CN112961919B - Circular RNA hsa _ circ _0059706, specific amplification primer thereof and application - Google Patents
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Abstract
The invention relates to a circular RNA hsa _ circ _0059706 and a specific amplification primer thereof, wherein the cDNA nucleotide sequence corresponding to the circular RNA is shown as SEQ ID NO. 1, the specific amplification primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3. The circular RNA is low expressed in bone marrow mononuclear cells of patients with acute myelogenous leukemia, so that the circular RNA can be used as a molecular marker for screening the acute myelogenous leukemia.
Description
Technical Field
The invention belongs to the field of tumor molecular biology, and particularly relates to a circular RNA for diagnosing acute myeloid leukemia.
Background
Acute Myeloid Leukemia (AML) is a malignant clonal disease of hematopoietic system originating in myeloid progenitor cells, and is the most common type of hematological malignancy in which leukemia cells proliferate in large amounts in hematopoietic tissues such as bone marrow to inhibit normal hematopoiesis. The pathogenesis of the AML involves abnormal changes in various aspects such as cell differentiation, proliferation and apoptosis, nonrandom chromosome abnormality, oncogene and cancer suppressor gene mutation and the like play an important role in the occurrence and development process of AML, and recent researches show that epigenetic changes play an important role in the process. Although the treatment means such as chemotherapy and hematopoietic stem cell transplantation combined with various medicines have great breakthroughs, the relapse and metastasis of acute myeloid leukemia still frequently occur, and the relapse and metastasis become the main cause of death of patients with advanced acute myeloid leukemia. The early diagnosis and the early treatment can greatly reduce the further development of the acute myelogenous leukemia deterioration, and with the development of the molecular level and the further research on the mechanism related to the development, the development and the treatment of the acute myelogenous leukemia, the screening and the research on the molecular markers of the targeted diagnosis and the targeted treatment become one of the most concerned fields of the research on the acute myelogenous leukemia.
Circular RNA (Circular RNA) is a covalently closed Circular RNA without 5 '-3' polarity and poly-adenine tail. circRNA may function as a miRNA sponge or competitive endogenous rna (cerna) to modulate expression of miRNA targets. circRNA is highly abundant and evolutionarily conserved among eukaryotes, and has specificity in specific cell types and at different stages.
In recent years, more and more researches show that the circular RNA is closely related to the occurrence and development of acute myeloid leukemia. Wang J reports that aberrant expression of circ _0075451 may lead to an independent risk factor for poor prognosis in AML and may also be a potential target for AML treatment. The expression of Circ _ HIPK2 in APL patients is obviously lower than that of normal peripheral mononuclear cells and other AML subtypes, and the CIc _ HIPK2 is used as a sponge of differentiation-related miR-124-3p to influence ATRA-induced killer cell differentiation, thereby indicating the potential function of the CIC _ HIPK2 as an APL biomarker. Therefore, the discovery and screening of the circRNA marker related to leukemia have important significance and value for early prevention and treatment of leukemia.
Disclosure of Invention
In order to solve the technical problem, the invention comprises the following aspects:
the invention provides a circular RNA, wherein a cDNA nucleotide sequence corresponding to the circular RNA is shown as SEQ ID NO. 1, wherein the first two nucleotides and the last two nucleotides are ring-forming binding sites.
The circular RNA is named as hsa _ circ _0059706 in a circ-RNA database, is called as circ _0059706 for short, is positioned in the human chromosome 20 sense strand 30193085-30194317 region, is formed by splicing and circularizing after being transcribed by an ID1 gene.
The invention also provides a specific amplification primer for amplifying the circular RNA, which comprises an upstream primer and a downstream primer, wherein,
the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
Specifically, the nucleotide sequence of the upstream primer is as follows: 5'-TGGTAAACTCTCATTCCACGTTC-3', respectively;
the nucleotide sequence of the downstream primer is as follows: 5'-CCACTGGCGACTTTCATGAT-3' are provided.
Preferably, the GC content of the upstream primer is 43%, and the GC content of the downstream primer is 50%;
preferably, the upstream primer has a TM value of 56.64, and the downstream primer has a TM value of 55.40.
Preferably, the most suitable TM value for RQ-PCR used for the upstream and downstream primers after the verification is 56.2.
