CN114634980A - Serum miRNA marker combination for lung cancer auxiliary diagnosis and detection kit thereof - Google Patents
Serum miRNA marker combination for lung cancer auxiliary diagnosis and detection kit thereof Download PDFInfo
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Abstract
The lung cancer is a common malignant tumor, and the latest statistical data show that the incidence rate is the first and the mortality rate is the first in the malignant tumors all over the world. Early diagnosis of lung cancer can significantly improve the survival of patients. The invention relates to a peripheral blood miRNA marker combination for lung cancer auxiliary diagnosis and a detection kit thereof, which are prepared from hsa-miR-545-5p, hsa-miR-514b-5p, hsa-miR-590-5p, hsa-miR-627-5p and hsa-miR-653-5p in human serum, wherein an internal reference gene is U6, and specific primers of the miRNA are provided. Also provides necessary reagents for detecting the positive quality control product, the negative quality control product, poly-A tailing enzyme, reverse transcriptase and other kits and a technical scheme for achieving the detection purpose. The invention can detect abnormal rise of some tumor-derived miRNAs in serum at an earlier stage, and is used for clinical early-stage lung cancer auxiliary diagnosis.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a serum miRNA marker combination for lung cancer auxiliary diagnosis and a detection kit thereof.
Background
Lung cancer is a common malignant tumor, which can be classified into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC) according to histopathology. The latest national cancer statistical data published by the national cancer center in 2019 shows that about 392.9 ten thousands of people suffer from malignant tumors and about 233.8 thousands of people die of the malignant tumors every year in China, wherein the lung cancer is the first of the total incidence rate and the first of the death rate of the malignant tumors in China. The incidence and mortality of lung cancer are the first in male malignant tumors, and the second in female malignant tumors. At present, the early diagnosis of the lung cancer mainly depends on an image technology and conventional pathological detection, except for liquid biopsy, the image detection technology can only detect tumor bodies larger than 5 mm, and tumor cells which do not form focuses can not be detected; detection with a fiberbronchoscope, mediastinoscope lavage fluid, or scraping tissue is prone to patient discomfort; x-ray, CT, nuclear magnetism, tissue biopsy, etc. are more traumatic. The detection of the serum tumor marker has important functions in screening and diagnosing early lung cancer because of safety, no wound, low cost, simplicity and feasibility. Early symptoms of lung cancer often can not cause the attention of patients, and many lung cancer patients are diagnosed in middle and advanced stages, so that the significance of early screening of lung cancer is great.
Micro RNA (microRNA, miRNA) is a highly conserved, endogenous and non-coding single-stranded small molecule RNA in peripheral blood, has the length of about 18-25 nt, is mainly involved in gene transcription and regulation of the level after transcription, can regulate different biological processes such as cell proliferation, differentiation, apoptosis, death and the like, and can play a role in promoting cancer or inhibiting cancer genes in the process of cancer diseases. miRNA can enter blood circulation due to cell death or active secretion of cells, and the miRNA has the following advantages as a tumor marker for detection: miRNA can be abnormally expressed in cancer cells and stably exists in peripheral blood, saliva, urine and excrement, so that the miRNA can be detected through real-time quantitative PCR, and the change of an expression spectrum of the miRNAs is earlier and more specific than that of protein tumor markers, so that noninvasive diagnosis is realized. Therefore, the quantitative detection of miRNA in peripheral blood is very suitable for early diagnosis of lung cancer. At present, the types of formally named human miRNA are 2800, and the invention aims to screen accurate specific lung cancer miRNA markers.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide a serum miRNA lung cancer diagnostic marker combination and a detection kit thereof, which can detect lung cancer simply, effectively and non-invasively.
In order to realize the purpose of the invention, the overall technical scheme is as follows:
provides a serum miRNA lung cancer diagnosis marker combination, which consists of 1 or more of hsa-miR-545-5p, hsa-miR-514b-5p, hsa-miR-590-5p, hsa-miR-627-5p and hsa-miR-653-5p in human serum.
The miRNA marker combination is applied to lung cancer diagnosis.
The miRNA marker combination is applied to the preparation of a detection kit.
Further, primers and reagents in the miRNA marker combined detection kit are used for reverse transcription and qPCR amplification.
The sequence of each miRNA in hsa-miR-545-5p, hsa-miR-514b-5p, hsa-miR-590-5p, hsa-miR-627-5p and hsa-miR-653-5p related by the invention is known and disclosed.
TABLE 1 above miRNA sequences and public database information of U6
The primers in the detection kit are qPCR forward primers of 5 miRNA markers and qPCR amplification primers of U6 serving as an internal reference.
The reagent of the kit for detecting the serum miRNA lung cancer diagnostic marker combination is a Poly-A tailing method reverse transcription reagent and a qPCR method detection reagent.
As the invention uses the Poly-A tailing method to carry out reverse transcription on miRNA, the reverse transcription primer and the reverse primer of pPCR are both universal primers, and only the forward primer of qPCR is used for identifying 5 miRNA markers.
A detection kit of a serum miRNA lung cancer diagnostic marker combination further comprises but is not limited to a positive quality control product, a negative quality control product, poly-A tailing enzyme, reverse transcriptase, dNTPs, reverse transcription buffer, RNase-free water, qPCR buffer, magnesium chloride, DNA polymerase and SYBR Green fluorescent dye.
The reagents involved in the examples of the present invention are all commercially available products, and can be purchased from commercial sources, unless otherwise specified. In some embodiments, the invention uses universal reverse transcription primers and qPCR universal reverse primers, commercially available from all-grass company.
