CN107881238A - The miRNA marker related to colorectal cancer prognosis and its application - Google Patents
The miRNA marker related to colorectal cancer prognosis and its application Download PDFInfo
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Abstract
The invention discloses the miRNA marker related to colorectal cancer prognosis and its application, wherein, the miRNA marker related to colorectal cancer prognosis is miR 96 or miR 155 in blood plasma, is in the tissue miR 99b.The miRNA related to colorectal cancer prognosis that the present invention is filtered out can provide important theoretical foundation for its clinical practice as the noninvasive mark of colorectal cancer prognosis.
Description
Technical field
The present invention relates to biotechnology and medicine technology field.A kind of it is more particularly related to and colorectal cancer
The related miRNA marker of prognosis and its application.
Background technology
Colorectal cancer (Colorectal cancer, CRC) is one of common alimentary tract cancer, and is whole world cancer
3rd main cause of disease associated death.Colorectal cancer hepatic metastases is a multifactor and process for multi-step.About 20-
25% CRC patient is initially diagnosed as hepatic metastases, and up to 40-50% patient occurs after primary tumor resection
Hepatic metastases.Most of Colon and rectum Patients with Liver Metastasis are not suitable for radical surgery excision, five-year survival rate≤10%.
The generation of colorectal cancer patients hepatic metastases with proto-oncogene and expression of tumor suppressor gene situation exception or loss except having
Close, also with " epigenetics ", such as acetylation, methylate is closely related with genetic regulation by non-coding RNAs.There is research to confirm, it is straight in knot
The hepatic metastases of intestinal cancer occurs in progression, its regulation and control of miRNA wide participations, has this kind of miRNA of scholar to be referred to as
“metastamiR”。
Microrna (miRNA) is class length for the endogenous non-coding RNA of 18-25 nucleotides, its by with mRNA
3' non-translational regions (UTR) base combine, post-transcriptional level regulation target gene expression so that translation be suppressed or believe
Crack RNA.Up to the expression of 20-30% gene is adjusted by miRNA.By adjusting physiology and pathologic process, difference table
The miRNA reached plays an important role in the generation of tumour, progress, transfer and recurrence.2003, Michael proposed to tie first
The miRNA of expression is had differences in the carcinoma of the rectum.Research subsequently, regarding to the miRNA of colorectal cancer is more and more.Certain research table
It is bright, different phase differential expression (high-level adenomas-tumour early stage-tumour of the miRNA in colorectal carcinoma occurrence and development be present
Late period).MiR-543 suppresses the propagation of colorectal cancer cell system and transfer by targetting HMGA2, KRAS and MTA1, and has
Hope the new diagnostic and prognosis biomarker as metastatic colorectal carcinoma.It is higher in CRC patient compared with health volunteer
Horizontal miR-211 prompts poor prognosis.Substantial amounts of research shows that circulation miRNA can be used as potential diagnosis and prognosis mark
Will thing.In recent years, although miRNA is largely studied and verified, colorectal cancer hepatic metastases correlation miRNA is potential
Effect and function are largely still unknown, it is necessary to be further discovered that and verify.
The patient of colorectal cancer hepatic metastases, based on operation, it is aided with the Synthetic of chemicotherapy, PCI and targeted therapy
Treatment makes the survival of patients time be significantly improved.But with the continuous lifting of therapeutic strategy, it is new the problem of also continue to bring out.Root
The property controlled operative treatment can improve 5 years survival rates of colorectal cancer hepatic metastases to 50%, for the high colorectal cancer liver of selectivity
Shift patient, it might even be possible to reach 60%;It is but postoperative still to have nearly 50% for resectable colorectal cancer hepatic metastases patient
Tumor recurrence occurs in patient.
Traditional screening methods, such as laparoscope, fine needle aspirate cytology and Imaging Method, such as strengthen computerized tomography and sweep
(CT), magnetic resonance imaging (MRI) are retouched as invasive, complication is high or typical indication deficiency, therefore, it is difficult to as colorectal cancer
The effective ways of Patients with Liver Metastasis early screening.Therefore, there is an urgent need to find the Noninvasive essence of early diagnosis and prognosis evaluation
Accurate biomarker, diagnosis and treatment for colorectal cancer patients.
The content of the invention
It is an object of the invention to provide the miRNA marker related to colorectal cancer prognosis and its application, is believed by biology
Cease method and excavate the miRNA marker relevant with colorectal cancer liver prognosis, important theoretical foundation is provided for its clinical practice.
In order to realize according to object of the present invention and further advantage, there is provided related to colorectal cancer prognosis
MiRNA marker, the mark are miR-96 or miR-155 in blood plasma, are in the tissue miR-99b, by blood plasma
MiR-99b carries out colorectal cancer prognosis in miR-96 or miR-155, or tissue.
The described miRNA marker related to colorectal cancer prognosis is in colorectal cancer prognostic agent, kit is prepared
Application.
The present invention comprises at least following beneficial effect:
First, blood plasma is easier to obtain, and compared with traditional screening methods, belongs to noninvasive test, is very easy to cure
The use for the treatment of personnel, alleviate the pain of patient.
Second, miRNA reflections in blood plasma are the overall pathology of body, physiological conditions, and its testing result has accurate
And detailed directive significance.
3rd, the miRNA related to colorectal cancer prognosis filtered out can be as the noninvasive marks of colorectal cancer prognosis
Thing, important theoretical foundation is provided for its clinical practice.
