CN105063183A - Application of ubiquitin pathway-related gene as biomarker for predicting effect of breast cancer neoadjuvant chemotherapy - Google Patents
Application of ubiquitin pathway-related gene as biomarker for predicting effect of breast cancer neoadjuvant chemotherapy Download PDFInfo
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Abstract
The invention discloses application of a ubiquitin pathway-related gene as a biomarker for predicting effect of breast cancer neoadjuvant chemotherapy. The ubiquitin pathway-related gene is at least one of HECTD3 gene with base sequence as shown in SEQ ID NO. 1, PSMB10 gene with base sequence as shown in SEQ ID NO.2, UBD gene with base sequence as shown in SEQ ID NO.3, UBE2C gene with base sequence as shown in SEQ ID NO.4 and UBE2S gene with base sequence as shown in SEQ ID NO.5. In the five genes, the HECTD3 and PSMB10 have quite significant expression difference (less than 0.01) in pCR (pathologic complete remission) group and NpCR (non- pathologic complete remission) group, while the other three genes have remarkable expression difference (less than 0.05), indicating that the five genes can be used for predicting the effect of breast cancer neoadjuvant chemotherapy, so as to avoid the blindness of neoadjuvant chemotherapy.
Description
Technical field
The invention belongs to biomedical sector, being specifically related to ubiquitination pathway genes involved in the application as the biomarker for predicting neoadjuvant chemotherapy in breast curative effect.
Background technology
Neoadjuvant chemotherapy in breast (neoadjuvantchemotherapy, NACT) also known as staging tomography (primarychemotherapy), general, systematic cytotoxic drug treatment that non-metastatic tumour is carried out before topical therapeutic is referred to.This embolic chemotherapy not only to use under increasing pathological state in early days for advanced breast cancer.NACT has following 3 effects to operable patient with breast cancer: the long-term survival phase that (1) reaches pathology complete incidence graph (pathologiccompleteremission, pCR) patient significantly improves.PCR is an Efficient Evaluation index of mammary cancer NACT, and the international expert council recommends pCR to be defined as: after new adjuvant chemotherapy, at mammary gland and axillary gland all without infiltrating cancer tissue residue.And after new adjuvant chemotherapy, block of swelling as long as former is or/and metastatic lesion still has infiltrating cancer tissue residue to be namely divided into non-pCR (Non-pathologiccompleteremission, NpCR) group.(2) reduce neoplasm staging, local advanced breast cancer can improve resection rate through new adjuvant chemotherapy and increase protects newborn chance.(3) preoperative drug sensitive test, prevents the appearance of Drug escape clones.
The clinical application of NACT has the history of more than 30 year, although achieve comparatively much progress in recent years, but deeply have also discovered problem demanding prompt solution and deficiency along with what study: the patient with breast cancer of about 20% is insensitive to NACT, for this part patient, if the NACT insensitive and course for the treatment of on long opportunity may incuring loss through delay topical therapeutic, this reason makes part doctor and patient too conservative when carrying out NACT just, and pCR leads lower than 10%.This just needs a kind of accurate outcome prediction index, in order to predict the curative effect of NACT before chemotherapy, avoids the blindness for the treatment of.There is the research of the index of correlation of predicting function to contribute to selecting the patient that may benefit from NACT to carry out staging tomography to NACT curative effect, improve pathology complete remission rate and the long-term survival rate of mammary cancer NACT, avoid the patient invalid to chemotherapy to carry out unnecessary staging tomography, in order to avoid incur loss through delay the therapic opportunity of its best simultaneously.NACT can obtain the information of mammary cancer to chemotherapy regimen susceptibility in a short time, and this feature determines it and have higher efficiency in the curative effect of prediction adjuvant chemotherapy.Therefore the structure of mammary cancer NACT outcome prediction system can be predicted making us the curative effect of how new chemotherapeutics in mammary cancer adjuvant chemotherapy within the shorter time.
Owing to there is no the generally acknowledged single index effectively can predicting mammary cancer NACT curative effect at present, therefore combine the curative effect of multiple potential prediction index to mammary cancer NACT and predict accuracy by effectively improving prediction.Along with the maturation of biochip technology develops, let us effectively can be analyzed, to obtain large-scale gene expression information simultaneously to heterogeneic expression thousands of in sample.Current chip technology has been applied to the outcome prediction of mammary cancer NACT.Gene chip can detect some known mammary cancer biomarker gene and express change in mammary cancer biological tissue or clone.So utilize biochip technology can obtain the gene expression profile of the front mammary cancer biopsy specimen for the treatment of, then with NACT after the dependency of pCR carry out single factor analysis, filter out the gene relevant to NACT curative effect, combine those simultaneously and be considered to the gene that predictive value is more weak under a single state, thus set up the Multi-factor estimation system of gene level, the curative effect of mammary cancer NACT is effectively judged.
