CN105018594A - Early-diagnosis marker for colorectal cancer and related kit - Google Patents
Early-diagnosis marker for colorectal cancer and related kit Download PDFInfo
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Abstract
The invention relates to an early-diagnosis marker for the colorectal cancer and a kit constructed by using the early-diagnosis marker for the colorectal cancer. The early-diagnosis marker for the colorectal cancer is composed of a plurality of nucleic acid molecules which include nucleic acid molecules coding blood plasma miR-1183, miR-938, miR-490-3p, miR-490-5p, miR-592, miR-193b and miR-1290, and the nucleic acid molecules have differential expression in at least one target blood plasma and at least one contrast blood plasma. According to the invention, through research on expressional differences of the blood plasma miRNAs in patients with the colorectal cancer, patients with colorectal high-grade intraepithelial neoplasia and healthy people, it is determined that a combination of the blood plasma miRNAs can be used as a potential early-diagnosis marker for the colorectal cancer. The kit provided by the invention can accurately distinguish patients with the colorectal cancer and patients with colorectal high-grade intraepithelial neoplasia from contrast healthy people.
Description
Technical field
The present invention relates to a kind of colorectal cancer early diagnosis marker, the colorectal cancer early diagnosis marker be particularly made up of blood plasma microRNA, and the test kit built by this colorectal cancer early diagnosis marker.
Background technology
Colorectal cancer is one of global modal malignant tumour, and according to the World Health Organization (WHO) report, colorectal cancer is the male sex the 3rd and the common malignant tumour of women's second.Within 2012, global male sex's colorectal cancer new cases are 746000 examples, account for all malignant tumours 10%; Women's colorectal cancer new cases are 614000 examples, account for all malignant tumours 9.2%.Constantly rise at the M & M of China in recent years, annual new cases is more than 170,000 people, and dead nearly 100,000 people, sickness rate occupies China's malignant tumour the three, Zhi Zhui American-European countries.According to Incidence and the mortality statistics result of Guangzhou in September, 2013 announcement, men and women's colorectal cancer incidence rate rises 53.31% and 39.35% respectively, and sickness rate occupies second, and mortality ratio is arranged in all malignant tumours the 3rd.
Although the past is surgery for colorectal carcinoma mode and auxiliary treatment means development over 50 years, after colorectal cancer radical surgery, survival rate is without too large change, finally dead because of local recurrence or transfer after about 50% operation in patients.When the one of the main reasons of prognosis mala is medical, Most patients belongs to middle and advanced stage, only the feasible radical excision of 56% patient.The cause of the death directly has important association with by stages more late.The up-to-date TNM Staging System of formulation is combined for standard with International Union Against Cancer (UICC) and tumour federation of the U.S. (AJCC), according to existing statistical information, 5 years survival rates of I, IIa, IIb, IIIa, IIIb, IIIc and IV phase colorectal cancer patients are respectively 93.2%, 84.7%, 72.2%, 83.4%, 64.1%, 44.3% and 8.1%, the importance of visible early stage diagnosis and treatment.
Colorectal cancer experienced by the evolution of normal mucosa ~ adenoma ~ gland cancer mostly, i.e. Nomal-Adenoma-Adenocarcinoma (N.A.AC) theory.Gland cancer is the topmost histological type of colorectal cancer, and adenoma is acknowledged as the most important precancerous lesion of Colon and rectum gland cancer at present.Colorectal cancer not easily finds in early days, most patient occurring having blood in stool, the symptom such as stomachache just goes to see a doctor after three months to half a year.Cancer cells has spread and has even distally shifted when the time comes, loses best operative treatment opportunity.In addition, if there is intestinal obstruction or intestinal perforation, 5 years survival rates of patient can reduce further; Simultaneously histological difference (except MSI-H sample tumour), lymphatic vessel/vascular invasion, nerve infiltration, or incisxal edge is close, uncertain or the positive is all considered to the factor of prognosis mala in NCCN guide.Although Clinics development, 5 years survival rates after operation in patients are also not improved, lack effective diagnostic method to realize the early diagnosis of Colon and rectum gland cancer and precancerous lesion thereof being one of chief reason, if energy early discovery also excises Colon and rectum gland cancer and adenoma pathology thereof, will contribute to reducing its sickness rate and mortality ratio.
