CN104120185A - Method and kit for diagnosing liver cancer by use of the ratio of change in serum miRNA quantity - Google Patents
Method and kit for diagnosing liver cancer by use of the ratio of change in serum miRNA quantity Download PDFInfo
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Abstract
The invention relates to the technical field of immunology and molecular biology, and particularly relates to a method for diagnosing liver cancer by use of the ratio of change in serum miRNA quantity. The method comprises the following steps: collecting a blood sample; separating Mir-RNA; performing quantitative determination of two or more Mir-RNAs in blood; calculating the proportion of the two or more Mir-RNAs according to the determination result; and measuring the proportion range of multiple groups of Mir-RNAs to determine the index of Mir-RNA change as the diagnosis basis. In the method provided by the invention, the liver cancer is diagnosed by measuring two or more Mir-RNAs and the ratio thereof, and the accuracy in liver cancer diagnosis can be effectively increased.
Description
Technical field
The present invention relates to immunology and technical field of molecular biology, especially a kind ofly utilize the variation of serum miRNA amount than the method for diagnosing liver cancer and test kit.
Background technology
In hepatocellular carcinoma (HCC), be one of modal malignant tumour, liver cancer survival rate is very low.In China and 1 Asian countries, most liver cancer comes from hepatitis B.The further understanding of carcinobiology and technical progress can have been signed the potential biological marker of not multiple liver cancer and learn.The early diagnosis of liver cancer is treatment at present and the key that extends survival rate.That the detection that biological marker is learned not only contributes to prognosis or recurrence prediction, also can aid in and determine methods for the treatment of or intervening measure.At present, the diagnosis of liver cancer mainly leans on the amount of the AFP of serum to judge, but its positive diagnosing rate is very low, and specificity also has certain limitation.
In text, at present the most frequently used biological marker is alpha-fetoprotein (AFP), has regular physical checkups together with the application with ultrasonic technique in instant every 6 to 12 months, does not also reach our expectation far away.The serum afp that surpasses 400 ngs/ml is considered to diagnosing liver cancer etc. foundation, and still, high like this numerical value is to display at sub-fraction patient.Along with being familiar with the development of deep and cell and the Protocols in Molecular Biology of oncobiology research, early discovery and metastases biomarker have been obtained significant progress.
MicroRNA (MicroRNA, miRNA) is the gene of a conservative family, has important biological action, as the growth of cell, and differentiation, propagation, apoptosis and metabolism.MiRNA expresses and has been proved to be and hepatic fibrosis at the tissue abnormalities of liver, and liver cancer is relevant with hepatitis.Recently, Liang Ge independent studies group finds that miR-122 does vital effect (Tsai W et al JCI 122:2884-2897,2012) to the growth of liver function and anti-liver tumor.The discovery that the expression of miRNA in several cancers reduces, promotes the research that miRNA acts in cancer.MiRNA is regulating tumorigenic very important latent effect to determine.Specificity miRNA is as crucial oncogene and tumor suppressor gene and brought crucial problem: " how to utilize the variation of the expression of miRNA to assist the influence prognosis of diagnosis and the treatment of liver cancer.Current research result shows, miRNA express spectra can be used for Accurate Diagnosis liver cancer and individual provides the prognosis of specific treatment.Some can, according to the effect of miRNA, can design the medicine of the miRNA of Hepatoma therapy.
Utilize miRNA as new biomarker, the transfer of prediction hepatocellular carcinoma (HCC) and treatment ground prognosis can supplement the deficiency of existing diagnosis and treatment prognosis.MiRNA in blood in circulation is the very promising disease marker that a class is new.In patients serum, have higher miR-1 and miR-122 level, its survival time is than the patient long (K berle V et al., Eur J Cancer, 49:3442-3449,2013) with the serum-concentration of lower miR-1.In serum, the amount of miR-122 is very relevant to liver cancer serum chemical parameters and function.At present, in document, report that the serological variation of a plurality of miRNA and liver tumor has significant relation, comprise Let7, M ir-1, Mir-15b, Mir-16, Mir-17, Mir-18, Mir-21, Mir-25, miR-26a, Mir-92, Mir-130, Mir-122, Mir-146, Mir-183, Mir-199, Mir-206, Mir-215, Mir-215, Mir-221, Mir-222, Mir-223, Mir-224, Mir-375, Mir-574, (the Chai S et al such as Mir-885, Chin J.Can, 32:419-426,2013).These miRNA lower some to raise, and in the article analysis of delivering, the miRNA that not only each article is found is different, and because the amount of quantivative approach and miRNA is relatively difficult to purify separated in serum, the amount of report also differs widely.Although miRNA has the value of diagnosing cancer, but restriction and difference due to quantivative approach, the data of delivering do not have comparability, do not reach the object of clinical diagnosis and treatment prognosis far away, in addition in Tumor Growth, the amount of the serum of miRNA may change with the growth of making tumour, and the amount of its corresponding special miRNA also changes this with regard to for to utilize the developmental stage of miRNA diagnosing cancer to increase more difficulty.
