CN105647923B - Serum miRNA marker related to liver cancer prognosis and application of detection kit thereof - Google Patents
Serum miRNA marker related to liver cancer prognosis and application of detection kit thereof Download PDFInfo
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Abstract
The invention discloses a serum miRNA marker related to liver cancer prognosis and application of a detection kit thereof. The tumor markers are serum miR-29a-3p and miR-192-5p related to a human liver cancer patient. According to the invention, early-stage work is carried out through high-throughput deep sequencing, a liver cancer related serum miRNA expression profile containing miR-29a-3p and miR-192-5p with obvious expression difference is screened, and quantitative PCR (qPCR) verification is carried out, so that the fact that the serum miR-29a-3p and miR-192-5p are tumor markers related to liver cancer risk (prognosis) is finally confirmed. The marker and the detection reagent can be used for preparing a rapid detection quantitative PCR kit, and have the characteristics of rapidness and convenience in detection, high accuracy, no wound and the like; and a prognosis model jointly constructed by miR-29a-3p, miR-192-5p and liver cancer Barcelona stage (BCLC stage) can be used for grading and layering the risk of liver cancer patients, so that the individual treatment of the liver cancer patients is guided.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a serum miRNA detection product and application of miR-29a-3p and miR-192-5p as liver cancer risk (prognosis) tumor molecular markers.
Background
Liver cancer is the second most common malignant tumor in our country, because it is a big country infected by hepatitis B, so there are many new patients each year, and the number of new patients is about 50% of the world, and it has the characteristics of unobvious early symptoms, rapid progress, high recurrence and transfer rate, etc. The existing TNM staging and Alpha Fetoprotein (AFP) can not well guide the prognosis of patients, a plurality of early patients are easy to relapse and transfer even after an operation, and a new molecular marker is urgently needed to guide the prognosis of the patients, so that the purpose of grading and layering the degree of danger is achieved, and the individualized treatment is guided.
Tumor Markers (TM) are substances synthesized and secreted by the gene expression of tumor cells or abnormally produced and/or elevated by the body's response to tumors during the occurrence and proliferation of malignant tumors, which reflect the existence and growth of tumors, including proteins, hormones, enzymes (isoenzymes), polyamines, oncogene products, etc., and exist in the blood, body fluids, cells or tissues of patients, and can be measured by biochemical, immunological, and molecular biological methods, etc., and have certain values for the auxiliary diagnosis, differential diagnosis, observation of therapeutic effects, monitoring of recurrence, and prognosis evaluation of tumors. Some markers play a role in predicting the disease course of a patient by monitoring the possibility of easy recurrence of tumors, rapid progression of natural disease course and the like, and are called tumor prognosis markers.
The prognosis of liver cancer refers to the progress of liver cancer patients from the beginning of diagnosis and establishment to the end (death), for example, the progress is fast and slow, the recurrence is early and late, and the survival time is short, in short, the final outcome is evaluated from the whole state of disease progression.
The prognosis marker of the liver cancer is a biomarker for revealing or predicting the progress of the disease to be developed in a good way or in a bad way, is divided into a poor prognosis marker and a good prognosis marker, carries the poor prognosis marker and prompts a patient that the disease progresses rapidly, the recurrence and transfer conditions are more early and the survival time is shorter in the progress of the disease development; on the contrary, the medicine carries a good prognosis marker, which indicates that the disease condition develops slowly, the recurrence and the metastasis are less late, and the survival time is longer. In short, the prognosis markers are based on the general summary of the disease condition of the tumor patients, and externally reveal the trend of high or low risk of disease condition; in the aspect of internal extension, the difference of the biological heterogeneity of tumor disease is revealed, and the difference of the malignant degree of the biological behavior of the tumor is caused.
