CN104372087A - Liver cancer diagnosis markers composed of serum microRNA (microribonucleic acid) and diagnosis kit - Google Patents
Liver cancer diagnosis markers composed of serum microRNA (microribonucleic acid) and diagnosis kit Download PDFInfo
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Abstract
The invention discloses a liver cell liver cancer diagnosis combination composed of serum microRNA (microribonucleic acid) and a kit the combination. The liver cancer diagnosis combination comprises microRNAs:hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, has-miR-145, hsa-miR-192 and hsa-miR-505. The diagnosis combination and kit can be used for distinguishing a liver cancer patient from a healthy person, hepatitis virus carrier, chronic hepatitis patient or hepatocirrhosis patient.
Description
Technical field
The present invention relates to a diagnosing cancer of liver combination be made up of serum microRNA, described serum microRNA comprises hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192 and hsa-miR-505, be specifically related to one for the application of primary hepatocyte hepatocarcinoma early warning and early diagnosis kit, belong to biomedical diagnostic field.
Background technology
Primary hepatocarcinoma is high incidence common in world wide, the malignant tumour of high lethality rate, and wherein hepatocellular carcinoma (being called for short liver cancer, Hepatocellular Carcinoma, HCC) accounts for more than 90% of primary hepatocarcinoma." global cancer report 2014 " that the World Health Organization delivers points out, global liver cancer new cases in 2012 rank the 5th of malignant tumour, and its lethality rate then occupies second, and wherein Chinese liver cancer new is sent out and death all accounts for the half of global case.Owing to lacking effective examination means, be in late period when causing the liver cancer patient of 60%-70% to be diagnosed, miss radical excision chance.Visible, early diagnosis, early treatment increases liver cancer patient survival rate, reduces the most effective approach of mortality of liver cancer.
The generation evolution of liver cancer is the process of multifactor, multistage, multi-step.The liver cirrhosis that any reason causes is the modal Hazard Factor of liver cancer.In Asia, the hepatitis that chronic HBV (HBV) causes, liver cirrhosis are the topmost risk factors of induced hepatocellular carcinoma.Most liver cancer patient is with hepatitis and liver cirrhosis pathological change, and going through hepatitis, after the liver cirrhosis stage, finally developing into liver cancer, people are referred to as " liver cancer trilogy ".According to estimates, Chinese hepatitis B carriers quantity nearly 1.2 hundred million, wherein 20% will develop into chronic hepatitis B patient, and the chronic hepatitis B patient of 15%-40% then will be further development of liver cirrhosis.Therefore, long term monitoring is carried out to liver cancer high risk population, find early and early warning early hepatocarcinoma, be convenient to determine individuation control prece targetedly, delay the generation even preventing liver cancer, be conducive to improving life in patients.
The early detection research network (Early Detection Research Network, EDRN) that National Cancer Institute is founded clearly proposes the five steps of screening, assessment of cancer early sign thing: 1) screening finds possible marker molecules; 2) large-scale retrospective study analysis; 3) Detection results of retrospective analysis candidate markers in high risk population's sample of perspective collection; 4) Prospective Study candidate markers is to the Detection results of disease; 5) Prospective Clinical random test, examination general population.Detect or imaging examination examination liver cancer mainly through serum alpha-fetoprotein (AFP) clinically at present.Although AFP is used for Hepatocarcinoma screening for many years, the susceptibility of its diagnosis is not high enough: the AFP level of about 35-50% liver cancer patient is still lower than warning value (20ng/ml); The recall rate of AFP in early days in Hepatocarcinoma screening only 22%, is even low to moderate 3% (200ng/ml is threshold value) within diagnosis the previous year or several moon.Although what the Image Examinations such as ultrasonic, CT, MRI can detect as AFP supplements, but for long term follow-up examination then somewhat expensive, and the recall rate of these methods depends on the experience of tumor size and operator, the situation of thus may occur mistaken diagnosis, failing to pinpoint a disease in diagnosis.Therefore, find new hypersensitivity, high specific, and the cheap hepatocarcinoma early diagnosis mark being easy to again to detect has important clinical meaning.
