CN103616518B - A kind of miRNA molecule is as the purposes of the biomarker of prognosis in hcc - Google Patents
A kind of miRNA molecule is as the purposes of the biomarker of prognosis in hcc Download PDFInfo
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- CN103616518B CN103616518B CN201310606743.9A CN201310606743A CN103616518B CN 103616518 B CN103616518 B CN 103616518B CN 201310606743 A CN201310606743 A CN 201310606743A CN 103616518 B CN103616518 B CN 103616518B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
Abstract
The present invention relates to the purposes of a kind of miRNA molecule as the biomarker of prognosis in hcc.Especially, the present invention relates to hsa-miR-1269a as the purposes in marker for prognosis of liver cancer, and detect hsa-miR-1269a test kit used.
Description
Technical field
The present invention relates to the field of liver cancer cell marker.Especially, the present invention relates to a kind of miRNA molecule as the purposes in marker for prognosis of liver cancer, described miRNA molecule is hsa-miR-1269a.
Background technology
Liver cancer is that a kind of grade of malignancy is high, the kinds of tumor of poor prognosis, various treatment recur afterwards and the probability that shifts up to more than 50%.How the relapse and metastasis of early prediction liver cancer is current study hotspot.More existing possibilities can be used to refer to the index of patients with hepatocellular carcinoma prognosis.These indexs are detect from peripheral blood a bit, and some is then obtain from the tumor tissues of excision.It is mostly some nucleic acid (as miRNA etc.) and protein.Below lift some examples: find that some albumen work in the developing of hepatocellular carcinoma by search document.Author chooses one of them and is studied.Detect the liver cancer tissue of some patients with hepatocellular carcinomas and the enhancing conjugated protein 4 (AP-4) of cancer beside organism's discovery activation and AP-4mRNA relative to cancer beside organism's high expression level in liver cancer tissue, and show that the prognosis of all the other patients is correlated with by the immunohistochemical methods of relatively large tumor tissues, expressing high person, to organize 5 years survival rates be that relatively to hang down 5 years survival rates be 67.3% in 33.3% expression, see Hu, B.S. people HighexpressionofAP-4predictspoorprognosisforhepatocellul arcarcinomaaftercurativehepatectomy.Tumourbiology:thejou rnaloftheInternationalSocietyforOncodevelopmentalBiology andMedicine34 is waited, 271-276, doi:10.1007/s13277-012-0547-4.In patients with hepatocellular carcinoma postoperative one week peripheral blood, the expression of AFPmRNA also can as the good index of prediction postoperative recurrence, see, RoleofAFPmRNAexpressioninperipheralbloodasapredictor.pdf and the expression discovery by contrasting liver cancer tissue and cancer beside organism miRNA-221, its expression is relevant to the recurrence of patients with hepatocellular carcinoma, the relatively low expression group of nothing recurrence survival time of high expression level group is short, see, Gramantieri, L. people .MiRNA-221targetsBmfinhepatocellularcarcinomaandcorrelat eswithtumormultifocality.ClinCancerRes15 is waited, 5073-5081, doi:10.1158/1078-0432.CCR-09-0092.
As before report, the result that a lot of index relevant with prognosis draws in different research is not quite similar, the possible cause of generation bias is this type of research of major part is all retrospective study, the hepatocellular carcinoma of different pathogeny may be different with the hepatocellular carcinoma that different crowd occurs molecular mechanism wherein, for example, see, Singhal, A., Jayaraman, M., Dhanasekaran, D.N. & Kohli, V.Molecularandserummarkersinhepatocellularcarcinoma:pred ictivetoolsforprognosisandrecurrence.CritRevOncolHematol 82, 116-140, doi:10.1016/j.critrevonc.2011.05.005 (2012). because great majority research is all retrospective study, this type of marker (index) also needs to carry out the effect that prospective clinical trial district confirms it.There is not the appearance of the marker relevant to prognosis doing extensive prospective clinical trial before.
