CN105238863A - Application of miR-197 as liver cancer detection marker - Google Patents

Abstract

The invention discloses an application of miR-197 as a liver cancer detection marker. Based on a real-time fluorescent quantitative PCR (polymerase chain reaction) technique, the serum miR-197 of a patient with liver cancer is used as a detection subject, and the comparison of miR-197 levels in the serum of normal people, patients with liver cancer and patients with other several domestic common cancers (liver cancer, esophageal cancer, gastric cancer, colon cancer and lung cancer) shows that the miR-197 has a high specific expression in the serum of patients with liver cancer. Therefore, the miR-197 stably presenting in the serum is used as a serum marker and molecular target for liver cancer and applicable to clinical diagnosis of liver cancer; and the miR-197 can be used in the preparation of liver cancer diagnostic products based on serum detection, and the products are of great significance to the treatment of tumors and have a promising application prospect.

Description

MiR-197 is detecting the application in mark as liver cancer

Technical field

The invention belongs to biological technical field, be specifically related to miR-197 and detecting the application in mark as liver cancer.

Background technology

Hepatocellular carcinoma (HepatocellularCarcinoma, HCC, hereinafter referred to as liver cancer) is one of modal malignant tumour in world wide, and its grade malignancy is high, invasiveness strong, poor prognosis, and mortality ratio occupies world's tumor mortality second.China is High Phc Incidence Area, sends out patient's number according to statistics every year and accounts for the whole world about 55%.Onset of liver cancer compares concealment, the clinical manifestation that early stage shortage is special, often belongs to middle and advanced stage during discovery.The principle improving liver cancer patient long-term survival rate is early discovery, early diagnosis, early treatment, and key is wherein early diagnosis.

At present, the most frequently used liver cancer routine diagnostic method has imaging examination, liver impedance rheograph or cytolgical examination and tumour serum mark inspection.But, imaging examination resolving power is poor and there is the blind area being difficult to detect, and needle biopsy of liver sampling process is complicated, easily causes larger wound and easily produces false negative result, therefore, current optimal diagnosing cancer of liver method is the tumour serum mark inspection of non-wound.The liver cancer serum mark known is many, as alpha-fetoprotein (AFP), glypican-3 (GPC-3), α-L fucosidase (AFU) etc.But these marks ubiquity susceptibility or specific degree among application process be not high, lack the limitation such as effective forewarning function in high risk population's examination and recurrence monitoring.With regard to Present clinical present situation, AFP is still application liver cancer widely and detects mark, and positive rate is the highest is also only 66.7% for it.

Research in recent years finds, abundant and stable miRNA is there is in blood, its express spectra has obvious tissue specificity, and in various disease, different Serum of Cancer Patients, also there is special express spectra, therefore peripheral blood miRNA has clear superiority (PNAS as potential mark in diagnosing tumor, 2008,105 (30): 10513-18), report that the circulation miRNA in serum has higher specificity and sensitivity (MolecularCancer in the diagnosis of kinds of tumors at present, 2010,9 (306): 1-9).Just occur that as far back as 2009 the miRNA blood serum designated object about liver cancer is studied, so far the miRNA possessing diagnosing cancer of liver potential value reported just has the multiple (Biomarkers such as miR-21, miR-122, miR-210,2009,14 (7): 529-38; MolCarcinog, 2011,50 (2): 136-42; EurJCancer, 2013,49 (16): 3442-9).But above-mentioned research generally adopts the nucleic acid originally just existed in sample serum such as, as endogenous reference, RNU6B, RNU44, RNU48, miR-16 etc.Due to miR-16, this plays function in tumour generating process, and its expression level is also unstable; And the short data records RNA such as RNU6B content in serum is extremely low not easily detects, the error (BMCClinicalPathology, 2014,14:27) that the individual difference between sufferer and experimentation must be avoided to cause.Therefore, the endogenous object of reference how choosing serum miRNA experiment also there is much controversy (J.Cancer, 2012,3:432-448) so far.

Cel-miR-39 is one of a collection of nematode miRNA be found the earliest (Science, 2001,294:858-62).Because nematode and Mammals gap in heredity is too large, cel-miR-39 sequence does not exist in the high Animal genomes such as Mammals, and it does not also have an impact to Mammals vital process.Thus, in Mammals miRNA testing process, the cel-miR-39 of additional synthetic is control the reliable way of the one of experimental error between sample at present as outer source reference.

It is a kind of general gene quantification detection method that real-time fluorescence quantitative PCR detects, it immediately measures the amount of specific product during PCR exponential amplification by the change of monitoring fluorescent signal power continuously, and infer the original bulk of goal gene accordingly, and do not need taking-up PCR primer to be separated.Real-time quantitative PCR is highly sensitive, and versatility is good, repeatable strong, is specially adapted to large flux and detects, be widely used in the every field of molecular biology research.

