CN104313171A - MicroRNA molecular marker for diagnosing glioma and application of microRNA molecular marker - Google Patents
MicroRNA molecular marker for diagnosing glioma and application of microRNA molecular marker Download PDFInfo
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Abstract
The invention relates to a microRNA marker for detecting glioma and application of the microRNA marker in preparing a detection kit for the glioma. With the adoption of the microRNA marker, content of microRNA-29 family in blood of a detected person is detected by virtue of a quantitative PCR method, and the glioma is diagnosed by comparing the content of the microRNA-29 family with a normal microRNA level. The microRNA molecular marker of the glioma is high in sensitivity, high in specificity, convenient to operate, safe without wounds and easily screened massively.
Description
Technical field
The present invention relates to Medical Molecular Biology technical field, is a kind of microRNA in blood is used for gliomatous diagnosis as biomarker.
Background technology
Neurospongioma is called for short glioma, also referred to as glioma, be modal primary central nervous system tumour, account for the half of all encephalic primary tumo(u)rs, broad sense refers to the tumour of all neuroepithelial origin, and narrow sense refers to the tumour coming from all kinds of spongiocyte.Astrocytoma, few branched oligodendrocyte knurl, ependymoma, Mixed Gliomas, choroid plexus knurl, uncertain neuroepithelial tissue knurl of originating, neurone and neurone neuroglia mixed tumor, pineal gland parenchymal tumor, embryonal tumors, neuroblastoma tumour is divided into according to the World Health Organization (WHO) classification schemes of 1999.
Current diagnosis and examination, in conjunction with on the basis of clinical manifestation, are diagnosed and are depended on neuroimaging and pathological examination.But neuroimaging inspection depends on instrument and price is high, although pathological examination is gold standard, belong to invasive, have the risk bringing sequela.Low price, convenient and swift and safe early diagnosis and examination are the main problems that neurospongioma runs into, and when often having clinical manifestation and find neuroimaging and pathological abnormalities too late, its five year survival rate only has 3%.
Therefore, clinically in the urgent need to one very accurately, low price, conveniently can realize the molecular marked compound of early diagnosis or examination.Regret so far, in neurospongioma early diagnosis and examination, not yet find believable molecular marked compound.Although methylguanine methyl transferase, PTEN/MMAC1 are put forward the marker as neurospongioma early diagnosis and examination by people, but it seems these markers (McNamara MG all not fully up to expectations in specificity, accuracy etc. at present, Sahebjam S, Mason WP (2013) Emerging biomarkers in glioblastoma.Cancer5 (3): 1103 – 1119.doi:10.3390/cancers5031103).
MicroRNAs (miRNAs) is the endogenic non-coding RNA with adjusting function of a class found in eukaryote, and its size is about 20 ~ 25 Nucleotide.Ripe miRNAs is produced by the shearing of longer primary transcript through a series of nuclease, be assembled into silencing complex (the RNA-induced silencing complex of RNA induction subsequently, RISC), by the mode identification said target mrna of base pair complementarity, and instruct silencing complex degraded said target mrna according to the difference of complementarity or check the translation of said target mrna.Nearest research shows that miRNA participates in various adjustment approach, comprises growth, virus defense, hematopoiesis, orga-nogenesis, cell proliferation and apoptosis, metabolism of fat etc.
Nearest research finds, miRNA expresses relevant to kinds cancer, and about 50% miRNAs obtaining explaining is positioned the fragile site (fragile site) relevant to tumour on genome.This illustrates that miRNAs plays vital effect in tumour generating process, and these miRNAs roles are similar to the function of cancer suppressor gene and oncogene, have researchist by miRNA called after " oncomirs ".
MiRNA can as the marker of a kind of potential tumour early discovery and diagnosis to have had research to think, and miRNA is stable in the body fluid such as serum, blood plasma, urine, its possibility as tumor marker is strengthened again further, and along with the progress of current technology, miRNA detection technique is more and more convenient, fast and cheap.
In the middle of the miRNA reported, the high expression level of microRNA-29 has been considered to develop relevant to the generation of some tumour, such as with colorectal cancer, its sensitivity and tolerance range are 69.0% and 89.1% (Huang Z respectively, Huang D, Ni S, Peng Z, Sheng W, Du X (2010) Plasma microRNAs are promising novel biomarkers for early detection of colorectal cancer.Int J Cancer J Int Cancer 127 (1): 118 – 126.doi:10.1002/ijc.25007).For another example in acute leukemia, the high expression level of microRNA-29 develops relevant to its generation, its sensitivity is respectively 86.7% and 95.0% (Wang F et al., 2012.miR-29a and miR-142-3p downregulation and diagnostic implication in human acute myeloid leukemia.Mol Biol Rep 39 (3): 2713 – 2722.doi:10.1007/s11033-011-1026-5) with the tolerance range after optimizing.