Existing application number is patent of invention microRNA biomarker of 201010220241.9 and uses thereof, a kind of microRNA biomarker is disclosed, for any 1 in miR-21, miR-25, miR-29c, miR-93, miR-198, miR-221 and miR-222, its sequence corresponds to SEQ ID NO: 1～SEQ ID NO: 7.This invention also discloses the purposes of above-mentioned miRNA biomarker simultaneously: for the preparation of the test kit with liver cancer detectivity.This invention also discloses a kind of test kit with liver cancer detectivity simultaneously, and this test kit is comprised of reverse transcription system, amplification system and primer system; Primer system comprises stem ring primer and the amplimer of any 1 in miR-21, miR-25, miR-29c, miR-93, miR-198, miR-221 and miR-222, can be used for Hepatocarcinoma screening and diagnosis.It is only the C by single miRNA
tvalue judges makes a definite diagnosis liver cancer, but in fact still has certain Error Diagnostics.Obviously, the otherness of the absolute magnitude that individual miRNA expresses is not considered in this application for a patent for invention, i.e. factor and the impact on the expression amount of the miRNA in serum thereof of tumor tissues size and developmental stage.In addition, the factors such as the Extraction and determination method of miRNA is various, unstable, poor repeatability, have caused the result comparison of different experiments chamber poor.As these factors are taken into account, the measuring method of this application has certain limitation.
Summary of the invention
For overcoming the above problem of background technology, improve a method of utilizing reliably the serodiagnosis liver cancer of MiRNA.
Studies have shown that, in cancer development process, some miRNA amount decreases than normally, and some is than high to some extent normally, especially liver cancer.
The present invention utilizes These characteristics, solving the technical scheme that its technical problem adopts is: a kind ofly utilize the variation of serum miRNA amount than the method for diagnosing liver cancer, its method is as follows: collect blood sample, separated miRNA, two or more miRNA in blood are carried out to quantitative assay, by measurement result, calculate the ratio of two or more miRNA, by measuring the proportional range of many group miRNA, determine the index that miRNA changes, as the foundation of diagnosis.
According to another embodiment of the invention, further comprise described collection blood sample, separated miRNA, to miRNA carry out quantitative assay, the concrete grammar of ratio that calculates miRNA is as follows:
(1), from hospital, collect separated blood sample, the liver cancer group that is divided into healthy group and has made a definite diagnosis, is two subgroups every component, is respectively healthy A1, A2, liver cancer group B1, the B2 of organizing; Each group sample is handled as follows: extract peripheral blood 2ml, under room temperature, place 1 hour, under 1000g condition centrifugal 10 minutes; Then, extraction blood plasma is transferred in 1.5ml pipe, under 16000g condition centrifugal 20 minutes to remove residual cell debris, subsequently supernatant liquor is transferred in clean pipe, do not use immediately the test tube can be-80 ℃ of storages;
(2), separated MicrosiRNA: utilize Trizol reagent and MiRNeasy reagent to distinguish extracted total RNA and purifying Mir-RNA;
(3), Mir-RNA is carried out the method employing quantitative RT-PCR of quantitative assay;
(4), the ratio of A1/A2 of health group of take is contrast, calculates respectively the ratio that B1/B2 group Mir-RNA changes, and determines the index that Mir-RNA changes, as the foundation of diagnosis.