The present invention also provides a method for amplifying the circular RNA, comprising the steps of:
(1) synthesis of cDNA: the raw materials comprise total RNA, random primers and RNase free ddH2O, dNTP mixture, 5 XPrimeScript II Buffer, RNase inhibitor and reverse transcriptase; firstly, random primer, dNTP mixed solution, total RNA and RNase free ddH2Reacting O at 65 ℃ for 5min, and rapidly cooling on ice; adding 5 XPrimeScript II Buffer, RNase into the mixtureInhibitors, reverse transcriptase and RNase free ddH2O, reacting at 30 ℃ for 10min, at 45 ℃ for 45min, at 95 ℃ for 5min, and keeping at 4 ℃; performing reverse transcription by using an effendoff PCR amplification instrument, and storing the cDNA at-80 ℃ for later use;
(2) the obtained cDNA was amplified by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR): preparing an amplification system, wherein the amplification system comprises the cDNA prepared in the step (1), the upstream primer, the downstream primer and ddH2O and RQ-PCR amplified the fluorescent dye Mix (containing SYBGREEN dye and ROX).
Preferably, in the step (1), 1. mu.l of random primer, 1. mu.l of dNTP mixture, 2. mu.l of total RNA and 6. mu.l of RNase free ddH are added2O reacts for 5min at the temperature of 65 ℃.
Preferably, 4. mu.l of 5 XPrimeScript II Buffer, 0.5. mu.l of RNase inhibitor, 1. mu.l of reverse transcriptase and 4.5. mu.l of RNase free ddH are further added to the mixture in the step (1)2O。
Preferably, the amplification system in step (2) includes 2. mu.l cDNA, 0.4. mu.l forward primer, 0.4. mu.l reverse primer, and 7.2. mu.l ddH2O and 10. mu.l RQ-PCR amplified the fluorescent dye Mix.
Preferably, the temperature-programmed reaction in step (2) is as follows: pre-denaturation at 95 deg.C for 5min, reacting at 95 deg.C for 10s, at 56.2 deg.C for 32s, performing 40 cycles, reacting at 95 deg.C for 15s, reacting at 60 deg.C for 60s, reacting at 95 deg.C for 15s, and terminating the reaction.
The invention also provides a kit for screening acute myeloid leukemia, which comprises the specific amplification primer and the amplification system.
The invention also provides application of the kit in preparing a reagent for detecting the expression level of circ _0059706 in bone marrow mononuclear cells.
The invention also provides application of the kit in preparing a reagent for screening acute myeloid leukemia.
The invention has the following beneficial effects:
through bioinformatics prediction, the inventor finds that the circular RNA circ _0059706 has a binding site with miR-29b, and the miR-29b is obviously related to the occurrence, development, treatment and prognosis of acute myeloid leukemia. The research finds that the expression of the circular RNA circ _0059706 is remarkably reduced in a bone marrow specimen of an acute myelogenous leukemia patient relative to a healthy control group, so that the circular RNA circ _0059706 can be used for screening acute myelogenous leukemia. Secondly, the circular RNA circ _0059706 is amplified by using the specific amplification primer provided by the application, the amplification result is analyzed, and the area under the ROC curve is 0.841, which shows that the primer has high sensitivity and strong specificity. Finally, the amplification product obtained by using the specific amplification primer provided by the application is a single band, and has no non-specific amplification.
Drawings
FIG. 1 shows RQ-PCR assay circ _0059706 differential expression profiles in blood of patients with acute myeloid leukemia and bone marrow of healthy people;
FIG. 2 shows the ROC curve for circ _0059706 detection in acute myeloid leukemia tissue.
Detailed Description
Test example 1 RQ-PCR reaction to detect the expression of circ _0059706 in acute myeloid leukemia tissue
RQ-PCR amplification mixed system (Mix), qPCR (fluorescent dye-containing) amplification mixed system (Mix), reverse transcriptase, dNTP mixed solution were purchased from TAKARA.