MiRNA is the endogenous Microrna being about 19 to 25 units, and it is by the specific binding of the 3 ' UTR with target messenger RNA(mRNA) thus regulate the expression of target gene.A large amount of tests demonstrates its miRNA in the developing of tumour and has played great effect.
Summary of the invention
The present inventor utilizes two models affecting the patient learning the multiple occupy-place of diagnosing hepatic to analyze, and finds that the expression of a kind of miRNA molecule, hsa-miR-1269a is relevant to prognosis in hcc.And even do not report the relevant of hsa-miR-1269a and prognosis in hcc before this.
The sequence (CUGGACUGAGCCGUGCUACUGG) as shown in SEQIDNO:1 of hsa-miR-1269a
The invention provides a kind of miRNA molecule as the purposes of molecular marked compound in the reagent for the preparation of the prognosis of qualification liver cancer patient, described miRNA molecule is the hsa-miR-1269a as shown in SEQIDNO:1.
Further, described reagent is for detecting in the method as the hsa-miR-1269a of molecular marked compound.
Further, the method detected as the hsa-miR-1269a of molecular marked compound comprises southern trace, northern trace, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitatively PCR in real time (qRT-PCR), nano-array, grand array, radioautograph and in situ hybridization.
On the other hand, the invention provides one or more primers detecting the expression level of the hsa-miR-1269a as shown in SEQIDNO:1 specifically and/or the purposes of probe in the reagent for the preparation of the prognosis of qualification liver cancer patient.
Improve ground, comprises the nucleotide sequence with SEQIDNO:1 complementation, or be made up of the nucleotide sequence with SEQIDNO:1 complementation according to primer of the present invention and/or probe.
Further, according to the present invention, described purposes comprises further:
A) sample of liver cancer tissue from individuality and cancer beside organism is provided; With
B) expression level of the hsa-miR-1269a in sample as shown in SEQIDNO:1 is determined;
C) expression level of hsa-miR-1269a in the sample of cancerous tissue and cancer beside organism is compared.
Especially, the use of described step (b) be selected from grand array screening, array screening, nano-array screening, reverse transcription PCR, PCR in real time or In situPCR method carry out.
More particularly, high-caliber hsa-miR-1269a expression level is the indication without recurrence survival rate with reduction.
During being meant to compared with the patients with hepatocellular carcinoma of expressing with low-level hsa-miR-1269a of " reduction without recurrence survival rate ", the patients with hepatocellular carcinoma that high-caliber hsa-miR-1269a expresses is lower without the probability of recurrence survival, and such as described probability can reduce at least 0.05,0.1,0.2,0.3,0.4 or 0.5 or more.
" high-caliber hsa-miR-1269a expresses " refers to compared with cancer beside organism, the dual specificity protein kinase protein of cancerous tissue and/or the expression amount height at least 10% of mRNA, such as high by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 3000%, 4000%, 5000%, 10000% or more.
Different without statistically of the dual specificity protein kinase protein being meant to cancerous tissue of " low-level hsa-miR-1269a express " and/or the amount of mRNA and the amount in cancer beside organism.
On the other hand, the invention provides a kind of test kit for the identification of liver cancer patient prognosis, it comprises one or more primer detecting the expression level of the hsa-miR-1269a as shown in SEQIDNO:1 specifically and/or probes.
Accompanying drawing explanation
Fig. 1 represents the abdominal CT sheet of two the representative patients carrying out the order-checking of two generations.In figure, left is an abdominal CT sheet meeting the patients with hepatocellular carcinoma of " transfer theory ", and wherein the PI-P of arrow indication is the primary tumor of hepatocellular carcinoma, PI-M: the satellite stove of primary tumo(u)r.PI-V: be intravenously cancer stove; In figure, right is that an abdominal CT meeting " multicenter origin is theoretical " patient is unenhanced, and the region P II-L shown in arrow is the left liver cancer stove represented, what P II-R represented is right liver cancer stove.