At present, the main path of early discovery Colon and rectum intestinal cancer, using occult blood test (FOBT) as the main generaI investigation means of colorectal cancer, filters out high risk population and carries out enteroscopy.But FOBT lacks specificity, the intestines mirror examination rate only 30%-40% of primary dcreening operation conformability and high risk population, disease detection rate about about 19%, needs to be improved further.China is populous, and colorectal cancer incidence rate rises very obvious, filters out the crowd that intestines mirror need examine further more necessary.Utilize noninvasive tumor markers to carry out the examination of intestines mirror to high risk population, enteroscopy number can be reduced and further specificity and the sensitivity improving intestines mirror.Recommend there is CEA for diagnosing with the important tumor markers of Index for diagnosis clinically at present, but its specificity and the application in diagnosis of colorectal carcinoma enjoy query.Therefore, current colorectal cancer tumor markers can not meet clinical demand far away, finds desirable to have important clinical meaning for early diagnosis colorectal cancer tumor markers.
MiRNA is a kind of widely distributed endogenous non-coding RNA with adjusting function, be about 18 ~ 25 Nucleotide, it is by the mode identification said target mrna of base pair complementarity, and said target mrna can be made to degrade or check the translation of said target mrna, thus after transcribing, the expression level of target gene is regulated, participate in the functions such as cytodifferentiation, growth, apoptosis, metabolism.MiRNA expresses has tissue specificity and timing, plays multiple regulating effect, participates in various adjustment approach, especially malignant tumour relevant to numerous disease in Growth of Cells and growth course.There is a large amount of miRNA in blood plasma, in different tumour, the express spectra of miRNA is also different, and prompting blood plasma miRNA can be used as new tumor markers, and the diagnosis for tumour is even treated.As potential biomarker, miRNA has the incomparable advantage of protein marker, what have research display miRNA exists with nucleic acid-protein composite form in blood or is coated in the middle of outer row's particle, miRNA is tolerated and comprises the situations such as rnase, multigelation, PH change, long-term preservation, not easily be degraded, stability is very good in blood.In recent years, the tumour miRNA that is correlated with is found successively in the blood of tumour patient, and the non-invasive early diagnosis for tumour provides a new approach.Such as, in human serum, the high expression level of Let-7, miR-9, miR-15a, mi26a, miR-155, miR-34, miR-220 etc. is closely related with diseases such as carcinoma of the pancreas, mammary cancer, lung cancer, liver cancer, leukemia respectively.But, single miRNA is still difficult to meet clinical demand as the Sensitivity and Specificity of mark, difference miRNA combination is set up tumor specific expression spectrum and the single molecular marker of improvement is difficult to the muting sensitivity overcome, low specificity issues, become the effective means of early diagnosis.If specificity plasma of colorectal cancer miRNA combination express spectra can be obtained, can be its early diagnosis and a new approach is provided.
Summary of the invention
First aspect of the present invention is to provide a kind of colorectal cancer early diagnosis marker.
Colorectal cancer early diagnosis marker of the present invention is made up of 7 kinds of nucleic acid molecule, and described nucleic acid molecule comprises the nucleic acid molecule of coding blood plasma miR-1183, miR-938, miR-490-3p, miR-490-5p, miR-592, miR-193b and miR-1290; Described nucleic acid molecule contrasts differential expression in blood plasma with at least one at least one target blood plasma.
The further feature of colorectal cancer early diagnosis marker according to claim 1, coding blood plasma miR-1183, miR-938, miR-490-3p, miR-490-5p, miR-592, miR-193b are raised compared with the expression contrasted at least one in blood plasma with the expression of nucleic acid molecule at least one target blood plasma of miR-1290; And the expression of nucleic acid molecule at least one target blood plasma of coding blood plasma miR-490-5p with miR-592 is lowered compared with the expression contrasted at least one in blood plasma.
The present invention provides a kind of colorectal cancer early diagnosis kit comprising above-mentioned colorectal cancer early diagnosis marker simultaneously.
Second aspect of the present invention is to provide another kind of colorectal cancer early diagnosis marker.
Colorectal cancer early diagnosis marker of the present invention is made up of 5 kinds of nucleic acid molecule, and described nucleic acid molecule comprises the nucleic acid molecule of coding blood plasma miR-1183, miR-938, miR-490-3p, miR-490-5p, miR-592, miR-193b and miR-1290; The expression of described nucleic acid molecule at least one target blood plasma is raised compared with the expression contrasted at least one in blood plasma.
The present invention provides a kind of colorectal cancer early diagnosis kit comprising above-mentioned colorectal cancer early diagnosis marker simultaneously.
According to the further feature of colorectal cancer early diagnosis kit of the present invention, described test kit also comprises a regression model, set up logit (P) discrimination formula as follows: logit (P)=-8.653+49.783 × (miR-1183)+16.666 × (miR-938)+235.243 × (miR-490-3p)+0.690 × (miR-193b)+18.096 × (miR-1290), wherein, miR-1183, miR-938, miR-1290, the expression level of miR-490-3p and miR-193b is that internal reference detection obtains with U6, the expression of logit (P) value in model at least one target blood plasma is raised compared with the expression contrasted at least one in blood plasma.