Report multiple gene microarray analysis model relevant to mammary cancer NACT curative effect at present, wherein most is representational be MDAnderson tumor center containing the mammary cancer NACT multiple baseline images predictive model of 30 gene expression profiles, very high accuracy is had to the prediction aspect of pCR after anthracycline-containing medicine and taxanes medicine NACT, its susceptibility and negative predictive value are significantly higher than the relevant dlinial prediction factor (92%vs.61%) and (96%vs.86%), prediction can obtain in the 13 routine patients of pCR has 12 examples to obtain pathology complete incidence graph, its predictive value can be treated with ER Endocrine and Her2 compares favourably to the predictive value that Herceptin treats.This center is being carried out a randomized III phase clinical study and is being verified the value of this polygene predictive model in mammary cancer NACT outcome prediction further on this basis.
But, full-length genome chip has the following disadvantages for the gene screening NACT relevant: during chip design, itself covers FDR (FalseDiscoveryRate) value that gene probe within the scope of full genome and tumor specimen itself have very large heterogeneous result to cause chip data higher and is difficult to identify important gene.Therefore, analyze that NACT process curative effect is correlated with have the gene pathway of biological significance but not individual gene seems very feasible, and proved relevant to the result of NACT.
Such as notification number is that the Chinese patent literature of CN103602746A discloses a kind of biomarker predicting neoadjuvant chemotherapy in breast curative effect, this biomarker is a kind of long non-coding RNA, it there are differences (P<0.05) in pCR group and the fresh breast cancer tissue of NpCR group, and the expression in NpCR group breast cancer tissue is significantly higher than pCR group.Point out this biomarker can be used for predicting Efficacy of Neoadjuvant Chemotherapy, avoid new adjuvant chemotherapy blindness.
Continue to find and effectively can predict that the biomarker of neoadjuvant chemotherapy in breast curative effect is significant to neoadjuvant chemotherapy in breast.
Summary of the invention
The invention provides ubiquitination pathway genes involved in the application as the biomarker for predicting neoadjuvant chemotherapy in breast curative effect, this biomarker can effectively predict neoadjuvant chemotherapy in breast curative effect, avoids new adjuvant chemotherapy blindness.
Ubiquitination pathway genes involved is in the application as the biomarker for predicting neoadjuvant chemotherapy in breast curative effect, it is characterized in that, described ubiquitination pathway genes involved is base sequence if the HECTD3 gene of SEQIDNo.1, base sequence are if the PSMB10 gene of SEQIDNo.2, base sequence are if the UBD gene of SEQIDNo.3, base sequence are if the UBE2C gene of SEQIDNo.4 and base sequence are as at least one in the UBE2S gene of SEQIDNo.5.
The application first collects patient with breast cancer and carries out the puncture of the cancerous tissue before new adjuvant chemotherapy sample, then carries out new adjuvant chemotherapy to patient, and distinguishes pCR and NpCR group according to curative effect; High-flux sequence is carried out to the cancerous tissue puncture sample collected before new adjuvant chemotherapy, Analysis and Screening goes out the gene of differential expression in pCR and NpCR group, pathway analysis is carried out to the difference expression gene filtered out, be separated and obtain relevant five ubiquitination pathway genes involveds evident in efficacy to mammary cancer NACT, i.e. HECTD3 gene, PSMB10 gene, UBD gene, UBE2C gene and UBE2S gene, these five genes can be used as biomarker, for surveying newborn mammary cancer NACT curative effect.
Present invention also offers the biomarker for predicting neoadjuvant chemotherapy in breast curative effect, is base sequence if the HECTD3 gene of SEQIDNo.1, base sequence are if the PSMB10 gene of SEQIDNo.2, base sequence are if the UBD gene of SEQIDNo.3, base sequence are if the UBE2C gene of SEQIDNo.4 and base sequence are as at least one in the UBE2S gene of SEQIDNo.5.
