CN114369662A - Tumor gene diagnosis kit - Google Patents

Tumor gene diagnosis kit Download PDF

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CN114369662A
CN114369662A CN202210037464.4A CN202210037464A CN114369662A CN 114369662 A CN114369662 A CN 114369662A CN 202210037464 A CN202210037464 A CN 202210037464A CN 114369662 A CN114369662 A CN 114369662A
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黄小容
黄少冬
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Abstract

The invention discloses an application of an acute myelocytic leukemia marker in preparing a diagnosis product of acute myelocytic leukemia, wherein the marker is hsa _ circ _0000907 and hsa _ circ _ 0021346. In another aspect, the present invention discloses a kit for diagnosing acute myeloid leukemia, which comprises reagents for detecting the expression levels of hsa _ circ _0000907 and hsa _ circ _ 0021346. In yet another aspect, the invention also discloses a pharmaceutical composition for treating acute myeloid leukemia, comprising inhibitors of hsa _ circ _0000907 and hsa _ circ _ 0021346. The kit can be used as one of AML diagnosis means, and has the characteristics of quick and convenient detection, high detection sensitivity, good specificity, low cost and the like. The medicinal composition has a better inhibiting effect on AML, and has important referential significance and practical significance in AML treatment.

Description

Tumor gene diagnosis kit
Technical Field
The invention belongs to the field of clinical medical diagnosis, and particularly relates to a tumor gene diagnosis kit.
Background
Acute Myelogenous Leukemia (AML) is the most common acute leukemia in adults, and is a malignancy caused by immature hematopoietic cells of the myeloid lineage in the bone marrow. According to the National Institutes of Health (NIH), 2.4 out of 10 million people per year are diagnosed with acute myeloid leukemia. Acute myeloid leukemia is an aggressive hematological tumor with multiple genotypes, phenotypes, epigenetic characteristics, and resistance to cytotoxic therapies. It is characterized by high recurrence rate, which is the leading cause of treatment failure. Its treatment is still predominantly chemotherapy, but about 70% of patients who achieve remission eventually relapse and develop refractory leukemia, leading to treatment failure and death.
Circular RNA (circRNA) is a special non-coding RNA molecule, and is different from the traditional linear RNA (linear RNA), the circRNA molecule has a closed circular structure, is not influenced by RNA exonuclease, has more stable expression and is not easy to degrade. Functionally, recent researches show that the circRNA molecules are rich in microrna (miRNA) binding sites and play a role of miRNA sponge (miRNA sponge) in cells, so that the inhibition effect of miRNA on target genes of the circRNA molecules is relieved, and the expression level of the target genes is increased. Recent studies have shown that circRNA is generally characterized by a large number of species and quantities, widespread distribution, strong evolutionary conservation, tissue specificity, extremely high stability and high-abundance expression. The unique advantage of circRNA makes it a new marker useful for disease diagnosis, prognosis and prediction of response to treatment.
At present, the diagnosis of AML is mostly judged by the change of clinical manifestations, hemograms and bone marrow images, and is classified and typed by combining cell morphology, immunology, cytogenetics and molecular biology, but symptoms are not typical in some early onset stages, so that the diagnosis is difficult. And is difficult to distinguish from other diseases (such as aplastic anemia and the like). Therefore, finding a faster, more sensitive and more specific diagnostic index is of great clinical significance.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides a biomarker for detecting acute myeloid leukemia, a kit and application thereof. The detection method can detect trace annular RNA in human peripheral blood, and has high sensitivity and strong specificity.
Therefore, the invention discloses the application of the acute myelocytic leukemia marker in the preparation of a diagnostic product for acute myelocytic leukemia, wherein the acute myelocytic leukemia marker is hsa _ circ _0000907 and hsa _ circ _ 0021346.
In another aspect, the present invention also discloses a kit for diagnosing acute myeloid leukemia, which comprises reagents for detecting the expression levels of hsa _ circ _0000907 and hsa _ circ _ 0021346.
Preferably, the reagents of the invention are primers that specifically amplify hsa _ circ _0000907 and hsa _ circ _ 0021346.