But due to the tissue specificity of tumorigenesis, the situation of the low expression of microRNA-29 family has been there is in the middle of other tumour, in the middle of these tumours, the generation development of tumour is relevant to the low expression of microRNA-29 family, these tumours comprise liver cancer (SU et al..MicroRNA-101, down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity.Cancer Res 2009; 69:1135-1142.), mammary cancer (Iorio MV, et al.MicroRNA gene expression deregulation in human breast cancer.Cancer Res 2005; 65:7065-7070.), chronic leukemia (Pekarsky Y, et al.Tcl1expression in chronic lymphocytic leukemia is regulated by miR-29and miR-181.Cancer Res 2006; 66:11590 – 11593.).
Especially, in the middle of leukemia, in acute and chronic leukemia, microRNA-29 family plays diametrically opposite role.Therefore, in the middle of specific tumour, microRNA-29 family role how, do not have ready-made answer, also cannot provide instruction and enlightenment by the result of other tumours, needs deep to carry out specific research.
Summary of the invention
Technical problem to be solved by this invention there is provided a kind of for detecting neurospongioma and microRNA mark by stages thereof, by detecting the content of microRNA-29 family in measured's serum or blood plasma, and compare with the level of the microRNA-29 family in normal population serum or blood plasma and diagnose neurospongioma, sensitivity and the tolerance range of this microRNA are high; Early stage and late tumor can be distinguished by the level height difference of microRNA-29 family.
The technical scheme that the present invention solves the problems of the technologies described above is: a kind of examination and diagnosis neurospongioma and microRNA mark by stages thereof, described mark is microRNA-29 family (comprising miR-29a, miR-29b and miR-29c), and the sequence of miR-29a, miR-29b and miR-29c is respectively as shown in sequence SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
The invention has the beneficial effects as follows: the microRNA mark that examination goes out is as detecting gliomatous tolerance range and highly sensitive.
Further, on the basis of technique scheme, the present invention can also do following improvement.
The present invention also comprises and a kind ofly diagnoses the purposes of gliomatous microRNA mark in preparation neurospongioma diagnostic reagent.
Accompanying drawing explanation
The normalization method level of the serum miR-29 family of Fig. 1 compared with the level of internal reference microRNA-24
Control refers to normal population, and WHO I-II refers to early stage Response in Patients with Gliomas, and WHO III-IV refers to Response in Patients with Gliomas in late period.Significant difference implication is:
*p<0.05,
*p<0.01,
* *p<0.005.Early stage patient (WHO I-II) compares with normal (Control)
*p<0.01 is pole significant difference.Patients with terminal (WHO III-IV) compares with normal (Control)
* *p<0.005 is pole significant difference.Early stage patient (WHO I-II) compares with patients with terminal (WHO III-IV)
*p<0.05 is significant difference.
ROC curve (the Receiver operating characteristic curves of Fig. 2 Response in Patients with Gliomas serum miR-29 family, ROC curves) and area under curve (areas under the curve, AUC) figure in show early stage patient (WHO I-II), the ROC curve of patients with terminal (WHO III-IV) and all patients (WHO I-IV), area under curve, sensitivity (Sensitivity) and accuracy (Specificity).
Embodiment
Below in conjunction with accompanying drawing, present invention is described, and cited embodiment is only used to explain the present invention, not for limiting the scope of the invention.
The correlation research that the expression of embodiment 1 clinical serum sample microRNA-29 family and neurospongioma are fallen ill
One, material and specimen collection describe
Response in Patients with Gliomas serum specimen is collected into and is in different Response in Patients with Gliomas by stages from clinical, adds up to 83 examples.The serum specimen of normal population is collected into from normal health crowd, adds up to 69 examples.Gliomatous diagnosis is confirmed by histopathology or nuclear magnetic resonance, and standard is the classification based on WHO; Gliomatous be criteria for classification (Sobin LH according to WHO and TNM by stages, Fleming ID, 1997.TNM classification of malignant tumors, fifth edition (1997) .Union internationale contre le cancer and the American joint committee on cancer.Cancer 80 (9): 1803 – 1804).Response in Patients with Gliomas before getting blood not through the treatment of any anti-tumor aspects such as surgical operation, chemotherapy or radiotherapy.Each sample PAXgene Blood RNA tubes (BD Biosciences, Basel, Switzerland) total collects 10ml blood.
