CN105671178B - Serum microRNA diagnosing cancer of liver marker and kit - Google Patents
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Abstract
The invention discloses the hepatocellular carcinoma diagnosis markers being made of serum microRNA to combine, and the kit containing the combination of these markers.The diagnosing cancer of liver marker group is amounted to including following microRNA:has-miR-145, hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-192 and hsa-miR-505.The present invention can be used for diagnosing cancer of liver.
Description
Technical field
The invention belongs to biomedical diagnostic fields, and in particular to it is combined by the diagnosing cancer of liver that serum microRNA is formed,
The serum microRNA includes hsa-miR-145, hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-
MiR-192 and hsa-miR-505, and kit and its application for diagnosing cancer of liver.
Background technique
Primary carcinoma of liver is the malignant tumour of high incidence common in world wide, high lethality rate, wherein liver cell liver
Cancer (abbreviation liver cancer, Hepatocellular Carcinoma, HCC) accounts for 90% of primary carcinoma of liver or more.The World Health Organization
" the global cancer report 2014 " delivered points out that global liver cancer new cases ranked the 5th of malignant tumour in 2012, causes
Dead rate then occupies second, wherein the new hair and death of Chinese liver cancer account for about the half of global case.Have due to lacking
The screening means of effect cause the liver cancer patient of 60%-70% to be in advanced stage when diagnosing, miss radical excision chance.As it can be seen that
Early diagnosis, early treatment are to increase liver cancer patient survival rate, reduce the most effective approach of mortality of liver cancer.
The occurrence and development process of liver cancer be one multifactor, the multistage, multi-step process.Liver caused by any reason is hard
Change is the most common risk factor of liver cancer.In Asia, the hepatitis of chronic HBV (HBV) initiation, cirrhosis are to induce
The most important risk factors of liver cancer.Most liver cancer patients are with hepatitis and cirrhosis pathological change, hard after hepatitis, liver
After the change stage, final development is liver cancer, and people are referred to as " liver cancer trilogy ".It is estimated that Chinese hepatitis B carriers quantity
Nearly 1.2 hundred million, wherein 20% will develop into chronic hepatitis B patient, the chronic hepatitis B patient of 15%-40% then will be into
The development of one step is cirrhosis.Therefore, long term monitoring is carried out to liver cancer high risk population, discovery and early warning early liver cancer, are convenient for early
It determines targetedly individuation control prece, delays or even prevent the generation of liver cancer, be conducive to improve life in patients.
Clinically mainly pass through serum alpha-fetoprotein (AFP) detection or imageological examination screening liver cancer at present.Although AFP is
It is widely used in Hepatocarcinoma screening, but its sensibility for detecting liver cancer is not high enough: in the patient that clinical diagnosis goes out liver cancer, about 35-
The AFP level of 60% liver cancer patient is still below warning value (20ng/ml)；Sensibility in subclinical carcinoma of liver is then only 14-
40%.Although the Image Examinations such as ultrasound, CT, MRI can be used as the supplement of AFP detection, since long term follow-up sieves
Then somewhat expensive is looked into, and the recall rate of these methods depends on tumor size and the experience of operator, thus is likely to occur mistake
The case where examining, failing to pinpoint a disease in diagnosis.Thus, it is found that new hypersensitivity, high specific, and hepatocarcinoma early diagnosis mark that is cheap and being easy to detect
Will object has important clinical meaning.
MicroRNA is a kind of endogenous small molecule non-coding RNA being widely present in eucaryote, length 18-24
A nucleotide (nt).MicroRNA inhibits the expression of target gene, regulating cell differentiation, proliferation, apoptosis etc. by post-transcriptional level
Vital movement plays a significant role in a variety of physiology such as embryonic development, organism metabolism, disease development and pathologic process.
In recent years, researcher detects microRNA in a variety of body fluid such as blood, saliva, urine, proposes circulation microRNA's
Concept.The humoral specimens such as blood are easily obtained, and clinic can have strong operability and traumatic small, and it is good to recycle microRNA stability,
Detection convenience, therefore, circulation microRNA have the potential as the non-invasive biomarker of the diseases such as tumour, are suitable for high-risk
Mass screening.
