TRNA modified molecular marker for diagnosis of acute myelogenous leukemia and application of tRNA modified molecular marker

Abstract

The invention relates to a tRNA (transfer ribonucleic acid) modified molecular marker for diagnosis of acute myelogenous leukemia and application of the tRNA modified molecular marker. The molecular marker is modified by N6-threonyl carbamoyl adenosine of total tRNA. Research finds that compared with healthy people, the N6-threonyl carbamoyl adenosine modification level of total tRNA in a biological sample of an acute myelogenous leukemia patient is remarkably improved; a further diagnosis performance analysis result shows that the kit has relatively high diagnosis accuracy and value when being used for detecting acute myelogenous leukemia. Therefore, the N6-threonyl carbamoyl adenosine modification of the total tRNA can be used as a specific molecular marker for detecting the acute myelogenous leukemia to assist the accurate diagnosis of the acute myelogenous leukemia. Besides, blood and the like of a to-be-detected person can be adopted as a detection sample for N6-threonyl carbamoyl adenosine modification detection based on the total tRNA, sampling is convenient and noninvasive, operation is relatively simple, cost is low, speed is high, and TAT lasts for 2-4 days.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular detection, and in particular relates to a tRNA modified molecular marker for diagnosing acute myeloid leukemia and its application. Background Art

[0002] Acute myeloid leukemia (AML) is a group of hematopoietic tumors that originate from the myeloid system, in which immature cells proliferate malignantly in the bone marrow, peripheral blood or other tissues, and cause clinical manifestations such as fever, anemia, bleeding and tumor infiltration. The existing MICM assessment and stratification system mainly relies on morphology, immunology, cytogenetics and molecular biology to diagnose, treat and evaluate the prognosis of AML patients. Although it can effectively guide clinical diagnosis, it lacks molecular biological diagnostic markers of epigenetic modification and cannot meet the clinical needs for accurate diagnosis. In addition, AML diagnosis requires bone marrow puncture and biopsy, which is somewhat traumatic; the second-generation sequencing technology detects genetic abnormalities at the DNA or mRNA level and the chromosome karyotype detects cytogenetic abnormalities, which requires a long laboratory rotation time. The reporting time limit (TAT) of the laboratory test items is 7-14 days, and the experimental steps are cumbersome and costly.

[0003] With the application of molecular diagnostic technology in the field of leukemia, the diagnosis of leukemia has evolved from the past diagnosis system based on morphology to an individualized diagnosis system with precise molecular diagnosis as the core. Studies in recent years have shown that epigenetics affects the occurrence and development of leukemia. Therefore, epigenetic modifications such as DNA methylation, RNA methylation, and tRNA modification are expected to serve as new molecular markers to assist in the diagnosis of diseases and improve the survival rate of patients.

[0004] tRNA is a non-coding RNA, generally 76-90 nucleotides in length, and is the key to efficient and accurate protein translation. Nucleotides on tRNA are widely modified, and these modifications play an important role in the function of tRNA in cells. Clinically, we expect to discover more specific and sensitive tRNA modifications as molecular markers for accurate diagnosis of AML. Summary of the invention

[0005] Based on this, the purpose of the present invention is a specific molecular marker for the diagnosis of acute myeloid leukemia and its application in the detection of acute myeloid leukemia. The molecular marker is N6-threonylaminoformyladenosine modification of total tRNA. Based on the molecular marker, auxiliary diagnosis of acute myeloid leukemia can be simply and quickly achieved.

[0006] The first aspect of the present invention is to provide N6-threonylcarbamoyladenosine (t6-threonylcarbamoyladenosine, t 6 A) Application of modification as a molecular marker in the preparation of acute myeloid leukemia detection reagents.

[0007] The second aspect of the present invention is to provide a reagent for detecting the level of N6-threonylaminoformyladenosine modification of total tRNA and its use in preparing a kit for detecting acute myeloid leukemia.

[0008] In some of the embodiments, the detection kit is used for chromatography-mass spectrometry detection.

