or control shRNA) are obtained. The transfection efficiency is confirmed by RT-qPCR. Figure 5 The cells were further expanded for subsequent experiments. Results showed that in MOLM-13 cells, the relative expression level of LncRNA-MYLK-AS1 in the sh-LncRNA-MYLK-AS1 group was significantly lower than that in the sh-NC group (0.29±0.03 vs 1.00±0.02). p <0.0001), with a knockdown efficiency of 71%; in KG-1 cells, the relative expression level of LncRNA-MYLK-AS1 in the sh-LncRNA-MYLK-AS1 group was significantly lower than that in the sh-NC group (0.21±0.01 vs 1.00±0.14, p <0.01), the knockdown efficiency reached 79%, and the difference between the two groups was statistically significant.

[0049] The above results indicate that this study successfully constructed MOLM-13 and KG-1 cell models with stable LncRNA-MYLK-AS1 knockdown, which can be used for subsequent in vivo and in vitro functional experiments.

[0050] Example 5. CCK8 assay to verify the ability of knockdown of LncRNA-MYLK-AS1 to inhibit the in vitro proliferation of AML cells. (1) According to the experimental purpose and experimental group, collect the corresponding resuspended cells and count them, and evenly seed them into 96-well plates, with 20,000 cells seeded in each well; then place them in a cell culture incubator for culture.

[0051] (2) According to the experimental purpose, cells were taken out at different time points such as 0h, 48h, 72h and 96h, and 10μl of CCK-8 solution was added to each well. Then the cells were put back into the incubator for further culture.

[0052] (3) After incubating at 37°C and 5% CO2 for 2 hours, the absorbance was measured at 450 nm using a multi-functional microplate reader.

[0053] (4) Use the wells containing the appropriate amount of cell culture medium and CCK-8 solution as experimental wells, and use the wells without cells as zeroing wells. Organize the data and calculate cell viability.

[0054] The cell viability of MOLM-13 and KG-1 cells after LncRNA-MYLK-AS1 knockdown was detected at 48 h, 72 h, and 96 h post-infection using the CCK-8 assay. Figure 6 The results showed that at 48 h, there was no statistically significant difference in cell viability between the sh-LncRNA-MYLK-AS1 group and the sh-NC group of the same cell line; at 72 h, the cell proliferation activity of the sh-LncRNA-MYLK-AS1 group decreased. p <0.05); at 96h, the cell viability of the sh-LncRNA-MYLK-AS1 group decreased more significantly ( p <0.0001). Meanwhile, the same result was observed in KG-1 cells: the relative cell viability of the knockdown groups decreased to varying degrees at both time points (72h: p <0.05; 96h: p <0.001). The above results indicate that knockdown of LncRNA-MYLK-AS1 can inhibit the in vitro proliferation of MOLM-13 and KG-1 cells, and this inhibitory effect is time-dependent.

[0055] Example 6. Flow cytometry detection of apoptosis (1) Collect cell suspension in the logarithmic growth phase, 300g, centrifuge at 4℃ for 5min to collect cells.

[0056] (2) Wash the cells twice with pre-cooled PBS, 300g each time, centrifuge at 4℃ for 5min, and collect 5×10⁻⁶ cells. 5 cell.

[0057] (3) Remove and discard the PBS, and add 100 μl of 1×Binding Buffer to resuspend the cells.

[0058] (4) Add 5 μl Annexin V-FITC and 10 μl PI Staining Solution and mix gently.

[0059] (5) React at room temperature in the dark for 15 minutes.

[0060] (6) Add 400 μl of 1×Binding Buffer, mix well and place on ice. Detect the sample using a flow cytometer within 1 hour and analyze the data using Flowjo-V10 software.

[0061] The results are as follows Figure 7 The results showed that the apoptotic phenotypes of the two AML cell lines were consistent: compared with the sh-NC cell line, the proportions of early and late apoptotic cells were significantly increased in the sh-LncRNA-MYLK-AS1 group. Specifically, the total apoptosis rate of MOLM-13 cells in the sh-NC group was 3.49%, while that in the sh-LncRNA-MYLK-AS1 group increased to 10.68%, a highly significant difference. p <0.0001); The total apoptosis rate of KG-1 cells in the sh-NC group was 6.28%, while that in the sh-LncRNA-MYLK-AS1 group increased to 9.84%, with a significant difference. p <0.01), and knockdown of LncRNA-MYLK-AS1 had a more significant apoptosis-inducing effect on MOLM-13 cells.