Recently research is pointed out, liver cancer patient has different circulation microRNA express spectras from non-liver cancer control.For example,
The level of miR-21, miR-122, miR-223 in liver cancer patient and HBV hepatitis serum is above Healthy People；And
Raised miR-21 is lowered after patient's resection in liver cancer patient blood plasma；Li et al. people passes through detection 513 individual serum
MicroRNA express spectra, it is determined that distinguish the sort merge of HBV hepatitis and Healthy People and liver cancer patient and Healthy People；
Zhou et al. has detected the level of nearly thousand parts of plasma specimen microRNA, finds by miR-122, miR-192, miR-21, miR-
The categorization module that 223, miR-26a, miR-27a and miR-801 etc. seven circulation microRNA are constituted, can distinguish liver cancer and institute
Have non-liver cancer group summation (including Healthy People, hepatitis, liver cirrhosis patient), and the liver cancer of diagnosable AFP negative.These researchs mention
Show potential of the circulation microRNA as diagnosing cancer of liver marker.
However about research of the microRNA as diagnosing cancer of liver marker is recycled, there are still following deficiencies at present: (1) big
Part research only picks the microRNA in liver cancer tissue expression imbalance of forefathers' report as candidate index, possibly can not
The case where objectively responding circulation microRNA comprehensively；(2) research sample size in part is few, lacks multicenter verifying；(3) control setting
It is not perfect, it is difficult to illustrate that determining marker is the special diagnosis marker of liver cancer.Therefore, it is still necessary being found to have at present
The hepatocarcinoma early diagnosis marker of clinical value, for the monitoring of liver cancer high risk population, thus the early stage microhepatia of early warning early
Cancer takes appropriate control measure convenient for clinician in time.
In the patent application 201410623463.3 of Zhongshan University, disclose by hsa-miR-29a, hsa-miR-29c,
Hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192 and hsa-miR-505 totally seven microRNA
The diagnosing cancer of liver marker of composition has the characteristics that early diagnosis sensibility is high, specificity is good.But for molecular diagnosis
Speech, guarantee diagnostic sensitivity and specificity under the premise of, reduce diagnostic marker molecule quantity can be effectively reduced diagnosis at
The complexity of this and operation.Therefore, this field, which exists, provides the demand of other diagnosing cancer of liver markers combination.
Summary of the invention
The purpose of the present invention is aiming at the above technical problems to be solved, provide it is a kind of it is easy to operate, being capable of efficient diagnosis
The diagnosing cancer of liver marker of liver cancer.
To realize the above-mentioned technical purpose, the present invention provides a kind of diagnosing cancer of liver marker, the diagnosing cancer of liver markers
Nucleic acid molecules including being separately encoded following microRNA molecule combination:
1) group unification: hsa-miR-145；
2) two: hsa-miR-145, hsa-miR-29a, hsa-miR-133a, hsa-miR-192, hsa-miR- are combined
505；
3) three: hsa-miR-145, hsa-miR-29c, hsa-miR-133a, hsa-miR-192, hsa-miR- are combined
505。
In a preferred embodiment, level of the microRNA in liver cancer patient blood serum be higher than Healthy People,
Hepatitis B carriers, chronic hepatitis B patient or liver cirrhosis patient.Preferably, the liver cancer is primary liver cell liver
Cancer.
On the other hand, the present invention also provides the microRNA molecule combinations for diagnosing cancer of liver comprising following
MicroRNA combination:
1) group unification: hsa-miR-145；
2) two: hsa-miR-145, hsa-miR-29a, hsa-miR-133a, hsa-miR-192, hsa-miR- are combined
505；
3) three: hsa-miR-145, hsa-miR-29c, hsa-miR-133a, hsa-miR-192, hsa-miR- are combined
505。
In a preferred embodiment, level of the microRNA in liver cancer patient blood serum be higher than Healthy People,
Hepatitis B carriers, chronic hepatitis B patient or liver cirrhosis patient.Preferably, the liver cancer is primary liver cell liver
Cancer.
