CN113308543B - Application of hsa _ circ _0000284 in preparation of neuroblastoma prognosis preparation - Google Patents
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Abstract
The invention discloses an application of hsa _ circ _0000284 in preparation of a neuroblastoma prognosis preparation, and relates to the technical field of molecular biology. The invention also discloses the nucleotide sequence of hsa _ circ _0000284 as shown in SEQ ID NO. 1, and the nucleotide sequence of the specific primer as shown in SEQ ID NO. 2 and SEQ ID NO. 3. Survival curve analysis of 38 neuroblastoma patients revealed that high hsa _ circ _ 0000284-expressing patients had poor post-operative survival (P ═ 0.0107). According to the invention, the expression level of hsa _ circ _0000284 is detected, so that prognosis judgment is made for a patient with neuroblastoma, and hsa _ circ _0000284 can be used as an important index for clinical diagnosis and evaluation of neuroblastoma.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to application of hsa _ circ _0000284 in preparation of a neuroblastoma prognosis preparation.
Background
Neuroblastoma (NB) is the most common extracranial solid tumor in children, originating from primitive neuroepithelial cells of the embryonic neural crest, which can develop anywhere along the sympathetic nervous system, most commonly adrenal medulla. The number of NB patients accounts for 8-10% of the number of children tumor patients, about 15% of the number of children tumor patients die, and about 50% of the number of children tumor patients have tumor metastasis at the time of diagnosis. Early NB can regress spontaneously or can achieve a good prognosis by simple surgical resection, but infants with late NB have a five-year survival rate of less than 40% despite intensive treatment measures. The prognosis judgment of the patient with neuroblastoma is carried out so as to select the optimal treatment scheme, the survival rate of the patient is obviously improved, and the method becomes an important subject to be solved urgently in the field of pediatric tumor surgery.
Circular RNA (circRNA) is a special RNA molecule, and is different from the traditional linear RNA, and the circRNA molecule has a closed circular structure, is not easy to degrade by RNA exonuclease and has more stable expression. Recent studies have shown that circRNA is involved in the progression of many diseases, especially tumorigenesis. The CircRNA has the characteristics of cell expression specificity, good stability, rich expression and the like, so that the CircRNA can be used as a biomarker and an accurate treatment target for tumor diagnosis and prognosis.
Therefore, how to accurately determine the prognosis of neuroblastoma by using circular RNA is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
Accordingly, the present invention provides an application of cyclic RNAhsa _ circ _0000284 in preparing a neuroblastoma prognosis preparation.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of hsa _ circ _0000284 in preparing a neuroblastoma prognosis preparation, wherein the nucleotide sequence of hsa _ circ _0000284 is shown as SEQ ID NO: 1.
Preferably, the prognostic formulation is a kit.
Preferably, the kit comprises primers specific to hsa _ circ _0000284, and the nucleotide sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3.
Forward primer 5'-TATGTTGGTGGATCCTGTTCGGCA-3', SEQ ID NO 2;
reverse primer 5'-TGGTGGGTAGACCAAGACTTGTGA-3', SEQ ID NO 3.
Preferably, the kit also comprises a GAPDH internal reference primer, and the nucleotide sequence of the GAPDH internal reference primer is shown as SEQ ID NO. 4 and SEQ ID NO. 5.
GAPDH internal reference primer:
forward primer 5'-TGCACCACCAACTGCTTAGC-3', SEQ ID NO 4;
reverse primer 5'-GGCATGGACTGTGGTCATGAG-3', SEQ ID NO: 5.
Preferably, the kit further comprises reagents for extracting total RNA from neuroblastoma tissue, reagents for reverse transcription of hsa _ circ _0000284 into cDNA using total RNA as a template, and reagents for real-time quantitative PCR of cDNA.
More preferably, the reagent for extracting total RNA from neuroblastoma tissue comprises RNA stabilizing solution, Trizol reagent, chloroform, isopropanol and enzyme-free water;
the reagent for reverse transcription of hsa _ circ _0000284 into cDNA by using total RNA as a template comprises a reverse transcription buffer solution, base triphosphate deoxynucleotides, an RNase inhibitor, reverse transcriptase and an hsa _ circ _0000284 primer;
preferably, further comprising a dnase;
the reagent for real-time quantitative PCR of cDNA comprises real-time fluorescent quantitative SYBR dye and amplification enzyme.
Another object of the present invention is to analyze the feasibility assessment of hsa _ circ _0000284 as a prognostic marker for neuroblastoma, comprising the following steps:
s1: freezing and storing the collected neuroblastoma tissue sample to be detected, and extracting total RNA;
s2: removing the genome;
s3: reverse transcription of total RNA;
s4: real-time quantitative PCR amplification of the expression difference of hsa _ circ _0000284 in different samples;
s5: patient survival was followed and the feasibility of hsa _ circ _0000284 as a prognostic marker was analyzed.