Summary of the invention

An object of the present invention is to provide the novelty teabag of the material detecting miR-197 expression amount.

Whether the invention provides the material that detects miR-197 expression amount preparation diagnosis or auxiliary diagnosis patient to be measured is the application in the product of liver cancer patient.

Present invention also offers the application of material in the product preparing detection or auxiliary detection liver cancer detecting miR-197 expression amount.

In above-mentioned application, the nucleotide sequence of described miR-197 is as shown in sequence in sequence table 1.

In above-mentioned application, the material of described detection miR-197 expression amount be following a) or b) or c):

A) increase the primer of described miR-197;

B) containing described PCR reagent group a);

C) containing described a) or described test kit b).

In above-mentioned application, described primer is made up of the single strand dna shown in sequence 3 in the single strand dna shown in sequence in sequence table 2 and sequence table.

Another object of the present invention is to provide a kind of material detecting miR-197 expression amount.

The material of detection miR-197 expression amount provided by the invention is following 1) or 2):

1) whether diagnosis or auxiliary diagnosis patient to be measured are the product of liver cancer patient;

2) product of detection or auxiliary detection liver cancer;

The nucleotide sequence of described miR-197 is as shown in sequence in sequence table 1.

In above-mentioned substance, the material of described detection miR-197 expression amount be following a) or b) or c):

A) increase the primer of described miR-197;

B) containing described PCR reagent group a);

C) containing described a) or described test kit b);

Described primer is made up of the single strand dna shown in sequence 3 in the single strand dna shown in sequence in sequence table 2 and sequence table.

A further object of the invention is to provide and a kind ofly detects the test kit whether patient to be measured is liver cancer patient.

Whether detection provided by the invention or auxiliary detection patient to be measured are the material that the test kit of liver cancer patient comprises above-mentioned detection miR-197 expression amount.

In mentioned reagent box, described test kit also comprises the diagnostic card recording following content:

1) if the expression amount of the miR-197 of patient to be measured is higher than Healthy People, and the two there were significant differences, then patient to be measured is or candidate is liver cancer patient; 2) if patient to be measured does not meet above-mentioned steps 1) condition, then patient to be measured be not or candidate for liver cancer patient.

In mentioned reagent box, the expression amount of the miR-197 of described patient to be measured higher than Healthy People, and the two there were significant differences is that the expression amount of miR-197 in patients serum to be measured is higher than the expression amount of miR-197 in Healthy Human Serum 18.8 times.

Last object of the present invention is to provide the novelty teabag of mentioned reagent box.

Whether the invention provides mentioned reagent box in preparation diagnosis or auxiliary diagnosis patient to be measured is the application in the product of liver cancer patient.

Present invention also offers the application of mentioned reagent box in the product preparing detection or auxiliary detection liver cancer.

Above-mentioned miR-197 expression amount is the expression amount of miR-197 in serum.

The present invention is based on Real-Time Fluorescent Quantitative PCR Technique, with the serum miR-197 of liver cancer patient for detected object, nematode tiny RNA cel-miR-39 is adopted to be outer source reference, by comparison normal people, liver cancer patient and other several domestic common cancer (liver cancer, esophagus cancer, cancer of the stomach, colorectal carcinoma, lung cancer) level of miR-197 in case patients serum, find the specificity overexpression of miR-197 in liver cancer patient blood serum, therefore, the miR-197 of stable existence in serum is applied to liver cancer clinical diagnosis as the blood serum designated object of liver cancer and molecular target, for the preparation of the diagnosing cancer of liver product based on Virus monitory, this product is by significant to the treatment of tumour, have broad application prospects.

Accompanying drawing explanation

Fig. 1 is that real-time fluorescence quantitative PCR detects serum miRNA.Figure 1A is pcr amplification curve; Figure 1B is amplified production solubility curve; Fig. 1 C is that amplified production dissolves peak.

Fig. 2 is that each sample miR-197 expression level compares.

Fig. 3 is that the AUC of miR-197 differential expression between liver cancer patient and normal people analyzes.

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Embodiment 1, miR-197 are as the application detected in liver cancer marker

One, the acquisition of test serum sample and information

The serum sample that the present invention detects provides by Gansu, medical scientific center, Gansu Province tumour serum resources bank, and every routine sufferer serum all has corresponding idagnostic logout and definite case information.The sampling of serum allows via giving advance notice and obtaining patient, meets social ethics relevant criterion.The present invention detects serum sample 66 example of healthy volunteer altogether, liver cancer patient blood serum sample 70 example, esophageal cancer patients serum sample 20 example, Serum Obtained From Advance Gastric Cancer sample 22 example, colorectal cancer patients serum sample 20 example, Serum of Patients with Lung Cancer sample 21 example.Each cancer patients's Information Statistics refer to table 1.