Two, sample preparation and RNA extract
Blood after blood coagulation, 500 × g, get after centrifugal 60 minutes under 4 degrees Celsius upper serum be stored in-80 degrees Celsius under for subsequent use.RNA in serum by MiRNeasy Kit (Qiagen GmbH, Hombrechtikon, Switzerland) to specifications operation steps carry out Isolation and purification.
Three, quantitative PCR
Quantitative PCR experiment is with reference to method (the Wei W of the people such as W Wei, et al..miR-29targets Akt3to reduce proliferation and facilitate differentiation of myoblasts in skeletal muscle development.Cell Death and Disease (2013) 4, e668; Doi:10.1038/cddis.2013.184).The height that in serum, microRNA-29 family expresses is assessed by Ct value (Cycle threshold), and method of calculation are 2
-△ △ Ct.In this experiment, microRNA-24 is as internal reference, and the Ct value of microRNA-29 family carrys out normalization method by the amount of internal reference microRNA-24.Each sample does three and repeats experiment.
Four, statistical analysis technique
Significant difference t inspection (student ' s t test), Mann – Whitney check (Mann – Whitney test) and chi square test (χ test).ROC curve (Receiver operating characteristic curves, ROC curves) and area under curve (areas under the curve, AUC) be used as the index of prediction miR-29 family marker in neurospongioma.The sensitivity optimized and tolerance range calculate acquisition according to inspection prior probability (pre-test probability) and the value ratio (cost ratio) that obtained by ROC.Statistical software used is SPSS 19.0, thinks remarkable when P value is less than 0.05.
Five, experimental result
1, the expression of miR-29 family in each phase Response in Patients with Gliomas such as morning and evening and normal population
As shown in Figure 1, the normalization method level of the serum miR-29 family compared with the level of internal reference microRNA-24 has significant difference (P<0.01) between all neurospongioma groups and normal people group.And between patients with terminal group and normal people group, have pole significant difference (P<0.005).Compare early stage patient and patients with terminal group again, between group, also there were significant differences (P<0.05).
2, in neurospongioma the level of miR-29 family as the predictor of biomarker
Different Response in Patients with Gliomas serum miR-29 family is by stages known by ROC curve (Fig. 2) as the prediction performance of biomarker.The Response in Patients with Gliomas serum miR-29 family in all stages (I-IV phase) is medium as the prediction performance of biomarker, its AUC is 0.74 (95%CI, 0.67 – 0.82), the sensitivity of optimization and accuracy are respectively 68.5% and 77.3%.Patients with terminal serum miR-29 family is best as the prediction performance of biomarker, and AUC is up to 0.81 (95%CI, 0.73 – 0.89).Early stage patient serum miR-29 family as biomarker prediction performance still can, AUC is 0.66 (95%CI, 0.58 – 0.76).
Six, conclusion
1, the level of Xue Zhong miR-29 family can come examination and diagnosis neurospongioma as biomarker;
2, the level of Xue Zhong miR-29 family can be carried out by stages Response in Patients with Gliomas as biomarker.
Claims (5)
1. for diagnosing a gliomatous microRNA molecule marker, it is characterized by, described microRNA molecule marker is microRNA-29 family.
2. as claimed in claim 1 a kind of for diagnosing gliomatous microRNA molecule marker, it is characterized by, described microRNA-29 family comprises microRNA-29a, microRNA-29b and microRNA-29c.
3. as claimed in claim 2 a kind of for diagnosing gliomatous microRNA molecule marker, it is characterized by, the sequence of described microRNA-29a, microRNA-29b and microRNA-29c is respectively SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
4. described in claim 1-3 any one a kind of for diagnose gliomatous microRNA molecule marker preparation gliomatous diagnostic reagent in purposes.
5. a kind of for diagnosing the purposes of gliomatous microRNA molecule marker in preparation neurospongioma early staging diagnosis reagent in late period described in claim 1-3 any one.
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|---|---|---|---|---|
| CN107604060A (en) * | 2017-08-31 | 2018-01-19 | 复旦大学附属华山医院北院 | Purposes and its related application of the estrogen-related receptor α as the diagnostic marker of glioma |
| CN109762883A (en) * | 2019-01-31 | 2019-05-17 | 温州医科大学 | Plasma/serum excretion body hsa-miRNA-29-3p is as the application in diagnosis of glaucoma marker |
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