MicroRNA is the endogenous small molecules non-coding RNA that a class is extensively present in eukaryote, and length is 18-24 Nucleotide (nt).MicroRNA suppresses the expression of target gene by post-transcriptional level, and the vital movement such as regulating cell differentiation, propagation, apoptosis, plays a significant role in the multiple physiology such as fetal development, organism metabolism, disease development and pathologic process.In recent years, researchist detects microRNA in the multiple body fluid such as blood, saliva, urine, proposes the concept of circulation microRNA.The humoral specimens such as blood are easy to obtain, and the clinical property grasped is strong and traumatic little, and circulation microRNA good stability, and it is convenient to detect, and therefore, circulation microRNA has the potential as the non-invasive biomarker of the diseases such as tumour, is applicable to high risk population's examination.
Recently research is pointed out, liver cancer patient and non-liver cancer contrast has different circulation microRNA express spectras.Such as, miR-21, miR-122, miR-223 level in liver cancer patient and HBV hepatitis serum is all higher than Healthy People; And the miR-21 raised in liver cancer patient blood plasma lowers after patient's surgical blanking; The people such as Li, by the routine individual serum microRNA express spectra of detection 513, determine the sort merge distinguishing HBV hepatitis and Healthy People and liver cancer patient and Healthy People; The people such as Zhou have detected the level of nearly thousand parts of plasma specimen microRNA, find by miR-122, miR-192, miR-21, miR-223, miR-26a, the sort module that seven circulation microRNA such as miR-27a and miR-801 are formed, liver cancer and all non-liver cancer colonies summation (comprising Healthy People, hepatitis, liver cirrhosis patient) can be distinguished, and the liver cancer of diagnosable AFP negative.These research prompting circulations microRNA is as the potential of diagnosing cancer of liver mark.
But still have the following disadvantages as the research of diagnosing cancer of liver mark about circulation microRNA at present: 1) major part research just picks the microRNAs alternatively index expressing imbalance at liver cancer tissue of forefathers' report, possibly cannot objectively respond the situation of circulation microRNA comprehensively; 2) part research sample size is few, lacks multicenter checking; 3) contrast arranges imperfection, is difficult to illustrate that the mark determined is the special diagnosis marker of liver cancer; 4) current all research is all the retrospective analysiss carried out in the sample being diagnosed as liver cancer, lacks the perspective study in high risk population, fails the value of microRNA for early hepatocarcinoma early warning that clearly circulates.Therefore, be still necessary the hepatocarcinoma early diagnosis mark finding to have clinical value at present, for the monitoring of liver cancer high risk population, thus the early stage small liver cancer of early warning early, be convenient to clinician and take appropriate prophylactico-therapeutic measures in time.
Summary of the invention
The object of the invention is the technical problem for solving above, a kind of effective diagnosing cancer of liver mark is provided.
For realizing above-mentioned technical purpose, the invention provides a kind of diagnosing cancer of liver mark, it is made up of the nucleic acid molecule of the following microRNA that encodes respectively: hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192 and hsa-miR-505.
In a preferred embodiment, the level of described microRNA in liver cancer patient blood serum is higher than Healthy People, hepatitis B carriers, chronic hepatitis B patient or liver cirrhosis patient.Preferably, described liver cancer is primary hepatocyte hepatocarcinoma.
On the other hand, present invention also offers a kind of microRNA molecule for diagnosing cancer of liver to combine, it is made up of hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192 and hsa-miR-505.
In a preferred embodiment, the level of described microRNA in liver cancer patient blood serum is higher than Healthy People, hepatitis B carriers, chronic hepatitis B patient or liver cirrhosis patient.Preferably, described liver cancer is primary hepatocyte hepatocarcinoma.