[0062] The above results indicate that knocking down LncRNA-MYLK-AS1 can significantly induce apoptosis in AML cells, verifying that LncRNA-MYLK-AS1 has a positive regulatory effect on the malignant proliferation phenotype of AML cells.

[0063] Example 7. In vivo functional study of LncRNA-MYLK-AS1 on the growth of subcutaneous xenografts in nude mice 1. Animal grouping Researchers randomly divided mice that met the weight and age requirements into 8 groups according to weight and experimental needs 1-3 days before the experiment: ① sh-NC group; ② sh-LncRNA-MYLK-AS1 group; ③ sh-NC+DNR group; ④ sh-LncRNA-MYLK-AS1+DNR group; ⑤ sh-NC+Ara-C group; ⑥ sh-LncRNA-MYLK-AS1+Ara-C group; ⑦ sh-NC+HTT group; ⑧ sh-LncRNA-MYLK-AS1+HTT group, with 6 mice in each group. For in vivo experiments, daunorubicin (DNR, 5 mg / kg, T20025, Yuanye) was administered via tail vein injection on days 1, 4, and 9, for a total of 3 times; cytarabine (Ara-C 2.5 mg / kg, SY004943, Shaoyuan) was administered intraperitoneally once daily for 14 days; and homoharringtonine (HTT, 0.15 mg / kg, A10149, Yuanye) was administered intraperitoneally twice daily for 14 days. All of the above drugs were administered to nude mice after the subcutaneous xenograft model was successfully established (approximately 14 days), and tissue samples were collected 14 days after the start of drug administration.

[0064] 2. Animal husbandry Female BALB / c nude mice, 4-6 weeks old and weighing 18-22g, were purchased and housed in a laboratory animal facility at a temperature of 22±2℃ and humidity of 55±5%. Bedding was changed twice weekly. The facility provided 12 hours of light and 12 hours of darkness daily, with 3-5 mice per cage, and free access to food and water. All mice were housed in the animal facility for one week after purchase to allow them to acclimatize before experiments were conducted.

[0065] (1) Construction of a nude mouse subcutaneous xenograft model and verification of the efficiency of LncRNA-MYLK-AS1 knockdown Nude mouse subcutaneous xenograft model: MOLM-13 cells (approximately 1 × 10⁻⁶) from lentivirus-transfected sh-LncRNA-MYLK-AS1 or sh-NC negative controls in logarithmic growth phase were used. 7(100 cells) were suspended in 1xPBS and subcutaneously injected into the right axilla of mice. Mice in groups ①③⑤⑦ were subcutaneously injected with 100 μl of sh-NC empty MOLM-13 cells, while mice in groups ②④⑥⑧ were subcutaneously injected with 100 μl of sh-LncRNA-MYLK-AS1 knocked-down Molm-13 cells. Immediately after inoculation, mice were returned to their cages for free movement and feeding. Tumor growth was observed every 2 days after tumor inoculation in nude mice, and tumor volume was measured every 2 days starting after 7-10 days. Tumor length and width were measured using calipers. Tumor volume was calculated as tumor length × tumor width × tumor width / 2; a value greater than 100 mm³ was considered successful model establishment. Tumor weight was measured using a precision balance. Drug treatment was initiated in nude mice approximately 14 days after successful model establishment. The endpoint of the mouse experiment was the end of drug treatment, or when the tumor volume reached 1000 mm³ and the mouse weight decreased by ≥20%.

[0066] LncRNA-MYLK-AS1 knockdown efficiency verification: To verify that the targeted shRNA can stably inhibit the expression of LncRNA-MYLK-AS1 in vivo and ensure the reliability of subsequent in vivo functional phenotypes, we sacrificed nude mice at the experimental endpoint, dissected the transplanted tumor tissue, extracted total RNA from the tumor tissue, and used RT-qPCR to detect the mRNA expression level of LncRNA-MYLK-AS1.