According to another embodiment of the invention, further comprise that Mir-RNA that described liver cancer group B1 comprises is for any one or more below: Let7C, Mir-1, Mir15b, Mir-17, Mir-18, Mir-25, Mir-26a, Mir-92, Mir-130, Mir-146, Mir-183, Mir-206, Mir-215, Mir-221, Mir-222, Mir-223, Mir-224, Mir-375, Mir-574, Mir-885;
Mir-1：UGGAAUGUAAAGAAGUAUGUAU
Mir15b：UAGCAGCACAUGGUUUACA
Mir-17：CAAAGUGCUUACAGUGCAGGUAG
Mir-18：UAAGGUGCAUCUAGUGCAGAUAG
Mir-25：AGGCGGAGACUUGGGCAAUUG
Mir-26a：CCUAUUCUUGGUUACUUGCAC
Mir-92： AGGGACGGGACGCGGUGCAGUG
Mir-130：ACUCUUUCCCUGUUGCACUAC
Mir-146：CCUCUGAAAUUCAGUUCUUCAG
Mir-183：UAUGGCACUGGUAGAAUUCACU
Mir-206：UGGAAUGUAAGGAAGUGUGUGG
Mir-215：AUGACCUAUGAAUUGACAGAC
Mir-221：AGCUACAUUGUCUGCUGGGUUUC
Mir-222：AGCUACAUCUGGCUACUGGGU
Mir-223：CGUGUAUUUGACAAGCUGAGUU
Mir-224：CAAGUCACUAGUGGUUCCGUU
Mir-375：UUUGUUCGUUCGGCUCGCGUGA
Mir-574：UGAGUGUGUGUGUGUGAGUGUGU
Mir-885：UCCAUUACACUACCCUGCCUCU
The Mir-RNA that described liver cancer group B2 comprises is for any one or more below: Mir-16, Mir-21, Mir-122, Mir-122a, Mir-125a, Mir-139, Mir-150, Mir-145, Mir-199, Mir-200, Mir-214;
Mir-16：UAGCAGCACGUAAAUAUUGGCG
Mir-21：UAGCUUAUCAGACUGAUGUUGA
Mir-122：AACGCCAUUAUCACACUAAAUA
Mir-122a：UGGAGUGUGACAAUGGUGUUUG
Mir-125a：UCCCUGAGACCCUUUAACCUGUGA
Mir-139：UCUACAGUGCACGUGUCUCCAGU
Mir-150：UCUCCCAACCCUUGUACCAGUG
Mir-145：GUCCAGUUUUCCCAGGAAUCCCU
Mir-199：CCCAGUGUUCAGACUACCUGUUC
Mir-200：CAUCUUACCGGACAGUGCUGGA
Mir-214：UGCCUGUCUACACUUGCUGUGC
According to another embodiment of the invention, further comprise that first miRNA selecting in described liver cancer group B1 is Mir-222, the miRNA in second the liver cancer group B2 selecting is Mir-122a.
According to another embodiment of the invention, the ratio that further comprises described Mir-222 and Mir-122a is between 1-4.
According to another embodiment of the invention, further comprise that Mir-RNA extracts test kit.
According to another embodiment of the invention, further comprise that described Mir-RNA box is Mir-122 box and Mir-222 specificity quantification kit.
The invention has the beneficial effects as follows, the present invention, by measuring two or more Mir-RNA and their ratio carrys out diagnosing liver cancer, can effectively improve the accuracy rate of diagnosing liver cancer.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the present invention is further described.
Fig. 1 is the direct comparison of two groups of data in the degree that declines of Mir-122a serum.
Fig. 2 is the direct comparison of two groups of data in the degree that raises of Mir-222 serum.
Fig. 3 is the not direct comparison of Mir-122a, two groups of data of Mir-222 in patients serum on the same group.
Fig. 4 is the direct comparison of Mir-122a or two groups of data of Mir-222 in same group of patients serum.
Embodiment
Basic goal of the present invention is to carry out diagnosing liver cancer by measuring the ratio of two groups or more miRNA, to improve the accuracy rate of diagnosing cancer of liver.The amount of measuring every group of miRNA can adopt different methods, and it is as follows that the present invention measures the embodiment mono-of miRNA metering method:
Step 1: blood sample is gathered from hospital in advance, is divided into healthy group and has made a definite diagnosis liver cancer group.Every component, being two groups, is respectively healthy group: A1, A2, and liver cancer group: B1, B2, each group sample all adopts the following step 2,3,4 to measure the amount of its miRNA.