According to the invention, the tumor tissues and the para-carcinoma tissues of 38 neuroblastoma patients are analyzed by fluorescent quantitative PCR, and hsa _ circ _0000284 has significant expression difference (P is less than 0.001) in the tumor tissues and the para-carcinoma tissues; survival curve analysis of 38 neuroblastoma patients revealed that high hsa _ circ _ 0000284-expressing patients had poor post-operative survival (P ═ 0.0107). According to the invention, the expression level of hsa _ circ _0000284 is detected, so that prognosis judgment is made for a patient with neuroblastoma, and hsa _ circ _0000284 can be used as an important index for clinical diagnosis and evaluation of neuroblastoma.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a graph showing the fluorescent quantitative PCR assay of hsa _ circ _0000284 expression levels in tumor and paracancerous tissues of neuroblastoma patients.
FIG. 2 is a graph showing the relationship between the expression level of hsa _ circ _0000284 in the tumor tissue of 38 patients with neuroblastoma and the survival time of the patients with neuroblastoma.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
preparation of a kit for the prognosis of neuroblastoma patients with hsa _ circ _0000284 (20 reactions)
50ml RNA stabilizing solution
2. Isopropanol 30ml
3. Chloroform 20ml
4.Trizol 30ml
5. Enzyme-free water 20ml x 2
6. Ethanol 5ml
7.gDNA Eraser 30μl
8. 5×gDNA EraserBuffer 60μl
9.RT Primer Mix 30μl
10.5 XPrimeScript Buffer 2 (containing dNTP mix) 90ml
PrimeScript RT Enzyme Mix I (containing RNase inhibitor) 20. mu.l
12.TB Green Premix Ex Taq II 50μl
13.10 μ M hsa _ circ _0000284 specific primer 30 μ l
Forward primer 5'-TATGTTGGTGGATCCTGTTCGGCA-3', SEQ ID NO 2;
reverse primer 5'-TGGTGGGTAGACCAAGACTTGTGA-3', SEQ ID NO 3.
14.10 μ M GAPDH specific primer 30 μ l:
forward primer 5'-TGCACCACCAACTGCTTAGC-3', SEQ ID NO 4;
reverse primer 5'-GGCATGGACTGTGGTCATGAG-3', SEQ ID NO: 5.
Example 2
Detection of hsa _ circ _0000284 in tumor and para-carcinoma tissues of neuroblastoma patients
1. Sample pretreatment: respectively collecting the neuroblastoma tissue to be detected and the tissue beside the cancer, storing the neuroblastoma tissue and the tissue beside the cancer in a freezing storage tube containing an RNA stable solution, and placing the neuroblastoma tissue and the tissue beside the cancer in a refrigerator at minus 80 ℃ for standby.
2. Extraction of RNA: taking a proper amount of specimen, adding liquid nitrogen into a mortar baked for 6-8h at 180 ℃, grinding the specimen into powder, adding 1ml of Trizol mortar specimen into the mortar, grinding the powder into liquid, transferring the liquid to an EP (EP) tube, adding 200 mu l/ml of Trizol of trichloromethane into the EP, rapidly and violently shaking for 15-30s, standing the mixture on ice for 5min, and centrifuging the mixture at 12000rpm at 4 ℃ for 15 min; carefully taking the uppermost layer of the water phase, transferring the uppermost layer of the water phase into a new EP tube, adding isopropanol with equal volume, slightly reversing the mixture up and down, uniformly mixing the mixture, and standing the mixture on ice for 10 min; centrifuging at 12000g at 4 deg.C for 10 min; discarding the supernatant, adding 1ml of ethanol diluted by 75% DEPC water, and mixing uniformly; centrifuging at 12000g at 4 deg.C for 10 min; the supernatant was discarded as much as possible, dried at room temperature for 5-10min, and when the white RNA precipitate was translucent, 10. mu.l of DEPC water was added to dissolve the RNA. The concentration and the quality of RNA are measured by spectrophotometry, the OD260/280 ratio is between 1.8 and 2.0, and the RNA is stored at the temperature of minus 80 ℃.
3. Genome removal: using the TAKARA genome ablation reverse transcription kit, 20. mu.L of the reverse transcription system was as follows:
4. reverse transcription:
5. performing fluorescent quantitative PCR by using hsa _ circ _0000284 specific primers: the hsa _ circ _0000284 specific primer sequence and the GAPDH internal reference primer sequence were synthesized by Biotechnology engineering (Shanghai) GmbH.
The reaction system is as follows:
hsa _ circ _0000284 specific primers:
forward primer 5'-TATGTTGGTGGATCCTGTTCGGCA-3', SEQ ID NO 2;
reverse primer 5'-TGGTGGGTAGACCAAGACTTGTGA-3', SEQ ID NO 3.
GAPDH specific primers 30 μ l:
forward primer 5'-TGCACCACCAACTGCTTAGC-3', SEQ ID NO 4;
reverse primer 5'-GGCATGGACTGTGGTCATGAG-3', SEQ ID NO: 5.