Micro RNA (microRNA, miRNA) is derived from a long-chain RNA initial transcription product (Pri-miRNA), the Pri-miRNA is subjected to enzyme digestion in cell nucleus to form a miRNA precursor with a stem-loop structure, and the miRNA precursor is further processed into miRNA after being transferred to cytoplasm; mature miRNA is combined with lipid or lipoprotein in a specific host cell to form microvesicle, is secreted to the outside of a cell in an ectosome form, and is endocytosed into a specific receptor cell to play a specific regulation function through body fluid microcirculation; the tumor-associated miRNA may be derived from miRNA released by tumor cell death, lysis or tumor cells to the surrounding environment; miRNA can cause the degradation or the inhibition of the translation of a target gene through the complementary pairing of the specific base of the target gene at the level of the transcription, regulate the expression of the oncogene and/or the cancer suppressor gene, participate in the proliferation, the apoptosis, the differentiation and the invasion of tumor cells, the generation of migration vessels and the like widely, and have close relation with the occurrence, the development and the prognosis of various tumors. Therefore, researchers have proposed the idea of "cancer micrornas" (OncomiRs), that is, the abnormal expression of some mirnas is thought to play a role in the development of tumors, similar to oncogenes.
The serum content is much lower than that of the tissue, and a very sensitive and specific detection method is needed; and because the primer sequence is short, the primer design is difficult, no targeted software can be directly utilized, and no labeled internal reference gene is used as a reference, the primer, the reaction system and the internal reference system all need to be screened and optimized. At present, a chip technology is generally adopted for high-throughput screening of miRNA, the cost is high, the specificity and the sensitivity are limited, and the real-time detection requirement of scattered specimens cannot be met.
Disclosure of Invention
The invention aims to provide a detection kit which is rapid and convenient to detect, high in accuracy and free of wound and can realize liver cancer risk (prognosis) classification, and an application of miR-29a-3p and miR-192-5p in combined use as a liver cancer serum molecular marker.
The invention provides a technical scheme for solving the technical problems, which comprises the following steps:
the first aspect of the invention relates to a marker for prognosis of liver cancer, the early-stage work of the invention screens out a miRNA expression profile of liver cancer related serum containing miR-29a-3p and miR-192-5p with obvious expression difference through high-throughput deep sequencing, and finally confirms that the miR-29a-3p and miR-192-5p in the serum are tumor markers related to liver cancer risk (prognosis) through quantitative PCR (qPCR) verification.
The marker for prognosis of liver cancer is a combination of miR-29a-3p and miR-192-5p, and the sequence information of miR-29a-3p is shown in SEQ ID NO. 1; the sequence information of miR-192-5p is shown in SEQ ID NO. 2.
The prognostic risk model based on the combination of the two markers is formulated as:
Y＝2.19*ABCLC stage+1.45*BmiR-192-5p+1.81*CmiR-29a-3p；
ABCLC stagevalues of 0 or 1, 0 representing early and 1 representing late;
BmiR-192-5pthe value of 0 or 1, 0 represents that miR-192-5p is low in expression and the expression quantity is 2-△△CT<2.24; 1 represents that it is highly expressed, and the expression level is 2-△△CT≥2.24；
CmiR-29a-3pThe value of 0 or 1, 0 represents that miR-29a-3p is low in expression and the expression quantity is 2-△△CT<1.37; 1 represents that it is highly expressed, and the expression level is 2-△△CT≥1.37；
When the Y value is less than 1.81, the survival risk of the patient is low, and when the Y value is more than 1.81, the survival risk of the patient is high.
Wherein, 2 is-△△CTExpressed as the expression level of microRNA in corresponding serum, and the calculation method belongs to the common knowledge in the field.
The second aspect of the invention relates to the application of the combination of miR-29a-3p and miR-192-5p, which comprises the following steps:
the application of the polypeptide as a molecular marker for evaluating the risk of prognosis of liver cancer; or
The application in preparing the liver cancer serum miRNA detection kit for evaluating the liver cancer prognosis risk degree.
The invention relates to a third aspect of the invention, which relates to a liver cancer serum miRNA detection kit for evaluating the risk of prognosis of liver cancer, wherein the detection system comprises a reverse transcription reaction system, a qPCR reaction system and an internal reference system, and the detection kit comprises a reagent for preparing the reverse transcription reaction system, a reagent for preparing the qPCR reaction system and a reagent for preparing the internal reference system. Wherein:
the reagent for preparing the reverse transcription reaction system at least comprises specific stem-loop reverse transcription primer solutions aiming at miR-29a-3p, miR-192-5p and nematode cel-miR-39 respectively; also comprises reverse transcriptase liquid, dNTP mixed liquid, ribonuclease inhibitor liquid, reverse transcription buffer liquid and nuclease-free pure water.