Step 2: extract the peripheral blood 2ml of each group sample, place under 1000g condition centrifugal 10 minutes under room temperature 1 hour.Then, extract blood transfer in 1.5ml pipe, under 16000g condition centrifugal 20 minutes to remove residual cell debris.Supernatant liquor is transferred in clean pipe subsequently, immediately do not used the test tube of use to store at-80 ° of C.
Step 3: the MicrosiRNA in separation of supernatant: first, the operation instruction of utilizing Trizol extraction agent (from Invitrogen company) to provide is put on the total RNA in clear liquid; Then, the operation instruction of utilizing MiRNeasy reagent (from Qiagen company) to provide is carried out the Mir-RNA in the total RNA of purifying.Trizol extraction agent and MiRNeasy reagent are all ready-made test kits, can directly from the buying of above-mentioned company, obtain.
Step 4: add the 3 ' end of the Mir-RNA that Ploy A crosses to purifying, carry out reverse transcription and PCR.
Below in conjunction with further method, the product of above-mentioned steps is analyzed to mensuration.
As to want the total Mir-RNA of purifying in the product of analytical procedure 3 be Yeast Nucleic Acid (as siRNA, miRNA), make with the following method
(1) with Poly A polysaccharase, at 3 ' end of small nucleic acids, add the preceding paragraph Poly A, method is as follows:
Blood plasma or tissue homogenate after 1.25ul dilution, then add respectively in the following order:
0.5ul 5X reaction solution
0.225ul 25mM MgCl
2
0.1ul ATP
0.05ul Poly A polysaccharase
0.35ul water
37 degree incubations 15 minutes
Its product should be: 5 ' UAGCUUAUCAGACUGAUGUUGAAAAAAAAAAAAAA ... 3 '
The principle of responsive transcription and reaction conditions
(2) 3 ' end in above-mentioned steps 3 has been added to the small nucleic acids of Poly A, added oligomerization dT following and with a primer
TTTTTTTTTTTTGAACCTAGCTGGCCAA, with the Poly A tail generation specific binding of small nucleic acids, as follows in conjunction with result:
5’ UAGCUUAUCAGACUGAUGUUGAAAAAAAAAAAAAA……3’
TTTTTTTTTTTTTTGAACCTAGCTGGCCAA
The method that adds oligomerization dT is: in the reaction completing after step 3, add following reagent:
0.625ul reverse transcription damping fluid
(this damping fluid can be selected and purchased from different manufacturers, all can realize this test effect)
1.875ul is containing the primer of oligomerization dT
65 degree are hatched 5 minutes
With the oligomerization dT of primer and the Poly A tail generation specific binding of small nucleic acids,
Then add respectively:
6.25ul 2X reaction solution
1.25ul ThermoScript II
50 degree are hatched 5 minutes, and 85 degree are hatched 5 minutes, synthetic cDNA
(3) cDNA dilution synthetic in above-mentioned steps is added to 5 ' and 3 ' primer: 5 ' TCCGAATAAACTCCAGGCCTA 3 ' and 5 ' AACCGGTCGATCCAA 3 ', and by the amount of following every pipe, use API7500 quantitative real time PCR Instrument, quantitative pcr amplification carrys out the amount of small nucleic acids in determination step 3 products.
The amount of every pipe:
The cDNA(of 6ul dilution is from said process (3))
10ul 2X reaction solution
0.4ul 50X SYBR green
1.5ul PCR primer (6uM)
2.1ul water
95 degree 15 minutes, 40 circulations: 95 degree 15 seconds, 55 degree 30 seconds, 72 degree 30 seconds.
The specific primer sequence table of Mir-RNA quantitative RT-PCR above-mentioned.