[0009] In some embodiments, the reagent is N6-threonylaminoformyladenosine. N6-threonylaminoformyladenosine is used as a reference substance.

[0010] In some embodiments, the reagent also includes a mobile phase, and the mobile phase includes a phase A and a phase B, wherein the phase A is a 0.05% to 2.0% formic acid aqueous solution, preferably a 0.1% to 2.0% formic acid aqueous solution, and more preferably a 0.1% to 1.0% formic acid aqueous solution; the phase B is a 0.05% to 2.0% formic acid methanol solution, preferably a 0.1% to 2.0% formic acid methanol solution, and more preferably a 0.1% to 1.0% formic acid methanol solution.

[0011] The third aspect of the present invention is to provide a method for detecting acute myeloid leukemia for non-diagnostic purposes, comprising the following steps: obtaining total tRNA of a sample to be tested, and detecting the N6-threonylaminoformyladenosine modification level of the total tRNA.

[0012] In some embodiments, chromatography-mass spectrometry is used to detect the level of N6-threonylaminoformyladenosine modification of the total tRNA.

[0013] In some embodiments, the chromatographic detection conditions include: mobile phase: including phase A and phase B, wherein phase A is 0.05% to 2.0% formic acid aqueous solution, preferably 0.1% to 2.0% formic acid aqueous solution, and more preferably 0.1% to 1.0% formic acid aqueous solution; phase B is 0.05% to 2.0% formic acid methanol solution, preferably 0.1% to 2.0% formic acid methanol solution, and more preferably 0.1% to 1.0% formic acid methanol solution.

[0014] In some of the embodiments, the chromatographic detection conditions include: chromatographic column: Waters ACQUITY BEH C18; column temperature: 35°C to 55°C; injection volume: 1 μL to 40 μL; gradient elution.

[0015] In some embodiments, the sample to be tested includes a tissue sample or a body fluid sample containing leukocytes; preferably, the body fluid sample is blood.

[0016] The present invention has been found through research that N6-threonylaminoformyladenosine modification of total tRNA can be used as a specific molecular marker for the detection of acute myeloid leukemia. Compared with healthy people, the level of N6-threonylaminoformyladenosine modification of total tRNA in biological samples of patients with acute myeloid leukemia is significantly improved; further diagnostic performance analysis results show that when it is used for the detection of acute myeloid leukemia, when the threshold is 0.0555, the AUC is 0.955, the sensitivity is 96.77%, and the specificity is 85.29%, which has high diagnostic accuracy and value. Therefore, N6-threonylaminoformyladenosine modification of total tRNA can be used as a specific molecular marker for the detection of acute myeloid leukemia, assisting the accurate diagnosis of acute myeloid leukemia.

[0017] In addition, the detection of N6-threonylaminoformyladenosine modification based on total tRNA can use the blood of the test subject as the detection sample. The sampling is convenient and non-invasive, the operation is relatively simple, low-cost, and fast, with a TAT of 2-4 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The t6A modification catalyzes the formation of ms 2 Pathway diagram of t6A or m6t6A.

[0019] Figure 2 This is a ROC curve analysis diagram based on the t6A modification detection results in Example 1.

[0020] Figure 3 This is a diagram of the experimental results of tRNA nucleotide fragment analysis in Example 2.

[0021] Figure 4 This is a graph showing the detection results of the t6A modification level of total tRNA in clinical samples in Example 3.

[0022] Figure 5 The clinical samples in Example 3 are based on t 6 A is the ROC curve analysis diagram of the modification detection results. DETAILED DESCRIPTION

[0023] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0024] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0025] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0026] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0027] The present invention is further described in detail below with reference to specific embodiments.