Preferably, the specific stem-loop reverse transcription primer solution has a trade name of: Bulge-LoopTMRT Primer, supplier: the reverse transcription primer solution of the specific stem-loop method comprises cel-miR-39 specific stem-loop method reverse transcription primer solution (the primer package is numbered as miRQ0000010-1-1) and miR-29a-3p specific stem-loop method reverse transcription primer solutionPrimer solution (the serial number of the primer set is miRQ0000086-1-1) and miR-192-5p specific stem-loop reverse transcription primer solution (the serial number of the primer set is miRQ 0000222-1-1); trade names of the reverse transcriptase liquid and reverse transcription buffer: reverse Transcriptase M-MLV, supplier: TaKaRa, trade number: code No. 2641A; the trade name of the dNTP mixture is: dNTP mix, supplier: TaKaRa, trade number: code No. 4030; trade name of the ribonuclease inhibitor liquid: combinant RNase Inhibitor, supplier: TaKaRa, trade number: code No. 2131A.
The total volume of the reverse transcription reaction system is 25 mu l; among them, preferably, 2 μ l of total RNA sample, 2 μ l of specific stem-loop method reverse transcription primer solution, 0.5 μ l of reverse transcriptase solution, 2 μ l of dNTP mixed solution, 0.5 μ l of ribonuclease inhibitor solution, 5 μ l of reverse transcription buffer solution and 13 μ l of nuclease-free pure water; the total RNA sample contains an internal reference cel-miR-39 liquid.
Preferably, the total RNA sample before reverse transcription is prepared by adding 2 ul of internal reference cell-miR-39 liquid during the process of extracting 250 ul of serum total RNA, wherein the concentration of the internal reference cell-miR-39 liquid is 0.05 pmol/ul.
The reagent for preparing the qPCR reaction system at least comprises forward primer liquid and reverse universal primer liquid aiming at miR-29a-3p, miR-192-5p and nematode cel-miR-39 respectively; also comprises SYBR Green mixed liquor, ROX Reference Dye II liquor and pure water.
Preferably, the SYBR Green mixture has a trade name:
Premix Ex TaqTMII, supplier: TaKaRa, trade number: code No. RR820A; trade name of the forward primer solution: Bulge-LoopTMForward Primer, supplier: the forward primer solution comprises cel-miR-39 forward primer solution (the number of a primer set is mirQ0000010-1-1), miR-29a-3p forward primer solution (the number of the primer set is mirQ0000086-1-1) and miR-192-5p forward primer solution (the number of the primer set is mirQ 0000222-1-1); the trade name of the reverse universal primer solution is: Bulge-LoopTMReversePrimer, supplier: rib(s)The reverse universal primer solution comprises cel-miR-39 reverse universal primer solution (the number of a primer set is mirQ0000010-1-1), miR-29a-3p reverse universal primer solution (the number of the primer set is mirQ0000086-1-1) and miR-192-5p reverse universal primer solution (the number of the primer set is mirQ 0000222-1-1).
The commodity with the primer package number of 'miRQ 0000010-1-1' comprises reverse transcription primer solution, forward primer solution and reverse general primer solution of a specific stem-loop method of cel-miR-39. The commodity of the primer package with the number of miRQ0000086-1-1 comprises reverse transcription primer solution, forward primer solution and reverse general primer solution of miR-29a-3p by a specific stem-loop method. The commodity of the primer set with the meal number of 'miRQ 0000222-1-1' comprises reverse transcription primer liquid, forward primer liquid and reverse general primer liquid of miR-192-5p by a specific stem-loop method.
The total volume of the qPCR reaction system is 20 mu l; wherein, preferably, the volume of the SYBR Green mixture is 9 μ l, the volume of the forward primer solution is 0.8 μ l, the volume of the reverse universal primer solution is 0.8 μ l, the volume of the ROX Reference Dye II solution is 0.4 μ l, the volume of the cDNA template is 4 μ l, and the rest is pure water; the using concentration of the forward primer solution is 5 mu M, and the using concentration of the reverse universal primer solution is 5 mu M; the cDNA template is a product after the reverse transcription reaction system is reacted.