Present invention also offers the application of described biomarker in the PCR kit for the preparation of prediction neoadjuvant chemotherapy in breast curative effect.
As preferably, described PCR kit is PCR kit for fluorescence quantitative.
Present invention also offers two kinds for predicting the PCR kit for fluorescence quantitative of neoadjuvant chemotherapy in breast curative effect.
One: based on the PCR kit for fluorescence quantitative of Taqman probe method, comprises the Auele Specific Primer and Taqman probe that design for described biomarker, described Auele Specific Primer and Taqman probe be 1. following ~ 5. at least one group:
1. for Auele Specific Primer and the Taqman probe of HECTD3 gene design:
Upstream primer: 5'-CCACCTCCCGATCATCGA-3';
Downstream primer: 5'-ACGAACATCAATCCCATCATCTC-3';
Taqman probe: 5'-ATCCGCATCGTGGAGTG-MGB-3';
2. for Auele Specific Primer and the Taqman probe of PSMB10 gene design:
Upstream primer: 5'-GAAAGCGAAAGCAGGAAGTACAG-3';
Downstream primer: 5'-CGTGAAGCCTAGCAGAG-3';
Taqman probe: 5'-CGTGAAGCCTAGCAGAG-MGB-3';
3. for Auele Specific Primer and the Taqman probe of UBD gene design:
Upstream primer: 5'-TGAGGAGCTGCCCTTGTTTC-3';
Downstream primer: 5'-TGTGGAGTCAGGTGATG-3';
Taqman probe is: 5'-TGTGGAGTCAGGTGATG-MGB-3';
4. for Auele Specific Primer and the Taqman probe of UBE2C gene design:
Upstream primer: 5'-GAGGCTGCTGCTGGAGCTT-3'
Downstream primer: 5'-TCAATGTTGGGTTCTGGCATT-3'
Taqman probe: 5'-TCTGCAACTGTTTCTC-MGB-3';
5. for Auele Specific Primer and the Taqman probe of UBE2C gene design:
Upstream primer: 5'-ACCTACCCCCGCACATCAT-3';
Downstream primer: 5'-GTGGGTCTGCGGTCAGTGT-3';
Taqman probe: 5'-CTGGTGTACAAGGAGGTG-MGB-3'.
Its two: based on the PCR kit for fluorescence quantitative of SYBRGreen fluorescent dye determination, the Auele Specific Primer comprising fluorescence dye and design for described biomarker, described Auele Specific Primer be 6. following ~ 10. at least one pair of:
6. the Auele Specific Primer for the HECTD3 gene that increases is:
Upstream primer: 5'-CTCATAGCAGCCACCTTCATTC-3';
Downstream primer: 5'-ACCGCTTCTTTGGGACACTTG-3';
7. the Auele Specific Primer for the PSMB10 gene that increases is:
Upstream primer: 5'-CCCATAGCCAATCAGTAGCC-3';
Downstream primer: 5'-TTAGGTCTCAACTCTTCCGTCAT-3';
8. the Auele Specific Primer for the UBD gene that increases is:
Upstream primer: 5'-CAACAGCGGAACCTCCAGTCTC-3';
Downstream primer: 5'-CGGTCCTCCTTGAATGCCAC-3';
9. the Auele Specific Primer for the UBE2C gene that increases is:
Upstream primer: 5'-AGCATTCCACTGCCCAAAGG-3';
Downstream primer: 5'-CGCTCCACCGTAACCACAGATAG-3';
10. the Auele Specific Primer for the UBE2S gene that increases is:
Upstream primer: 5'-TTGGTAGCCGTGGTTATCTCT-3';
Downstream primer: 5'-ACACTCTCAGCCAGTCCCTTAG-3'.
Two kinds of PCR kit for fluorescence quantitative include the Auele Specific Primer for the reference gene β-actin that increases all further, and the Auele Specific Primer of described reference gene β-actin is:
Upstream primer: 5'-GCATACACGAAACTACCTTCAACTCC-3';
Downstream primer: 5'-ATGGTGGTGCCGCCAGACA-3'.
In addition, also probe can be related to for biomarker of the present invention, in order to the biochip for the preparation of prediction neoadjuvant chemotherapy in breast curative effect.