[0028] Example 1 Screening of clinical samples for differential tRNA modification

[0029] Inclusion samples: (1) AML group: 15 patients with acute myeloid leukemia who were initially diagnosed with clinical symptoms, excluding patients with leukemia transformed from myelodysplastic syndrome or secondary to other malignant tumors. The diagnostic criteria for acute myeloid leukemia are: FAB diagnostic criteria for acute myeloid leukemia or the 2022 version of the WHO guidelines for acute myeloid leukemia. (2) Healthy person group: 19 healthy people aged 18 years and above were used as controls.

[0030] The basic clinical information of the two groups of samples is shown in Table 1.

[0031] Table 1 Clinical sample information

[0032]

[0033] 1. Sample collection and leukocyte separation

[0034] Collect EDTA anticoagulated whole blood samples and use red blood cell lysis buffer to separate white blood cell samples.

[0035] 1) Take a freshly collected EDTA anticoagulated whole blood sample, add 3 times the volume of red blood cell lysis buffer EL (QIAGEN), and mix well.

[0036] 2) Incubate on ice for 10-15 min until red blood cells are lysed. Centrifuge at 800×g for 10 min at 4°C and discard the supernatant.

[0037] 3) Add 2 times the volume of red blood cell lysis buffer EL to the precipitate, shake briefly, centrifuge at 4°C, 800×g for 10 min, and completely discard the supernatant.

[0038] 4) Add an appropriate amount of TRIzol reagent (Thermo Invitrogen), pipette up and down to mix thoroughly, and store at -80℃ for later use.

[0039] 2. tRNA extraction and separation

[0040] Total tRNA was extracted according to the RNA Clean&Concentrator-25 kit (ZYMO) operating procedures.

[0041] 1) Take 1 mL of TRIzol solution of the above sample, add 1 / 5 volume of chloroform, mix well, centrifuge at 12000 rpm for 10 min, and aspirate the supernatant.

[0042] 2) Add an equal volume of anhydrous ethanol to the supernatant, mix well, and pipette onto the filter. Centrifuge at 12000 rpm for 30 seconds and discard the filtrate.

[0043] 3) Add 400 μL RNA Prep Buffer to the filter cartridge, centrifuge at 12000 rpm for 30 seconds, and discard the filtrate.

[0044] 4) Add 700 μL RNA Wash Buffer to the filter, centrifuge at 12000 rpm for 30 seconds, and discard the filtrate.

[0045] 5) Add 400 μL RNA Wash Buffer to the filter cartridge, centrifuge at 12000 rpm for 1 min, discard the filtrate, and transfer the filter cartridge to a new RNase-free centrifuge tube.

[0046] 6) Add 100 μL DNase / RNase-Free Water to the filter, centrifuge at 12,000 rpm for 1 min, and collect the filtrate.

[0047] 7) Add 2 times the volume (200 μl) of Adjusted RNA Binding Buffer (i.e., dilute 1:1 with anhydrous ethanol) to the filtrate, mix well, and aspirate onto a new filter. Centrifuge at 12000 rpm for 30 seconds and collect the filtrate.

[0048] 8) Add an equal volume of anhydrous ethanol to the filtrate, mix well, and absorb into a new filter. Centrifuge at 12000 rpm for 30 seconds and discard the filtrate.

[0049] 9) Add 400 gL RNA Prep Buffer to the filter cartridge, centrifuge at 12000 rpm for 30 seconds, and discard the filtrate.

[0050] 10) Add 700 μL RNA Wash Buffer to the filter cartridge, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0051] 11) Add 400 μL RNA Wash Buffer to the filter cartridge, centrifuge at 12,000 rpm for 1 min, discard the filtrate, and transfer the filter cartridge to a new RNase-free centrifuge tube.

[0052] 12) Add 50 μL DNase / RNase-Free Water to the filter cartridge, centrifuge at 12,000 rpm for 1 min, and collect the filtrate.

[0053] 13) The obtained tRNA was quality controlled by urea SDS-PAGE electrophoresis, and no large fragments remained and no degradation was found.

[0054] 3. Sample hydrolysis

[0055] The total tRNA sample was fully hydrolyzed into oligonucleotides using endonuclease RNaseI, phosphodiesterase I, phosphodiesterase II and shrimp alkaline phosphatase (rSAP).