Fig. 2 A represents ROC curve, and it analyzes all patient's cancerous tissues of detection and the hsa-miR-1269a expression values of cancer beside organism.
Fig. 2 B represents the survival curve drawn by Kaplan-Meier method.As can be seen from survival rate curve, the higher patient's group of hsa-miR-1269a expresses low patient relative to hsa-miR-1269a, and to organize survival rate lower.
Should understand, appended accompanying drawing not shows the technique of painting slightly simplified of the exemplifying various feature of ultimate principle of the present invention pari passu.Specific design feature of the present invention disclosed herein comprises such as concrete size, direction, position and profile and will partly be determined by the environment specifically will applied and use.
In these figures, run through several figures of accompanying drawing, Reference numeral quotes equally or equivalent part of the present invention.
Embodiment
Contriver first has to comparing the researchs that two the typical clinical cases representing meaning have carried out many groups.The feature of these two clinical cases, for being the multiple occupy-place of imaging diagnosis liver (as shown in Figure 1) equally, confirms to be all HBV-associated hepatocellular carcinoma after excision of performing an operation.First patient has the patient sending out stove in low differentiation liver, its 3 months after operation is namely dead, and this patient meets the theory of metastasis model clinically, and second patient has differentiated patients with hepatocellular carcinoma, within its postoperative 2 years, still without recurrence, this patient meets the theory of multicenter origin.
We are to multiple knurl stoves (comprising two independent cancer stoves of the primary tumor of first patient, metastasis, Venous cancerous embolus and second patient), their cancer beside organism's totally 7 tissue samples under this two corrective surgeries excision, wherein, two everyone each cancer beside organism's samples of patient, first patient have two cancerous tissue samples of 3 cancerous tissue samples and second patient, 7 cancerous tissues altogether, and the germ cell line of background has carried out miRNA-seq (Hua Da gene).Concrete operation step is as follows: extracted total RNA from tissue also carries out sample detection to total serum IgE, after sample detection is qualified, the small RNA fragments of 18-30 or 40nt is reclaimed by PAGE, two ends add 5 ' and 3 ' after joint (building library), reverse transcription becomes cDNA and pcr amplification (RT-PCR), rear CS preparation also HiSeq2000 order-checking (50SE).
Carry out the expression analysis of miRNA subsequently, from the analysis of miRNA sequencing data, obtain 125 miRNA: the first stdn of miRNA expression values: by the mapread number of miRNA each in each sample except in this sample map to genomic total read number, as the expression values of this gene.We have chosen the miRNAs of mapread number maximum value more than 1000 in 7 samples and carry out subsequent analysis, obtain 125 miRNAs altogether.
According to acquired results, by setting the following value screening miRNA relevant to prognosis in hcc.
1) miRNA(first selecting 7 sample average expression amounts higher on average reads several 100
76 above miRNA) carry out next step research;
2) compared with cancer beside organism, at least 3 cancer samples consistently raise or lower more than 2 times, and the change multiple of other samples in cancerous tissue is between rise 2 times to downward 2 times; Also namely, if having 3-5 sample rise or lower more than 2 times, even if residue 2-0 sample is lowered on the contrary or raise, its change multiple also must not more than 2 times, and the difference raised between 2 times to downward 2 times is construed to not obvious.
3) read up the literature and database (as including the database miR2disease of miRNA research conditions), delete the known miRNA relevant to liver cancer.
4) to average expression amount and mean difference multiple integrated ordered, choose the hsa-miR-1269a clearly not studying report.
In order to confirm the dependency of hsa-miR-1269a and prognosis in hcc further, contriver utilizes the method for qRT-PCR in the cancer and cancer beside organism of the patient of 96 routine hepatitis B related Hepatocellular Carcinomas, detect the expression of hsa-miR-1269a.Concrete grammar is as follows: the expression amount detecting cancerous tissue and cancer beside organism hsa-miR-1269a with the Taqmanassay test kit purchased from lifetechnologies according to the specification sheets of manufacturer, then, with U6 as reference gene, to the hsa-miR-1269a expression amount normalization method obtained.