Compared with prior art, beneficial effect of the present invention is:
The present invention screens one group of ubiquitination pathway genes involved, wherein there is extremely significant differential expression (P<0.01) in HECTD3 gene and PSMB10 gene in pCR group and NpCR group, and there is significant differential expression (P<0.05) in UBD, UBE2C and UBE2S gene in pCR group and NpCR group, be presented as compared with pCR group, in NpCR group, the expression of PSMB10 gene, UBD gene, UBE2C gene and UBE2S gene is significantly lowered, and the expression of HECTD3 gene is then significantly raised; Point out these five ubiquitination pathway genes involveds as biomarker, can be used for predicting neoadjuvant chemotherapy in breast curative effect, to avoid new adjuvant chemotherapy blindness.
Accompanying drawing explanation
Figure 1A is pCR and NpCR group gene expression profile;
Wherein, " log
2(readcountsforeachgene) inpCR " represent gene in pCR group read long log
2value; " log
2(readcountsforeachgene) inNpCR " represent gene in NpCR group read long log
2value; After pCR represents new adjuvant chemotherapy, at mammary gland and axillary gland all without the sample group of infiltrating cancer tissue residue, after NpCR represents new adjuvant chemotherapy, block of swelling as long as former or/and metastatic lesion still has the sample group of infiltrating cancer tissue residue, lower with;
Figure 1B is the reads number distribution of the uniqueness coupling be assigned in pCR and NpCR group on people's transcript;
Wherein, " log
2(Numberofreadsmappedtoagene) log that effectively read long number of comparison to transcript fragment " is represented
2value;
Fig. 2 is the cluster analysis of digital gene express spectra order-checking difference expression gene;
Fig. 3 is the expression that fluorescent quantitation verifies differential gene in pCR group and NpCR group;
Wherein, " RelativeQuantification " represents relative expression quantity.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
The screening of embodiment 1 difference expression gene
Connect subject 20 examples in Zhejiang Prov. Tumor Hospital and obtain breast cancer tissue's sample without the patient with breast cancer of the NACT cancerous tissue that punctures, entering to organize patient requests has clear and definite cytodiagnosis, and operation consent without any treatment, and has complete pathological information.
Sample and rear new adjuvant chemotherapy has been carried out to patient, and distinguished pCR group and NpCR group according to curative effect; Breast cancer tissue's sample is handled as follows simultaneously:
1, breast cancer tissue's Total RNAs extraction
(1) Total RNAs extraction
1) the clear area that less RNase disturbs (1 ‰ DEPC water wipings all used by all apparatus), mortar needs 200 DEG C of sterilizings 5 hours and cools 4 hours, take in vitro mammary tissue sample and be about 20mg to having in the mortar of Liquid nitrogen precooler, be ground to Powdered (output of the RNA that grinding thoroughly then can not have a strong impact on) with pestle;
2) add 700 μ lQIAzol lysates in mortar, continue to be ground to the uniform liquid without obvious tissue block, be then transferred in the 1.5mleppendorf pipe without RNA enzyme;
3) refrigerated centrifuge 4 DEG C, 12000 × g, centrifugal 10min are to remove uncracked complete organization and cell debris in solution;
4) careful draw centrifugal after supernatant liquor, move to another new eppendorf and manage, be sure not to draw lower floor's cell debris residue and precipitate;
5) add 140 μ l chloroforms, turn upside down, after thermal agitation, leave standstill 2min;
6) refrigerated centrifuge 4 DEG C is centrifugal, 12000 × g, 15min, and is transferred to by supernatant liquid one new in RNA enzyme eppendorf pipe;
7) add 1.5 times of dehydrated alcohols, vortex mixes, and completes Tissue Lysis;
8) be all transferred in adsorption column by all liquid, and be placed in the collection tube of 2ml by adsorption column, the centrifugal 15s of 8000 × g, abandons the filtrate in collection tube;
9) get the RWT solution of 700 μ l in adsorption column, the centrifugal 15s of 8000 × g, abandons filtrate;
10) bufferRPE getting 500 μ l washs to adsorption column, and the centrifugal 15s of 8000 × g, abandons filtrate;
11) again wash adsorption column with the bufferRPE of 500 μ l, the centrifugal 2min of 8000 × g, abandons filtrate and underlying collection sleeve pipe;
12) adsorption column is transferred in a new 2ml collection tube, at full speed centrifugal 1min;
13) abandon underlying collection pipe, adsorption column is moved in new 1.5mleppendorf pipe, add 45 μ lRNase-freewater wash-out adsorption films, leave standstill the centrifugal 1min of 1min, 8000 × g;
14) at full speed centrifugal 1min again, obtains total serum IgE.