[0056] 1) Take an appropriate amount of total tRNA sample and place it in a 1.5mL centrifuge tube, add 1 / 7 of the total sample volume Buffer, pre-incubate at 37℃ for 10 min.

[0057] 2) Prepare a mixed enzyme solution according to the volumes specified in Table 2 below:

[0058] Table 2

[0059] Stock solution of reagent Volume added (μL) IOU / gL RNase I 50 0.025U / μL phosphodiesterase I 100 0.025U / μL phosphodiesterase II 100 <![CDATA[RNase-free H20]]> 250 Total volume 500

[0060] 3) Add 10 μL of the mixed enzyme solution to the total tRNA, vortex to mix, and react in a constant temperature incubator at 37°C for 2.5-3 hours.

[0061] 4) Add 2 μL rSAP (1 U / μL) and continue incubation for 1 h.

[0062] 5) After incubation at 70°C for 10 min, terminate the reaction, centrifuge at 10,000 rpm for 10 min, and transfer the supernatant to a sample injection bottle for testing.

[0063] 4. Chromatography-mass spectrometry detection

[0064] The base modifications were detected by electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS).

[0065] 1) Chromatographic conditions:

[0066] Chromatographic column: Waters ACQUITY BEH C18 (100 mm × 2.1 mm, 1.7 μm);

[0067] Mobile phase: 0.1% formic acid-methanol (phase B); 0.1% formic acid in water (phase A);

[0068] Flow rate: 0.2 mL / min;

[0069] Column temperature: 40°C;

[0070] Injection volume: 15 μL;

[0071] Elution gradient: as shown in Table 3.

[0072] Table 3

[0073] Time (min) Mobile phase A% Mobile phase B% Flow rate 1 0.00min 98.00％ 2.00％ 0.200mL / min 2 6.00min 68.00％ 32.00％ 0.200mL / min 3 8.00min 20.00％ 80.00％ 0.200mL / min 4 9.00min 98.00％ 2.00％ 0.200mL / min 5 12.00min 98.00％ 2.00％ 0.200mL / min

[0074] 2) Mass spectrometry conditions:

[0075] The ESI ion source was used in the positive ion mode and the multiple reaction monitoring mode (MRM) was used for detection. Source gas parameters: drying gas temperature 250°C; drying gas flow rate 14 L / min; nebulizing gas pressure 50 psi; sheath gas temperature 300°C; sheath gas flow rate 11 L / min;

[0076] MRM method: as shown in Table 4.

[0077] Table 4

[0078] Nucleosides Rt(min) Q1(m / z) Q3(m / z) Dwell time Fragmentor CE(V) A 3.30 268.1 136.1 10 380 5 Am 4.14 282.1 136.1 15 380 18 <![CDATA[cm 5 In]]> 3.46 303.1 171.1 15 380 5 <![CDATA[cmnm 5 with 2 In]]> 4.32 348.1 216.1 15 380 18 <![CDATA[cmnm 5 In]]> 8.36 332.1 200.1 15 380 5 G 3.45 284.1 152.1 10 380 10 Gm 4.29 298.1 152.1 15 380 18 <![CDATA[ho 5 In]]> 8.55 261.1 129.1 15 380 5 <![CDATA[i 6 A]]> 8.88 336.1 204.1 10 380 18 <![CDATA[m 6 , 6 Am]]> 3.12 310.1 164 15 380 18 <![CDATA[m 6 Am]]> 5.44 296.1 150 15 380 18 mxD 7.78 427.2 295.1 15 380 18 <![CDATA[mcm 5 with 2 In]]> 5.72 333.1 201.1 15 380 5 <![CDATA[mcm 5 In]]> 4.54 317.1 185.1 15 380 5 <![CDATA[mcm 5 One]]> 5.72 331.1 185.1 15 380 5 <![CDATA[mnm 5 with 2 In]]> 2.06 304.1 172.1 15 380 18 <![CDATA[mnm 5 to 2 In]]> 3.42 352.1 220.1 15 380 18 <![CDATA[ms 2 i 6 A]]> 9.24 382.2 250.1 15 380 18 <![CDATA[ms 2 I 6 A]]> 3.42 398.2 266.1 15 380 18 <![CDATA[ms 2 t 6 A]]> 7.90 459.1 327.1 15 380 18 U5 2.32 302.1 170.1 15 380 5 <![CDATA[t 6 A]]> 6.75 413.1 281.1 10 380 18