Specifically, the present invention has obtained above-mentioned three kinds of diagnosing cancer of liver being made of serum microRNA by following steps
Combination:
1. screening the candidate serum of liver cancer patient and chronic hepatitis B patient difference by high throughput qPCR Array
microRNA；
2. real-time fluorescence quantitative PCR verifies candidate serum microRNA；
3. establishment can distinguish liver cancer and non-cancer pair in the training group being made of Healthy People, hepatitis, cirrhosis, liver cancer patient
According to the serum microRNA combination of (including Healthy People, hepatitis and liver cirrhosis patient)；
4. in the effect for the serum microRNA combined diagnosis liver cancer that two individual authentication group verification steps 3 are established；
5. the serum microRNA combination that analytical procedure 3 is established is in the liver cancer patient of small liver cancer, BCLC early stage and AFP negative
In diagnosis effect.
Carry out analysis to experimental result and ASSOCIATE STATISTICS is shown: in above-mentioned steps 1, inventor passes through high throughput qPCR
Array screening has determined 19 candidate microRNA, and demonstrates 19 candidate microRNA in step 2 in liver cancer patient blood
Level in clear increases.Level of the candidate microRNA in training group (sample size is 257 parts) is further had detected, and really
Differentiation liver cancer has been found to combine with the optimal serum microRNA that non-cancer compares.And then in 2 (sample of independent validation group 1 and validation group
This amount is respectively 352,139 parts) in demonstrate serum microRNA combination and can distinguish liver cancer and compareed with non-cancer.It is same with this
When, traditional Hepatocarcinoma screening means are compared in inventor's discovery, such as AFP, serum microRNA combination small liver cancer, BCLC early stage and
There is better diagnosis effect in the liver cancer patient of AFP negative.
On the other hand, the invention also discloses a kind of diagnostic kit for liver cancer, it includes be respectively used to examine
Survey following microRNA molecule reagent horizontal in serum:
1) group unification: hsa-miR-145；
2) two: hsa-miR-145, hsa-miR-29a, hsa-miR-133a, hsa-miR-192, hsa-miR- are combined
505；
3) three: hsa-miR-145, hsa-miR-29c, hsa-miR-133a, hsa-miR-192, hsa-miR- are combined
505。
In one embodiment, described for detecting microRNA molecule reagent horizontal in serum as real-time fluorescence
Quantitative PCR related reagent.
In another preferred embodiment, the diagnostic kit further includes serum RNA extraction system and reverse transcription
System.In preferred embodiments, which further includes the analysis method for assessing whether suffering from hepatic cancer.
In a preferred embodiment, the diagnostic kit includes that can be respectively used to detect following microRNA water
Flat LNA Mdification primer:
1) group unification: hsa-miR-145；
2) two: hsa-miR-145, hsa-miR-29a, hsa-miR-133a, hsa-miR-192, hsa-miR- are combined
505；
3) three: hsa-miR-145, hsa-miR-29c, hsa-miR-133a, hsa-miR-192, hsa-miR- are combined
505。
Preferably, described to combine reagent horizontal in serum for detecting microRNA molecule as real time fluorescent quantitative
PCR related reagent.
It is furthermore preferred that the diagnostic kit further includes the LNA Mdification primer for detecting outer source reference NC67.Joined by external source
It is calibrated according to NC67, obtains level 2 of above-mentioned 6 target genes in serum specimen to be checked-ΔCt(Δ Ct=Cttaget-
Ctreference).It will test sample microRNA level according to microRNA threshold value and be assigned a value of 1 or 0 respectively, realize discretization.
For a group unification, the liver cancer that is diagnosed as being 1 with hsa-miR-145 discretization threshold value, the diagnosis that discretization threshold value is 0
For non-liver cancer.