| MicroRNA | Sequence (5 '-3 ') |
| Let7C | UGAGGUAGUAGGUUGUAUGGUU |
| Mir-1 | TGGAATGTAAAGAAGTATGTAT |
| Mir15b | TAGCAGCACATCATGGTTTACA |
| Mir-17 | CAAAGTGCTTACAGTGCAGGTAG |
| Mir-18 | TAAGGTGCATCTAGTGCAGATAG |
| Mir-25 | AGGCGGAGACTTGGGCAATTG |
| Mir-26a | CCTATTCTTGGTTACTTGCAC |
| Mir-92 | AGGGACGGGACGCGGTGCAGTG |
| Mir-122 | AACGCCATTATCACACTAAATA |
| Mir-130 | ACTCTTTCCCTGTTGCACTAC |
| Mir-146 | CCTCTGAAATTCAGTTCTTCAG |
| Mir-183 | TATGGCACTGGTAGAATTCACT |
| Mir-206 | TGGAATGTAAGGAAGTGTGTGG |
| Mir-215 | ATGACCTATGAATTGACAGAC |
| Mir-221 | AGCTACATTGTCTGCTGGGTTTC |
| Mir-222 | AGCTACATCTGGCTACTGGGT |
| Mir-223 | CGTGTATTTGACAAGCTGAGTT |
| Mir-224 | CAAGTCACTAGTGGTTCCGTT |
| Mir-375 | TTTGTTCGTTCGGCTCGCGTGA |
| Mir-574 | TGAGTGTGTGTGTGTGAGTGTGT |
| Mir-885 | TCCATTACACTACCCTGCCTCT |
| Mir-16 | TAGCAGCACGTAAATATTGGCG |
| Mir-21 | TAGCTTATCAGACTGATGTTGA |
| Mir-122a | TGGAGTGTGACAATGGTGTTTG |
| Mir-125a | TCCCTGAGACCCTTTAACCTGTGA |
| Mir-139 | TCTACAGTGCACGTGTCTGGAGT |
| Mir-150 | TCTCCCAACCCTTGTACCAGTG |
| Mir-145 | GTCCAGTTTTCCCAGGAATCCCT |
| Mir-199 | CCCAGTGTTCAGACTACCTGTTC |
| Mir-200 | CATCTTACCGGACAGTGCTGGA |
| Mir-214 | TGCCTGTCTACACTTGCTGTGC |
| Mir-223 | CGTGTATTTGACAAGCTGAGTT |
After mensuration finishes, just can find out that the product in every group of sample is how many, determine the amount of small nucleic acids Mir-RNA, two groups or more Mir-RNA is compared.
The Mir-RNA that the present invention compares can be for following one or more: Let7C, Mir-1, Mir15b, Mir-17, Mir-18, Mir-25, Mir-26a, Mir-92, Mir-130, Mir-122, Mir-146, Mir-183, Mir-206, Mir-215, Mir-221, Mir-222, Mir-223, Mir-224, Mir-375, Mir-574, Mir-885, Mir-16, Mir-21, Mir-122a, Mir-125a, Mir-139, Mir-150, Mir-145, Mir-199, Mir-200, Mir-214.Under different illumination conditions, some risings, some reductions, must adopt same measuring method, see that whether its ratio is the same, and ratio is more effective with respect to single value, and this is a ultimate principle of the present invention.
Alternative 1 of the present invention: the direct comparison to two groups of data of the degree that declines in Mir-122a serum.As shown in Figure 1, the Mir-122a of test group one amount is between 0～1, and the Mir-122a of test group two amount is between 2～4, and the data of two groups differ greatly, and singly sees that a Mir-122a value can not make a definite diagnosis liver cancer well.
Alternative 2 of the present invention: the direct comparison to two groups of data of the degree that raises in Mir-222 serum.As shown in Figure 2, the Mir-222 of test group one amount is between 0.8～2, and the Mir-222 of test group two amount is between 4～8, and it is equally very large that the data of two groups differ, therefore can not make a definite diagnosis well liver cancer only according to a Mir-222 value.
Alternative 3 of the present invention: look for different patients' blood sample, measure the amount of two kinds of miRNA separately according to above-mentioned steps method, compare.The Mir-122a measuring in the present embodiment and Mir-222, as shown in Figure 3, measure first patient's Mir-122a and Mir-222, and its result is shown as the column of Mir-122a test group one, the column of Mir-222 test group one; Then separately look for a collection of patient, second batch patient's Mir-122a and Mir-222 are measured, its result is shown as the column of Mir-122a test group two, the column of Mir-222 test group two.Than being easier to, find out, the Mir-122a of the Mir-122a of test group one and Mir-222 ratio, test group two and Mir-222 ratio are not very approaching, because be not same test, from initial serum, are separated to last quantitative PCR, it has certain error, and the comparative effectiveness of this scheme is general.