[0079] 5. Results Analysis

[0080] The mass spectrometry peak area was normalized by AG response and analyzed using the Student's t-test statistical method.

[0081] 1) In the qualitative analysis software, extract the ion currents of A and G of all samples to see if the chromatographic peaks of A and G are saturated and whether the retention time is normal. In the quantitative analysis software, check the retention time and integration of each compound one by one to exclude compounds with too low signal-to-noise ratio.

[0082] 2) Export the peak area of ​​each compound, sort it (A is divided into one group, G is divided into one group), and perform normalization analysis through AG response.

[0083] 3) The Student's t-test statistical test method was used to compare the patient group and the healthy control group for each compound.

[0084] The results are shown in Table 5. 6 A.ms 2 t6A and m 6 The fold difference of t6A is large, the p value is significant, and these three modifications are in the same synthetic pathway, which deserves further study.

[0085] Table 5 tRNA modification detection results of AML and healthy subjects

[0086]

[0087] Compared with the healthy control group, the total tRNA in the AML group 6 A.ms 2 t6A and m 6 The t6A modification level was significantly upregulated. 6 A modification will further catalyze the formation of ms 2 t 6 A or m 6 t 6 A, so in this pathway, t 6 A modification is key ( Figure 1 ).

[0088] Further based on t 6 The detection results of A were analyzed by ROC curve, and the results are as follows Figure 2 As shown, the AUC of t6A modification of total tRNA for AML diagnosis was 0.9333, and when the threshold value was 0.05594, the sensitivity was 94.74% and the specificity was 86.67%, indicating that it can be used as a specific molecular marker for AML diagnosis.

[0089] Example 2 tRNA nucleotide fragment analysis experiment

[0090] Studies have shown that 6 The A modification is located at the 37th adenine of the tRNA responsible for decoding the ANN codon. In Example 1, the extracted tRNA is completely hydrolyzed into oligonucleotides and then the t6A modification level is detected. In order to avoid the influence of adenine in oligonucleotides formed by hydrolysis of other small molecule RNAs that may remain in the extracted tRNA, it is further confirmed that the t6A modification detected in Example 1 occurs on the tRNA. In this example, the extracted tRNA is incompletely digested to obtain nucleotide fragments, and the t6A modification of the nucleotide fragments is analyzed.

[0091] The extracted tRNA samples of 15 healthy people and 15 tRNA samples of AML patients were mixed into NC and AML, and 10 μg of each sample was added with RNase T1 (50U / μgRNA) and ammonium acetate buffer (220mM), and digested at 37°C for 3h to obtain incompletely digested nucleotide fragments. The reaction was terminated by incubation at 70°C for 10min. The reaction mixture was lyophilized, resuspended in 60 μL ion pair mobile phase A (containing 100mM HFIP, 15mM TEA in methanol aqueous solution, pH=8.5), and then chromatographic and mass spectrometric detection (detection method is the same as in Example 1) was performed on the machine for tRNA modification positioning.