Further advantage, target and the feature of the present invention embodies part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Brief description of the drawings
Fig. 1 is technical method of the present invention and data analysis process block diagram;
Fig. 2 is normal liver tissue otherness miRNA cluster analysis express spectras by liver cancer tissue of the present invention and its cancer
Figure;
Fig. 3 is total serum IgE gel electrophoresis figure in blood plasma of the present invention;
Fig. 4 is the amplification of miRNAs real-time fluorescence quantitative PCRs and melting curve figure in blood plasma of the present invention;
Fig. 5 is colorectal cancer patients of the present invention without DISTANT METASTASES IN and Patients with Liver Metastasis, human normal plasma miRNA
Expression figure;
Fig. 6 is the hepatic metastases discriminating efficiency that ROC curve of the present invention analyzes miRNA;
Fig. 7 is that Kaplan-Meier methods of the present invention analyze miR-96 and miR-155 and colorectal cancer hepatic metastases trouble
The graph of a relation of person's overall survival phase;
Fig. 8 is total serum IgE gel electrophoresis figure in tissue of the present invention;
Fig. 9 is the amplification of miRNAs real-time fluorescence quantitative PCRs and melting curve figure in tissue of the present invention;
Figure 10 is that colorectal cancer patients of the present invention are fixed without DISTANT METASTASES IN patient and Patients with Liver Metastasis formalin
MiRNA expression figure in paraffin-embedded tissue;
Figure 11 is miRNA in Liver Metastases from Colorectal Cancer intestinal cancer tissue of the present invention and corresponding diagnosis of hepatic metastases tissue
Expression figure;
Figure 12 is the ROC curve of diagnostics of the analysis miRNA of the present invention between colorectal cancer patients difference group;
Figure 13 is that Kaplan-Meier methods of the present invention analyze miR-99b and Liver Metastases from Colorectal Cancer is totally raw
Deposit the graph of a relation of phase.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be noted that experimental method described in following embodiments, is conventional method unless otherwise specified, institute
Reagent and material are stated, unless otherwise specified, is commercially obtained.
Part I:Colorectal cancer shifts related miRNA bioinformatic analysis and screening
Materials and methods
1. technical method and data analysis process (see Fig. 1)
2.miRNA and gene expression data acquisition
Search key of this research in GEO databases for " Colorectal cancer ", " Colon cancer ",
" rectal cancer ", " Colonic carcinoma ", " Metastasis ", " Hepatic metastasis ", " liver
Metastasis ", " MicroRNAs ".And supplement and retrieved Google Scholar and Baidu's science, in different time point retrieval
Twice, chipset is included after 2010, is limited without language.Include chipset and be shown in Table 1-1.
The related chipset of table 1-1 colorectal cancers transfer
Wherein, *:Chipset type is the miRNA chips of Agilent brand.
3. normal liver tissue miRNA chip datas processing by liver cancer tissue and its cancer
This research choose miRNA chip datas come from this seminar early-stage Study (1. Gao B, Ning S, Li J,
et al.Integrated analysis of differentially expressed mRNAs and miRNAs
between hepatocellular carcinoma and their matched adjacent normal liver
tissues.Oncol Rep.2015.34(1):325-33.
2. normal liver tissue by Gao Bing, Ning Shufang, Tang Yanping, bang continent, niche woods, tonogram human liver cancer tissues and cancer
MRNA differential expressions spectrum world Chinese digest magazine .2014. (31):2 pairs of liver cancer groups of liver cancer patient in 4734-4744.)
Knit and cancer side normal liver tissue, tissue are derived from Guangxi Medical Uneversity Cancer Hospital's sample storehouse, all patients are to participation section
Learn and study equal informed consent, all samples are the first operation excision sample of primary lesion, are not received chemotherapy, radiotherapy
Deng.The tissue specimen of excision gives -80 DEG C of preservations immediately after liquid nitrogen flash freezer.
MiRNA array experiments data are extracted and analysis, chip are scanned by Agilent chip scanners, are obtained miscellaneous
Intersection graph piece.Hybridize picture using Agilent Feature Extraction (v10.7) software analysis and obtain data, use
Data are normalized AgilentGeneSpring softwares and variance analysis.
4.PUBMED literature searches
In order to which overall merit above difference Microrna (microRNA, miRNA) is examined colorectal cancer transfer/hepatic metastases
Disconnected and prognostic value.This research has carried out comprehensive literature search (including Pub Med, The Cochrane to differential gene
Library, ISI Web of Science, CNKI, all places and the general database of dimension), collect each number with exclusion standard according to including
According to the storehouse document related to tumour on different miRNA that in March, 2017 delivered from 2010 to.We press in literature search
According to:Difference miRNA it is related to metastases (including participate in regulation and control EMT, cell propagation and invasion and attack;Participate in some oncogene
Regulation and control, such as P53, KRAS etc.), thus select five genes of miR-96, miR-99b, miR-155, let-7a, let-7b and do in detail
Thin literature search form simultaneously lists the differential expression multiple for being related to miRNA in document.
5. statistical method
5.1 principal statistical softwares
R-3.3.3 network address:https://www.r-project.org/
Fun rich network address:http://www.funrich.org/
The network address of GraphPad Prism 7:http://www.graphpad.com/
The standardization of 5.2 chip datas
After data acquisition, the comment file of RNA initial data and related chip is combined by we first, and is divided
Group, then it is standardized with the limma bags of R language and is analyzed with differential expression, as included clinic in the affiliated document of fruit chip
Data, we will be to wherein information renaming, and differential gene and clinical information are compared, and finds the chip of complete information
Collection includes research.
The miRNA of differential expression is screened, the SAM methods that we generally use using studying both at home and abroad at present are smooth by this
Good fortune university develops, and is widely used, be it is a kind of by t examine based on screening differential gene algorithm.The screening of this research
Condition is P<0.1.That is miRNA is statistically significant in transfer stove and primary tumor differential tissue expression.This seminar early-stage Study
Liver cancer miRNA chip datas differentiation screening be same as above.
As a result:
The miRNA of differential expression in 1.GEO data sets
1.1 the related differential expression miRNA of colorectal cancer primary tumor/transfer stove tissue
Selected data set is GSE72199, and chip sample is respectively from colorectal cancer primary tumor and hepatic metastases stove, application
The normalized expression data of 28 colorectal cancer primary tumors and 8 hepatic metastases stove check samples, Analysis and Screening is carried out, according to whether
Statistically significant and its fold differences, the miRNA of qualified differential expression share 83, wherein in transfer stove tissue
33 of up-regulation, 50 of downward, up-regulation, the miRNA expressions lowered are shown in Table 1-2.Due to the miRNA data of differential expression
Relatively more, follow-up we reduce research range again with reference to early-stage Study and document analysis.