Specifically, the present invention obtains the above-mentioned diagnosing cancer of liver combination be made up of 7 serum microRNAs by following steps:
1. the candidate serum microRNAs of liver cancer patient and chronic hepatitis B patient difference is screened by high-throughput qPCR Array;
2. real-time fluorescence quantitative PCR checking candidate serum microRNAs;
3. establish in the training group be made up of Healthy People, hepatitis, liver cirrhosis, liver cancer patient and can distinguish liver cancer and contrast the serum microRNA of (comprising Healthy People, hepatitis and liver cirrhosis patient) with non-cancer and combine;
4. in the effect of the serum microRNA combined diagnosis liver cancer of two individual authentication group verification steps 3 establishments;
5. the serum microRNA that analytical procedure 3 is established is combined in small liver cancer, the diagnosis effect of TNM in early days and in the liver cancer patient of AFP negative.
Experimental result is analyzed and ASSOCIATE STATISTICS display: in above-mentioned steps 1, contriver determines 19 candidate microRNAs by high-throughput qPCR Array screening, and demonstrates the level of 19 candidate microRNAs in liver cancer patient blood serum in step 2 and all raise.Have detected the level of candidate microRNAs in training group (sample size is 257 parts) further, and establish and distinguish the optimum serum microRNA that contrasts with non-cancer of liver cancer and combine.And then in independently checking group 1 and checking group 2 (sample size is respectively 352,139 parts), demonstrate this serum microRNA combination can distinguish liver cancer and contrast with non-cancer.Meanwhile, contriver finds to compare traditional Hepatocarcinoma screening means, and as AFP, serum microRNA is combined in small liver cancer, TNM has better diagnosis effect in early days and in the liver cancer patient of AFP negative.
Contriver carries out nido contrast case research further in the high risk population of perspective collection (comprising chronic hepatitis B patient, liver cirrhosis patient) sample.By monitoring 1484 chronic hepatitis Bs or liver cirrhosis patient PD for a long time, contriver finds that in checking group 3 27 examples are new and sends out liver cancer case, collect each case diagnosing cancer of liver and (be decided to be zero point, M0) and diagnosis first 12,9,6,3 months (M-12,-9,-6 ,-3) sequence serum specimen, collects the sequence serum specimen of the corresponding time point of chronic hepatitis/liver cirrhosis patient of 135 sex age-matched in contrast simultaneously.
Experimental result is analyzed and ASSOCIATE STATISTICS display: serum microRNA is combined in hepatocarcinoma early diagnosis has good prediction effect, show: As time goes on the susceptibility of this combined prediction liver cancer, and to promote all higher than AFP gradually at each time point; In addition, the area under curve (AUC) that serum microRNA combines all is greater than AFP.
In yet another aspect, the invention also discloses a kind of test kit of diagnosing cancer of liver, it comprises the reagent for detecting following microRNA molecule level in serum: hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192 and hsa-miR-505.
In one embodiment, the described reagent for detecting microRNA molecule level in serum is real-time fluorescence quantitative PCR related reagent.
In another preferred embodiment, described test kit also comprises serum RNA extraction system and reverse transcription system.In preferred embodiments, this test kit also comprises the analytical procedure of the suffering from hepatic cancer for assessment of whether.In a preferred embodiment, described test kit comprises 7 to the LNA Mdification primer that can be used for detecting following microRNA level: hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-505.Preferred, described test kit also comprises the LNA Mdification primer detecting outer source reference NC67.Calibrated by outer source reference NC67, obtain the level 2 of above-mentioned 7 target genes in serum specimen to be checked
-Δ Ct(Δ Ct=Ct
taget-Ct
reference).According to microRNAs threshold value by detection sample microRNAs level respectively assignment be 1 or 0, realize discretize.Further according to logistic regression methods analyst serum microRNA combined evaluation person to be measured whether suffering from hepatic cancer: Logit (p=HCC)=-0.356-0.264*hsa-miR-29a-0.081*hsa-miR-29c-0.518*hsa-miR-133a-0.250*hsa-miR-143-0.489*hsa-miR-145-0.466*hsa-miR-1 92-0.417*hsa-miR-505, wherein hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-505 is the numerical value after corresponding serum microRNA detection level discretize, and with 0.5 for diagnostic threshold, liver cancer is diagnosed as higher than 0.5, non-liver cancer is diagnosed as lower than 0.5.Thus, described diagnostic kit can be applicable to liver cancer early warning.