Preferably, the primer sequences of the specific amplification hsa _ circ _0000907 of the invention are shown in SEQ ID NO.1 and SEQ ID NO. 2; the primer sequence of the specific amplification hsa _ circ _0021346 is shown in SEQ ID NO.3 and SEQ ID NO. 4.
Preferably, the determination formula of the kit of the present invention is Y ═ 0.058 × Ahsa _ circ _0000907+0.068 × Bhsa _ circ _ 0021346; wherein, Ahsa _ circ _0000907 is the expression quantity of hsa _ circ _0000907, and Bhsa _ circ _0021346 is the expression quantity of hsa _ circ _ 0021346; when Y is more than or equal to 0.45, the result is positive, and the possible AML is represented; when Y is less than 0.45, the result is negative, and the result indicates normal.
In yet another aspect, the invention also discloses a pharmaceutical composition for treating acute myeloid leukemia, comprising inhibitors of hsa _ circ _0000907 and hsa _ circ _ 0021346.
Preferably, the inhibitors of hsa _ circ _0000907 and hsa _ circ _0021346 according to the invention are si-hsa _ circ _0000907 and si-hsa _ circ _ 0021346.
Preferably, the sequences of si-hsa _ circuit _0000907 and si-hsa _ circuit _0021346 according to the invention are shown in SEQ ID NO.5 and SEQ ID NO. 6.
The invention has the following beneficial effects:
the invention screens circular RNA markers for AML diagnosis and treatment by means of bioinformatics and existing molecular biology techniques. We chose hsa _ circ _0000907 and hsa _ circ _0021346 as combination markers (in AML, both hsa _ circ _0000907 and hsa _ circ _0021346 were up-regulated). On the basis, the invention provides application of reagents for jointly detecting the expression levels of hsa _ circ _0000907 and hsa _ circ _0021346 in preparing products for diagnosing AML and related detection kits. The kit can be used as one of AML diagnosis means, and has the characteristics of quick and convenient detection, high detection sensitivity, good specificity, low cost and the like.
In addition, the invention also provides application of the potential pharmaceutical composition for treating AML and the related pharmaceutical composition. The medicinal composition has a better inhibiting effect on AML, and has important referential significance and practical significance in AML treatment.
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FIG. 1 is a dissolution profile of real-time fluorescent quantitative PCR reactions of hsa _ circ _0000907 and hsa _ circ _ 0021346.
FIG. 2 statistics of survival for leukemia mouse model.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 detection of circular RNA in peripheral blood of Acute Myeloid Leukemia (AML)
Collecting samples: 5 normal peripheral blood and 5 AML peripheral blood were collected. All participants signed informed consent and were approved by the hospital ethics committee. All participants retained 5ml of peripheral venous blood in the fasting state in the morning and stored in a refrigerator at-70 deg.C.
RNA extraction: extracting total RNA of peripheral blood by using a Trizol (15596026, Invitrogen, Car, Cal and USA) one-step method, detecting the purity and the concentration by using a NanoDrop ND-2000 instrument, wherein the ratio of A260 to A280 is 1.8-2.0.
RNA library establishment and sequencing: circular RNA was enriched and pre-treated using the CircRNA Enrichment Kit (from Cloud-seq, USA) and the TruSeq Stranded Total RNA Library Prep Kit (from Illumina, USA), generating sequencing libraries and testing for quality. Sequencing was performed on an Illumina HiSeq 4000 sequencer.
Bioinformatics analysis: sequencing by a sequencer, and processing by using cutadapt software to obtain high-quality clean reads. Clear reads were compared and circular RNA was detected and identified using DCC software. Differential circular RNAs were identified in both sets of samples using the t-test using normalized reads numbers with fold change > 2.0 and P-value < 0.05 as thresholds for differential circular RNAs, i.e., differentially expressed circular RNAs. The results showed that 275 circular RNAs were up-regulated and 84 were down-regulated in the AML group compared to the normal group; wherein hsa _ circ _0000907 and hsa _ circ _0021346 are both up-regulated in expression and are prognostic-related; therefore, hsa _ circ _0000907 and hsa _ circ _0021346 were selected as the major circular RNAs for this study. Wherein the sequences of hsa _ circ _0000907 and hsa _ circ _0021346 are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.