The reagent for preparing the internal reference system comprises an internal reference cel-miR-39 liquid, and the sequence information of cel-miR-39 is shown in SEQ ID NO. 3. The internal reference cel-miR-39 solution is a nematode cel-miR-39 solution (Access Number: AJ487564) which is synthesized by England Weiji Shanghai trade Co., Ltd, and is packaged in a small bottle, wherein the prepared concentration is 20 mu M and 10 mu l in total, and the use concentration is 0.05 pmol/mu l.
Preferably, the kit further comprises an analysis method for evaluating the risk of prognosis of liver cancer, the analysis method comprises a prognosis risk model formula, which is:
Y＝2.19*ABCLC stage+1.45*BmiR-192-5p+1.81*CmiR-29a-3p；
ABCLC stagevalues of 0 or 1, 0 representing early and 1 representing late；
BmiR-192-5pThe value of 0 or 1, 0 represents that miR-192-5p is low in expression and the expression quantity is 2-△△CT<2.24; 1 represents that it is highly expressed, and the expression level is 2-△△CT≥2.24；
CmiR-29a-3pThe value of 0 or 1, 0 represents that miR-29a-3p is low in expression and the expression quantity is 2-△△CT<1.37; 1 represents that it is highly expressed, and the expression level is 2-△△CT≥1.37；
When the Y value is less than 1.81, the survival risk of the patient is low, and when the Y value is more than 1.81, the survival risk of the patient is high.
The fourth aspect of the invention relates to the application of the kit in evaluating the prognosis risk of liver cancer, and the kit has the characteristics of rapidness and convenience in detection, high accuracy, no wound and the like.
The invention has the beneficial effects that:
(1) according to the invention, miR-29a-3p and miR-192-5p are selected as combined molecular markers, and clinical verification experiments of big data show that the kit has outstanding advantages in terms of the risk level of liver cancer. The two molecular markers are applied to the development of a liver cancer serum miRNA detection kit for the first time, and the liver cancer serum miRNA detection kit can realize the hierarchical layering of the liver cancer risk degree by using a real-time fluorescence quantification method, so that the individual treatment is guided, and the clinical application value is high.
(2) Compared with the early warning and auxiliary diagnosis effects of Panel in the prior art, the prognosis model PI (prognosic index) has breakthrough innovation, and has higher innovative significance because biomarkers in the aspect of liver cancer prognosis, particularly serum prognosis markers, are rarely reported.
From the technical route, the invention screens candidate targets by a second-generation sequencing technology and has the advantages of high sensitivity, high flux and the like. The method adopts deep sequencing and QPCR technology, and builds a model PI (local { Y (1) X } (beta) through survival analysis and COX (COX) multifactor regression0+ΣβixiI.e. PI ═ β1x1+β2x2+β3x3Finally determining a prognostic model PIOSAs 2.19 × bclstage +1.45 × miR-192-5p +1.81 × miR-29a-3p was implicated in survival analysis and COX regression, the follow-up survival data for patients enrolled in the present invention began, at the earliest, in 2009.
(3) Compared with the early warning and auxiliary diagnosis effects of Panel in the prior art, the prognosis model PI (prognosic index) has more obvious economic and social significance.
At present, the relatively mature aspect of the liver cancer prognosis internationally is the Barcelona stage, namely BCLC stage, the invention incorporates two miRNA based on the stage, and subdivides (stratifies) liver cancer patients with different risks through a prognosis model constructed by the two miRNA, thereby guiding doctors to adopt different treatment strategies, means and measures to the liver cancer patients with different risks, not only avoiding over-treatment, but also avoiding insufficient treatment intensity, thereby improving the liver cancer treatment effect, saving medical resources and cost and having social significance.
(4) From the perspective of convenience in clinical application, through big data clinical verification experiments, compared with the simultaneous detection of the Panel consisting of more than 2 miRNAs, the model of the invention only needs to detect the Panel consisting of 2 miRNAs, thereby obviously reducing the errors and complexity of detection data.
(5) From the aspect of health economy, compared with the method for simultaneously detecting the Panel consisting of more than 2 miRNAs, the method for detecting the Panel consisting of 2 miRNAs obviously reduces the economic cost and accords with the simple, practical, scientific, economic and other standards required by clinical application.
(6) The invention provides a basis for developing a medicament for inhibiting two targets of miR-192-5p and miR-29a-3p in the future.