Serum microRNA combines the superiority being used for liver cancer detection and early diagnosis and is: (1) sample is easy to obtain, and the clinical property grasped is strong and traumatic little, and serum microRNA good stability, and it is convenient to detect.(2) experimental technique is very ripe, and testing process is easy, be easy to repetition, all can be completed by common technician.(3) the present invention adopts high flux screening, multicenter verify and study in high risk population's sample of perspective collection, the effect combine serum microRNA and diagnostic kit have carried out comprehensive assessment, the application of above methods and strategies ensure that the potential using value of the present invention in liver cancer clinical diagnosis, and provides referential methods and strategies for the development of other diseases biomarker.(4) serum microRNA diagnosing liver cancer test kit can reflect the morbid state of liver cancer patient in time, and avoid previously numerous and diverse detection, save time human cost, is convenient to clinician and takes personalized control prece in time.
Accompanying drawing explanation
Fig. 1 is the ROC graphic representation of the embodiment of the present invention 4,5 in training group, checking group.Training group (A), checking group 1 (B) and the middle serum microRNA of checking group 2 (C) combine and AFP distinguishes liver cancer contrasts (upper figure) and high risk population's (figure below) ROC graphic representation with non-cancer.
Fig. 2 is the ROC graphic representation of the embodiment of the present invention 5 in nido contrast case research.When liver cancer patient diagnosis front 12 (A), 9 (B), 6 (C) or individual month of 3 (D) and diagnosis, (E) serum microRNA combination and AFP distinguish the ROC graphic representation that liver cancer contrasts with hepatitis/liver cirrhosis.
Fig. 3 is the ROC graphic representation of the embodiment of the present invention 6 in small liver cancer patient (tumour≤3cm).Training group (A), checking group 1 (B), checking group 2 (C) and training group combine with serum microRNA in all checking groups (D) and AFP distinguishes small liver cancer patient contrasts (left figure) and high risk population (right figure) ROC graphic representation with non-cancer.
Fig. 4 is the ROC graphic representation of the embodiment of the present invention 7 in different TNM by stages liver cancer patient.Serum microRNA combines and AFP distinguishes TNM I phase (A), and TNM II phase (B) or TNM III/IV phase (C) liver cancer patient contrast the ROC graphic representation of (upper figure) and high risk population's (figure below) with non-cancer.
Fig. 5 is the ROC graphic representation of the embodiment of the present invention 8 in AFP negative liver cancer patient (AFP≤20ng/ml).Training group (A), checking group 1 (B), checking group 2 (C) and training group combine with serum microRNA in all checking groups (D) and distinguish AFP negative liver cancer patient contrasts (left figure) and high risk population (right figure) ROC graphic representation with non-cancer.
Embodiment
For making the present invention easier to understand, below in conjunction with the drawings and specific embodiments, set forth the present invention further.Should be understood that these embodiments are only for illustration of the present invention, and be not used in and limit the scope of the invention; Described accompanying drawing is also only schematic, is considered to nonrestrictive.
Embodiment 1: the collection of serum specimen and preparation
Contriver acquires the blood preparation of Healthy People (HC), hepatitis B carriers (IHC), chronic hepatitis B patient (CHB), liver cirrhosis patient (LC) and liver cancer patient (HCC) year August in August, 2009 to 2014, these crowds meet into group standard (table 1), and according to the principle of sex, age-matched, setting liver cancer and control group sample thereof.