Table 1, cancer patients's information summary sheet

Two, real-time fluorescence quantitative PCR measures the expression level of miR-197

The present invention, detects the expression level of miR-197 in the test serum sample serum in step one for outer ginseng gene with synthetic nematode 39miRNA (cel-miR-39).The ripe nucleotides sequence of miR-197 is classified as CGGGUAGAGAGGGCAGUGGGAGG (sequence 1).

1, the extraction of serum RNA

Experimental program in reference literature " Zhang Xiaojuan etc., Nanjing Medical University's journal (natural science edition), 2011,31 (4): 529-31 ", adopts the full RNA of TRIzol method to serum sample to extract.Concrete steps are as follows:

(1) by each test serum sample of step one at thawed on ice, use the centrifugal force of 1000g until completely dissolved at 4 DEG C centrifugal 10 minutes, to remove in serum the impurity such as hemocyte relic, collect supernatant.

(2) every routine serum sample gets the supernatant that 400 μ L steps (1) obtain, join the TRIzol (ThermoFisherScientific of 1mL, Waltham, MA, USA) in, the concentration simultaneously adding 10uL is nematode miRNAcel-39 analogue (Guangzhou FulenGen Co., Ltd. of the synthetic of 1mM, Guangzhou, Guangdong, China), fully leave standstill 5 minutes after mixing.

(3) product step (2) obtained, by after chloroform extraction and isopropanol precipitating, obtains RNA, is dissolved into 20 μ L and removes in the aqua sterilisa of RNA enzyme, obtain the RNA of test serum.

2, RNA reverse transcription

3 ' the end of RNA is adopted to add the reverse transcription that A (VITAMIN B4) method carries out RNA.The RNA sample that step 1 is obtained, POLYA polysaccharase, M-MLV ThermoScript II and 5 × polyreaction Buffer mixing, be configured to reverse transcription system, 37 DEG C of reactions 60 minutes, obtain reverse transcription product.In reaction system, each component concentration and specific implementation process are with reference to miRNAqRT-PCRDetectionKit (Guangzhou FulenGen Co., Ltd., Guangzhou, Guangdong, China) specification sheets.

3, real-time fluorescence quantitative PCR

The reverse transcription product obtained with above-mentioned steps 2 is for template, Hsa-miR-197 specificity amplification primer and cel-miR-39 specificity amplification primer is adopted to carry out real-time fluorescence quantitative PCR respectively, obtain amplified production respectively, the amount of the template that each reaction system uses is consistent.Hsa-miR-197 specificity amplification primer:

Sence:5 '-CGGGUAGAGAGGGC-3 ' (sequence 2);

Antisense:5 '-TTTTTTTTTTTTTTTT-3 ' (sequence 3);

Cel-miR-39 specificity amplification primer:

Sence：5’-TCACCGGGTGTAAATC-3’；

antisense：5’-TTTTTTTTTTTTTTTT-3’。

As shown in Figure 1A, curve and baseline intersection are the cycle number (Ct value) that PCR reacts to the amplification curve of the real-time fluorescence quantitative PCR of each sample to be tested, and this value can reflect the initial amount of detected miRNA.Respectively as illustrated in figures ib and 1 c, as can be seen from the figure, it is unimodal for dissolving peak for the solubility curve of the PCR primer of each sample to be tested and dissolving peak, and illustrate that quantitative fluorescent PCR reaction is single product, PCR mensuration is with a high credibility.Also illustrate that conventional methods (TRIzol method extraction RNA, real-time fluorescence quantitative PCR carry out miRNA detection) can realize extraction and the detection by quantitative of the miRNA of test serum sample.

4, statistical analysis judges miRNA relative expression quantity

Reference literature " WangGK, ZhouJ, EuropeanHeartJournal, 2010,31:659-66; JOURNALOFCLINICALONCOLOGY, 2011,29 (36): 4781-8 " the statistics strategy in; be outer source reference with the cel-miR-39 of synthetic; use the Ct value of cel-miR-39 to remove sample room error; with normal people miRNA express change multiple average for reference point, the relative expression that employing Δ Δ Ct method calculates all kinds of cancer patients's serum miRNA changes multiple.Calculation formula is as follows: miRNA relative expression changes multiple=2 ^{-Δ Δ Ct}, the wherein average of Δ Δ Ct=sample Δ Ct – normal people Δ Ct; Sample Δ Ct=sample miRNACt Zhi – sample cel-miR-39Ct value; The arithmetical av of (the miRNACt Zhi – cel-miR-39Ct value) of average=all normal people's samples of normal people's Δ Ct.Give up Ct value higher than the result of 40, change multiple by the relative expression of the miR-197 in each sample to be tested serum of above-mentioned formulae discovery, adopt significant difference between Z test decision data, when p<0.05 is judged to be that difference has significance.And use SPSS19.0 to carry out Receiver operating curve (ROC, ReceiverOperatingCharacteristicCurve) analysis, as AUC value >90%, be then judged to be that accuracy is high.