Example 2 detection of hsa _ circ _0000907 and hsa _ circ _0021346 in AML peripheral blood
Enlarging and collecting a sample: a total of 40 specimens were collected, 20 of which were AML patients and 20 of which were normal peripheral blood, and stored in a refrigerator at-70 ℃. All participants signed informed consent.
RNA extraction: extracting total RNA of peripheral blood by using a Trizol (15596026, Invitrogen, Car, Cal and USA) one-step method, detecting the purity and the concentration by using a NanoDrop ND-2000 instrument, wherein the ratio of A260 to A280 is 1.8-2.0.
Reverse transcription: RNA was reverse transcribed into cDNA in 20. mu.l of the experimental system according to the EasyScript First-Strand cDNA Synthesis SuperMix (catalog number AE301-02, Beijing Quanyu, China).
Primer design and synthesis: primer5.0 was used to design primers, the sequences of hsa _ circ _0000907 and hsa _ circ _0021346 are shown in example 1, with U6 being chosen as the internal control. The primers were designed and synthesized by Nanjing Kingsrei science and technology, Inc. The specific primer sequences are shown in the following table:
Figure BDA0003469020550000041
real-time fluorescent quantitative PCR reaction: the PCR reaction was carried out using a qPCR SYBR Green master mix (purchased from Shanghai Yunji Biotech Co., Ltd.) in a real-time fluorescent quantitative PCR apparatus (ABI 7500, USA) in the following reaction system: 2 × master mix 5 μ l, upstream primer 0.5 μ l, downstream primer 0.5 μ l, cDNA 2 μ l, RNase Free dH2O2. mu.l. Reaction conditions are as follows: 10min at 95 ℃; the temperature of 95 ℃ for 15s and the temperature of 60 ℃ for 1min are continuously carried out for 40 cycles. The fluorescence was collected. Setting the temperature at 95 ℃ for 10s after the amplification reaction; 60 ℃ for 60 s; 95 ℃ for 15 s; and slowly heated from 60 c to 99 c. Ct values were recorded for each well with U6 as an internal reference.
The results show that: in AML, hsa _ circ _0000907 and hsa _ circ _0021346 were significantly higher than in the normal group, with P < 0.01, consistent with high throughput sequencing results; and the design of the primer meets the detection requirement. Specific results are shown in fig. 1 and the following table:
name of Gene AML (average 2)-△△CT) Normal (average 2)-△△CT) P value
hsa_circ_0000907 13.82±5.12 3.27±1.58 ＜0.001
hsa_circ_0021346 10.59±2.47 1.59±0.49 ＜0.001
The two indexes are fitted through a Logistic regression equation, and the sensitivity and specificity after fitting are higher than those of the single indexes. Through data analysis, a judgment formula (regression equation) is obtained as follows:
Y＝0.058*Ahsa_circ_0000907+0.068*Bhsa_circ_0021346(ii) a Wherein A ishsa_circ_0000907Is the expression level of hsa _ circ _0000907, Bhsa_circ_0021346Is the expression level of hsa _ circ _ 0021346.
When Y is more than or equal to 0.45, the result is positive, and the possible AML is represented; when Y is less than 0.45, the result is negative, and the result indicates normal.
Example 3 Assembly of the kit and detection thereof
On the basis of example 2, a diagnostic kit for detecting AML was assembled, comprising the following components: (1)3 sets of primer pairs: primer pairs hsa _ circ _0000907, hsa _ circ _0021346 and U6 respectively, wherein the primer pairs hsa _ circ _0000907 and hsa _ circ _0021346 are shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively; the primer pair of U6 is shown as SEQ ID NO.9 and SEQ ID NO. 10. (2) SYBR Green polymerase chain reaction system: qPCR SYBR Green master mix. (3) Positive and negative controls: the positive control is cDNA sample mixture of hsa _ circ _0000907 and hsa _ circ _0021346, and the negative control is RNase Free dH2O。
The reaction systems identified are shown in the following table:
components Volume of
2×master mix 5μl
Upstream primer 0.5μl
Downstream primer 0.5μl
cDNA 2μl
RNase Free dH2O 2μl
Total up to 10μl
The optimal reaction conditions are as follows: 10min at 95 ℃; the temperature of 95 ℃ for 15s and the temperature of 60 ℃ for 1min are continuously carried out for 40 cycles. The fluorescence was collected.