Training group: 257 parts of serum specimens of the Healthy People (51 example) that year March in August, 2009 to 2012 gathers, chronic hepatitis B patient (51 example), liver cirrhosis patient (47 example) and liver cancer patient (108 example).
352 parts of serum specimens of the Healthy People (60 example) that year April in checking group 1:2012 April to 2013 gathers, chronic hepatitis B patient (68 example), liver cirrhosis patient (71 example) and liver cancer patient (153 example).
139 parts of serum specimens of the Healthy People (48 example) that year August in checking group 2:2013 May to 2013 gathers, hepatitis B carriers (42 example) and liver cancer patient (49 example).
The checking group 3:2009 year chronic hepatitis B of collection in August in August to 2014 or 668 parts of sequence serum specimens of liver cirrhosis patient (135 examples totally 568 parts) and liver cancer patient (27 examples totally 100 parts), for nido contrast case research.Particular case is: recruit 1484 chronic hepatitis Bs or liver cirrhosis patient is followed the trail of for a long time, within every 3 months, carries out a Hepatocarcinoma screening, detects the imaging examinations such as biochemical indicator and B ultrasonic such as AFP, AST, ALT, gathers serum specimen simultaneously and is stored in-80 DEG C.To in August, 2014, follow the trail of for 1484 and newly in patients send out liver cancer 27 example, wherein 15 examples are made a definite diagnosis for histopathology, and 12 examples are imaging diagnosis.
The Clinical symptoms of above-mentioned group of participants is in table 2 and table 3.
Table 1. group of participants enters group standard
enter set condition with reference to hepatopathy research association of the U.S. (AASLD) practice guideline in 2009.
with reference to liver biopsy Metavir system.
It is imaging diagnosis that § newly sends out a liver cancer patient except 12 in checking group 3, and all the other liver cancer cases are histopathology and make a definite diagnosis.
The specifying information reference table 3 of these 12 cases.
The clinical pathologic characteristic of table 2. training group and checking group participant
Table 3. checking group 3 newly sends out the clinical pathologic characteristic of 27 routine liver cancer patients
Extract liver cancer patient preoperative, Healthy People, hepatitis B carriers, chronic hepatitis B patient and each 4ml of liver cirrhosis patient peripheric venous blood, in dry heparin tube more than 4 DEG C of standing half hours.400g subsequently, 4 DEG C of centrifugal 10min get supernatant, further 1800g, and 4 DEG C of centrifugal 10min get supernatant, obtain serum, save backup after packing in-80 DEG C.
Embodiment 2:qPCR Array and data analysis thereof
Contriver chooses preoperative and 8 chronic hepatitis B patients of 6 liver cancer patients and checks that the serum specimen in more than at least one years screens for qPCR Array apart from lasts.These patients are the male sex, and its age average, distribution all without significant difference (table 4).
The sample clinical pathologic characteristic that table 4. is analyzed for qPCR Array
The present invention adopts Applied Biosystems company
the microRNAs of Array Human MicroRNA method screening liver cancer and chronic hepatitis B difference, the altogether level of the mankind microRNAs that detection 754 is known.Concrete steps are see Applied Biosystems website.The raw data obtained is after calibration, contriver adopts Significant Analysis ofMicroarray (SAM) analytical procedure to select difference microRNAs, and final screening obtains 19 candidate microRNAs (table 5) for subsequent authentication.
The candidate microRNAs that table 5. the present invention adopts and outer source reference information
exiqon Products is numbered
Embodiment 3: real-time fluorescence quantitative PCR detects training group sample microRNAs level
1.1 serum RNA extract
The present invention adopts Trizol reagent to extract, and helps heavy method to obtain serum RNA through phenol/chloroform purifying, isopropanol precipitating, glycogen, and concrete steps are as follows:
1. get 200 μ l serum, preferably add and be isopyknicly mixed with cel-miR-67 (NC67, there is no designed by the ripe body sequence of the nematode miR-67 of homology a double-stranded RNA based on human genomic sequence, final concentration is 0.2nM, sequence is in table 5) Trizol lysate, abundant vibration mixing, ice bath 15min.