U6 is a nucleoid microRNA, and existing document confirms that it exists in human plasma and expression is constant, the present invention by it as reference gene, to detect relative expression quantity (the △ CT=CT of miRNA
to be measured miRNA-CT
internal reference).
According to the further feature of diagnostic kit of the present invention, described early stage colorectal cancer is Colon and rectum intraepithelial neoplasia.
According to the further feature of diagnostic kit of the present invention, described early stage colorectal cancer is the colorectal cancer of TNM I phase.
3rd aspect of the present invention is to provide a kind of test kit of blood plasma of blood plasma He at least one healthy individuals for distinguishing at least one colorectal cancer patients.
The test kit of the blood plasma of a kind of blood plasma He at least one healthy individuals for distinguishing at least one colorectal cancer patients of the present invention, comprises above-mentioned colorectal cancer early diagnosis marker, and wherein, at least one contrast blood plasma is from healthy individuals.
According to the further feature of test kit of the present invention, described test kit also comprises a regression model, set up logit (P) discrimination formula as follows: logit (P)=-8.653+49.783 × (miR-1183)+16.666 × (miR-938)+235.243 × (miR-490-3p)+0.690 × (miR-193b)+18.096 × (miR-1290), wherein, miR-1183, miR-938, miR-1290, the expression level of miR-490-3p and miR-193b is that internal reference detection obtains with U6, the expression of logit (P) value in model at least one target blood plasma is raised compared with the expression contrasted at least one in blood plasma.
The present invention by unconventionality expression in Colorectal Carcinoma obviously and more constant miRNA is defined as preliminary study object, have found the closely related miRNA with Colorectal Cancer.The present invention have selected 7 may in plasma of colorectal cancer the miRNA of obvious unconventionality expression, i.e. miR-1183, miR-938, miR-1290, miR-490-3p, miR-490-5p, miR-592, miR-193b.The present invention is by the differential expression of these blood plasma miRNA of research in colorectal cancer patients, Colon and rectum intraepithelial neoplasia patient and healthy population, and determining the combination of this blood plasma miRNA may as the potential of colorectal cancer early diagnosis marker.The present invention's application real time fluorescence quantifying PCR method is verified in 79 routine CRC (colorectal cancer) patients, 9 routine Colon and rectum intraepithelial neoplasia patients and 15 routine healthy volunteer's blood plasma, detect its expression level, observe its express spectra in the plasma sample of different clinical characters, and the value of analysed for plasma specificity miRNA combination in colorectal cancer early diagnosis.After empirical tests, the present invention obtains specific plasma 5-miRNA and combines, i.e. miR-1183, miR-938, miR-1290, miR-490-3p, miR-193b, the susceptibility that this group biomarker combinations detects colorectal cancer is 98.7%, specific degree is 100%, and the area under curve (area under the curve, AUC) that can produce maximum Receiver operating curve (receiver operator characteristic curve, ROC) is 0.997.Therefore, blood plasma miR-1183, miR-938, miR-1290, miR-490-3p, miR-193b can be used as the potential tumor markers of colorectal cancer, the 5-miRNA combination built accurately can distinguish colorectal cancer, Colon and rectum intraepithelial neoplasia and normal healthy controls person, for miRNA has carried out new effectively supplementing as the potential existing Laboratory evidence of colorectal cancer early diagnosis biomarker.
Accompanying drawing explanation
Fig. 1 is U6 amplification and solubility curve.
Fig. 2 is miR-1183 amplification and solubility curve.
Fig. 3 is miR-938 amplification and solubility curve.
Fig. 4 is miR-490-3p amplification and solubility curve.
Fig. 5 is miR-490-5p amplification and solubility curve.
Fig. 6 is miR-592 amplification and solubility curve.
Fig. 7 is miR-193b amplification and solubility curve.
Fig. 8 is miR-1290 amplification and solubility curve.
Fig. 9 is the expression of miR-1183.
Figure 10 is the expression of miR-938.
Figure 11 is the expression of miR-490-3p.
Figure 12 is the expression of miR-490-5p.
Figure 13 is the expression of miR-592.
Figure 14 is the expression of miR-193b.
Figure 15 is the expression of miR-1290.
Figure 16 is the ROC graphic representation of miR-1183 Colorectal Cancer Diagnosis.
Figure 17 is the ROC graphic representation of miR-938 Colorectal Cancer Diagnosis.
Figure 18 is the ROC graphic representation of miR-490-3p Colorectal Cancer Diagnosis.
Figure 19 is the ROC graphic representation of miR-490-5p Colorectal Cancer Diagnosis.
Figure 20 is the ROC graphic representation of miR-592 Colorectal Cancer Diagnosis.
Figure 21 is the ROC graphic representation of miR-193b Colorectal Cancer Diagnosis.