Human colon cancer is with respect to the miRNA that its hepatic metastases histological difference is expressed in table 1-2GSE72199
2. the miRNA of liver cancer tissue and differential expression in the Carcinoma side normal tissue that matches in early-stage Study
Using miRNA chip technologies, after handling initial data, we have obtained 33 differential expressions
MiRNA, wherein the miRNA raised has 21, the miRNA of downward has 12, the results are shown in Table 1-3.
Table 1-3 human liver cancer tissues are with respect to its normal liver tissue differential expression miRNA
Cluster analysis is carried out to the miRNA of otherness, as a result show liver cancer tissue (C) and its Para-cancerous tissue (N) have it is bright
Aobvious different express spectra, is shown in Fig. 2, and in Fig. 2, C1 and C2 represent liver cancer tissue, and N1 and N2 represent Para-cancerous tissue.
3. differential expression miRNA and tumour associative key text mining and literature search
In pubmed databases, we in the miRNA of differential expression, by miR-96, miR-99b, miR-155,
Let-7a, let-7b and " Neoplasms (disease) ", " Colorectal Neoplasms (alias
COLORECTALCANCER) " keyword is retrieved jointly.The result of miR-96 and colorectal cancer associative search find its with liver cancer,
The keywords such as metastases, existence and prognosis have close association;MiR-99b is not all sent out with tumour, colorectal cancer retrieval-by-unification
Now it is relevant with other keywords;MiR-155 and tumour retrieval-by-unification find its with cell migration, neoplasm staging, existence and
The keywords such as TP53 have close association;Let-7a has found its expression regulation with tumour, first with colorectal cancer associative search result
Base, DNA are repaired, and the keyword such as intra-abdominal chemotherapy and TP53, MYC, ras family gene is closely related;Let-7b and Colon and rectum
The result of cancer associative search finds its expression regulation with tumour, metastases, Victibix, existence and prognosis, KRAS genes
It is closely related Deng keyword.
Using above-mentioned 5 miRNA of text mining, entering of meeting that we further study group requires.We are to wherein one
A little emphasis documents are retrieved, and it the results are shown in Table 1-4.Wherein, research generally believe miR-96, miR-155, let-7a,
Tetra- genes of let-7b take part in the transfer of tumour, and be considered as potential prognostic marker;Wherein miR-96, let-
7a, let-7b expression are considered as and TP53, ras family gene, KRAS gene-correlations;Due to miR-99b in GEO databases and
High expression is found to be in early-stage Study, but its report in tumour and colorectal cancer is relatively fewer, in order to probe into its
Latent effect mechanism and value in colorectal cancer, therefore, we include miR-99b in the index of our analysis and research.
MiR-96, miR-99b, miR-155, let-7a, let-7b expression list in table 1-4 documents
Note:* the expression in cancerous tissue/cell/circulating.
In summary, by bioinformatics method, we are analyzed and screening has been obtained in colorectal cancer with shifting phase
5 miRNA closed, are miR-96, miR-99b, miR-155, let-7a and let-7b respectively, respective sequence is shown in Table 1-5.It
May the generation of colorectal cancer, development or transfer in play an important role, can carry out further molecular biosciences for this supposition
Learn experiment.
Table 1-5miR-96, miR-99b, miR-155, let-7a and let-7b sequence
Part II:Hepatic metastases correlation miRNA expression and its diagnosis and prognostic value in colorectal cancer patients blood plasma
1. experiment material
Case selection and specimen collection
This research is equal by the approval of Guangxi Medical Uneversity Cancer Hospital's Medical Ethics Committee, every subject
Signed informed consent form is agreed to use sample and clinical data.On December 30th, 2008 is collected to during 30 days December in 2011
In the CRC patient 90 that Guangxi Medical Uneversity Cancer Hospital goes to a doctor, while collect 1 day to 2016 September 30 of September in 2016
The Healthy People 20 of day physical examination.Patient's the median age is 55.9 years old (the range of age 20-84 year).Male 56, women 34;It is left
Half colon 24, right hemicolon 24,42, rectum;Low differentiation tumor 26, middle differentiation tumor 53, differentiated tumour 11
Example;57 of diameter of tumor < 5cm, 33 of diameter of tumor >=5cm;T1+T2 has 11, and T3+T4 has 79;N0 has 22,
N1+N2 has 68;Clinical stages, has 20 for I+II's, and clinical stages III+IV's has 70;Without DISTANT METASTASES IN patient 41,
Patients with Liver Metastasis 49.Clinical stages assessment is carried out by UICC and AJCC.Refer to table 2-1.
Liver Metastases from Colorectal Cancer inclusion criteria:During collection all patients do not receive chemotherapy, radiotherapy, targeted therapy
Deng complex treatment, the blood plasma of collection is preoperative blood plasma;Do not merge DISTANT METASTASES IN and/or the recurrence of other positions, do not suffer from simultaneously
There are other benign diseases.
Colorectal cancer is without DISTANT METASTASES IN patient's inclusion criteria:It is simultaneously not benign with other without DISTANT METASTASES IN and/or recurrence
Disease.
Health volunteer's inclusion criteria:Without intestines problem history, without tumour medical history.
90 colorectal cancer patients of table 2-1 and the clinical pathology essential characteristic of 20 health volunteers
2. experimental method
2.1 human normal plasma collections of specimens and preliminary treatment
2.1.1 healthy human peripheral blood 2mL is collected, is immediately placed in vacuum blood collection tube containing EDTA, is stored at room temperature about 30min;
2.1.2 the plasma specimen that will be stored at room temperature, 15min is centrifuged with 3000rpm, room temperature, takes 1mL supernatants;
2.1.3 by gained supernatant, 15min is centrifuged in 4 DEG C with 12000rpm, takes supernatant to dispense, often the μ L of pipe 200, are posted
Label, be placed in -80 DEG C it is stand-by.