2. add 100 μ l precooling chloroforms, vibration mixing, 4 DEG C, the centrifugal 15min of 12000g.
3. shift supernatant, add equal-volume precooling phenol/chloroform (1:1), vibration mixing, 4 DEG C, the centrifugal 10min of 14000g.And repeat this step once.
4. shift supernatant, add equal-volume precooling chloroform, vibration mixing, 4 DEG C, the centrifugal 15min of 14000g.
5. shift supernatant, preferably add equal-volume Virahol and glycogen (final concentration is 200 μ g/ml), vibration mixing, 4 DEG C, the centrifugal 30min of 16000g.
6. carefully topple over supernatant, precipitate once by 1ml 70% washing with alcohol, 4 DEG C, the centrifugal 10min of 16000g.
7. abandon supernatant, after ethanol volatilization, add 10 μ l DEPC water dissolution, be placed in-80 DEG C and save backup.
1.2 real-time fluorescence quantitative PCRs (RT-qPCR)
The present invention preferably adopts Universal cDNA Synthesis Reverse Transcriptase kit, and the serum RNA of reciprocity volume carries out reverse transcription.Further preferably adopt SYBR Green qPCR master mix test kit, to dilute the cDNA of 20 times for template, the primer that LNA modifies carries out RT-qPCR detection.Described Reverse Transcriptase kit, qPCR detection kit and LNA Mdification primer are all purchased from Exiqon company (Denmark).
Calibrated by outer source reference NC67, obtain the expression values 2 of target microRNA
-Δ Ct(Δ Ct=Ct
taget-Ct
reference).Result shows, and the level of 19 candidate microRNAs in liver cancer patient blood serum all significantly raises.
Embodiment 4: determine in training group that optimum serum microRNA combines
By training group sample according to 19 microRNAs separately detection level arrange from big to small, and value (then only getting once if any repetition values) successively, according to value, sample is judged to be positive or negative monoid, given class analysis in conjunction with sample obtains the Sensitivity and Specificity of each value, draws ROC curve (Receiver Operating Characteristic Curve) further.Searching makes, and (susceptibility+specificity)/2 values reach maximum point, and the expression numerical value of this some correspondence is the threshold value of microRNA discretize.Further by be more or less than threshold value sample respectively assignment be 1 or 0, realize discretize, for further model construction.MicroRNAs discretize threshold value (table 5) that the present invention adopts will be used for the discretize of training corresponding microRNA data in group, checking group, thus changes continuous variable into two classified variables.
Modeling result shows, 7 serum microRNA that Logic Regression Models builds are combined as optimum combination, its formula assessing whether suffering from hepatic cancer is: Logit (p=HCC)=-0.356-0.264*hsa-miR-29a-0.081*hsa-miR-29c-0.518*hsa-miR-133a-0.250*hsa-miR-143-0.489*hsa-miR-145-0.466*hsa-miR-1 92-0.417*hsa-miR-505, wherein hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-505 is the numerical value after corresponding serum microRNA detection level discretize.This is combined in training group can distinguish liver cancer and contrast or high risk population with non-cancer, there is good diagnosing cancer of liver effect, show AUC that serum microRNA combines all be greater than AFP20 or AFP400 (using 20 or 400ng/ml as AFP threshold value) AUC (Figure 1A).
Embodiment 5: the effect verifying serum microRNA combined diagnosis liver cancer in checking group
The serum microRNA combination established in training group is used to checking group diagnosing liver cancer.Similarly, Trizol extraction and real-time fluorescence quantitative PCR detection method is adopted to test.Described being combined in checking group 1,2 still can be distinguished liver cancer and contrast or high risk population with non-cancer, has good diagnosing cancer of liver effect, shows that AUC that serum microRNA combines all is greater than the AUC (Figure 1B, C) of AFP20 or AFP400.