Figure 22 is the ROC graphic representation of miR-1290 Colorectal Cancer Diagnosis.
Figure 23 is the ROC graphic representation of 5-miRNA combined diagnosis colorectal cancer.
Figure 24 is the ROC graphic representation of the high-level intraepithelial neoplasia of 5-miRNA combined diagnosis Colon and rectum
Figure 25 is the ROC graphic representation of 5-miRNA combined diagnosis I phase colorectal cancer.
Embodiment
1, experiment sample
1.1 sampling flow processs: strictly observe 1998 by " mankind's inheritance resources management tentative method " issued by the State Council and 2005 " Chinese mankind's inheritance resources disease flesh tissue compiles Techniques of preserving code " and as benchmark.The peripheral blood of the 79 routine colorectal cancers that collection Guangzhou medical university the 3rd affiliated hospital 2010-09/2013-09 accepts for medical treatment and 9 routine Colon and rectum intraepithelial neoplasia patients, patient all makes a definite diagnosis through pathologic finding, without other tumour medical histories, before sample collection, patient is without any Radiotherapy chemotherapy, and all patients all have complete clinical, pathological data.The male sex 51 example, women 37 example; Age 38-82 year, average 64.16 ± 12.36 years old; TNM is carried out by stages, I phase 10 example, II phase 22 example, III phase 38 example and IV phase 10 example according to american cancer federation (American Joint Committee on Cancer, AJCC) the 7th edition standard.In addition, collect 15 routine physical examination of healthy population peripheral bloods, wherein the male sex 10 example, women 5 example; Age 53-87 year, average 67.67 ± 10.31 years old.The age of colorectal cancer patients and physical examination of healthy population and gender matched (P>0.05).This research is ratified by the examination & verification of Guangzhou medical university the 3rd affiliated hospital's Medical Ethics Committee, and the collection of all samples all obtains the informed consent of patient.
The registration of 1.2 sample related datas: after sample is deposited, carries out the registration work of sample related data in time, and the register content of sample related data comprises sample numbering, name, admission number, tumor type, quantity of drawing materials, pathological data and pathological examination etc.
2, laboratory apparatus and reagent
2.1 key instrument equipment
SWC-J100 type Bechtop, real-time PCR (ABl7500), Heal Force Biohazard Safety Equipment (HF safe 1200), normal temperature low speed centrifuge, refrigerated centrifuge (German Eppendorf company), 4 DEG C of refrigerators ,-20 DEG C of refrigerators, Ultralow Temperature Freezer, UV detector NanoDrop ND-2000, micro oscillator, ice-making machine, BioRad S1000Thermal cycler PCR instrument, vertical mixed instrument, mini whizzer.
2.2 main agents
RNA extracts reagent: Trizol, purchased from TAKARA company;
RT-PCR reagent, purchased from DBI company;
DEPC, purchased from American Sigma company;
RNasin, purchased from American Promega company;
Reverse Transcriptase kit, purchased from DBI company;
MiRNA primer, purchased from Sheng Gong bio tech ltd, Shanghai;
DEPC water, purchased from Shanghai biotechnology company limited;
TEMED, available from Sigma;
Water-saturated phenol, purchased from TBD company;
Chloroform, purchased from Beijing Qing Shengda Chemical Engineering Technology company limited;
DMSO, purchased from Biolabs company;
Other: dehydrated alcohol, Tween 20, Tris, EDTA etc. is laboratory conventional reagent.
3, solvent preparation
DEPC process water: ultrapure water, sterilizing, add the DEPC of 0.1%, process is spent the night, and DEPC is removed in sterilizing in second day.As: (1) preparation DEPC aqueous solution (being used for soaking rifle head, centrifuge tube etc.): add 1mL DEPC in 1000mL sterilizing deionized water, cover tightly stopper jolting 5min, leave standstill 4 hours, pour into fill rifle head and centrifuge tube container in, seal vessel port with preservative film to volatilize to prevent DEPC, room temperature hold over night, outwells solution, and 30min is to remove residual DEPC for the sterilizing of process article.The DEPC aqueous solution autoclaving 30min poured out abandons again.(2) DEPC process water (being used for joining RNA electrophoretic buffer) is prepared: in 1000mL deionized water, add 1mL DEPC, cover tightly stopper jolting, hold over night, adds plastics bag and is tamping bottleneck, and autoclaving 30min is to remove residual DEPC.(3) join the DEPC process water of 1000ml, add 1mlDEPC.Magnetic stirrer is spent the night.Second day high pressure steam sterilization 121c, 30min DEPC resolves into carbonic acid gas and alcohol, closes refrigeration for subsequent use.