2.2 colorectal cancer patients plasma specimen Collecting and dealings (being same as above)
The extraction of 2.3 blood plasma total serum IgEs
2.3.1 200 μ L plasma samples are taken out from -80 DEG C of refrigerators, in thaw at RT;
2.3.2 5 volumes (1mL) lysate is added into the blood plasma to have thawed, abundant mixing is beaten with pipettor.Often
Temperature stands about 5min;
2.3.3 200 μ L chloroform is added in above-mentioned mixed liquor, mixes 15s with vortice, normal temperature stands about 5min.This
The abundant mixing at place is the guarantee that follow-up reverse transcription and polymerase chain reaction run smooth;
2.3.4 low temperature (4 DEG C) centrifuge 12000rpm is used, is centrifuged 15 minutes;
2.3.5 carefully supernatant (about 600 μ L) is transferred in new 1.5mL centrifuge tube, adds 1.5 times of volumes (about 900
μ L) absolute ethyl alcohol, fully mixed with pipettor;
2.3.6 take the above-mentioned mixed liquors of 700 μ L to add in centrifugal column, 4 DEG C, 1min is centrifuged with 12000rpm, abandons waste liquid;
2.3.7 above step is repeated, remaining mixed liquor is added in centrifugal column and centrifuged;
2.3.8 700 μ L initial flush buffer solutions are added in centrifugal column, 12000rpm, 4 DEG C, 1min is centrifuged, abandons waste liquid;
2.3.9 500 μ L, bis- dcq buffer liquid are added in centrifugal column, 12000rpm, 4 DEG C, 1min is centrifuged, abandons waste liquid;
2.3.10 dose volume fraction is 80% coke diethyl phthalate alcohol, and 500 μ L Jiao is added in each centrifugal column
Charcoal diethyl phthalate alcohol, 5min is centrifuged in refrigerated centrifuge 12000rpm, abandons waste liquid;Centrifugal column is taken out, avoids alcohol residue;
2.3.11 centrifugal column is positioned in new centrifuge tube, unlided, 4 DEG C, 12000rpm, centrifuged 5 minutes, dry
Pipe interior diaphragm, abandons waste liquid;
2.3.12 centrifugal column is placed in new centrifuge tube, adds 14 μ L and stand 5min without enzyme water, normal temperature;
2.3.13 by centrifugal column with 12000rpm, 5min is centrifuged, it is stand-by.
MiRNA expressions in 2.4 measure blood plasma
2.4.1 the reverse transcription of blood plasma total serum IgE
The 12 above-mentioned total serum IgEs of μ L are taken, reverse transcription is carried out according to Kai Jie companies tiny RNA Reverse Transcriptase kit.Whole process for preparation
Carry out in 4 DEG C, reacted 60 minutes in 37 DEG C, carry out Poly (A) tailings and reverse transcription reaction, 5 minutes inactivators are small in 95 DEG C
RNA reverse transcription systems.Reaction system is shown in Table 2-2:
Table 2-2 reaction systems
200 μ L are added in 20 obtained μ L reverse transcription products without enzyme water, products therefrom cDNA is subjected to concentration and purity
- 20 DEG C of refrigerators are placed in after detection to save backup.It is stored in stand-by in -80 DEG C of refrigerators.
2.4.2 quantitative fluorescent PCR
Using the cDNA after reverse transcription as template, prepared according to the specification of tiny RNA polymerase chain reaction dye method kit
PCR reaction systems, primer are all from Tiangeng.Every primer repeats 3 multiple holes, and experiment every time is repeated 3 times.Sense primer is Tiangeng
The primer of company's design, anti-sense primer are the universal primer of Kai Jie companies design.The condition of reaction is set according to the requirement of kit
Put:15min in 95 DEG C of pre-degeneration;15s in 94 DEG C of denaturation;30s in 55 DEG C of annealing extension;40 circulations, drawn at 70 DEG C molten
Solution curve.Reaction system is shown in Table 2-3.Primer sequence is shown in Table 2-4.
Table 2-3 reaction systems
Table 2-4 primer sequences
Our differential expression levels using miR-16 as each miRNA of criterion calculation, each miRNA expression useMethod is calculated, wherein-△ △ CT=(reference of CT experimental group-CT standards)-(reference of CT control group-CT standards), point
Analyse differential expression situations of the miRNA in colorectal cancer patients and normal healthy controls crowd.
2.5 survival analysis
Collect and gone to a doctor to during 30 days December in 2011 in Guangxi Medical Uneversity Cancer Hospital on December 30th, 2008
Colorectal cancer patients, using phone and the form regular follow-up visited, using death as endpoints, end to during last follow-up
Endpoints do not occur or lost to follow-up are all included into censored data.Follow-up deadline is in September, 2016, and the median follow-up time time is 47.8
Month.
2.6 statistical analysis
Data analysis is carried out using SPSS 20.0, GraphPad Prism5.0 or Medcalc12.2.1.Normal distribution
Data is represented using mean ± standard deviation;Examined using t, one-way analysis of variance or non-parametric test are assessed between different groups
Each miRNA differential expressions;MiRNA Combining diagnosis is assessed using receiver operating curves (ROC) and binary linear regression
Efficiency;Each miRNA prognostic value is assessed using single factor test and multifactor survival analysis;Analyzed by bioinformatics method
MiRNA target gene and its signal path being related to.P values are considered as statistically significant less than 0.05.
As a result:
The relation of 1 colorectal cancer patients blood plasma miRNA s expressions and clinicopathologic features
The total serum IgE Quality Identification and PCR amplifications and melting curve figure of 1.1 extractions
Detected through trace dna analyzer, 260/280 OD values of each sample total serum IgE are between 1.8-2.0;Coagulated through agarose
Gel electrophoresis are tested, and each sample 28S, 18S, 5S band are clear, and the brightness of 28S bands is more than 18S and 5S, therefore the RNA mass of extraction
Reliable complete, available for studying in next step, as shown in figure 3,1-3 is colorectal cancer patients from left to right in Fig. 3,4-6 is health
People.
In this research, the amplification curve of internal reference and purpose miRNA quantitative fluorescent PCR shows S types, out of shape smooth,
There are obvious amplification phase and plateau, represent that amplification is preferable;The melting of internal reference and purpose miRNA quantitative fluorescent PCR is bent
Line is a narrow unimodal curve, and between 76 DEG C -80 DEG C, nothing is obvious bimodal at fusing point peak, shows amplified production specificity
It is good.As shown in figure 4, A in Fig. 4, miR-16;B, miR-96;C, miR-99b;D, miR-155;E, let-7a;F, let-7b.