First, test method
1. Design of specific amplification primers
The sequence and related information of the specific amplification primer designed by the invention are shown in Table 1:
TABLE 1 circular RNA circ _0059706 specific amplification primer sequences and related information
2. Separation of bone marrow nucleated cells
(1) Centrifuging dipotassium ethylenediaminetetraacetate (EDTA-K2) anticoagulation at 2000r/min for 5min, injecting 5ml of erythrocyte lysate into erythrocyte lysate at the lower layer, reversing and mixing uniformly until the liquid in the test tube is clear, centrifuging in a low-speed centrifuge for 10min at the rotating speed of 2000r/min, and centrifuging and reversing to remove the supernatant;
(2) continuously adding 5ml of erythrocyte lysate into the precipitate, turning upside down until the mixture is uniformly mixed, and standing at room temperature until the liquid is transparent;
(3) centrifuging in a centrifuge at 2000r/min for 10min to remove supernatant;
(4) adding 1ml NS into the test tube, blowing and uniformly mixing by using a 1ml gun head, and sucking 1ml mixed solution into a 1.5ml EP tube;
(5) centrifuging the mixed solution at high speed for 3min at 13000r/min, sucking and discarding supernatant, and storing the white precipitate in a refrigerator at-80 deg.C for use.
3. Extraction of Total RNA
(1) Adding 1000 mu l of TRIZOL lysate into the white precipitate obtained in the step (5) in the step (2), fully blowing the liquid by using a 1ml pipette gun until the liquid in an EP tube is clear, has no visible precipitate and is a transparent pink water sample, and placing the liquid in an ice box after blowing to fully lyse cells;
(2) adding 200 μ l of chloroform, allowing liquid to stratify, covering the cover of the EP tube, oscillating on an oscillator for 30s, standing for visible stratification, oscillating the EP tube again, and standing at room temperature for 10 min;
(3) centrifuging at high speed for 10min at 12000r/min, wherein the liquid of EP tube is divided into three layers, the upper layer is colorless and transparent, the middle layer is white film layer, and the lower layer is pink transparent layer, wherein RNA is above the white film, and protein is below the white film;
(4) absorbing the colorless transparent layer above the white film layer and adding the colorless transparent layer into a new EP tube; adding 600 mu l of isopropanol;
(5) gently inverting and mixing the RNA tubes, centrifuging for 10min, 12000r/min, discarding the liquid, and leaving the precipitate as RNA;
(6) adding 1000 μ l of 70% ethanol, slightly inverting to suspend the precipitate in the liquid, inverting several times, centrifuging at high speed 12000r/min for 10min, and discarding the supernatant; adding 1000 μ l 100% ethanol, and absorbing ethanol to obtain white precipitate;
(7) drying the white precipitate obtained in the above step in a metal dry bath at 37 deg.C, baking for about 5-10min, and mixing with appropriate amount of diethyl pyrocarbonate (DEPC) water; incubating and cracking on ice for 30 min;
(8) detecting the purity and concentration of total RNA of the samples by using a NanoDrop One spectrophotometer, wherein DEPC is used as a blank control for zero setting, and the RNA in each sample is diluted to the same concentration according to the measured concentration and the volume of the residual liquid; the diluted sample can be subjected to reverse transcription experiment, or the RNA can be stored in a refrigerator at-80 ℃.
4. Reverse transcription method
The reverse transcription system of the two-step method comprises the following operation steps: firstly, taking a PCR tube to configure a reverse transcription system, wherein the reverse transcription system is as follows: 1 μ l random primer, 1 μ l dNTP mix, 2 μ l total RNA, 6 μ l RNase free ddH2O, reacting for 5min at 65 ℃, and rapidly cooling on ice; mu.l of 5 XPrimeScript II Buffer, 0.5. mu.l RNase inhibitor, 1. mu.l reverse transcriptase, 4.5. mu.l RNase free ddH were added to the above mixture2O, reacting at 30 ℃ for 10min, at 45 ℃ for 45min, at 95 ℃ for 5min, and keeping at 4 ℃. Reverse transcription was performed using an effendoff PCR amplifier and the cDNA was stored at-80 ℃ until use. Reverse transcription kit, available from Takara.
5. RQ-PCR amplification reaction of circ _0059706 gene
The obtained cDNA was amplified by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR): preparing an amplification system, wherein the amplification system comprises the cDNA prepared in the step, the upstream primer, the downstream primer and the ddH2O, RQ PCR amplified fluorescent dye Mix (containing SYBGREEN dye and ROX) and the reaction was temperature controlled according to the following procedure. The preparation system is as follows: mu.l cDNA, 0.4. mu.l forward primer, 0.4. mu.l reverse primer, 7.2. mu.l ddH2O, 10 μ l qPCR amplifies the fluorescent dye Mix. The amplification procedure was: pre-denaturation at 95 deg.C for 5min, reacting at 95 deg.C for 10s, at 56.2 deg.C for 32s, performing 40 cycles, reacting at 95 deg.C for 15s, reacting at 60 deg.C for 60s, reacting at 95 deg.C for 15s, and terminating the reaction. The amplification reaction is carried out on a real-time fluorescent quantitative PCR instrument ABI 7500, ABL1 is amplified as an internal reference control while the target gene is amplified, and the amplification reaction is carried out by a 2(-△△CT)The relative expression level of the gene is calculated.