For combination two, combination three, then serum microRNA combination is further analyzed according to logistic regression method respectively and commented
Estimate person under test whether suffering from hepatic cancer:
1) two: Logit (p=HCC)=- 0.356-0.536*hsa-miR-145-0.335*hsa-miR-29a- is combined
0.559*hsa-miR-133a-0.477*hsa-miR-192-0.445*hsa-miR-505；
2) three: Logit (p=HCC)=- 0.351-0.597*hsa-miR-145-0.300*hsa-miR-29c- is combined
0.536*hsa-miR-133a-0.486*hsa-miR-192-0.500*hsa-miR-505。
Wherein, hsa-miR-145, hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-192,
Hsa-miR-505 is the numerical value after corresponding serum microRNA detection level discretization, and is with Logit (p=HCC)=0.5
Diagnostic threshold, is diagnosed as liver cancer higher than 0.5, is diagnosed as non-liver cancer lower than 0.5.The kit can be applied to liver cancer and examine
It is disconnected.
Serum microRNA combination of the invention, which is detected and early diagnosed for liver cancer, to be advantageous in that: (1) sample is more
It is easily obtained, the clinic property grasped is stronger and traumatic smaller, and serum microRNA stability is more preferable, detects more convenient；(2)
Experimental method is highly developed, and detection process is easier, is easy to repeat, and can be completed by common technician；(3) present invention adopts
It is studied with high flux screening, multicenter verifying and in people at highest risk's sample, to the effect of serum microRNA combination
And diagnostic kit is fully assessed, the application of above method and strategy ensure that the present invention in liver cancer clinical diagnosis
Potential using value, and provide referential methods and strategies for the development of other diseases biomarker；(4) serum
MicroRNA diagnosing liver cancer kit can reflect the morbid state of liver cancer patient much sooner, avoid previously many and diverse detection, section
It makes an appointment human cost, takes personalized control prece in time convenient for clinician.
Detailed description of the invention
Fig. 1 is ROC curve figure of the embodiment of the present invention 4,5 in training group, validation group.Specifically, training group (A), test
Serum microRNA combination and AFP distinguish liver cancer and compare (above) and high-risk people with non-cancer in card group 1 (B) and validation group 2 (C)
The ROC curve figure of group's (following figure).
Fig. 2 is ROC curve figure of the embodiment of the present invention 6 in small liver cancer patient (tumour≤3cm).Specifically, training group
(A), validation group 1 (B), validation group 2 (C) and training group are combined with serum microRNA in all validation groups (D) and AFP is distinguished
Small liver cancer patient compares the ROC curve figure of (left figure) and people at highest risk's (right figure) with non-cancer.
Fig. 3 is ROC curve figure of the embodiment of the present invention 7 in different BCLC by stages liver cancer patient.Specifically, serum
MicroRNA combination and AFP distinguish BCLC 0+A phase (A), BCLC B phase (B) or BCLC C phase (C) liver cancer patient and compare with non-cancer
(above) and the ROC curve figure of people at highest risk's (following figure).
Fig. 4 is ROC curve figure of the embodiment of the present invention 8 in AFP negative liver cancer patient (AFP≤20ng/ml).Specifically
Ground, training group (A), validation group 1 (B), validation group 2 (C) and training group are combined with serum microRNA in all validation groups (D)
Distinguish the ROC curve figure that AFP negative liver cancer patient compares (left figure) and people at highest risk's (right figure) with non-cancer.
Specific embodiment
To keep the present invention easier to understand, in the following with reference to the drawings and specific embodiments, the present invention is further explained.Ying Li
Solution, these embodiments are merely to illustrate the present invention, rather than limit the scope of the invention；Described attached drawing is also only signal
Property, it is considered to be it is unrestricted.
Embodiment 1: the acquisition and preparation of serum specimen
Inventor acquires Healthy People (HC), hepatitis B carriers (IHC), chronic in August, 2009 in August, 2014
The blood preparation of hepatitis B patient (CHB), liver cirrhosis patient (LC) and liver cancer patient (HCC), these crowds meet into a group mark
Quasi- (table 1), and according to gender, the principle of age-matched, set liver cancer and its control group sample.
Training group: Healthy People (51) that in August, 2009 is acquired in March, 2012, chronic hepatitis B patient (51),
257 parts of serum specimens of liver cirrhosis patient (47) and liver cancer patient (108).