(7) The liver cancer serum miRNA detection kit selects two molecular markers miR-29a-3p and miR-192-5p to combine, is developed into the detection kit, is quick and convenient to detect, has high detection sensitivity and specificity and low cost, can meet the detection requirements of most tumor patients, has a wide application range, and is high in prediction accuracy rate through clinical verification.
(8) According to the liver cancer serum miRNA detection kit, a stable exogenous RNA sequence cel-miR-39 (from nematodes) is particularly introduced as an internal reference control aiming at the factors which are used as internal references of quantitative reaction in serum and have great differences in different samples, and an internal reference forward primer aiming at one is optimally designed, so that the accuracy of relative quantification in detection is greatly improved.
Drawings
FIG. 1 is a melting curve diagram of miR-29a-3p for detecting a sample by using the kit of the invention
FIG. 2 is a melting curve diagram of miR-192-5p for detecting samples by using the kit of the invention
FIG. 3 is a melting curve diagram of cel-miR-39 of a sample detected by using the kit of the invention
FIG. 4 is a graph of ROC (survival risk prediction) curves for performing miR-29a-3p and miR-192-5p single indexes and two indexes combined in Barcelona staging respectively by using the kit disclosed by the invention.
FIG. 5 is a prognostic risk model of the present invention.
Detailed Description
The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure. In the following examples, the reagents used were all analytical grade and were commercially available unless otherwise indicated. Experimental procedures not specifically identified herein are generally carried out under conventional conditions such as those described in the molecular cloning guidelines published by scientific Press, J. SammBruk et al, or under conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.
Example 1
Composition of kit
The liver cancer serum miRNA detection kit for evaluating the liver cancer prognosis risk degree comprises a reagent for preparing a reverse transcription reaction system, a reagent for preparing a qPCR reaction system and a reagent for preparing an internal reference system.
1. Internal reference system
The reagent for preparing the internal reference system comprises internal reference cel-miR-39 liquid. The internal reference solution was nematode cel-miR-39 solution (Access Number: AJ487564) synthesized by England Weiji Shanghai trade Co., Ltd, and packaged in a vial at a concentration of 20. mu.M and a total of 10. mu.l, using a concentration of 0.05 pmol/. mu.l, as shown in Table 1.
TABLE 1 ingredient table of internal reference system
| Components | Volume of | Concentration of use |
| cel-miR-39 solution | 2μl | 0.05pmol/μl |
2. Reverse transcription reaction system:
the reagent for preparing the reverse transcription reaction system comprises a reverse transcription primer solution (Bulge-Loop) of a specific stem-Loop methodTMRT Primer, supplier: ribobio), Reverse Transcriptase solution and buffer kit (Reverse Transcriptase M-MLV, supplier: TaKaRa, trade number: code No.2641A), dNTP mix (dNTP mix, supplier: TaKaRa, trade number: code No.4030), RNase Inhibitor solution (Recombinant RNase Inhibitor, supplier: TaKaRa, trade number: code No.2131A) and nuclease-free pure water. For preparing reverse transcription reaction systemThe reagent is packaged bottle by bottle, when in use, a reverse transcription reaction system is prepared according to a certain proportion, and the reverse transcription reaction system is 25 mu l/time, as shown in table 2.
TABLE 2 reverse transcription reaction System Components Table
| Components | Volume of | Final concentration |
| Total RNA samples | 2μl | |
| Specific stem-loop reverse transcription primer working solution (500nM) | 2μl | 40nM |
| Nuclease-free pure water | 7μl | |
| Reverse transcription buffer solution 5 | 5μl | 1× |
| dNTP mix (2.5mM) | 2μl | 0.2mM |
| Ribonuclease inhibitor solution (40U/. mu.L) | 0.5μl | |
| Reverse transcriptase (200U/. mu.L) | 0.5μl | |
| Nuclease-free pure water | 6μl |
Mixing the above systems, centrifuging instantly, standing at 70 deg.C for 10min, incubating with ice for 2min, adding the following reagents to perform Reverse Transcription (RT) reaction, and performing RT reaction: 60min at 42 ℃ and 10min at 70 ℃.