4, experimental technique
The collection of 4.1 plasma samples: use every example person's peripheral blood to be checked 2mL that EDTA test tube gathers, at 4 DEG C, the centrifugal 10min of 1900g.Draw supernatant to manage to clean 1.5mL DEPC, 4 DEG C, the centrifugal 10min of 16000g, careful supernatant of drawing, in new DEPC pipe, is placed in-80 DEG C of refrigerators and saves backup.Aforesaid operations completes in 1h under room temperature (22 DEG C-25 DEG C).
The extraction of 4.2 blood plasma total serum IgE: use TRIzol method, gets appropriate blood plasma, adds 1mL TRIzol, and concussion mixing room temperature places 5min; Add 0.2mL chloroform, concuss 15s, leave standstill 3min; 4 DEG C of centrifugal 10min of 10000r/min, upper water phase transition in new pipe; Add equal-volume Virahol, mixing, leave standstill 20min; 4 DEG C of centrifugal 10min of 10000r/min, remove supernatant; Precipitate by 1mL 75%DEPC washing with alcohol; 4 DEG C of centrifugal 5min of 5000r/mim, discard liquid; After room temperature is dried, add the ddH2O water of 30 μ L DEPC process, dissolve RNA, get 1 μ L RNA at NanoDropND-2000 (NanoDrop company of the U.S.) upper mensuration RNA concentration and A260/280 ratio.If ratio >1.7, thinks and reaches specification of quality.The RNA that extracting obtains is placed in one 80 DEG C of refrigerators and saves backup.
Table 1: part sample rna extracting concentration and spectrophotometer ratio result
4.3cDNA synthesizes: with 2 μ g total serum IgE for template, according to Bestar qPCR RT Kit specification sheets preparation reverse transcription reaction system, and 5 × RT damping fluid 4.0 μ L; RT Enzyme Mix 1.0 μ L; Primer Mix RnaseFree dH2O 4.0 μ L is totally 20 μ L, is placed in and carries out .37 DEG C, 60min on ice; 98 DEG C, 10min, synthesis cDNA, after collecting ,-20 DEG C save backup.
As shown in the table:
Table 3: reverse transcription system is detailed
RNA 2.0ug
Add up to 10.0ul;
65 DEG C of 5min, on ice chillings;
4.4 fluorescence quantitative RT-RCRs detect miRNA:
Specific primer sequence is retrieved in miRBASE, miRNA and U6 sequence is as follows:
Table 3: each miRNA to be measured and internal reference specific primer sequence
Above-mentioned primer is synthesized by Shanghai Sheng Gong company.
The condition optimizing of fluorescence quantitative RT-RCR is to Mg
2+, primer isoconcentration is optimized.
The reaction system of Real time pcr amplification is 20 μ L (DBI
sybrGreen qPCRmaster Mix) be specially
sybrGreen qPCR master Mix 10 μ L; PCR forward primer (10 μm of ol/L) 0.5 μ L; PCR reverse primer (10 μm of ol/L) 0.5 μ L; DNA profiling (above-mentioned cDNA) 1.0 μ L; DH
2o (sterile purified water) 8.0 μ L.
Reaction conditions is: 94 DEG C of 2min, 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 20s, 40 circulations, and adopt Agilent Stratagene quantitative real time PCR Instrument Mx3000P to carry out fluorescent quantitation detection, reaction terminates the Ct value automatically calculated by computer in each reaction tubes.Each reaction all repeats 3 times, gets its mean value.
As shown in the table:
Table 4:PCR reaction system is detailed
5. experimental result
5.1miRNA dissolves and amplification curve analysis:
As shown in Figures 1 to 8, the Realtime pcr amplification curve of miRNA and U6 to be detected is all in " S " type; PCR primer melting curve is unimodal, and the good and reliable results of goal gene specificity of amplification is described.
5.2miRNA expression level
The relative expression levels of miRNA is with U6 as internal reference, and result adopts △ △ Ct method to analyze:
Δ CT tumour=CT tumour-CT internal reference, Δ CT is normal=normal-CT internal reference of CT, Δ Δ CT=Δ CT tumour-Δ CT is normal.Its expression level of the higher expression of Δ CT value is lower, and namely Δ Δ CT value was greater than for 0 (2-Δ Δ CT is less than 1), then represent that this miRNA is low expression in tumour; Δ Δ CT value is less than 0 (2
-Δ Δ CTbe greater than 1), then represent that this miRNA is high expression level in tumour; Δ Δ CT value equals 0 (2
-Δ Δ CTequal 1), then represent that this miRNA is identical with the expression level in control group in tumour, result histogram is described.