6. Amplification results and prognosis determination
2-ΔΔCtIndex measurement: the experimental data was analyzed by a relatively quantitative analysis method using GAPDH as an internal reference gene and using GraphPad Prism 8.0 software. Analysis shows that the expression level of hsa _ circ _0000284 in the para-carcinoma tissues is significantly different from that in the tumor tissues in 38 patients with neuroblastoma (see figure 1). And the 38 patients were followed for 5 years, and the survival curves were analyzed, and it was found that the patients with low expression of hsa _ circ _0000284 (less than 3.145) in neuroblastoma tissue had a longer prognosis survival time than the patients with high expression of hsa _ circ _0000284 (more than 3.145) in neuroblastoma tissue, with a significant difference (P ═ 0.0107) (see fig. 2).
The above results indicate that hsa _ circ _0000284 can be used as a specific molecular marker for prognosis of neuroblastoma patients.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Children hospital for women in Qingdao city
Application of <120> hsa _ circ _0000284 in preparation of neuroblastoma prognosis preparation
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1099
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtatggcctc acaagtcttg gtctacccac catatgttta tcaaactcag tcaagtgcct 60
tttgtagtgt gaagaaactc aaagtagagc caagcagttg tgtattccag gaaagaaact 120
atccacggac ctatgtgaat ggtagaaact ttggaaattc tcatcctccc actaagggta 180
gtgcttttca gacaaagata ccatttaata gacctcgagg acacaacttt tcattgcaga 240
caagtgctgt tgttttgaaa aacactgcag gtgctacaaa ggtcatagca gctcaggcac 300
agcaagctca cgtgcaggca cctcagattg gggcgtggcg aaacagattg catttcctag 360
aaggccccca gcgatgtgga ttgaagcgca agagtgagga gttggataat catagcagcg 420
caatgcagat tgtcgatgaa ttgtccatac ttcctgcaat gttgcaaacc aacatgggaa 480
atccagtgac agttgtgaca gctaccacag gatcaaaaca gaattgtacc actggagaag 540
gtgactatca gttagtacag catgaagtct tatgctccat gaaaaatact tacgaagtcc 600
ttgattttct tggtcgaggc acgtttggcc aggtagttaa atgctggaaa agagggacaa 660
atgaaattgt agcaatcaaa attttgaaga atcatccttc ttatgcccgt caaggtcaaa 720
tagaagtgag catattagca aggctcagta ctgaaaatgc tgatgaatat aactttgtac 780
gagcttatga atgctttcag caccgtaacc atacttgttt agtctttgag atgctggaac 840
aaaacttgta tgactttctg aaacaaaata aatttagtcc cctgccacta aaagtgattc 900
ggcccattct tcaacaagtg gccactgcac tgaaaaaatt gaaaagtctt ggtttaattc 960
atgctgatct caagccagag aatattatgt tggtggatcc tgttcggcag ccttacaggg 1020
ttaaagtaat agactttggg tcggccagtc atgtatcaaa gactgtttgt tcaacatatc 1080
tacaatctcg gtactacag 1099
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tatgttggtg gatcctgttc ggca 24
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tggtgggtag accaagactt gtga 24
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggcatggact gtggtcatga g 21
Claims (5)
1. The application of the reagent or the product for detecting the expression quantity of hsa _ circ _0000284 in preparing a neuroblastoma prognosis preparation is characterized in that the nucleotide sequence of hsa _ circ _0000284 is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein the prognostic formulation is a kit.
3. The use according to claim 2, wherein the kit comprises primers specific for hsa _ circ _0000284 and has the nucleotide sequences shown in SEQ ID NO 2 and SEQ ID NO 3.
4. The use of claim 3, wherein the kit further comprises a GAPDH reference primer, and the nucleotide sequence is shown as SEQ ID NO. 4 and SEQ ID NO. 5.
5. The use of claim 4, wherein the kit further comprises reagents for extracting total RNA from neuroblastoma tissue, reagents for reverse transcription of hsa _ circ _0000284 into cDNA using total RNA as a template, and reagents for real-time quantitative PCR of cDNA.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108064176A (en) * | 2015-04-22 | 2018-05-22 | 库瑞瓦格股份公司 | For treating the composition containing RNA of tumor disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108064176A (en) * | 2015-04-22 | 2018-05-22 | 库瑞瓦格股份公司 | For treating the composition containing RNA of tumor disease |
Non-Patent Citations (4)
| Title |
|---|
| CircRNAs: From anonymity to novel regulators of gene expression in cancer;Katherine L. Harper等;《International Journal of Oncology》;20191024;第55卷(第6期);第1183-1193页 * |
| Circular RNA HIPK3 is a prognostic and clinicopathological predictor in malignant tumor patients;Gao Wenzhe;《Journal of Cancer》;20200427;第11卷;第2753-2760页 * |
| 环状RNAs在肿瘤防治中的研究进展;王兵等;《临床肿瘤学杂志》;20201231(第04期);第362-367页 * |
| 环状RNA在头颈部肿瘤中的研究进展;杨丽可等;《医学综述》;20201231(第12期);第2238-2342+2348页 * |
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