Data processing and calculation: after the detection, the CT value of each sample was counted and calculated according to the company Y ═ 0.058 × ahsa_circ_0000907+0.068*Bhsa_circ_0021346(ii) a Wherein A ishsa_circ_0000907Is the expression level of hsa _ circ _0000907, Bhsa_circ_0021346Is the expression level of hsa _ circ _ 0021346. When Y is more than or equal to 0.45, the result is positive, and the possible AML is represented; when Y is less than 0.45, the result is negative, and the result indicates normal.
Detection of the kit: taking peripheral blood of a detected person, extracting RNA according to a conventional method, carrying out inversion rate to obtain cDNA, carrying out real-time fluorescence quantitative PCR detection on the cDNA by using a reagent in the kit, calculating and processing a detection result according to the method, wherein the detection result can be used for AML possibility risk assessment or disease diagnosis and natural prognosis of the individual; can also be used for evaluating the curative effect and treating prognosis of AML treatment for the individual.
The kit of the invention detects the expression condition of the circRNA gene through the simplest and specific primer pair, is stable and accurate, is convenient to detect, and greatly improves the sensitivity and specificity for diagnosing AML, so that the kit can help to guide early diagnosis and more effective individualized treatment when put into practice.
Example 4 AML mice in vitro treatment study
Preparation of inhibitors of hsa _ circ _0000907 and hsa _ circ _ 0021346: designing the interference RNA of hsa _ circ _0000907 and hsa _ circ _0021346 according to the sequence of the circular RNA, wherein the sequences of the interference RNA of hsa _ circ _0000907 and hsa _ circ _0021346 are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.
The cell culture and sorting (see the preparation of Chinese patent 2020102945010) specifically comprises: human AML cell line KG1a (ATCC, Manassas, VA) was cultured in suspension in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 100U/ml penicillin and streptomycin. The Leukemia Stem Cells (LSCs) are CD34+CD38-KG1a cells (K), all KG1a cells were CD34+LSCs were therefore enriched by indirect labeling with CD38-FITC antibody and anti-FITC magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, the cell suspension was centrifuged at 125g for 10min and washed with PBS. The cell pellet was then suspended in isolation buffer (PBS containing 0.5% BSA) and mixed with CD38-FITC antibody was incubated for 30 minutes. After PBS washing, the cell pellet was incubated for 15 minutes in separation buffer containing anti-FITC microspheres. After washing, the filtrate (CD 34)+CD38-Cells) were collected using LD column of the mitimacs separator system. Throughout the sorting process, cells were placed at 4 ℃ and analyzed by flow cytometry. The remaining cells after depletion of LSCs were defined as AML cells and used for co-culture studies.
Nude mice transplantation tumor experiment: NOD/SCID/IL2r gamma (null) mice (NSG) (6-9week-old) (available from Schleicklanda laboratory animals, Inc. of Hunan province). NOD/SCID mice were randomly divided into 4 groups of 10 mice each. The 4 groups were irradiated with 1.8Gy of whole body X-ray. The experimental groups were as follows: (1) blank control group, AML tail vein injection of stable transfection physiological saline at logarithmic growth phase of 5 × 10 after 24h6One/one; (2) set si-hsa _ circ _0000907, stably transfected si-hsa _ circ _0000907 at logarithmic growth phase taken 24h laterAML tail vein injection of 5 x 106One/one; (3) in si-hsa _ circ _0021346 group, AML tail vein of stably transfected si-hsa _ circ _0021346 in logarithmic growth phase was injected at 24h after 5 × 106One/one; (4) in si-hsa _ circ _0000907+ si-hsa _ circ _0021346, 5X 10 AML tail vein injection of stably transfected si-hsa _ circ _0000907 and si-hsa _ circ _0021346 in logarithmic growth phase was performed 24h later6One/only. The survival rates of two groups of mice are observed and detected. All animal experiments in this study were in accordance with local laboratory animal management.