5.2.1miR-1183 the expression amount in expression normal healthy controls group blood plasma is 0.026 (0.005,0.047); In intraepithelial neoplasia group, expression amount is 0.225 (0.125,0.840); In colorectal cancer group, expression amount is 0.236 (0.038,1.899).Blood plasma miR-1183 at the expression amount of intraepithelial neoplasia group and colorectal cancer group higher than normal healthy controls group (see Fig. 9).
5.2.2miR-938 expression
Expression amount in normal healthy controls group blood plasma is 0.007 (0.001,0.067); In intraepithelial neoplasia group, expression amount is 0.045 (0.008,0.324); In colorectal cancer group, expression amount is 0.066 (0.006,0.527).The expression amount of blood plasma miR-938 in intraepithelial neoplasia group and colorectal cancer group is higher than normal healthy controls group (see Figure 10).
5.2.3miR-490-3p expression
Expression amount in normal healthy controls group blood plasma is 0.002 (0.001,0.004); In intraepithelial neoplasia group, expression amount is 0.010 (0.003,0.151); In colorectal cancer group, expression amount is 0.115 (0.004,0.326).Blood plasma miR-490-3p at the expression amount of intraepithelial neoplasia group and colorectal cancer group higher than normal healthy controls group (see Figure 11).
5.2.4miR-490-5p expression
Expression amount in normal healthy controls group blood plasma is 0.169 (0.047,0.591); In intraepithelial neoplasia group, expression amount is 0.000 (0.000,0.009); In colorectal cancer group, expression amount is 0.000 (0.000,0.326).Blood plasma miR-490-5p at the expression amount of intraepithelial neoplasia group and colorectal cancer group lower than normal healthy controls group (see Figure 12).
5.2.5miR-592 expression
Expression amount in normal healthy controls group blood plasma is 0.073 (0.004,0.268); In intraepithelial neoplasia group, expression amount is 0.001 (0.000,0.004); In colorectal cancer group, expression amount is 0.001 (0.000,0.326).Blood plasma miR-592 at the expression amount of intraepithelial neoplasia group and colorectal cancer group lower than normal healthy controls group (see Figure 13).
5.2.6miR-193b expression
Expression amount in normal healthy controls group blood plasma is 1.948 (1.065,5.092); In intraepithelial neoplasia group, expression amount is 30.695 (8.124,145.889); In colorectal cancer group, expression amount is 17.447 (0.326,100.677).Blood plasma miR-193b at the expression amount of intraepithelial neoplasia group and colorectal cancer group higher than normal healthy controls group (see Figure 14).
5.2.7miR-1290 expression
Expression amount in normal healthy controls group blood plasma is 0.007 (0.000,0.042); In intraepithelial neoplasia group, expression amount is 0.305 (0.151,0.929); In colorectal cancer group, expression amount is 0.432 (0.070,1.386).Blood plasma miR-1290 at the expression amount of intraepithelial neoplasia group and colorectal cancer group higher than normal healthy controls group (see Figure 15).
5.3 statistical procedures results
Application SPSS20.0 statistical software software carries out Mann-Whitney U inspection and Kruskal-Wallis inspection to real-time fluorescence quantitative PCR detected result; By Receiver operating curve (receiveroperator characteristic curve, ROC) judge the sensitivity of each miRNA expression level in diagnosis of colorectal carcinoma and specific degree, find best miRNA combination by Multivariate logistic regression analysis.P<0.05 is that difference has statistical significance.
5.3.1 the relation between the expression level of blood plasma miRNA and colorectal cancer patients Clinical symptoms
Expression in colorectal cancer patients blood plasma and clinical and pathological data are analyzed, the expression level of miRNA and the sex of examined person in result display blood plasma, age all irrelevant (P>0.05), but it is closely related with colorectal diseases, intraepithelial neoplasia group and normal healthy controls group, colorectal cancer group and normal healthy controls group all have significant difference (P<0.05), but intraepithelial neoplasia group is compared with colorectal cancer group, difference not statistically significant (P>0.05) between the two, in table 5.
Table 5: the relation between blood plasma miRNA expression amount and clinical characteristic
5.3.2 single blood plasma miRNA is as the usefulness of diagnosis of colorectal carcinoma mark
ROC tracing analysis result shows, sensitivity when alone blood plasma miR-1183 relative quantification value carries out diagnosis of colorectal carcinoma (threshold value is 0.132) is 92.2%, specific degree is 65.4%, and area under curve (area underthe curve, AUC) is 0.794; Sensitivity when alone blood plasma miR-938 relative quantification value carries out diagnosis of colorectal carcinoma (threshold value is 0.028) is 85.7%, and specific degree is 69.2%, AUC is 0.780; Sensitivity when alone blood plasma miR-490-3p relative quantification value carries out diagnosis of colorectal carcinoma (threshold value is 0.004) is 98.7%, and specific degree is 65.4%, AUC is 0.829; Sensitivity when alone blood plasma miR-193b relative quantification value carries out diagnosis of colorectal carcinoma (threshold value is 3.748) is 98.7%, and specific degree is 53.8%, AUC is 0.702; Sensitivity when alone blood plasma miR-1290 relative quantification value carries out diagnosis of colorectal carcinoma (threshold value is 0.056) is 100%, and specific degree is 57.7%, AUC is 0.805; The equal >0.7 of AUC of miR-1183, miR-938, miR-490-3p, miR-193b and miR-1290, and difference all has statistical significance (p<0.01), these 5 blood plasma miRNA are pointed out all to have certain accuracy and feasibility as the biomarker of colorectal cancer separately.See Figure 16-22 and table 6.