Alternative 4 of the present invention: look for patient's blood sample on the same group, measure the amount of two kinds of Mir-RNA separately according to above-mentioned steps method, compare.In the present embodiment, that same mensuration is Mir-122a and Mir-222, and as shown in Figure 4, test group one patient's Mir-222 is 3 than the ratio of Mir-122a, and test group two patients' Mir-222 is between 2.5～3 than the ratio of Mir-122a.As can be seen from the above results, the odds ratio of two Mir-222 and Mir-122a is more fixing.Thus, we can learn, when detecting Mir-222 that sample compares and the ratio of Mir-122a, are greater than 2.5, are even greater than at 3 o'clock, can be diagnosed as liver cancer patient; When the ratio of Mir-222 and Mir-122a is significantly less than 2.5, can be diagnosed as normal people.By that analogy, can measure other two or more Mir-RNA and their ratio carrys out diagnosing liver cancer by same method.
By several schemes above, can be found out, alternative 4 its ratios of the present invention are more firm, and more effective, can significantly improve the accuracy rate of diagnosing liver cancer.
To sum up, the present invention passes through to extract patients serum, and therefrom extracts Mir-RNA, utilizes the method for quantitative RT-PCR, determine the amount of MiRNA, analyze each MiRNA amount change, according to their increasing and drop-out value, calculate the ratio row of its variation, the index that is changed to according to its ratio, rather than be changed to index according to its absolute magnitude, as the foundation of diagnosing liver cancer, can improve the accuracy rate of diagnosing liver cancer.
The nucleotide sequence table of microRNA above-mentioned.
| MicroRNA | Sequence (5 '-3 ') |
| Let7C | UGAGGUAGUAGGUUGUAUGGUU |
| Mir-1 | UGGAAUGUAAAGAAGUAUGUAU |
| Mir15b | UAGCAGCACAUGGUUUACA |
| Mir-17 | CAAAGUGCUUACAGUGCAGGUAG |
| Mir-18 | UAAGGUGCAUCUAGUGCAGAUAG |
| Mir-25 | AGGCGGAGACUUGGGCAAUUG |
| Mir-26a | CCUAUUCUUGGUUACUUGCAC |
| Mir-92 | AGGGACGGGACGCGGUGCAGUG |
| Mir-122 | AACGCCAUUAUCACACUAAAUA |
| Mir-130 | ACUCUUUCCCUGUUGCACUAC |
| Mir-146 | CCUCUGAAAUUCAGUUCUUCAG |
| Mir-183 | UAUGGCACUGGUAGAAUUCACU |
| Mir-206 | UGGAAUGUAAGGAAGUGUGUGG |
| Mir-215 | AUGACCUAUGAAUUGACAGAC |
| Mir-221 | AGCUACAUUGUCUGCUGGGUUUC |
| Mir-222 | AGCUACAUCUGGCUACUGGGU |
| Mir-223 | CGUGUAUUUGACAAGCUGAGUU |
| Mir-224 | CAAGUCACUAGUGGUUCCGUU |
| Mir-375 | UUUGUUCGUUCGGCUCGCGUGA |
| Mir-574 | UGAGUGUGUGUGUGUGAGUGUGU |
| Mir-885 | UCCAUUACACUACCCUGCCUCU |
| Mir-16 | UAGCAGCACGUAAAUAUUGGCG |
| Mir-21 | UAGCUUAUCAGACUGAUGUUGA |
| Mir-122a | UGGAGUGUGACAAUGGUGUUUG |
| Mir-125a | UCCCUGAGACCCUUUAACCUGUGA |
| Mir-139 | UCUACAGUGCACGUGUCUCCAGU |
| Mir-150 | UCUCCCAACCCUUGUACCAGUG |
| Mir-145 | GUCCAGUUUUCCCAGGAAUCCCU |
| Mir-199 | CCCAGUGUUCAGACUACCUGUUC |
| Mir-200 | CAUCUUACCGGACAGUGCUGGA |
| Mir-214 | UGCCUGUCUACACUUGCUGUGC |
| Mir-223 | CGUGUAUUUGACAAGCUGAGUU |
The specific primer sequence of MiRNA quantitative RT-PCR above-mentioned.