Healthy People (60) that validation group 1:2012 April acquires in April, 2013, chronic hepatitis B patient (68
Example), 352 parts of serum specimens of liver cirrhosis patient (71) and liver cancer patient (153).
Healthy People (48) that validation group 2:2013 May in August, 2013 acquires, hepatitis B carriers (42) with
And 139 parts of serum specimens of liver cancer patient (49).
The inclusion criteria of 1. group of participants of table
Hepatopathy research association, the Ru Zu conditioned reference U.S. (AASLD) practice guideline in 2009.
With reference to liver biopsy Metavir system.
The Clinical symptoms of above-mentioned group of participants is shown in Table 2.
Extract preoperative liver cancer patient, Healthy People, hepatitis B carriers, chronic hepatitis B patient and liver cirrhosis patient
Each 4ml of peripheric venous blood is stood more than half an hour for 4 DEG C in dry heparin tube.Subsequent 400g, 4 DEG C of centrifugation 10min take
Clearly, further 1800g, 4 DEG C of centrifugation 10min take supernatant, obtain serum, save backup after packing in -80 DEG C.
The clinical pathologic characteristic of 2. training group of table and validation group participant
Embodiment 2:qPCR Array and its data analysis
Choose the blood that 6 liver cancer patients are preoperative and 8 chronic hepatitis B patients are away from last inspection more than at least a year
Clear sample is screened for qPCR Array.These patients are male, and its age mean value, distribution are without significant difference (table 3).
Table 3. is used for the sample clinical pathologic characteristic of qPCR Array analysis
The present invention is using Applied Biosystems companyArray Human MicroRNA method sieve
The microRNA of liver cancer Yu chronic hepatitis B difference is selected, detects the level of 754 known mankind microRNA altogether.Specific step
Suddenly referring to the website Applied Biosystems.After obtained initial data is calibrated, using Significant Analysis
Of Microarray (SAM) analysis method selects difference microRNA, finally screens the candidate for obtaining 19 for subsequent authentication
MicroRNA (table 4).
The candidate microRNA and outer source reference information that 4. present invention of table uses
Products number
Embodiment 3: it is horizontal that real-time fluorescence quantitative PCR detects training group sample microRNA
1.1 serum RNA are extracted
The present invention is extracted using Trizol reagent, and by phenol/chloroform purifying, isopropanol precipitating, that glycogen helps is heavy
Method obtains serum RNA, the specific steps are as follows:
1. take 200 μ l serum, it is preferably added and isometric be mixed with cel-miR-67 (NC67 is based on and human genome
Sequence does not have double-stranded RNA designed by the nematode miR-67 maturation body sequence of homology, final concentration of 0.2nM, 4) sequence is shown in Table
Trizol lysate, sufficiently oscillation mix, ice bath 15min.
2. 100 μ l, which are added, is pre-chilled chloroform, oscillation is mixed, and 4 DEG C, 12000g is centrifuged 15min.
3. shifting supernatant, isometric pre-cooling phenol/chloroform (1:1) is added, oscillation mixes, and 4 DEG C, 14000g is centrifuged 10min.And
It is primary to repeat the step.
4. shifting supernatant, isometric pre-cooling chloroform is added, oscillation mixes, and 4 DEG C, 14000g is centrifuged 15min.
5. shifting supernatant, it is preferably added isometric isopropanol and glycogen (final concentration of 200 μ g/ml), oscillation mixes,
4 DEG C, 16000g is centrifuged 30min.
6. carefully toppling over supernatant, precipitated once with 70% ethanol washing of 1ml, 4 DEG C, 16000g is centrifuged 10min.
7. abandoning supernatant, after ethyl alcohol volatilization, the dissolution of 10 μ l DEPC water is added, is placed in -80 DEG C and saves backup.
1.2 real-time fluorescence quantitative PCRs (RT-qPCR)
Present invention preferably uses Universal cDNA Synthesis Reverse Transcriptase kit, the serum of reciprocity volume
RNA carries out reverse transcription.SYBR Green qPCR master mix kit is used, further preferably to dilute 20 times
CDNA is template, and the primer of LNA modification carries out RT-qPCR detection.The Reverse Transcriptase kit, qPCR detection kit and
LNA Mdification primer is purchased from Exiqon company (Denmark).