Table 6: blood plasma miRNA is as the specific degree of diagnosis of colorectal carcinoma mark, susceptibility and ROC area under curve
| Threshold value | Sensitivity | Specific degree | AUC | P value | |
| miR-1183 | 0.132 | 92.2％ | 65.4％ | 0.794 | 0.000 |
| miR-938 | 0.028 | 85.7％ | 69.2％ | 0.780 | 0.000 |
| miR-490-3p | 0.004 | 98.7％ | 65.4％ | 0.829 | 0.000 |
| miR-193b | 3.748 | 98.7％ | 53.8％ | 0.702 | 0.002 |
| miR-1290 | 0.056 | 100％ | 57.7％ | 0.805 | 0.000 |
5.3.3 the foundation of blood plasma miRNA combination
Current plasma of colorectal cancer miRNA research mostly is single or several miRNA expression study, its diagnostic is relatively low, if can screen from genomic level, thus the blood plasma miRNA specifically expressing setting up colorectal cancer differential expression composes the early diagnostic rate that will significantly improve colorectal cancer.
The present invention adopts the mode progressively verified, first differential expression miRNA is screened, and then adopt RT-qPCR technology to verify one by one the differential expression miRNA filtered out, finally find and confirm that miR-1183, miR-938, miR-490-3p, miR-490-5p, miR-592, miR-193b and miR-1290 have notable difference in colorectal cancer patients and healthy population blood plasma.In this research, RT-qPCR result display blood plasma miR-1183, miR-938, miR-490-3p miR-193b and miR-1290 high expression level in colorectal cancer; Blood plasma miR-490-5p, miR-592 be low expression in colorectal cancer, accurately can differentiate colorectal cancer and normal healthy controls group.
Further, the present invention is by miR-1183 wherein, miR938, miR-490-3p, miR-490-5P and miR-1290 is built into 5-miRNA combination, introduce multiple logistic regression equation simultaneously, the relative expression quantity be namely combined in by this 5-miRNA in blood plasma introduces multiple logistic regression equation, set up logit (P) discrimination formula as follows: logit (P)=-8.653+49.783 × (miR-1183)+16.666 × (miR-938)+235.243 × (miR-490-3p)+0.690 × (miR-193b)+18.096 × (miR-1290).
Based on the checking of above-mentioned logit (P) discrimination formula, surprisingly find that this 5-miRNA combination has higher diagnostic sensitivity and specific degree to colorectal cancer.
The AUC of this blood plasma of ROC curve display 5-miRNA combined diagnosis colorectal cancer is 0.997, and sensitivity is 98.7%, and specific degree is 100% (see Figure 23).This experimental result is significantly better than traditional tumour mark CEA (see " Duffy; M.J.; et al. " Clinical utility of biochemical markers in colorectalcancer:European Group on Tumour Markers (EGTM) guidelines. " European journalof cancer 39.6 (2003): 718-727. "), the document is reported, the susceptibility of CEA to diagnosis of colorectal carcinoma only has 30-40%, specificity about 87%, result is unsatisfactory.
Intraepithelial neoplasia refers to that structure and cytological abnormal expand to the first half of epithelium, and even holostrome, its cytology and weave construction have the epithelial lesion of malignant characteristics, but without any infiltrating the evidence of interstitial, comprise severe dysplasia and carcinoma in situ, continue development if let alone down, finally can become cancer, be most important precancerous lesion.Early discovery Colon and rectum intraepithelial neoplasia of clarifying a diagnosis, carrying out excision intraepithelial neoplasia is in time reduce one of the most effective means of colorectal cancer incidence rate.
TNM I phase colorectal cancer refers to that colorectal cancer has invaded submucosa (T1) or tumor invading intestines wall muscularis propria (T2), there is no regional lymph node metastasis (N0), there is no distant metastasis (M0), this phase colorectal cancer only needs to carry out tumor resection in treatment, without the need to complementary chemicotherapy, Post operation 5 years survival rates are up to more than 90%.