The experimental results show that: overall survival was significantly increased in si-hsa _ circ _0000907, si-hsa _ circ _0021346, and si-hsa _ circ _0000907+ si-hsa _ circ _0021346 mice compared to the control group (figure 2). The research results show that: in a leukemia mouse, si-hsa _ circ _0000907 and si-hsa _ circ _0021346 can inhibit AMLSC self-renewal, improve the survival rate of the leukemia mouse, and have better effect when the two are used together.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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actgatttct tgtttctgcc tctgactcaa gaagatactc aatagcacag ctataggaag 360
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caaatataca ttcatattca aagtcaacct cccattcaag tggtagtagt agaagggtat 960
aatgatcagc tattcctatt ccaaactcac tttattgtga agaagataag gtgtaaatga 1020
tttttcaatc agcttccaaa acactttccc cccaagttta gctttataaa taagaaacac 1080
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acaacccctt attctatgag ctacaagaag gaaaaaatat ccaacatcat ttttgtttca 1260
atttttatat gcaaaagcta atctggacat aagtggagac acagatgaaa aactaacaag 1320
gaaaaagcta acatgtatgc agtaacaact agccattgaa attgtaaaac tattcactca 1380
tctgatagag aatacatagt attaagtcct aatgtgatct tttcctttaa aaatattgaa 1440
actggcagtt ttctcttcta tttgcattac gataagcaaa taaggtgtta atccttctgg 1500
gctatgctta aaaaattatt taacattcag gccattattt agcatacagc tggttagcag 1560
ttttccacta aagtactggc tcatatccca catttttttc ttctggcact tacctggagc 1620
tgtaaagggc tgagtccaga agcagcagca gctgccattg ttgatggaat gaactgtacg 1680
gggtagttat cac 1693
<210> 9
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ctcgcttcgg cagcaca 17
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aacgcttcac gaatttgcgt 20

Claims (8)

1. The application of the acute myelocytic leukemia marker in preparing the diagnosis product of the acute myelocytic leukemia is characterized in that the acute myelocytic leukemia marker is hsa _ circ _0000907 and hsa _ circ _ 0021346.
2. A kit for diagnosing acute myeloid leukemia, comprising reagents for detecting the expression levels of hsa _ circ _0000907 and hsa _ circ _ 0021346.
3. The kit of claim 2, wherein the reagents are primers that specifically amplify hsa _ circ _0000907 and hsa _ circ _ 0021346.
4. The kit according to claim 3, wherein the primer sequence for specific amplification hsa _ circ _0000907 is shown in SEQ ID No.1 and SEQ ID No. 2; the primer sequence of the specific amplification hsa _ circ _0021346 is shown in SEQ ID NO.3 and SEQ ID NO. 4.
5. According to the rightThe kit according to claim 2, wherein the determination formula of the kit is Y-0.058 ahsa_circ_0000907+0.068*Bhsa_circ_0021346(ii) a Wherein A ishsa_circ_0000907Is the expression level of hsa _ circ _0000907, Bhsa_circ_0021346Is the expression level of hsa _ circ _ 0021346; when Y is more than or equal to 0.45, the result is positive, and the possible AML is represented; when Y is less than 0.45, the result is negative, and the result indicates normal.
6. A pharmaceutical composition for the treatment of acute myeloid leukemia, comprising inhibitors of hsa _ circ _0000907 and hsa _ circ _ 0021346.
7. The pharmaceutical composition of claim 6, wherein the inhibitors of hsa _ circ _0000907 and hsa _ circ _0021346 are si-hsa _ circ _0000907 and si-hsa _ circ _ 0021346.
8. The pharmaceutical composition of claim 7, wherein the sequences of si-hsa _ circ _0000907 and si-hsa _ circ _0021346 are set forth in SEQ ID No.5 and SEQ ID No. 6.
CN202210037464.4A 2022-01-13 2022-01-13 Tumor gene diagnosis kit Withdrawn CN114369662A (en)

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