The present invention adopts nido to contrast case research serum analysis microRNA further and is combined in prediction effect in early hepatocarcinoma.Contriver is by long-term monitoring 1484 chronic hepatitis Bs or liver cirrhosis patient PD, in checking group 3, find that 27 examples are new send out liver cancer case, have collected each case diagnosing cancer of liver and (be decided to be zero point, and first 12,9,6,3 months (M-12 M0),-9,-6 ,-3) sequence serum specimen, collects the sequence serum specimen of the corresponding time point of chronic hepatitis/liver cirrhosis patient of 135 sex age-matched in contrast simultaneously.For avoiding contrast case to there is subclinical carcinoma of liver situation, contriver only chooses the patient that last inspection does not find liver cancer yet, and is set to the time point that distance last checks more than at least one year its zero point (M0).Result shows, described serum microRNA combination has good hepatocarcinoma early diagnosis effect, can be used for liver cancer early warning and early diagnosis, show: the susceptibility of described serum microRNA combined prediction liver cancer at each time point all higher than AFP, in diagnosis first 12 months, the susceptibility of this combination was 44.4%, As time goes on, its susceptibility promotes gradually, and reaches 70.4% zero point in diagnosis; And the time point susceptibility of AFP before diagnosis is low to moderate 0-25%, even diagnosis is also only 40.7% (AFP20) or 18.5% (AFP400) (table 6) zero point.In addition, the AUC of this combination is all greater than AFP20, and especially before diagnosis, 12,9 months gaps are more obvious, are respectively 0.626vs 0.536,0.654vs 0.506; The AUC of this combination is then at any time all much larger than AFP400 (Fig. 2).
Table 6. serum microRNA is combined in the prediction effect in nido contrast case research
Embodiment 6: serum microRNA is combined in the diagnosis effect in small liver cancer patient (tumour≤3cm)
The present invention further demonstrates the effect that serum microRNA combination has good diagnosis of small hepatic cell carcinoma.In training group, checking group 1, checking group 2, the AUC of this combined diagnosis small liver cancer is all significantly greater than AFP; Training group is same with three checking group case combined analysis results shows the AUC of this combined diagnosis small liver cancer much larger than AFP20 or AFP400, its differentiation small liver cancer contrasts with non-cancer or the AUC of high risk population is respectively 0.837vs 0.729 or 0.616,0.831vs 0.711 or 0.615 (Fig. 3).
Embodiment 7: serum microRNA is combined in the diagnosis effect of different TNM by stages in liver cancer patient
Serum microRNA is combined in different TNM all has preferably diagnosis effect in liver cancer patient by stages, the AUC of this combination is all significantly greater than the AUC of AFP: with all non-cancer contrast/high risk population for contrast, in TNM I, TNM II, TNM III/IV phase diagnosing cancer of liver, the AUC of this combination is respectively 0.822/0.816,0.816/0.810 and 0.860/0.852, AFP20 or AFP400 be then respectively 0.727/0.706 or 0.654/0.653,0.741/0.717 or 0.641/0.639,0.820/0.795 or 0.720/0.719 (Fig. 4).
Embodiment 8: serum microRNA is combined in the diagnosis effect in AFP negative (AFP≤20ng/ml) liver cancer patient
Serum microRNA is combined in AFP negative liver cancer patient has good diagnosis effect equally: with all non-cancer contrast/high risk population for contrast, this is combined in training group, checking group 1, the AUC of the prediction AFP negative liver cancer of each group of checking group 2 and all center combination is respectively 0.826/0.820,0.820/0.824,0.856/0.817 and 0.826/0.819 (Fig. 5).