The relation of 1.2 blood plasma miRNA s expressions and clinicopathologic features
We have detected 90 CRC patients and 5 miRNA of 20 health volunteers expression, and itself and clinic are special
The relation of sign such as following table 2-5.From 2-5 tables, in this research, blood plasma miR-96, miR-155 and let-7a are in hepatic metastases group table
Up to up-regulation, and miR-99b and let-7b is expressed in hepatic metastases group and lowered, and difference is statistically significant (P < 0.05).In addition, this
The N of 5 miRNA expression and tumour is by stages in research and clinical stages is relevant, and difference is statistically significant (P < 0.05);
MiR-96, miR-99b and miR-155 are relevant with diameter of tumor, and difference is statistically significant (P < 0.05);miR-96、miR-
99b, miR-15 and Let-7b and tumour T are by stages relevant, and difference is statistically significant (P < 0.05);Our research is not observed
To this 5 miRNA and the relation of other clinicopathologic features, no significant difference (P > 0.05).
Table 2-5 colorectal cancer patients miRNA expression and the relation (mean ± standard deviation) with clinical pathologic characteristic
Wherein, * one-way analysis of variances;* rank tests.
2 colorectal cancer patients and health volunteer's blood plasma miRNA s expressions
In order to explore in this research 5 miRNA between CRC patient and health volunteer's blood plasma whether variant expression,
We detect expressions of this 5 miRNA in blood plasma using real-time fluorescence quantitative PCR (qRT-PCR).As shown in table 2-6,
Statistical analysis shows that miR-96, miR-155 and let-7a are high in CRC patient blood plasma to express, and is presented in health volunteer
Low expression;MiR-99b and let-7b low expressions in CRC patient blood plasma, high expression, difference are presented in health volunteer's blood plasma
Statistically significant (P < 0.05).
Table 2-6 Colon and rectums patient and human normal plasma miRNA expression
In order to further find the relation of the blood plasma miRNA of above-mentioned 5 differential expressions and hepatic metastases, we are by 90 CRC
Patient is divided into no DISTANT METASTASES IN group (clinical stages I-III) and hepatic metastases group (clinical stages IV), more each blood plasma miRNA
Whether expression is variant.Statistical analysis shows that blood plasma miR-96, miR-155 and let-7a are high in hepatic metastases group to express,
The low expression in without DISTANT METASTASES IN group, difference are statistically significant (P < 0.05);And blood plasma miR-99b and let-7b turns in liver
Low expression in shifting group, the high expression in without DISTANT METASTASES IN group, difference is statistically significant (P < 0.05), as shown in figure 5, Fig. 5 is
Expression of the colorectal cancer patients without DISTANT METASTASES IN (n=41), Patients with Liver Metastasis (n=49), human normal plasma (n=20) miRNA
Level (A:miR-96;B:miR-99b;C:miR-155;D:Let-7a;E:Let-7b.Note:* it is P < 0.05;* is P <
0.01;* * are P < 0.001).
MiRNA diagnostic analysis in 3 colorectal cancer patients blood plasma
In order to detect whether the blood plasma miRNA of above-mentioned differential expression has diagnostic value in terms of hepatic metastases is detected, we
Carry out analyzing diagnosis effects of each miRNA between each group using receiver operating curves (ROC) and two variable linear regression
Can, it is shown in Table 2-7.First, we analyze the blood plasma miRNA of above-mentioned 5 differential expressions between CRC patient and health volunteer
Diagnostic, ROC and TG-AUC (AUC) show that 5 blood plasma miRNAs have preferable diagnostic in this research,
MiR-96, miR-99b, miR-155, let-7a and let-7b TG-AUC are respectively AUC=0.823 (CI:0.738-
0.889), 0.86 (CI:0.781-0.919), 0.858 (CI:0.778-0.917),
0.908(CI:0.837-0.954), 0.869 (CI:0.792-0.926), difference is statistically significant.
MiRNA diagnostic analysis in table 2-7 colorectal cancer patients blood plasma
On the basis of the studies above, we further analyze the above-mentioned miRNA with more excellent diagnostic value differentiate and
Diagnosis is without the efficiency in DISTANT METASTASES IN (clinical stages I-III) and hepatic metastases (clinical stages IV) patient, such as table 2-7.
This research have detected CEA expression simultaneously, it is intended to compare 5 miRNA and CEA of our candidates diagnosis effect
Energy.The result of ROC curve analysis shows that blood plasma miR-96 TG-AUC is 0.937 (0.866-0.978), blood plasma miR-
99b TG-AUC is 0.929 (0.855-0.973), and blood plasma miR-155 TG-AUC is 0.806 (0.710-
0.882), blood plasma let-7a TG-AUC is 0.843 (0.751-0.911), and blood plasma let-7b TG-AUC is
0.782 (0.682-0.862), CEA TG-AUC are 0.693 (0.587-0.786), in this 6 blood plasma Testing index,
MiR-96, miR-99b and let-7a are being diagnosed without the independent diagnostic value highest in DISTANT METASTASES IN patient and Patients with Liver Metastasis, and
CEA diagnostic is less than other 5 indexs of this research.
Then, we analyze miRNA Combining diagnosis efficiency, miR-96, miR-99b by two variable linear regression
With let-7a Combining diagnosis efficiency:TG-AUC is 0.971 (CI:0.911-0.995), optimal section value is 0.565, quick
Perception is 91.84%, and specificity is 80.49%.Thus, our result indicate that, blood plasma miR-96, miR-99b, let-7a tri-
The value of the Combining diagnosis of individual index is better than CEA and other indexs, sees Fig. 6, in Fig. 6, A:MiRNAs differentiate without DISTANT METASTASES IN and
The ROC curve of hepatic metastases colorectal cancer patients;B:MiR-96, mR-99b and Let-7a joint differentiate without DISTANT METASTASES IN and hepatic metastases
The ROC curve of colorectal cancer patients.Result of study shows that miR-96, miR-99b and let-7a combination are possibly as knot in blood plasma
The new non-invasive diagnostic of rectal cancer patient, particularly Liver Metastases from Colorectal Cancer and the biomarker of monitoring, Ke Nengzuo
For the molecular target for the treatment of of colorectal cancer.