Therefore, high-level for Colon and rectum intraepithelial neoplasia patient and I phase colorectal cancer patients bring in research by the present invention further, to realizing the early diagnosis to colorectal cancer precancerous lesion.Experimental result shows, above-mentioned blood plasma 5-miRNA combination also has higher higher diagnostic sensitivity and specific degree to Colon and rectum high-level intraepithelial neoplasia patient (see Figure 24) and the colorectal cancer patients of TNM I phase (see Figure 25).In fig. 24,5-miRNA combination differentiates the senior intraepithelial neoplasia (cin) of colorectal cancer, sensitivity=88.7%, specific degree=86.5%, AUC=0.734 from different grouping.In fig. 25,5-miRNA combination differentiates I phase colorectal cancer, sensitivity=90.5%, specific degree=89.8%, AUC=0.855 from different grouping.
In sum, the test kit constructed by this blood plasma 5-miRNA combination is expected to for the diagnosis to early stage colorectal cancer and precancerous lesion thereof.
This blood plasma 5-miRNA combination also can be used for building the test kit distinguishing the blood plasma of at least one colorectal cancer patients and the blood plasma of at least one healthy individuals.This test kit comprises above-mentioned colorectal cancer early diagnosis marker, and wherein, at least one contrast blood plasma is from healthy individuals.
Claims (10)
1. a colorectal cancer early diagnosis marker, it is characterized in that: described diagnosis marker is made up of 7 kinds of nucleic acid molecule, described nucleic acid molecule comprises the nucleic acid molecule of coding blood plasma miR-1183, miR-938, miR-490-3p, miR-490-5p, miR-592, miR-193b and miR-1290; Described nucleic acid molecule contrasts differential expression in blood plasma with at least one at least one target blood plasma.
2. colorectal cancer early diagnosis marker according to claim 1, is characterized in that: coding blood plasma miR-1183, miR-938, miR-490-3p, miR-490-5p, miR-592, miR-193b are raised compared with the expression contrasted at least one in blood plasma with the expression of nucleic acid molecule at least one target blood plasma of miR-1290; And the expression of nucleic acid molecule at least one target blood plasma of coding blood plasma miR-490-5p with miR-592 is lowered compared with the expression contrasted at least one in blood plasma.
3. for differentiating a diagnostic kit for early stage colorectal cancer, it is characterized in that: described diagnostic kit comprises colorectal cancer early diagnosis marker as claimed in claim 1.
4. a colorectal cancer early diagnosis marker, it is characterized in that: described diagnosis marker is made up of 5 kinds of nucleic acid molecule, described nucleic acid molecule comprises the nucleic acid molecule of coding blood plasma miR-1183, miR-938, miR-490-3p, miR-490-5p, miR-592, miR-193b and miR-1290; The expression of described nucleic acid molecule at least one target blood plasma is raised compared with the expression contrasted at least one in blood plasma.
5. for differentiating a diagnostic kit for early stage colorectal cancer, it is characterized in that: described diagnostic kit comprises colorectal cancer early diagnosis marker as claimed in claim 4.
6. colorectal cancer early diagnosis kit according to claim 5, it is characterized in that, described test kit also comprises a regression model, set up logit (P) discrimination formula as follows: logit (P)=-8.653+49.783 × (miR-1183)+16.666 × (miR-938)+235.243 × (miR-490-3p)+0.690 × (miR-193b)+18.096 × (miR-1290), wherein, miR-1183, miR-938, miR-1290, the expression level of miR-490-3p and miR-193b is that internal reference detection obtains with U6, the expression of logit (P) value in model at least one target blood plasma is raised compared with the expression contrasted at least one in blood plasma.
7. according to the diagnostic kit one of claim 3,5 and 6 Suo Shu, it is characterized in that: described early stage colorectal cancer is Colon and rectum intraepithelial neoplasia.
8. according to the diagnostic kit one of claim 3,5 and 6 Suo Shu, it is characterized in that: described early stage colorectal cancer is the colorectal cancer of TNM I phase.
9. the test kit for the blood plasma of the blood plasma He at least one healthy individuals of distinguishing at least one colorectal cancer patients, it is characterized in that: comprise colorectal cancer early diagnosis marker according to claim 4, wherein, at least one contrast blood plasma is from healthy individuals.
10. test kit according to claim 9, it is characterized in that, described test kit also comprises a regression model, set up logit (P) discrimination formula as follows: logit (P)=-8.653+49.783 × (miR-1183)+16.666 × (miR-938)+235.243 × (miR-490-3p)+0.690 × (miR-193b)+18.096 × (miR-1290), wherein, miR-1183, miR-938, miR-1290, the expression level of miR-490-3p and miR-193b is that internal reference detection obtains with U6, the expression of logit (P) value in model at least one target blood plasma is raised compared with the expression contrasted at least one in blood plasma.
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