It is calibrated by outer source reference NC67, obtains the expression value 2 of target microRNA-ΔCt(Δ Ct=Cttaget-
Ctreference).The results show that horizontal significant raising of 19 candidate microRNA in liver cancer patient blood serum.
Embodiment 4: optimal serum microRNA combination is determined in training group
By training group sample, according to 19 microRNA, respectively detection level arranges from big to small, and successively value (if any
Repetition values then only take once), sample is determined as by positive or negative monoid according to value, is obtained in conjunction with the given class analysis of sample
The sensibility and specificity of each value is obtained, ROC curve (Receiver Operating is further drawn
Characteristic Curve).Searching makes (sensibility+specificity)/2 values reach maximum point, the corresponding expression number of the point
Value is the threshold value of microRNA discretization.Further the sample for being more or less than threshold value is assigned a value of 1 or 0 respectively, realized discrete
Change, is used for further model construction.The microRNA discretization threshold value (table 4) that the present invention uses will be used for training group, verifying
The discretization of corresponding microRNA data in group, so that continuous variable is changed into two classified variables.
Modeling result shows that combination has the effect of good diagnosing liver cancer below:
1) group unification: hsa-miR-145；
2) two: hsa-miR-145, hsa-miR-29a, hsa-miR-133a, hsa-miR-192, hsa-miR- are combined
505；
3) three: hsa-miR-145, hsa-miR-29c, hsa-miR-133a, hsa-miR-192, hsa-miR- are combined
505。
For a group unification, the liver cancer that is diagnosed as being 1 with hsa-miR-145 discretization threshold value, the diagnosis that discretization threshold value is 0
For non-liver cancer.
For combination two, combination three, assess whether that the formula of suffering from hepatic cancer is respectively as follows:
(1) two: Logit (p=HCC)=- 0.356-0.536*hsa-miR-145-0.335*hsa-miR-29a- is combined
0.559*hsa-miR-133a-0.477*hsa-miR-192-0.445*hsa-miR-505；
(2) three: Logit (p=HCC)=- 0.351-0.597*hsa-miR-145-0.300*hsa-miR-29c- is combined
0.536*hsa-miR-133a-0.486*hsa-miR-192-0.500*hsa-miR-505。
Wherein, hsa-miR-145, hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-192,
Hsa-miR-505 is the numerical value after corresponding serum microRNA detection level discretization.
Said combination can distinguish liver cancer in training group and compare with non-cancer or people at highest risk, have good diagnosing cancer of liver
Effect shows that the AUC of serum microRNA combination is all larger than AFP20 or AFP400 (using 20 or 400ng/ml as AFP threshold
Value) AUC (Figure 1A).
It is disclosed by hsa-miR-29a, hsa-miR- compared in the patent application 201410623463.3 of Zhongshan University
29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192 and hsa-miR-505 totally seven
The diagnosing cancer of liver marker combination of microRNA composition, these three combinations can guarantee diagnosing cancer of liver sensibility and specificity
Under the premise of (table 5), as far as possible reduce diagnostic marker molecule quantity, be effectively reduced diagnosis cost and operation complicated journey
Degree.Particularly point out, group unification can satisfy clinical diagnosis requirement: the sensibility and specificity of single hsa-miR-145 is
More existing AFP diagnoses ideal (sensibility 82.4%/69.4%)；Combination two and combination three are then single hsa-miR-145's
On the basis of increase other miRNA, further improve sensibility and specificity, for liver cancer molecular diagnosis provide it is different
Selection.
The effect of liver cancer and non-cancer group is distinguished in table 5.microRNA combination
Embodiment 5: the effect of serum microRNA combined diagnosis liver cancer is verified in validation group
The serum microRNA combination established in training group is used for validation group diagnosing liver cancer.Similarly, it uses
Trizol is extracted and real-time fluorescence quantitative PCR detection method is tested.The combination can still distinguish liver in validation group 1,2
Cancer is compareed with non-cancer or people at highest risk, has good diagnosing cancer of liver effect, shows that the AUC of serum microRNA combination is big
In the AUC (B, C in Fig. 1) of AFP20 or AFP400.