3. qPCR reaction system:
the reagent for preparing the PCR reaction system comprises SYBR Green mixed solution (
Premix Ex TaqTMII, supplier: TaKaRa, trade number: code No. RR820A), ROX Reference Dye II liquid (
Premix Ex TaqTMII attached), forward primer solution (Bulge-Loop)TMForward Primer supplier: ribobio), reverse universal primer solution (Bulge-Loop)TMReverse Primer supplier: ribobio), reverse transcription product cDNA template, and nuclease-free purified water. The reagents for preparing the reaction system were packaged bottle by bottle, and were used in a proportion of 20. mu.l/time, as shown in Table 3.
TABLE 3 qPCR reaction System Components Table
The forward primers used for preparing the reaction system are respectively a forward primer aiming at miR-29a-3p, a forward primer aiming at miR-192-5p and a forward primer aiming at cel-miR-39 of an internal reference nematode.
Second, using method of kit
The detection kit of the embodiment comprises the following specific detection steps:
1. and (4) extracting miRNA. By using
LS reagen method (supplier: Life Technologies, USA, trade number: 10296-: a, adding 750 mu l of Trizol LS into 250 mu l of serum, uniformly mixing (shaking), and standing at room temperature for 5 minutes for denaturation; b, adding exogenous internal reference nematode Cel-miR-39 with the volume of 2 mu l (the concentration is 0.05pmol/ul), immediately mixing the mixture, adding 200 mu l of chloroform (covering the mixture and shaking the mixture for 15 seconds), standing and incubating the mixture for 10 to 15 minutes at room temperature, and centrifuging the mixture for 15 minutes at 12000g and 4 ℃. And c, sucking about 500UL of the supernatant to a new tube, adding 50UL of sodium acetate and 4UL of nucleic acid precipitation aid, adding 500UL of isopropanol, uniformly mixing, standing at room temperature for 10 minutes, and centrifuging at 12000G for 10 minutes at 4 ℃. d, centrifuging and removing the supernatant to leave a precipitate (RNA floccule can be seen), adding 1 ml of 75% ethanol (prepared by DEPC treated water after sterile disinfection), shaking and washing, centrifuging at 7500g for 5 minutes at 4 ℃, and removing the supernatant. e, after removing the supernatant, the RNA was dried at room temperature for 20-30 minutes in 30. mu.l ddH2O (DEPC treatment and nuclease-free water after high temperature) dissolved RNA and stored at-20 ℃.
After the RNA sample is extracted, the sample quality is controlled by measuring the ratio of the concentration to the OD 260/280, and the sample is finally added into a reverse transcription reaction system, so that the optimal reaction result can be obtained when the OD 260/280 ratio is between 1.8 and 2.0.
2. Reverse transcription reaction (same as Table 2, total volume of reaction system is 25. mu.l)
| Components | Volume of | Final concentration |
| Total RNA samples | 2μl | |
| Specific stem-loop reverse transcription primer working solution (500nM) | 2μl | 40nM |
| Nuclease-free pure water | 7μl | |
| Reverse transcription buffer solution 5 | 5μl | 1× |
| dNTP mix (2.5mM) | 2μl | 0.2mM |
| Ribonuclease inhibitor solution (40U/. mu.L) | 0.5μl | |
| Reverse transcriptase (200U/. mu.L) | 0.5μl | |
| Nuclease-free pure water | 6μl |
Mixing the above systems, centrifuging instantly, standing at 70 deg.C for 10min, incubating with ice for 2min, adding the following reagents to perform Reverse Transcription (RT) reaction, and performing RT reaction: 60min at 42 ℃ and 10min at 70 ℃.
3. Fluorescent quantitative qPCR reaction (same as table 3, total volume of qPCR reaction system is 20. mu.l)
Fluorescent quantitative qPCR reaction and dissolution curve conditioning program
a，95℃，20sec；
b，95℃，10sec；
c，60℃，34sec；
d, repeating the steps b to c for 40 cycles;
e，95℃，15sec；
f，60℃，60sec；
g，95℃，15sec；
h，60℃，15sec。
qPCR reaction results:
when the liver cancer serum miRNA detection kit is used for detecting miR-29a-3p, miR-192-5p and Nematoda cel-miR-39, the amplification specificity of corresponding primers is good, and the primers are all single peaks. FIG. 1 is a melting curve diagram of miR-29a-3p of a detection sample; FIG. 2 is a melting curve diagram of miR-192-5p of a detection sample; FIG. 3 is a melting curve diagram of a reference cel-miR-39 in a detection sample.