[0092] Previous studies have built an internal database of human tRNA digestion products based on the public transfer RNA databases tRNAdb and Modomics and the Mongo-Oligo mass calculator. The molecular formula of the tRNA digestion products was calculated by a simple program written in R language. tRNA mapping is processed using the "Search by Formula" algorithm in the Agilent MassHunterWorkstation software. The mass accuracy between the theoretical mass and the calculated mass of the multiply charged ion is less than 5ppm. Sequence information is mainly deduced from the c- and y-type series ions in the collision-induced dissociation (CID) spectrum, and is additionally confirmed by the "Fixed Sequence Modification Mapping" in RNAModmapper. The maximum tolerance for fragment ions is 0.5Da [PMID: 33290562]. In this example, [m1A]AUCCCACUUCUG (SEQ IN NO.1) was used as a reference to calibrate the data. The results show that the t 6 The modified nucleotide fragment is a tRNA nucleotide fragment, including t 6 A modified nucleotide fragment UU[t6A]CCG (represents tRNA A sn), t6A-modified nucleotide fragment ψCU[Cm]AU[t 6 A]AψCUG (represents tRNA Met ), wherein ψ represents pseudouracil (Pseudouridine, ψ). Compared with NC, the nucleotide fragment is highly expressed in the AML patient group. Figure 3 The results of this example further confirm that the present invention detects the t6A modification level on tRNA.

[0093] Example 3 t based on total tRNA 6 Validation of the performance of A modification abundance in diagnosing AML

[0094] In this example, blood samples were collected from 34 AML patients (diagnostic criteria were the same as in Example 1) and 31 healthy subjects from Yuhuangding Hospital of Yantai University. The sample information is shown in Table 6. 6 A modification level was detected and analyzed.

[0095] Table 6 Clinical sample information

[0096]

[0097] The results are as follows Figure 4 As shown in Figure 2, compared with the healthy control group, the t6A modification level of total tRNA in the AML group was significantly increased, and the p value was less than 0.0001. Further ROC curve analysis was performed, as shown in Figure 2. Figure 5 As shown, the AUC is 0.955, the threshold value = 0.0555, the sensitivity is 96.77%, and the specificity is 85.29%.

[0098] The results of this example once again verified that the t6A modification level of total tRNA can be used as a specific detection molecular marker for AML, and can be used to assist in the accurate diagnosis of AML.

[0099] The detection of t6A modification of tRNA can use blood or other materials as the detection sample. Sampling is convenient and non-invasive, the operation is relatively simple, low-cost, and fast, with a TAT of 2-4 days.

[0100] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of N6-threonylaminoformyladenosine modification of total tRNA as a molecular marker in the preparation of acute myeloid leukemia detection reagents.

2. Application of a reagent for detecting the level of N6-threonylaminoformyladenosine modification of total tRNA in the preparation of an acute myeloid leukemia detection kit.

3. The use according to claim 2, characterized in that The detection kit is used for chromatography-mass spectrometry detection.

4. The use according to any one of claims 2 to 3, characterized in that: The reagent is N6-threonylaminoformyladenosine.

5. The use according to claim 4, characterized in that The reagent also includes a mobile phase, which includes a phase A and a phase B. The phase A is a 0.05% to 2.0% formic acid aqueous solution, and the phase B is a 0.05% to 2.0% formic acid methanol solution.

6. A method for detecting acute myeloid leukemia for non-diagnostic purposes, characterized in that: The following steps are involved: The total tRNA of the sample to be tested is obtained, and the N6-threonylaminoformyladenosine modification level of the total tRNA is detected.

7. The method according to claim 6, characterized in that The N6-threonylaminoformyladenosine modification level of the total tRNA was detected using chromatography-mass spectrometry.

8. The method according to claim 7, characterized in that The chromatographic detection conditions include: mobile phase: including phase A and phase B, the phase A is 0.05% to 2.0% formic acid aqueous solution, and the phase B is 0.05% to 2.0% formic acid methanol solution.

9. The method according to claim 7 or 8, characterized in that Chromatographic detection conditions include: chromatographic column: Waters ACQUITY BEH C18; column temperature: 35°C to 55°C; injection volume: 1 μL to 40 μL; gradient elution.

10. The method according to claim 6, characterized in that The sample to be tested includes a tissue sample and a body fluid sample containing leukocytes; preferably, the body fluid sample is blood.

Images