[0067] The results are as follows Figure 8 As shown, compared with the negative control sh-NC group, the expression level of LncRNA-MYLK-AS1 in sh-LncRNA-MYLK-AS1 xenograft tissue was significantly downregulated ( p The knockdown efficiency was <0.0001%, with an in vivo knockdown efficiency of approximately 50%, confirming that the knockdown system we constructed can function stably in an in vivo xenograft model of AML.

[0068] (2) The inhibitory effect of knockdown of LncRNA-MYLK-AS1 on the growth of AML subcutaneous xenografts in nude mice Starting one week after tumor-bearing mice, the long and short diameters of the tumor were measured and recorded every 2 days. The tumor volume was calculated and a volume growth curve was plotted. Figure 9 After the tumor-bearing model was successfully established (approximately day 14 of tumor bearing), drug treatment began. Mice were sacrificed 14 days after drug administration, and the subcutaneous transplanted tumor was completely dissected, weighed, and recorded. Figure 10 ).

[0069] Tumor growth curve results show ( Figure 9In the CTR group (control group without chemotherapy), knockdown of LncRNA-MYLK-AS1 mildly inhibited the in vivo growth of AML xenografts. At the endpoint of administration, the tumor volume in the sh-LncRNA-MYLK-AS1 group was lower than that in the sh-NC group at the same time point, but the difference was not statistically significant. However, in the Ara-C, DNR, and HTT chemotherapy groups, knockdown of LncRNA-MYLK-AS1 significantly inhibited the proliferation of xenografts. From day 7 after the start of administration (day 14 of tumor bearing), the tumor volume in the sh-LncRNA-MYLK-AS1 group showed a statistically significant difference compared to the sh-NC group at the same time point, and this difference continued to increase with the duration of administration. By the endpoint of administration, the tumor volume in the sh-LncRNA-MYLK-AS1 group in all three chemotherapy groups was nearly 50% less than that in the sh-NC group under the same conditions. This indicates that LncRNA-MYLK-AS1 has a positive regulatory effect on the malignant proliferation phenotype of AML cells, suggesting that knockdown of LncRNA-MYLK-AS1 enhances chemosensitivity.

Claims

1. The application of LncRNA-MYLK-AS1 as a biomarker in the preparation of kits for the diagnosis and / or prognosis of acute myeloid leukemia, characterized in that, The gene for LncRNA-MYLK-AS1 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, If the relative expression level of LncRNA-MYLK-AS1 in the peripheral blood of patients is significantly higher than that in the peripheral blood of healthy controls, the patients have acute myeloid leukemia or have a poor prognosis; if there is no significant difference between the relative expression level of LncRNA-MYLK-AS1 in the peripheral blood of patients and the relative expression level in the peripheral blood of healthy controls, the patients do not have acute myeloid leukemia or have a good prognosis.

3. A kit for diagnosing and / or predicting the prognosis of acute myeloid leukemia, characterized in that, The kit includes an upstream primer and a downstream primer. The sequence of the upstream primer is shown in SEQ ID NO.2, and the sequence of the downstream primer is shown in SEQ ID NO.

3.

4. The kit according to claim 3, characterized in that, The kit also includes primer combinations for qPCR amplification of Mix, ddH2O, and amplification of GAPDH internal control.

5. A recombinant acute myeloid leukemia cell line, characterized in that, The cell line knocked down LncRNA-MYLK-AS1, and the gene for LncRNA-MYLK-AS1 is shown in SEQ ID NO.

1.

6. The cell line according to claim 5, characterized in that, The cell line mentioned is either MOLM-13 or KG-1.

7. An inhibitor of LncRNA-MYLK-AS1 expression, characterized in that, The inhibitor is a shRNA that knocks down LncRNA-MYLK-AS1.

8. The inhibitor according to claim 7, characterized in that, The shRNA gene is a hairpin structure prepared from SEQ ID NO.6 and SEQ ID NO.

7.

9. The use of the LncRNA-MYLK-AS1 of claim 1, the cell line of claim 5 or 6, or the expression inhibitor of claim 7 or 8 in the preparation of products that enhance the sensitivity of chemotherapeutic drugs.

10. The application according to claim 9, characterized in that, Chemotherapy drugs include cytarabine, daunorubicin, or homoharringtonine.

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