4 survival analysises
In order to assess in this research miRNA and the relation of Liver Metastases from Colorectal Cancer prognosis in 5 blood plasma, we are by 49
Patient is divided into high expression group and low expression group by example Patients with Liver Metastasis according to the median of each miRNA expressions, is passed through
Kaplan-Meier methods, Log-Rank are examined and expression and the overall survival phase of the multifactor analysis of regression model blood plasma miRNAs of Cox
Relation.The results of univariate logistic analysis shows the miR-96 of high expression, and miR-155 patient's Overall survival is short compared with low expression patient
(log-rank, which is examined, shows that P is respectively 0.031,0.047);And miR-99b patient's Overall survival of high expression is compared with low expression
Patient's length (log-rank examine shows that P is respectively 0.025), be shown in Table 2-8.However, other miRNA and clinical pathology ginsengs are not found
Number, such as statistically significant (the P > of difference of CEA expressions, age, sex and diameter of tumor etc. and Overall survival
0.05).Multifactor Cox regression models show that the expression of miR-96, miR-155 in blood plasma are Liver Metastases from Colorectal Cancer prognosis
Independent hazard factor, see Fig. 7.Show that miR-96 and miR-155 have prognosis evaluation to Liver Metastases from Colorectal Cancer in blood plasma
Value.
The single factor test and multiplicity of table 2-8 colorectal cancer patients Overall survivals
CI credibility intervals, RR Relative hazards
Hepatic metastases correlation miRNA expression and its diagnosis and prognosis in Part III colorectal cancer patients paraffin-embedded tissue
Value
1. experiment material
Case selection and specimen collection
This research is equal by the approval of Guangxi Medical Uneversity Cancer Hospital's Medical Ethics Committee, every subject
Signed informed consent form is agreed to use sample and clinical data.It is collected in the date in December 30 30 days to 2011 December in 2008
Between Guangxi Medical Uneversity Cancer Hospital go to a doctor colorectal cancer patients 58.Patient's the median age is 57 years old (fluctuation model
Enclose for 33-89 year).Male 28, women 30;Left hemicolon 13, right hemicolon 23,22, rectum;Low differentiation tumor
15, middle differentiation tumor 26,17, differentiated tumour;35 of diameter of tumor < 5cm, 23 of diameter of tumor >=5cm;T1
+ T2 has 27, and T3+T4 has 31;N0 has 20, and N1+N2 has 38;Clinical stages, has 14 for I+II's, clinical stages III
+ IV's has 44;Without DISTANT METASTASES IN patient 25, Patients with Liver Metastasis 33.Clinical stages assessment is carried out by UICC and AJCC.
Refer to table 3-1.
Liver Metastases from Colorectal Cancer inclusion criteria:During collection all patients do not receive chemotherapy, radiotherapy, targeted therapy
Deng complex treatment, the tumor tissues of collection are preoperative tissue;DISTANT METASTASES IN and/or the recurrence of other positions are not merged, not together
When suffer from other benign diseases.
Colorectal cancer is without DISTANT METASTASES IN patient's inclusion criteria:It is simultaneously not benign with other without DISTANT METASTASES IN and/or recurrence
Disease.
The Clinical symptoms of table 3-1 colorectal cancer patients
* P < 0.05
2 experimental methods
The processing of 2.1 corrective surgery samples
After tumor tissues underwent operative is cut, immediately it is positioned in the neutral formalin solution that volume fraction is 4% and fixes,
FFPE (FFPE) tissue is made through drawing materials, be dehydrated, after the processing such as transparent, waxdip and embedding, cuts into slices and dyes pathology through HE
Make a definite diagnosis, it is then long-term to preserve.Whether there is DISTANT METASTASES IN patient leaves and takes intestinal cancer tissue and the wax stone of intestines cancer beside organism;All livers turn
Move the wax stone that patient leaves and takes intestinal cancer tissue, intestines cancer beside organism and the diagnosis of hepatic metastases tissue to match.
The extraction of total serum IgE in 2.2 paraffin-embedded tissues
2.2.1 paraffin layer is pruned with scalpel;
2.2.2 it is cut into the sheet (if sample is had been exposed in air, head layer 2-3 is discarded) of 5-20 μ m-thicks;
2.2.3 deparaffnize operates:
2.2.3.1 plus 1mL dimethylbenzene, cinene or polypropylene, vortex concussion 10s, full speed centrifuge 2min;
2.2.3.2 supernatant carefully is drawn with pipette tips, does not encounter sediment when drawing supernatant;
2.2.3.3 1mL ethanol (volume fraction 96-100%) is added in precipitation, is vortexed and mixes, centrifuges 2min at full speed
(effect of ethanol is to remove the dimethylbenzene in sample);
2.2.3.4 supernatant carefully is drawn with pipette tips, does not encounter sediment when drawing supernatant, and carefully sopped up with small pipette tips
Heptane/methanol of residual;
2.2.3.5 uncap and be incubated (15-25 DEG C, highest does not exceed 37 DEG C) 10min at room temperature, or be incubated to all
Raffinate volatilizees;
2.2.3.6 150 μ L or 240 μ L (when number of sections is more than 2) paraffin specimen processing buffer solution are added, is vortexed and mixes;
2.2.3.7 10 μ L Proteinase Ks are added, is gently mixed with pipette tips and (is inhaled and beat repeatedly);
2.2.3. 15min is incubated at 56 DEG C, 15min is incubated at 80 DEG C.