The data statistics result of the expression amount of the miR-197 of each sample to be tested is as shown in table 2 and Fig. 2, median from table 2, in liver cancer patient blood serum sample, miR-197 expression amount increases 18.8 times relative to normal people, and the expression amount of miR-197 in cancer patients's sample serum such as esophagus cancer, cancer of the stomach, colorectal carcinoma, lung cancer is compared with normal people and do not had significant difference.Change multiple from the relative expression Fig. 2, in liver cancer patient blood serum sample, the relative expression of miR-197 changes multiple and is significantly higher than the cancer patientss such as normal people, esophagus cancer, cancer of the stomach, colorectal carcinoma, lung cancer.

The data statistics result of table 2, each sample miR-197 expression level

*: the data of Z test 1 are comparison result between each sample and normal people; The data of #:Z inspection 2 are comparison result between each sample and liver cancer.

As shown in Figure 3, the characteristic curve of miR-197 shows higher specificity and susceptibility to Receiver operating curve's analytical results, and its AUC reaches 0.946, is greater than 90%, illustrates that detected result accuracy of the present invention is high.

In sum, the present invention can confirm that liver cancer patient blood serum miR-197 calibration ordinary person has specificity overexpression, can be used as liver cancer clinical diagnosis mark.Therefore, by detecting the method for the expression amount of the miR-197 of patient to be measured to judge that whether patient to be measured is for liver cancer patient, concrete grammar is as follows:

1) if the expression amount of the miR-197 of patient to be measured is higher than Healthy People, and the two there were significant differences, then patient to be measured is or candidate is liver cancer patient;

2) if patient to be measured does not meet above-mentioned steps 1) condition, then patient to be measured be not or candidate for liver cancer patient.

Claims (10)

1. whether the material detecting miR-197 expression amount is the application in the product of liver cancer patient preparation diagnosis or auxiliary diagnosis patient to be measured;

The nucleotide sequence of described miR-197 is as shown in sequence in sequence table 1.

2. detect the application of material in the product preparing detection or auxiliary detection liver cancer of miR-197 expression amount;

The nucleotide sequence of described miR-197 is as shown in sequence in sequence table 1.

3. application according to claim 1 and 2, is characterized in that: the material of described detection miR-197 expression amount be following a) or b) or c):

A) increase the primer of described miR-197;

B) containing described PCR reagent group a);

C) containing described a) or described test kit b).

4., according to described application arbitrary in claim 1-3, it is characterized in that: described primer is made up of the single strand dna shown in sequence 3 in the single strand dna shown in sequence in sequence table 2 and sequence table.

5. detecting the material of miR-197 expression amount, is following 1) or 2):

1) whether diagnosis or auxiliary diagnosis patient to be measured are the product of liver cancer patient;

2) product of detection or auxiliary detection liver cancer;

The nucleotide sequence of described miR-197 is as shown in sequence in sequence table 1.

6. material according to claim 5, is characterized in that: the material of described detection miR-197 expression amount be following a) or b) or c):

A) increase the primer of described miR-197;

B) containing described PCR reagent group a);

C) containing described a) or described test kit b).

7. the material according to claim 5 or 6, is characterized in that: described primer is made up of the single strand dna shown in sequence 3 in the single strand dna shown in sequence in sequence table 2 and sequence table.

8. detect or whether auxiliary detection patient to be measured is the test kit of liver cancer patient, it comprises the material of arbitrary described detection miR-197 expression amount in claim 5-7.

9. test kit according to claim 8, is characterized in that: described test kit also comprises the diagnostic card recording following content:

1) if the expression amount of the miR-197 of patient to be measured is higher than Healthy People, and the two there were significant differences, then patient to be measured is or candidate is liver cancer patient;

2) if patient to be measured does not meet above-mentioned steps 1) condition, then patient to be measured be not or candidate for liver cancer patient.

10. whether the test kit described in claim 8 or 9 is the application in the product of liver cancer patient in preparation diagnosis or auxiliary diagnosis patient to be measured;

Or the application of test kit in the product preparing detection or auxiliary detection liver cancer described in claim 8 or 9.