Embodiment 6: serum microRNA combines the diagnosis effect in small liver cancer patient (tumour≤3cm)
The present invention, which further demonstrates serum microRNA combination, has the effect of good diagnosis of small hepatic cell carcinoma.In training
Group, validation group 1, in validation group 2, the AUC of three combined diagnosis small liver cancers is noticeably greater than AFP；Training group and three validation groups
Case combined analysis result equally shows that these combination differentiation small liver cancers are compareed with non-cancer or the AUC of people at highest risk is much larger than
AFP20 or AFP400 (Fig. 2), is respectively as follows:
Group unification: 0.881vs 0.750 or 0.571,0.810vs 0.750 or 0.571；
Combination two, combination three: 0.920vs 0.750 or 0.571,0.881vs 0.750 or 0.571.
Embodiment 7: serum microRNA combines the diagnosis effect in different BCLC by stages liver cancer patient
Serum microRNA combination has preferable diagnosis effect in different BCLC by stages liver cancer patient, these combinations
AUC is noticeably greater than the AUC of AFP, and especially in the BCLC 0+A phase, i.e. in early days, combined advantage is more obvious: with all non-by BCLC
Cancer control/people at highest risk is control, and in BCLC 0+A phase diagnosing cancer of liver, the AUC of said combination is respectively as follows: a group unification:
0.801/0.797；Combination two: 0.815/0.810；Combination three: 0.809/0.808；And AFP20 or AFP400 are then respectively
Or 0.649/0647 (Fig. 3) 0.735/0.709.
Embodiment 8: serum microRNA combines the diagnosis effect in AFP negative (AFP≤20ng/ml) liver cancer patient
Serum microRNA combination equally has good diagnosis effect in AFP negative liver cancer patient.With all non-cancer
Control/people at highest risk is control, and said combination is pre- training group, validation group 1,2 each group of validation group and all center combinations
The AUC (Fig. 4) for surveying AFP negative liver cancer is respectively as follows:
Group unification: 0.820/0.822,0.793/0.810,0.892/0.820 and 0.816/0.811；
Combine two: 0.844/0.847,0.792/0.798,0.881/0.842 and 0.822/0.817；
Combine three: 0.844/0.847,0.805/0.819,0.881/0.842 and 0.831/0.830.
Embodiment 9: the production of serum microRNA kit
Kit of the present invention be used for diagnosing cancer of liver, especially early liver cancer, by serum RNA extraction system, reverse transcription system,
Real-time fluorescence quantitative PCR system, primer system and the logistic regression analysis method for assessing whether suffering from hepatic cancer form.
In the serum RNA extraction system of the kit, inventor is extracted using Trizol reagent, and passes through phenol/chloroform
Extracting and purifying, isopropanol precipitating, glycogen help heavy method to obtain serum RNA.Inventor is repaired using a series of Exiqon company LNA
Primer system of the primer of decorations as the kit, for detecting following molecule: hsa-miR-145, hsa-miR-29a,
Hsa-miR-29c, hsa-miR-133a, hsa-miR-192, hsa-miR-505 and NC67 (outer source reference).
In real-time fluorescence quantitative PCR system, inventor uses the Reverse Transcriptase kit and SYBR of Exiqon company
Green qPCR master mix kit is detected.
For a group unification, the liver cancer that is diagnosed as being 1 with hsa-miR-145 discretization threshold value, the diagnosis that discretization threshold value is 0
For non-liver cancer.