2.2.4 after being incubated 3min on ice, 20,000 × g (13,500rpm) centrifugations 15min;
2.2.5 supernatant is transferred to a new centrifuge tube, is careful not to encounter sediment;
2.2.6 add about 1/10 sample volume genomic deoxyribonucleic acid remove buffer solution (about 16 μ L or 25 μ L-
Depending on sample size), and 10 μ L DNA digestive ferment.Positive and negative inversion centrifuge tube mixes liquid.Simple centrifugation with
Collect the raffinate on tube wall;
2.2.7 15min is incubated at room temperature;
2.2.8 320 μ L or 500 μ L (when sample is more than 2 sections) red blood cell buffer solution are added, is thoroughly mixed dissolving production
Thing;
2.2.9 1120 μ L or 1750 μ L ethanol (100%) are added into sample, are mixed with pipette tips, are carried out immediately in next step;
2.2.10 700 μ L sample (including precipitation that may be present) is transferred on micro ribonucleic acid extraction agent box
(micro ribonucleic acid extraction agent being placed in 2mL collecting pipe, provided in collecting pipe kit).Cover collecting pipe
Lid, 15s more than 8, is being centrifuged under the conditions of 000 × g (10,000rpm), is abandoning the collection liquid in collecting pipe;
2.2.11 above step is repeated, until sample before is all disposed;
2.2.12 500 μ L film combination ribonucleic acid flushing liquors are added to micro ribonucleic acid extraction agent box.Carefully cover
The lid of collecting pipe, more than 8,15s is centrifuged under the conditions of 000 × g (10,000rpm), abandons the collection liquid in collecting pipe;
2.2.13 500 μ L film combination ribonucleic acid flushing liquors are added to micro ribonucleic acid extraction agent box.Cover collection
The lid of pipe, more than 8,2min is centrifuged under the conditions of 000 × g (10,000rpm), abandons collecting pipe;
2.2.14 micro ribonucleic acid extraction agent is placed in new 2mL collecting pipe, open lid, at full speed from
Heart 5min, loses collecting pipe;
2.2.15 micro ribonucleic acid extraction agent is placed in new 1.5mL collecting pipe, directly adds 14-30 μ L
On film without enzyme water to kit, carefully close the lid, centrifuge 1min at full speed, collect eluent.It is collected into 1.5mL centrifuge tubes
Be extraction total serum IgE.
The reverse transcription and real-time fluorescence quantitative PCR of 2.3 total tissue RNAs
MiRNA expression assay method is the same as Part II 2.4 in this part FFPE tissues.We join by standard of U6
According to normalizing each miRNA expression.
2.4 survival analysis
Be collected on December 30th, 2008 to during 30 days December in 2011 in Guangxi Medical Uneversity Cancer Hospital just
The colorectal cancer patients examined, using phone and the form regular follow-up visited, using death as endpoints, end to last follow-up
When endpoints do not occur or lost to follow-up are all included into censored data.Follow-up deadline is in September, 2016, and the median follow-up time time is
53.8 months.
2.5 statistical analysis
Data analysis is carried out using SPSS 20.0, GraphPad Prism5.0 or Medcalc12.2.1.Normal distribution
Data is represented using mean ± standard deviation (Mean ± SD);Examined using t, one-way analysis of variance or non-parametric test are assessed
Each miRNA differential expressions between different groups;MiRNA is assessed using receiver operating curves (ROC) and binary linear regression
The diagnostic of combination;Each miRNA prognostic value is assessed using single factor test and multifactor survival analysis.P values are less than 0.05
It is considered as statistically significant.
As a result:
The relation of miRNAs expressions and clinicopathologic features in 1 colorectal cancer patients tissue
The total serum IgE Quality Identification and PCR amplifications and melting curve figure of 1.1 extractions
Detected through trace dna analyzer, 260/280 OD values of each sample total serum IgE are between 1.8-2.0;Coagulated through agarose
Gel electrophoresis are tested, and each sample 28S, 18S, 5S band are clear, and the brightness of 28S bands is more than 18S and 5S, therefore the RNA mass of extraction
It is reliable complete, available for studying in next step, see Fig. 8,1-3 is colorectal cancer patients cancerous tissue from left to right in Fig. 8, and 4-6 is
Colorectal cancer patients cancer beside organism, 7-9 are colorectal cancer patients diagnosis of hepatic metastases tissue.
In this research, the amplification curve of internal reference and purpose miRNA quantitative fluorescent PCR shows S types, out of shape smooth,
There are obvious amplification phase and plateau, represent that amplification is preferable;The melting of internal reference and purpose miRNA quantitative fluorescent PCR is bent
Line is a narrow unimodal curve, and between 76-80 DEG C, nothing is obvious bimodal, shows that amplified production specificity is good at fusing point peak.
See Fig. 9, A, U6 in Fig. 9;B, miR-96;C, miR-99b;D, miR-155;E, let-7a;F, let-7b.A-F in Fig. 4 and Fig. 9
Abscissa is:Cycle;Ordinate is:Fluorescence.A-F abscissas mark numerical values recited interval 2 by 0 in Fig. 4;
Ordinate marks numerical values recited interval 10000 by 0.A-D abscissas mark numerical values recited interval 2 by 0 in Fig. 9;
Ordinate marks numerical values recited interval 1000 by 0.E and F abscissas mark numerical values recited interval 2 by 0;Ordinate
By 0, numerical values recited interval 10000 is marked.
1.2 formalin fix the relation of miRNAs expressions and clinicopathologic features in paraffin-embedded tissue
We have detected 5 of 25 colorectal cancers without DISTANT METASTASES IN patient and 33 Liver Metastases from Colorectal Cancer
MiRNA expression, its relation such as following table 3-2 with Clinical symptoms.From table 3-2,5 miRNA table in this research
Up to the T with tumour by stages or the clinical parameter such as hepatic metastases is relevant, difference is statistically significant (P < 0.05).Table 3-2 colorectal cancers
Patient's intestinal cancer tissue miRNA expression and the relation of clinicopathologic features
* non-parametric test
MiRNAs expressions in 2 colorectal cancer patients cancerous tissues and cancer beside organism
In order to explore in this research 5 miRNA between colorectal cancer patients cancerous tissue and cancer beside organism it is whether variant
Expression, we detect expressions of this 5 miRNA in tissue using real-time fluorescence quantitative PCR (qRT-PCR).Such as table 3-3
Shown, statistical analysis shows, miR-96, miR-155 and let-7a high expression in CRC patient tissue, and in cancer beside organism
Low expression is presented;MiR-99b and let-7b low expressions in colorectal cancer patients tissue, high expression is presented in cancer beside organism,
Difference is statistically significant (P < 0.05), consistent with the expression trend in blood plasma.