(2) the quantitative and quality examination of total serum IgE
Nanodrop2000 is utilized to detect the content of mammary tissue RNA sample, through the quantitative assay of OD value, so that the volume of RNA solution required when calculating target gene reverse transcription; The RNA sample more serious with DNA pollution of having degraded is got rid of, to ensure the specificity of subsequent experimental through qualitative detection.Concrete detection method is as follows:
1) total serum IgE is quantitative: the OD value measuring 260nm and 280nm on ultraviolet spectrophotometer, and draws A260/A280, when its numerical value illustrates that between 1.8 ~ 2.0 the purity of total serum IgE is better; Remaining salt ion and the pollution of small molecules magazine is had, when being greater than the degraded that 2.0 illustrate possibility total serum IgE when being less than 2.0 explanations:
2) integrity detection of total serum IgE: 1% denaturing formaldehyde agarose gel electrophoresis is separated the ribosome-RNA(rRNA) (rRNA) in total serum IgE, two maximum band 28rRNA and the brightness of 18sRNA are roughly than during for 2:1, illustrate that the integrity of RNA is better, do not occur signs of degradation.
2, acquisition mRNA is separated
Use Sera-magMagneticOligo (dT) Beads of ThermoFisher company to be separated and obtain mRNA: in total serum IgE, add NaOH mRNA is broken into short-movie section, and as template, with RT primer synthesis Article 1 cDNA chain.Purifying connects sequence measuring joints (5 ' DNA/DNAadaptor) after reclaiming RT product, and pcr amplification cDNA, 6%TBEPAGEGel rubber tapping is reclaimed 300bp-500bp object product and checked order.
3, solexa order-checking
The RNA library built friendship is carried out Solexa order-checking to Hua Da gene.The sequenator adopted is HiSeq2000, and strategy is single end sequencing, surveys 50bp.The data analysis of high-flux sequence mainly comprises coupling (mapping), splicing (assembly), and sequencing sequence quantitatively and downstream functional analysis.Concrete process from raw data to data available comprises the following aspects:
(1) sequencing quality assessment:
The quality of high-flux sequence raw data is the important foundation of subsequent experimental, and base mass value is the important indicator weighing sequencing quality.Software FastQC is comparatively ripe software high-flux sequence data being carried out to quality control.Base mass value Q represents, its value is higher, and to represent base less by the probability (P) of makeing mistakes, and its calculation formula is Q=-10lgP, generally adopts the assessment of Q20 and Q30 these two value, the check order probability of makeing mistakes of the base that namely represents Q20 is that 1%, Q30 represents that the base probability of makeing mistakes that checks order is 1 ‰.What this experiment adopted is that single end (Single-end) strategy of 1 × 50 of HiSeq2000 carries out, and Illumina company commonly uses the base accuracy being greater than 70% and arrives Q30 and just can be used for follow-up experimental analysis.
(2) the read inferior quality that checks order is filtered
In view of sequencing data error rate is on the impact of result, we remove joint to raw data, go inferior quality, and the process such as depollute, and obtains the sequence data of Clean, and carry out length distribution statistics to Clean sequence.Quality of data pre-treatment step:
1) inferior quality read is removed: quality threshold 20 (error rate=1%), proportion threshold value 40%;
2) the larger sequence of N fraction is contained in removal Reads: proportion threshold value 4%;
3) joint sequence is removed.
By solexa sequencing technologies the data obtained, the gene of 715 unconventionality expressions detected altogether, wherein 342 genes only high expression level in NpCR group, and low expression in pCR group; Article 373, low expression in NpCR group, and in pCR group high expression level.The expression and distribution of pCR group and NpCR group sequenced genes is shown in Figure 1A and Figure 1B.
4, the screening of significant difference expressing gene
(1) difference expression gene GO enrichment is analyzed
The screening of difference expression gene will meet two standards: fold differences is more than 2 times, namely | and logFC|>1; Pvalue<0.05.The enrichment of GO function significance is analyzed and is provided compared with genome background, the GO function entry of significant enrichment in difference expression gene, thus provides difference expression gene and which biological function significant correlation.First this analysis maps all differences expressing gene each term to GeneOntology database (http://www.geneontology.org/), calculate the number gene of each term, then hypergeometry inspection is applied, find out compared with whole genome background, significant enrichment in difference expression gene.