Embodiment 9: the making of serum microRNA test kit
Test kit of the present invention is used for liver cancer early warning and diagnosis, especially early hepatocarcinoma, by serum RNA extraction system, reverse transcription system, real-time fluorescence quantitative PCR system, primer system and whether for assessment of the logistic regression analysis method of suffering from hepatic cancer forms.In the serum RNA extraction system of described test kit, contriver adopts Trizol reagent to extract, and helps heavy method to obtain serum RNA through phenol/chloroform purifying, isopropanol precipitating, glycogen.The primer that contriver adopts a series of Exiqon company LNA to modify as the primer system of described test kit, for detecting following molecule: hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-505 and NC67 (outer source reference).In real-time fluorescence quantitative PCR system, contriver adopts the Reverse Transcriptase kit of Exiqon company and SYBR Green qPCR master mix test kit to detect.Whether for assessment of, the logistic regression formula of suffering from hepatic cancer is then: Logit (p=HCC)=-0.356-0.264*hsa-miR-29a-0.081*hsa-miR-29c-0.518*hsa-miR-133a-0.250*hsa-miR-143-0.489*hsa-miR-145-0.466*hsa-miR-1 92-0.417*hsa-miR-505.Wherein, hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-505 are the numerical value after corresponding serum microRNA detection level discretize.With 0.5 for diagnostic threshold, being liver cancer patient higher than 0.5, is non-liver cancer patient lower than 0.5.
Claims (10)
1. a diagnosing cancer of liver mark, it is made up of the nucleic acid molecule of the following microRNA that encodes respectively: hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192 and hsa-miR-505.
2. diagnosing cancer of liver mark according to claim 1, the level of wherein said microRNA in liver cancer patient blood serum is higher than Healthy People, hepatitis virus carrier, chronic hepatitis patient or liver cirrhosis patient.
3., according to the diagnosing cancer of liver mark of claim 1 or 2, wherein said liver cancer is primary hepatocyte hepatocarcinoma.
4. the microRNA molecule for diagnosing cancer of liver combines, and it is made up of hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192 and hsa-miR-505.
5. microRNA molecule combination according to claim 4, the level of wherein said microRNA in liver cancer patient blood serum is higher than Healthy People, hepatitis virus carrier, chronic hepatitis patient or liver cirrhosis patient.
6. combine according to the microRNA molecule of claim 4 or 5, wherein said liver cancer is primary hepatocyte hepatocarcinoma.
7., for a diagnostic kit for diagnosing cancer of liver, it comprises the reagent for detecting following microRNA molecule level in serum: hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192 and hsa-miR-505.Preferably, the described reagent for detecting microRNA molecule level in serum is real-time fluorescence quantitative PCR related reagent.
8. diagnostic kit according to claim 7, its logistic regression formula is:
Logit(p＝HCC)＝-0.356-0.264*hsa-miR-29a-0.081*hsa-miR-29c-0.518*hsa-miR-133a-0.250*hsa-miR-143-0.489*hsa-miR-145-0.466*hsa-miR-192-0.417*hsa-miR-505,
Wherein hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-505 are the numerical value after corresponding serum microRNA detection level discretize, and with 0.5 for diagnostic threshold, be diagnosed as liver cancer higher than 0.5, be diagnosed as non-liver cancer lower than 0.5.
9. according to the diagnostic kit of claim 7 or 8, it is characterized in that, described diagnostic kit is applied to liver cancer early warning.Preferably, the colony of described liver cancer early warning is the liver cancer high risk population such as chronic hepatitis, liver cirrhosis patient, and preferably, described liver cancer is primary hepatocyte hepatocarcinoma.
10. according to the application of diagnostic kit in diagnosing cancer of liver of any one of claim 7-9.
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| WO2016070819A1 (en) * | 2014-11-06 | 2016-05-12 | 中山大学 | Hepatocellular carcinoma diagnostic marker consisting of serum micro-rna, and diagnostic kit |
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