MiRNAs expressions in table 3-3 colorectal cancer patients tissues
In order to further find the miRNA of above-mentioned 5 differential expressions and hepatic metastases relation, we are by 58 colorectal cancers
Patient is divided into no DISTANT METASTASES IN group (clinical stages I-III, n=25) and hepatic metastases group (clinical stages IV, n=33), compares two
Whether miRNA expression is variant in group.Statistical analysis shows, formalin fix miR-96 in paraffin-embedded tissue,
MiR-155 and let-7a high expression, the low expression in without DISTANT METASTASES IN group, statistically significant (the P < of difference in hepatic metastases group
0.05);And miR-99b low expressions in hepatic metastases group, the high expression in without DISTANT METASTASES IN group, statistically significant (the P < of difference
0.05) let-7b differential expressions between the two groups, are not found, see Figure 10, Tu10Zhong, A:miR-96;B:miR-99b;C:miR-
155;D:Let-7a;E:Let-7b.* it is P < 0.05;* is P < 0.01;* * are P < 0.001.
MiRNA expression in 3 Liver Metastases from Colorectal Cancer intestinal cancer tissues and the diagnosis of hepatic metastases that matches
In order to further analyze this expressions of 5 miRNA in Liver Metastases from Colorectal Cancer, we have to 33
The patients with bowel cancer of diagnosis of hepatic metastases is studied in detail.The intestinal cancer that we compare every patient by paired t-test is primary
Organize and the hepatic metastases stove tissue that matches in this 5 miRNA expression, see Figure 11, A in Figure 11:miR-96;B:miR-
99b;C:miR-155;D:Let-7a;E:Let-7b.It was found that compared with the hepatic metastases stove that matches, miR-96 and miR-155
The up-regulated expression in intestinal cancer primary tumor, and miR-99b expression lower, difference it is statistically significant (P 0.027, < 0.001,
0.032), but we do not have found that let-7a and let-7b have differences table in Patients with Liver Metastasis intestinal cancer and diagnosis of hepatic metastases tissue
Up to (P=0.098,0.131).
MiRNA diagnostic analysis in 4 colorectal cancer patients FFPE tissues
In order to detect whether the tissue miRNA of above-mentioned differential expression has diagnostic value in terms of hepatic metastases is detected, we
Carry out analyzing diagnosis effects of each miRNA between each group using receiver operating curves (ROC) and two variable linear regression
Can, it is shown in Table 3-4 and Figure 12.First, we analyze the blood plasma miRNA of above-mentioned 5 differential expressions in Colorectal Carcinoma and cancer
Diagnostic between other normal structure, ROC and TG-AUC (AUC) show in this research that 5 miRNA have and preferably examined
Disconnected efficiency, miR-96, miR-99b, miR-155, let-7a and let-7b ROC curve are as shown in figure 12, and difference has statistics
Learn meaning (P<0.0001).
Then, we compare the diagnosis of each miRNA in the intestinal cancer tissue of no DISTANT METASTASES IN patient and Patients with Liver Metastasis
Efficiency, it is found that miR-99b, miR-155 and let-7a have preferably diagnostic value, the statistically significant (P of difference<
0.0001), and miR-96 and let-7b diagnostic value is relatively low (P > 0.05);By 3 index miR- with statistical significance
99b, miR-155 and let-7a combine, and evaluate its Combining diagnosis efficiency, it is found that AUC=0.954 (0.864-0.992) is above
The independent diagnostic of this 3 indexs and other miRNAs Combining diagnosis efficiency, are shown in Figure 12.In Figure 12, A:5 miRNA mirror
ROC curve not without DISTANT METASTASES IN and hepatic metastases colorectal cancer patients;B:MiR-99b, miR-155 and let-7b joint differentiate nothing
The ROC curve of DISTANT METASTASES IN and hepatic metastases colorectal cancer patients.
The assessment of miRNA diagnostics in table 3-4 colorectal cancer patients FFPE tissues
5 survival analysises
In order to assess 5 miRNA and the relation of Liver Metastases from Colorectal Cancer prognosis in this research, we turn 33 livers
Move patient trouble is divided into by high expression group and low expression group according to the median of each miRNA expressions, by Kaplan-
MiRNA expression and the relation of overall survival phase in Meier methods and the multifactor analysis of regression model FFPE tissues of Cox, such as table 3-
5.The results of univariate logistic analysis shows that miR-99b, let-7b and N are by stages relevant with patient's Overall survival, P < 0.05), do not send out
Existing other miRNA and clinicopathologic features, such as the difference of age, sex and diameter of tumor etc. and Overall survival have statistics
Learn meaning (P > 0.05).Multifactor Cox regression models show that miR-99b, N are the only of Liver Metastases from Colorectal Cancer prognosis by stages
Vertical hazards, are shown in Figure 13.
The single factor test and multiplicity of table 3-5 Liver Metastases from Colorectal Cancer Overall survivals
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, it is of the invention and unlimited
In specific details and shown here as the legend with description.
<110>Guangxi Tumour Research Institute
<120>The miRNA marker related to colorectal cancer prognosis and its application
<130> 2017
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> RNA
<213> Homo sapiens
<400> 1
cacccguaga accgaccuug cg 22
<210> 2
<211> 23
<212> RNA
<213> Homo sapiens
<400> 2
uuuggcacua gcacauuuuu gcu 23
<210> 3
<211> 23
<212> RNA
<213> Homo sapiens
<400> 3
uuaaugcuaa ucgugauagg ggu 23
<210> 4
<211> 22
<212> RNA
<213> Homo sapiens
<400> 4
ugagguagua gguuguauag uu 22
<210> 5
<211> 22
<212> RNA
<213> Homo sapiens
<400> 5
ugagguagua gguugugugg uu 22
Claims (2)
1. the miRNA marker related to colorectal cancer prognosis, it is characterised in that the mark in blood plasma for miR-96 or
MiR-155, it is in the tissue miR-99b.
2. as claimed in claim 1 the miRNA marker related to colorectal cancer prognosis prepare colorectal cancer prognostic agent,
Application in kit.
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