2. difference expression gene KEGG enrichment is analyzed
We use fishertest to carry out KEGGPathway enrichment analysis to difference expression gene.The enrichment of KEGG function significance is analyzed and is provided compared with genome background, the metabolic pathway of the KEGG of significant enrichment in difference expression gene, thus provides difference expression gene and which biological function significant correlation.First this analysis maps all differences expressing gene each pathway to KEGG database (www.genome.jp/kegg/), calculate the number gene of each pathway, then hypergeometry inspection is applied, find out compared with whole genome background, significant enrichment in difference expression gene.
Go out five difference expression genes by solexa sequencing technologies, GO enrichment analysis and KEGG enrichment Analysis and Screening, the selection result is in table 1.
Table 1
From table 1, HECTD3 gene and PSMB10 gene, UBD gene, UBE2C gene and UBE2S gene are ubiquitination pathway genes involved, wherein HECTD3 gene high expression level and low expression in pCR group in NpCR group, other four genes then in NpCR group low expression and in pCR group high expression level.
The quantitative fluorescent PCR analysis of embodiment 2 difference expression gene
1, the design of Auele Specific Primer
For HECTD3 gene and PSMB10 gene, UBD gene, UBE2C gene and UBE2S gene, utilize primer5.0 to carry out fluorescence quantification PCR primer design, design of primers information is in table 2.
Table 2 is for the Auele Specific Primer of quantitative fluorescent PCR
2、Real-TimePCR
1) synthesis of cDNA first chain
The total serum IgE obtained with embodiment 1 respectively carries out reverse transcription reaction for template and synthesizes cDNA.
Following reagent is added successively: the SYBRPremixExTaqTM (2 ×) of 10 μ l, each 0.4 μ l of upstream and downstream primer (10 μMs), the ROXReferenceDye (50 ×) of 0.4 μ l in 200 new μ l optics PCR eight unions; The ddH of 7.8 μ l
2o, cumulative volume 20 μ l.
2) centrifugally a little in ABIPRISM7300, carry out PCR reaction after mixing, reaction conditions is set to:
Stage1:95 DEG C of denaturation 30s, 1 circulation;
Stage2:PCR reacts
Cycle number: 40
95℃5s
60℃31s。
3, the process of data and analysis
As shown in Figure 2, the present embodiment, is normalized target gene for reference gene with β-actin, to guarantee the expression amount of comparison object gene in the sample of equal quantity.The formula of the change of expression amount multiple is:
RQ＝2
-△△Ct，△△Ct＝(Ct
1-Ct
2)
pCR-(Ct
1-Ct
2)
NpCR；
Wherein, RQ represents relative expression quantity (RelativeQuantitation), Ct
1represent the Ct value of the target gene that fluorescent quantitation detects, Ct
2represent the Ct value of the reference gene β-actin that fluorescent quantitation detects; After pCR represents new adjuvant chemotherapy, at mammary gland and axillary gland all without the sample group of infiltrating cancer tissue residue, after NpCR represents new adjuvant chemotherapy, block of swelling as long as former is or/and metastatic lesion still has the sample group of infiltrating cancer tissue residue.
Arrange quantitatively and repeat experiment and negative control experiment for three times, in quantitative sample, each sample repeats 3 times, does not add sample form cDNA in negative control, but replaces with water, whether there is PCR pollution or the pollution of higher primer dimer for detecting.
The data that fluorescent quantitation draws carry out statistical analysis, adopt SPSS16.0 statistical analysis software.The relative expression component analysis of target gene in two samples adopts levene ' s chi square test and independent-samplet inspection, as P value < 0.05, thinks that result has significant difference statistically.As P value < 0.01, think that result has pole significant difference statistically.The visualize of the difference analysis that target gene is expressed in different patient with breast cancers tissue is realized (as Fig. 3) by the histogram drawn containing error line.
As shown in Figure 3, HECTD3 is low expression in pCR group, and PSMB10, UBD, UBE2C and UBE2S be equal high expression level in pCR group; And all there is statistical significance, wherein there is extremely significant differential expression (P<0.01) in HECTD3 gene and PSMB10 gene in pCR group and NpCR group, and UBD, UBE2C and UBE2S gene exists significant differential expression (P<0.05) in pCR group and NpCR group.
Embodiment 3 is based on the PCR kit for fluorescence quantitative of SYBRGreen fluorescent dye determination
For predicting a PCR kit for fluorescence quantitative for neoadjuvant chemotherapy in breast curative effect, comprise the primer shown in table 2, and the reagent such as Taq enzyme, dNTP.