And for combination two, combination three, then serum microRNA combination is further analyzed according to logistic regression method respectively
Assess person under test whether suffering from hepatic cancer:
Combine two: Logit (p=HCC)=- 0.356-0.536*hsa-miR-145-0.335*hsa-miR-29a-
0.559*hsa-miR-133a-0.477*hsa-miR-192-0.445*hsa-miR-505；
Combine three: Logit (p=HCC)=- 0.351-0.597*hsa-miR-145-0.300*hsa-miR-29c-
0.536*hsa-miR-133a-0.486*hsa-miR-192-0.500*hsa-miR-505。
Wherein, hsa-miR-145, hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143,
Hsa-miR-192, hsa-miR-505 are the numerical value after corresponding serum microRNA detection level discretization.With Logit (p=
It HCC)=0.5 is diagnostic threshold, being higher than 0.5 is liver cancer patient, is non-liver cancer patient lower than 0.5.
Claims (6)
1. nucleic acid molecules combination is preparing the purposes in diagnosing cancer of liver kit, the nucleic acid molecules combination is following by being separately encoded
The nucleic acid molecules composition of microRNA molecule combination:
1) group unification: hsa-miR-145, hsa-miR-29a, hsa-miR-133a, hsa-miR-192, hsa-miR-505；Or
2) two: hsa-miR-145, hsa-miR-29c, hsa-miR-133a, hsa-miR-192, hsa-miR-505 are combined；
The liver cancer is primary hepatocyte hepatocarcinoma.
2. purposes according to claim 1, wherein level of the microRNA in liver cancer patient blood serum is higher than health
People, hepatitis virus carrier, chronic hepatitis patient or liver cirrhosis patient.
3.microRNA molecular combinations are preparing the purposes in diagnosing cancer of liver reagent, and the microRNA molecule combination is by following
MicroRNA group is combined into:
1) group unification: hsa-miR-145, hsa-miR-29a, hsa-miR-133a, hsa-miR-192, hsa-miR-505；Or
2) two: hsa-miR-145, hsa-miR-29c, hsa-miR-133a, hsa-miR-192, hsa-miR-505 are combined；
The liver cancer is primary hepatocyte hepatocarcinoma.
4. purposes according to claim 3, which is characterized in that the microRNA in the microRNA combination suffers from liver cancer
Level in person's serum is higher than Healthy People, hepatitis virus carrier, chronic hepatitis patient or liver cirrhosis patient.
5. combining the diagnostic reagent of reagent horizontal in serum in preparation for diagnosing cancer of liver for detecting microRNA molecule
Purposes in box, the microRNA molecule combination are combined by following microRNA group:
1) group unification: hsa-miR-145, hsa-miR-29a, hsa-miR-133a, hsa-miR-192, hsa-miR-505；Or
2) two: hsa-miR-145, hsa-miR-29c, hsa-miR-133a, hsa-miR-192, hsa-miR-505 are combined；
The liver cancer is primary hepatocyte hepatocarcinoma.
6. purposes according to claim 5, described group of unification is respectively as follows: with the two logistic regression formula of combining
1) group unification: Logit (p=HCC)=- 0.356-0.536*hsa-miR-145-0.335*hsa-miR-29a-0.559*
hsa-miR-133a-0.477*hsa-miR-192-0.445*hsa-miR-505；
2) two: Logit (p=HCC)=- 0.351-0.597*hsa-miR-145-0.300*hsa-miR-29c-0.536* is combined
hsa-miR-133a-0.486*hsa-miR-192-0.500*hsa-miR-505；
Wherein hsa-miR-145, hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-192, hsa-miR-
505 be the numerical value after corresponding serum microRNA detection level discretization, and with Logit (p=HCC)=0.5 for diagnostic threshold,
It is diagnosed as liver cancer higher than 0.5, is diagnosed as non-liver cancer lower than 0.5.
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| CN104372087A (en) * | 2014-11-06 | 2015-02-25 | 中山大学 | Liver cancer diagnosis markers composed of serum microRNA (microribonucleic acid) and diagnosis kit |
| CN104450874A (en) * | 2014-04-11 | 2015-03-25 | 中国人民解放军军事医学科学院基础医学研究所 | Kit and method for detecting serum of liver cancer patient by taking miR-145 as marker |
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| CN104372087A (en) * | 2014-11-06 | 2015-02-25 | 中山大学 | Liver cancer diagnosis markers composed of serum microRNA (microribonucleic acid) and diagnosis kit |
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