4. Judgment Standard of kit
The method comprises the steps of fitting two indexes of miR-29a-3p and miR-192-5p through a Logistic regression equation and constructing a survival prognosis risk model of the liver cancer patient by combining with a liver cancer Barcelona stage (BCLC stage), wherein the sensitivity and specificity of the fitted prognosis risk model are higher than those of the single index. Through data analysis, a prognosis risk model formula is obtained, namely a regression equation is as follows:
Y＝PIOS＝2.19*ABCLC stage+1.45*BmiR-192-5p+1.81*CmiR-29a-3p；
PI, namely a prognotic index; OS, i.e., overall survival;
ABCLC stagevalues of 0 or 1, 0 representing early and 1 representing late;
BmiR-192-5pthe value of 0 or 1, 0 represents that miR-192-5p is low in expression and the expression quantity is 2-△△CT<2.24; 1 represents that it is highly expressed, and the expression level is 2-△△CT≥2.24；
CmiR-29a-3pThe value of 0 or 1, 0 represents that miR-29a-3p is low in expression and the expression quantity is 2-△△CT<1.37; 1 represents that it is highly expressed, and the expression level is 2-△△CT≥1.37.
When the Y value is less than 1.81, the survival risk of the patient is low, and when the Y value is more than 1.81, the survival risk of the patient is high.
Application example 1
By adopting the miRNA detection kit for liver cancer serum in example 1, miRNA in serum of 100 liver cancer patients and 70 healthy people (control) are detected, and expression quantity (2) of miR-192-5p and miR-29a-3p is detected-△△CT) The value is obtained. For the detection result, statistical analysis is carried out by adopting statistical software SPSS 17.0 and MedCalc12.2.1, the prediction result of each single index of miR-192-5p and miR-29a-3p, and the risk model jointly constructed by the miR-192-5p, miR-29a-3p and Barcelona staging (BCLC stage)The results of the joint prediction are shown in FIGS. 4 and 5 and tables 4 and 5. FIG. 5 shows that the prognostic risk model of the present invention can be used to subdivide (stratify) a large group of liver cancer patients into a low-risk group, a moderate-risk group and a high-risk group, each of which corresponds to three survival curves, and the difference between the three is significant (P value is less than 0.05) by the survival analysis Kaplan-Meier method, i.e., Kaplan-Meier curves of Overall Survival (OS) and Log-rank test, so as to guide the clinician to prepare an individualized treatment scheme for liver cancer patients with different risks.
TABLE 4 ROC Curve-bottom area of survival risk prediction for each variable
TABLE 5 results of pairwise comparisons of the areas under ROC curves for each variable prediction of survival Risk
As shown in tables 4 and 5, the risk model PI is jointly constructed by the miR-192-5p, the miR-29a-3p and the Barcelona staging (BCLC stage)OSThe combined prediction result of (A) has the maximum area under the ROC curve of 0.83, and is obviously higher than the area under the curve of any other single index (P values are all less than 0.01), which shows that miR-192-5P, miR-29a-3P and Barcelona staging (BCLC stage) are combined to construct a risk model PIOSThe survival risk advantage of the liver cancer patient is obviously predicted.
It should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And such obvious variations or modifications which fall within the spirit of the invention are intended to be covered by the scope of the present invention.
Claims (8)
1. An application of a marker for liver cancer prognosis in preparation of a liver cancer serum miRNA detection kit for evaluating risk of liver cancer prognosis is characterized in that the marker is a combination of miR-29a-3p and miR-192-5p, and the sequence information of miR-29a-3p is shown in SEQ ID No. 1; the sequence information of miR-192-5p is shown in SEQ ID NO. 2.