Adopt the test kit that the present embodiment provides, the using method based on the fluorescence quantitative PCR detection of SYBRGreen fluorescent dye determination is as follows:
Add successively in 0.2 new μ l optics PCR reaction tubes:
Slightly centrifugal after mixing, reaction parameter is set to:
The value of this test kit is the cancerous tissue RNA sample by patient, simple DNA dye method is utilized to detect the expression level of ubiquitination pathway genes involved, carried out the curative effect situation of NACT again by the difference degree prediction patient with breast cancer of this expression level, thus reduce unnecessary treatment.Therefore this test kit puts into production practice, can be applied in the therapeutic choice situation of the efficacy result of the NACT of mammary cancer and mammary cancer.
Embodiment 4 is based on the PCR kit for fluorescence quantitative of Taqman probe method
For predicting a PCR kit for fluorescence quantitative for neoadjuvant chemotherapy in breast curative effect, comprise 1. following ~ 5. at least one group-specific primers and Taqman probe:
1. for Auele Specific Primer and the Taqman probe of HECTD3 gene design:
Upstream primer: 5'-CCACCTCCCGATCATCGA-3';
Downstream primer: 5'-ACGAACATCAATCCCATCATCTC-3';
Taqman probe: 5'-ATCCGCATCGTGGAGTG-MGB-3';
2. for Auele Specific Primer and the Taqman probe of PSMB10 gene design:
Upstream primer: 5'-GAAAGCGAAAGCAGGAAGTACAG-3';
Downstream primer: 5'-CGTGAAGCCTAGCAGAG-3';
Taqman probe: 5'-CGTGAAGCCTAGCAGAG-MGB-3';
3. for Auele Specific Primer and the Taqman probe of UBD gene design:
Upstream primer: 5'-TGAGGAGCTGCCCTTGTTTC-3';
Downstream primer: 5'-TGTGGAGTCAGGTGATG-3';
Taqman probe: 5'-TGTGGAGTCAGGTGATG-MGB-3';
4. for Auele Specific Primer and the Taqman probe of UBE2C gene design:
Upstream primer: 5'-GAGGCTGCTGCTGGAGCTT-3'
Downstream primer: 5'-TCAATGTTGGGTTCTGGCATT-3'
Taqman probe: 5'-TCTGCAACTGTTTCTC-MGB-3';
5. for Auele Specific Primer and the Taqman probe of UBE2C gene design:
Upstream primer: 5'-ACCTACCCCCGCACATCAT-3';
Downstream primer: 5'-GTGGGTCTGCGGTCAGTGT-3';
Taqman probe: 5'-CTGGTGTACAAGGAGGTG-MGB-3';
Also comprise the Auele Specific Primer for the reference gene β-actin that increases:
Upstream primer: 5'-GCATACACGAAACTACCTTCAACTCC-3';
Downstream primer: 5'-ATGGTGGTGCCGCCAGACA-3'.
Claims (7)
1. ubiquitination pathway genes involved is in the application as the biomarker for predicting neoadjuvant chemotherapy in breast curative effect, it is characterized in that, described ubiquitination pathway genes involved is base sequence if the HECTD3 gene of SEQIDNo.1, base sequence are if the PSMB10 gene of SEQIDNo.2, base sequence are if the UBD gene of SEQIDNo.3, base sequence are if the UBE2C gene of SEQIDNo.4 and base sequence are as at least one in the UBE2S gene of SEQIDNo.5.
2. for predicting the biomarker of neoadjuvant chemotherapy in breast curative effect, it is characterized in that, be base sequence if the HECTD3 gene of SEQIDNo.1, base sequence are if the PSMB10 gene of SEQIDNo.2, base sequence are if the UBD gene of SEQIDNo.3, base sequence are if the UBE2C gene of SEQIDNo.4 and base sequence are as at least one in the UBE2S gene of SEQIDNo.5.
3. the application of biomarker as claimed in claim 1 in the PCR kit for the preparation of prediction neoadjuvant chemotherapy in breast curative effect.
4. apply as claimed in claim 3.It is characterized in that, described PCR kit is PCR kit for fluorescence quantitative.