Our result confirms hsa-miR-1269a high expression level in cancerous tissue really.The clinical data of itself and patient is comprehensively analyzed again by we, finds that hsa-miR-1269a expression level and patient's is relevant without recurrence survival rate.
Embodiment 1, hsa-miR-1269a and liver cancer are without the dependency recurring survival rate
The expression amount of cancerous tissue and cancer beside organism hsa-miR-1269a after the expression amount normalization method of respective reference gene U6, by ROC curve (as Fig. 2 A).Find out the threshold value that an energy distinguishes the expression values by cancer and cancer, the Sensitivity and Specificity in this threshold point is all maximum (as Fig. 2 A).Then according to this threshold value, height classification is carried out in patient's cancerous tissue hsa-miR-1269a expression, separate high expression level patient and organize n=67, low expression group n=29.
Our result shows, compared with the patient of low expression, the nothing recurrence survival rate of the patient of hsa-miR-1269a high expression level is lower, as shown in Figure 2 B.
So far we find that the expression amount of hsa-miR-1269a in detection patients with hepatocellular carcinoma can predict the prognosis of patient.
Claims (9)
1. miRNA molecule is as the purposes of molecular marked compound in the reagent for the preparation of the prognosis of qualification liver cancer patient, and described miRNA molecule is the hsa-miR-1269a as shown in SEQIDNO:1.
2. purposes according to claim 1, wherein said reagent is for detecting in the method as the hsa-miR-1269a of molecular marked compound.
3. purposes according to claim 2, the method wherein detected as the hsa-miR-1269a of molecular marked compound is selected from southern trace, northern trace, reverse transcription PCR, quantitatively PCR in real time, nano-array, grand array, radioautograph or in situ hybridization.
4. one or more detect primer and/or the purposes of probe in the reagent for the preparation of the prognosis of qualification liver cancer patient of the expression level of the hsa-miR-1269a as shown in SEQIDNO:1 specifically.
5. purposes according to claim 4, wherein said primer and/or probe comprise the nucleotide sequence with SEQIDNO:1 complementation.
6. purposes according to claim 4, wherein said primer and/or probe are made up of the nucleotide sequence with SEQIDNO:1 complementation.
7. the purposes according to any one of claim 1 to 6, it comprises further:
A) sample of liver cancer tissue from individuality and cancer beside organism is provided; With
B) expression level of the hsa-miR-1269a in sample as shown in SEQIDNO:1 is determined;
C) expression level of hsa-miR-1269a in the sample of cancerous tissue and cancer beside organism is compared.
8. purposes according to claim 7, wherein at described step b) in use be selected from grand array screening, array screening, nano-array screening, reverse transcription PCR, PCR in real time or In situPCR method carry out.
9. purposes according to claim 7, wherein high-caliber hsa-miR-1269a expression level is the indication without recurrence survival rate with reduction.
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| CN105441566B (en) * | 2016-01-04 | 2019-02-19 | 中南民族大学 | Kit and chemotherapy of hepatocellular carcinoma sensitizer for prognosis evaluation after Liver Cancer Operation |
| CN106048036A (en) * | 2016-07-05 | 2016-10-26 | 周重昌 | Coloresctal cancer anular RNA molecular marker and detection reagent |
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| AU2012358200B2 (en) * | 2011-12-22 | 2018-08-30 | Baylor Research Institute | Identification of metastasis-specific miRNA and hypomethylation signatures in human colorectal cancer |
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| CN102002490A (en) * | 2009-08-31 | 2011-04-06 | 上海市肿瘤研究所 | 9 microRNA markers for predicting whether primary liver cancer relapses |
| CN101798599A (en) * | 2010-04-02 | 2010-08-11 | 上海生物芯片有限公司 | Purpose of hsa-miR-150 |
| CN102002494A (en) * | 2010-07-08 | 2011-04-06 | 浙江理工大学 | microRNA biomarker and application thereof |
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