| MicroRNA | Sequence (5 '-3 ') |
| Let7C | TAGGTAGTAGGTTGTATGGTT |
| Mir-1 | TGGAATGTAAAGAAGTATGTAT |
| Mir15b | TAGCAGCACATCATGGTTTACA |
| Mir-17 | CAAAGTGCTTACAGTGCAGGTAG |
| Mir-18 | TAAGGTGCATCTAGTGCAGATAG |
| Mir-25 | AGGCGGAGACTTGGGCAATTG |
| Mir-26a | CCTATTCTTGGTTACTTGCAC |
| Mir-92 | AGGGACGGGACGCGGTGCAGTG |
| Mir-122 | AACGCCATTATCACACTAAATA |
| Mir-130 | ACTCTTTCCCTGTTGCACTAC |
| Mir-146 | CCTCTGAAATTCAGTTCTTCAG |
| Mir-183 | TATGGCACTGGTAGAATTCACT |
| Mir-206 | TGGAATGTAAGGAAGTGTGTGG |
| Mir-215 | ATGACCTATGAATTGACAGAC |
| Mir-221 | AGCTACATTGTCTGCTGGGTTTC |
| Mir-222 | AGCTACATCTGGCTACTGGGT |
| Mir-223 | CGTGTATTTGACAAGCTGAGTT |
| Mir-224 | CAAGTCACTAGTGGTTCCGTT |
| Mir-375 | TTTGTTCGTTCGGCTCGCGTGA |
| Mir-574 | TGAGTGTGTGTGTGTGAGTGTGT |
| Mir-885 | TCCATTACACTACCCTGCCTCT |
| Mir-16 | TAGCAGCACGTAAATATTGGCG |
| Mir-21 | TAGCTTATCAGACTGATGTTGA |
| Mir-122a | TGGAGTGTGACAATGGTGTTTG |
| Mir-125a | TCCCTGAGACCCTTTAACCTGTGA |
| Mir-139 | TCTACAGTGCACGTGTCTGGAGT |
| Mir-150 | TCTCCCAACCCTTGTACCAGTG |
| Mir-145 | GTCCAGTTTTCCCAGGAATCCCT |
| Mir-199 | CCCAGTGTTCAGACTACCTGTTC |
| Mir-200 | CATCTTACCGGACAGTGCTGGA |
| Mir-214 | TGCCTGTCTACACTTGCTGTGC |
| Mir-223 | CGTGTATTTGACAAGCTGAGTT |
Claims (7)
1. one kind is utilized the variation of serum miRNA amount than the method for diagnosing liver cancer, it is characterized in that, its method is as follows: collect blood sample, separated miRNA, two or more miRNA in blood are carried out to quantitative assay, by measurement result, calculate the ratio of two or more miRNA, by measuring the proportional range of many group miRNA, determine the index that miRNA changes, as the foundation of diagnosis.
2. according to claim 1ly utilize the variation of serum miRNA amount than the method for diagnosing liver cancer, it is characterized in that, described collection blood sample, separated miRNA, to miRNA carry out quantitative assay, the concrete grammar of ratio that calculates miRNA is as follows:
(1), from hospital, collect separated blood sample, the liver cancer group that is divided into healthy group and has made a definite diagnosis, is two subgroups every component, is respectively healthy A1, A2, liver cancer group B1, the B2 of organizing; Each group sample is handled as follows: extract peripheral blood 2ml, under room temperature, place 1 hour, under 1000g condition centrifugal 10 minutes; Then, extraction blood plasma is transferred in 1.5ml pipe, under 16000g condition centrifugal 20 minutes to remove residual cell debris, subsequently supernatant liquor is transferred in clean pipe, do not use immediately the test tube can be-80 ℃ of storages;
(2), separated MicrosiRNA: utilize Trizol reagent and MiRNeasy reagent to distinguish extracted total RNA and purifying Mir-RNA;
(3), Mir-RNA is carried out the method employing quantitative RT-PCR of quantitative assay;
(4), the ratio of A1/A2 of health group of take is contrast, calculates respectively the ratio that B1/B2 group Mir-RNA changes, and determines the index that Mir-RNA changes, as the foundation of diagnosis.