2. The use of the marker for the prognosis of liver cancer according to claim 1 in the preparation of a kit for detecting serum miRNA in liver cancer for the assessment of risk of prognosis of liver cancer, wherein the formula of a prognosis risk model based on the marker is as follows: Y2.19ABCLC stage+1.45*BmiR-192-5p+1.81*CmiR-29a-3p；
ABCLC stageValues of 0 or 1, 0 representing early and 1 representing late;
BmiR-192-5pthe value of 0 or 1, 0 represents that miR-192-5p is low in expression and the expression quantity is 2-△△CT<2.24; 1 represents that it is highly expressed, and the expression level is 2-△△CT≥2.24；
CmiR-29a-3pThe value of 0 or 1, 0 represents that miR-29a-3p is low in expression and the expression quantity is 2-△△CT<1.37; 1 represents that it is highly expressed, and the expression level is 2-△△CT≥1.37；
When the Y value is less than 1.81, the survival risk of the patient is low, and when the Y value is more than 1.81, the survival risk of the patient is high.
3. The use of the marker for liver cancer prognosis as claimed in claim 1 for preparing a kit for detecting serum miRNA for liver cancer for evaluating risk of liver cancer prognosis, wherein the kit comprises: the kit comprises a reagent for preparing a reverse transcription reaction system, a reagent for preparing a qPCR reaction system and a reagent for preparing an internal reference system; wherein, the reagent for preparing the reverse transcription reaction system at least comprises specific stem-loop reverse transcription primer solutions aiming at miR-29a-3p, miR-192-5p and nematode cel-miR-39 respectively; the reagent for preparing the qPCR reaction system at least comprises forward primer liquid and reverse universal primer liquid aiming at miR-29a-3p, miR-192-5p and nematode cel-miR-39 respectively; the reagent for preparing the internal reference system comprises an internal reference cel-miR-39 liquid, and the sequence information of cel-miR-39 is shown in SEQ ID NO. 3.
4. The use of the marker for liver cancer prognosis as claimed in claim 3 for preparing a kit for detecting serum miRNA in liver cancer for evaluating risk of liver cancer prognosis, wherein the kit comprises: the reagent for preparing the reverse transcription reaction system also comprises reverse transcriptase liquid, dNTP mixed liquid, ribonuclease inhibitor liquid, reverse transcription buffer liquid and nuclease-free pure water.
5. The use of the marker for liver cancer prognosis as claimed in claim 3 for preparing a kit for detecting serum miRNA in liver cancer for evaluating risk of liver cancer prognosis, wherein the kit comprises: the total volume of the reverse transcription reaction system is 25 mu l; wherein, the total RNA sample is 2 mul, the reverse transcription primer liquid of the specific stem-loop method is 2 mul, the reverse transcriptase liquid is 0.5 mul, the dNTP mixed liquid is 2 mul, the ribonuclease inhibitor liquid is 0.5 mul, the reverse transcription buffer liquid is 5 mul and the pure water without nuclease is 13 mul; the total RNA sample contains an internal reference cel-miR-39 liquid.
6. The use of the marker for liver cancer prognosis as claimed in claim 5 for preparing a kit for detecting serum miRNA for liver cancer for evaluating risk of liver cancer prognosis, wherein the kit comprises: the preparation of the total RNA sample is to add 2 mul of internal reference cell-miR-39 liquid in the process of extracting 250 mul of serum total RNA, and the using concentration of the internal reference cell-miR-39 liquid is 0.05 pmol/mul.
7. The use of the marker for liver cancer prognosis as claimed in claim 3 for preparing a kit for detecting serum miRNA in liver cancer for evaluating risk of liver cancer prognosis, wherein the kit comprises: the reagent for preparing the qPCR reaction system also comprises SYBR Green mixed liquor, ROX Reference Dye II liquor and pure water.
8. The use of the marker for liver cancer prognosis as claimed in claim 7 for preparing a kit for detecting serum miRNA in liver cancer for evaluating risk of liver cancer prognosis, wherein the kit comprises: the total volume of the qPCR reaction system is 20 mu l; wherein the volume of the SYBR Green mixed solution is 9 μ l, the volume of the forward primer solution is 0.8 μ l, the volume of the reverse universal primer solution is 0.8 μ l, the volume of the ROX Reference Dye II solution is 0.4 μ l, the volume of the cDNA template is 4 μ l, and the balance is pure water; the using concentration of the forward primer solution is 5 mu M, and the using concentration of the reverse universal primer solution is 5 mu M; the cDNA template is a product after the reverse transcription reaction system is reacted.
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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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| 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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