5. for predicting the PCR kit for fluorescence quantitative of neoadjuvant chemotherapy in breast curative effect, it is characterized in that, comprise for described in claim 2 biomarker design Auele Specific Primer and Taqman probe, described Auele Specific Primer and Taqman probe be 1. following ~ 5. at least one group:
1. for Auele Specific Primer and the Taqman probe of HECTD3 gene design:
Upstream primer: 5'-CCACCTCCCGATCATCGA-3';
Downstream primer: 5'-ACGAACATCAATCCCATCATCTC-3';
Taqman probe: 5'-ATCCGCATCGTGGAGTG-MGB-3';
2. for Auele Specific Primer and the Taqman probe of PSMB10 gene design:
Upstream primer: 5'-GAAAGCGAAAGCAGGAAGTACAG-3';
Downstream primer: 5'-CGTGAAGCCTAGCAGAG-3';
Taqman probe: 5'-CGTGAAGCCTAGCAGAG-MGB-3';
3. for Auele Specific Primer and the Taqman probe of UBD gene design:
Upstream primer: 5'-TGAGGAGCTGCCCTTGTTTC-3';
Downstream primer: 5'-TGTGGAGTCAGGTGATG-3';
Taqman probe is: 5'-TGTGGAGTCAGGTGATG-MGB-3';
4. for Auele Specific Primer and the Taqman probe of UBE2C gene design:
Upstream primer: 5'-GAGGCTGCTGCTGGAGCTT-3'
Downstream primer: 5'-TCAATGTTGGGTTCTGGCATT-3'
Taqman probe: 5'-TCTGCAACTGTTTCTC-MGB-3';
5. for Auele Specific Primer and the Taqman probe of UBE2S gene design:
Upstream primer: 5'-ACCTACCCCCGCACATCAT-3';
Downstream primer: 5'-GTGGGTCTGCGGTCAGTGT-3';
Taqman probe: 5'-CTGGTGTACAAGGAGGTG-MGB-3'.
6. for predicting the PCR kit for fluorescence quantitative of neoadjuvant chemotherapy in breast curative effect, it is characterized in that, comprise fluorescence dye and for described in claim 2 biomarker design Auele Specific Primer, described Auele Specific Primer be 6. following ~ 10. at least one pair of:
6. the Auele Specific Primer for the HECTD3 gene that increases is:
Upstream primer: 5'-CTCATAGCAGCCACCTTCATTC-3';
Downstream primer: 5'-ACCGCTTCTTTGGGACACTTG-3';
7. the Auele Specific Primer for the PSMB10 gene that increases is:
Upstream primer: 5'-CCCATAGCCAATCAGTAGCC-3';
Downstream primer: 5'-TTAGGTCTCAACTCTTCCGTCAT-3';
8. the Auele Specific Primer for the UBD gene that increases is:
Upstream primer: 5'-CAACAGCGGAACCTCCAGTCTC-3';
Downstream primer: 5'-CGGTCCTCCTTGAATGCCAC-3';
9. the Auele Specific Primer for the UBE2C gene that increases is:
Upstream primer: 5'-AGCATTCCACTGCCCAAAGG-3';
Downstream primer: 5'-CGCTCCACCGTAACCACAGATAG-3';
10. the Auele Specific Primer for the UBE2S gene that increases is:
Upstream primer: 5'-TTGGTAGCCGTGGTTATCTCT-3';
Downstream primer: 5'-ACACTCTCAGCCAGTCCCTTAG-3'.
7. the PCR kit for fluorescence quantitative as described in claim 5 or 6, is characterized in that, also comprises the Auele Specific Primer for the reference gene β-actin that increases, and the Auele Specific Primer of described reference gene β-actin is:
Upstream primer: 5'-GCATACACGAAACTACCTTCAACTCC-3';
Downstream primer: 5'-ATGGTGGTGCCGCCAGACA-3'.
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| WO2008082730A2 (en) * | 2006-09-19 | 2008-07-10 | Novartis Ag | Biomarkers of target modulation, efficacy, diagnosis and/or prognosis for raf inhibitors |
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| WO2008082730A2 (en) * | 2006-09-19 | 2008-07-10 | Novartis Ag | Biomarkers of target modulation, efficacy, diagnosis and/or prognosis for raf inhibitors |
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| BERTUCCI ET AL: "Gene Expression Profiling Shows Medullary Breast Cancer Is a Subgroup of Basal Breast Cancers", 《CANCER RESEARCH》 * |
| LICHTER DI ET AL: "NM_002801", 《GENBANK》 * |
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