Second, test results
1. Analysis of Gene expression
Results of the RQ-PCR method for detecting the level of circ _0059706 in bone marrow mononuclear cells of 6 control and 85 AML patients are shown in FIG. 1. Compared with a control group, the expression of the circular RNA circ _0059706 in the bone marrow mononuclear cells of the acute myeloid leukemia is remarkably reduced, and the suggestion that the circular RNA circ _0059706 can be used for screening the acute myeloid leukemia.
2. ROC curve analysis
The experimental data were analyzed using SPSS software to obtain ROC curves, as shown in fig. 2. Wherein, the area under the curve, AUC, is 0.841, which means that the detection target can be used as a specific marker for detecting the circular RNA, and the detection sensitivity can reach 83.3%, and the specificity can reach 87.1%.
Sequence listing
<110> first-person hospital in Zhenjiang city
<120> circular RNA hsa _ circ _0059706, specific amplification primer and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1232
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 1
actctcattc cacgttctta actgttccat tttccgtatc tgcttcgggc ttccacctca 60
tttttttcgc tttgcccatt ctgtttcagc cagtcgccaa gaatcatgaa agtcgccagt 120
ggcagcaccg ccaccgccgc cgcgggcccc agctgcgcgc tgaaggccgg caagacagcg 180
agcggtgcgg gcgaggtggt gcgctgtctg tctgagcaga gcgtggccat ctcgcgctgc 240
gccgggggcg ccggggcgcg cctgcctgcc ctgctggacg agcagcaggt aaacgtgctg 300
ctctacgaca tgaacggctg ttactcacgc ctcaaggagc tggtgcccac cctgccccag 360
aaccgcaagg tgagcaaggt ggagattctc cagcacgtca tcgactacat cagggacctt 420
cagttggagc tgaactcgga atccgaagtt ggaacccccg ggggccgagg gctgccggtc 480
cgggctccgc tcagcaccct caacggcgag atcagcgccc tgacggccga ggtgagatcc 540
agatccgacc actagatcat ccttataccg acggggaaac ggaggccaga gagggcgtgg 600
gcgcttgcac cacttccgtc ccatccttgc gggtacctgg ctatgcgggg gtgcctaagg 660
agcctggaaa aagcgctccc ccgtcgtgct tcctggggaa gggggcgttc gctgcgctcg 720
gagcggcgtc ccttccaacc cgccggtctc atttcttctc gttttcacag gcggcatgcg 780
ttcctgcgga cgatcgcatc ttgtgtcgct gaagcgcctc ccccagggac cggcggaccc 840
cagccatcca gggggcaaga ggaattacgt gctctgtggg tctcccccaa cgcgcctcgc 900
cggatctgag ggagaacaag accgatcggc ggccactgcg cccttaactg catccagcct 960
ggggctgagg ctgaggcact ggcgaggaga gggcgctcct ctctgcacac ctactagtca 1020
ccagagactt tagggggtgg gattccactc gtgtgtttct attttttgaa aagcagacat 1080
tttaaaaaat ggtcacgttt ggtgcttctc agatttctga ggaaattgct ttgtattgta 1140
tattacaatg atcaccgact gaaaatattg ttttacaata gttctgtggg gctgtttttt 1200
tgttattaaa caaataattt agatggtggt aa 1232
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tggtaaactc tcattccacg ttc 23
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccactggcga ctttcatgat 20
Claims (1)
1. The application of a specific amplification primer for amplifying a circular RNA in the preparation of a reagent for screening acute myeloid leukemia, wherein the specific amplification primer comprises an upstream primer and a downstream primer,
the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3;
the GC content of the upstream primer is 43 percent, and the GC content of the downstream primer is 50 percent;
the TM value of the upstream primer is 56.64, and the TM value of the downstream primer is 55.40;
the circular RNA is called hsa _ circ _0059706 in the circ-RNA database, and the corresponding cDNA nucleotide sequence is shown in SEQ ID NO. 1.
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