3. according to claim 2ly utilize the variation of serum miRNA amount than the method for diagnosing liver cancer, it is characterized in that,
The Mir-RNA that described liver cancer group B1 comprises is for any one or more below: Let7C, Mir-1, Mir15b, Mir-17, Mir-18, Mir-25, Mir-26a, Mir-92, Mir-130, Mir-122, Mir-146, Mir-183, Mir-206, Mir-215, Mir-221, Mir-222, Mir-223, Mir-224, Mir-375, Mir-574, Mir-885;
Mir-1：UGGAAUGUAAAGAAGUAUGUAU
Mir15b：UAGCAGCACAUGGUUUACA
Mir-17：CAAAGUGCUUACAGUGCAGGUAG
Mir-18：UAAGGUGCAUCUAGUGCAGAUAG
Mir-25：AGGCGGAGACUUGGGCAAUUG
Mir-26a：CCUAUUCUUGGUUACUUGCAC
Mir-92： AGGGACGGGACGCGGUGCAGUG
Mir-130：ACUCUUUCCCUGUUGCACUAC
Mir-146：CCUCUGAAAUUCAGUUCUUCAG
Mir-183：UAUGGCACUGGUAGAAUUCACU
Mir-206：UGGAAUGUAAGGAAGUGUGUGG
Mir-215：AUGACCUAUGAAUUGACAGAC
Mir-221：AGCUACAUUGUCUGCUGGGUUUC
Mir-222：AGCUACAUCUGGCUACUGGGU
Mir-223：CGUGUAUUUGACAAGCUGAGUU
Mir-224：CAAGUCACUAGUGGUUCCGUU
Mir-375：UUUGUUCGUUCGGCUCGCGUGA
Mir-574：UGAGUGUGUGUGUGUGAGUGUGU
Mir-885：UCCAUUACACUACCCUGCCUCU
The Mir-RNA that described liver cancer group B2 comprises is for any one or more below: Mir-16, Mir-21, Mir-122, Mir-122a, Mir-125a, Mir-139, Mir-150, Mir-145, Mir-199, Mir-200, Mir-214;
Mir-16：UAGCAGCACGUAAAUAUUGGCG
Mir-21：UAGCUUAUCAGACUGAUGUUGA
Mir-122：AACGCCAUUAUCACACUAAAUA
Mir-122a：UGGAGUGUGACAAUGGUGUUUG
Mir-125a：UCCCUGAGACCCUUUAACCUGUGA
Mir-139：UCUACAGUGCACGUGUCUCCAGU
Mir-150：UCUCCCAACCCUUGUACCAGUG
Mir-145：GUCCAGUUUUCCCAGGAAUCCCU
Mir-199：CCCAGUGUUCAGACUACCUGUUC
Mir-200：CAUCUUACCGGACAGUGCUGGA
Mir-214：UGCCUGUCUACACUUGCUGUGC。
4. according to claim 3ly utilize the variation of serum miRNA amount than the method for diagnosing liver cancer, it is characterized in that, first miRNA selecting in described liver cancer group B1 is Mir-222, select second for Mir-RNA in liver cancer group B2 be Mir-122a.
5. according to claim 4ly utilize the variation of serum miRNA amount than the method for diagnosing liver cancer, it is characterized in that, the ratio of described Mir-222 and Mir-122a is between 1-4.
6. for a quantification kit for method described in claim 1～5, it is characterized in that, comprise that Mir-RNA extracts test kit.
7. for a quantification kit for method described in claim 6, it is characterized in that, described Mir-RNA box is Mir-122 box and Mir-222 specificity quantification kit.
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| CN109706244A (en) * | 2019-02-22 | 2019-05-03 | 上海市中医医院 | Detect diagnosing cancer of liver kit and its application of microRNA expression |
| CN110184338A (en) * | 2019-05-31 | 2019-08-30 | 南方医科大学第三附属医院（广东省骨科研究院） | Application of the cerebrospinal fluid excretion body miRNA in MMD diagnosing and treating |
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| CN115029347B (en) * | 2022-05-11 | 2024-02-20 | 珠海中科先进技术研究院有限公司 | Molecular monitoring sequence for recognizing and regulating hepatic and renal cell fibrosis, recombinant plasmid and virus inhibition |
| CN117248029A (en) * | 2023-11-17 | 2023-12-19 | 北京热景生物技术股份有限公司 | Liver cancer diagnosis marker based on exosome miRNA and application thereof |
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