Specifically, the technical scheme of the invention comprises the following steps:
1. candidate marker screening protocol: and (3) establishing a lung cancer serum miRNA candidate marker by analyzing big data of an NCBI-GEO database and a TCGA database. The NCBI-GEO database selects data from serum sample sources, and the data content is the serum sample analysis results of 205 lung cancer patients and healthy volunteers; the TCGA database is data from tissue samples, and the data content is miRNA differential expression analysis of 477 lung cancer tissues and 51 paracarcinoma tissues. Finding miRNA expression profiles synchronously up-regulated in the serum of lung cancer population and lung cancer tissues, and then selecting miRNA with poor prognosis from the miRNA expression profiles to obtain 206 miRNA candidate markers. And then, adopting colinearity analysis to remove collinear miRNA, and reserving 22 non-collinear miRNA candidate markers. And detecting the expression conditions of the 5 miRNAs in the serum sample by adopting a tailing method reverse transcription and qPCR method.
2. Blood samples meeting the standard are collected by a Standard Operating Procedure (SOP), complete demographic data and clinical data are collected, and a standardized sample library and a database are established.
3. And detecting the collected sample, and verifying the miRNA candidate marker of the lung cancer serum by a reverse transcription-qPCR method. Detection index Δ Ct value = CtmiRNA- CtU6。
4. And judging the diagnosis efficiency of the miRNAs on the lung cancer by using an ROC curve, finally establishing a miRNA combined diagnosis marker combination and establishing a disease risk judgment formula.
5. Statistical methods used for statistical analysis include, but are not limited to χ2Checking,t-test, collinearity analysis, logistic regression, and Fisher's decision.
The invention has the beneficial effects that:
1. the detection of serum miRNA tumor markers belongs to the category of liquid biopsy, has the characteristics of high specificity, early detection, high sensitivity and minimal invasion compared with the clinical conventional blood tumor markers, and is a novel marker for diagnosing various diseases including tumors.
2. The invention uses the self-designed qPCR forward primer to carry out semi-quantitative analysis on the 5 miRNAs, and has the advantages that: compared with a stem-loop method, the tailed reverse transcription method has the advantages that the reverse transcription of multiple miRNAs can be completed only by one reverse transcription system, and has the advantages of high flux, less required blood sample amount, low cost and the like, and the defects that the specificity of a primer is generally poor. The self-designed qPCR forward primer has high specificity, sensitivity and reproducibility, fully makes up the defects of the tailing reverse transcription, and has great advantages in practical application.
3. The invention uses the self-designed qPCR forward primer, and has the advantages that:
4. the invention applies a reverse transcription-qPCR method, verifies and evaluates a plurality of miRNA in the serum of a large number of lung cancer and healthy people, and the miRNA combination has obvious difference in the serum of the lung cancer and the healthy people, thereby being an ideal and reliable lung cancer marker.
5. The detection result of the training set established by the invention on the test set shows that the diagnostic efficiency is up to 90.7%. Has important significance for the lung cancer diagnosis of the future clinical pair.
Drawings
Figure 1 is a flow diagram of an embodiment.
FIG. 2 Combined diagnostic ROC curves for the above 5 miRNAs.
Figure 3 expression of the 5 mirnas described above in all 116 samples. In the figure, N represents healthy people, T represents tumor people, and the expression level of miRNA in ordinate is Δ Ct, Ct = CtmiRNA-CtU6 。
Detailed Description
1. Sample collection criteria: the inventor collects a large number of serum samples of lung cancer patients and normal physical examination people from a Hospital affiliated to Chinese medical science and university in 2018 to 2019, and selects 116 samples from the serum samples by sorting data, wherein 56 samples of lung cancer and 60 samples of healthy people are detected. The selected lung cancer patients were diagnosed without surgery and medication history and were pathologically confirmed to be lung cancer patients after hospitalization. The healthy population is qualified for physical examination in the physical examination center. The system collects demographic and clinical data of these samples.
2. Serum sample preparation: collecting venous whole blood with 5 ml blood collecting tube with inert separating gel, transferring to centrifuge rapidly after completion, centrifuging at 3000 g-4 deg.C-10 min, sucking 1 ml supernatant, transferring to RNA enzyme-free EP tube, and storing in-80 deg.C refrigerator until use.
3. Sample pretreatment before RNA extraction: taking out serum from a refrigerator at the temperature of-80 ℃, standing at room temperature until the serum is completely melted, putting the serum into a centrifugal machine, taking 200 ul of supernatant under the centrifugal condition of 10000 g-6 ℃ to 15 minutes, and putting the supernatant into a new EP tube.
4. Full-automatic extraction of cell-free serum miRNA: the sample was placed in a fully automatic nucleic acid extractor, commercially available from QIAGEN, model QIAcube. The instrument uses QIAGEN centrifugal column Kit (miRNeasy Serum/Plasma Kit) to realize the full-automatic extraction of the miRNA from the cell-free Serum. The volume of the obtained RNA is 15 ul, and the concentration is more than 100 ng/ul.
5. And (3) carrying out reverse transcription on the miRNA by using a tailing reverse transcription kit. The total reverse transcription rate was 20 ul, and the amount of RNA added was 200 ng/20 ul. The reverse transcription system is as follows:
the reverse transcription was performed under conditions of 37 ℃ for 60 minutes and then 85 ℃ for 5 seconds for enzyme inactivation. The concrete operation refers to the specification of TransScript miRNA First-Strand cDNA Synthesis SuperMix.
6. Real-time fluorescent quantitative pcr (qpcr): the kit used in the amplification reaction is TransStart Tip Green qPCR SuperMix kit sold by the general gold company, and the reaction system is as follows:
the instrument used for the amplification reaction is carried out on a QuantStaudio 5 real-time fluorescence quantitative PCR instrument of Sammerfei, the reaction program is set as shown in the following table, and other settings are system default values:
and (3) acquiring results by using QuantStaudio Design & Analysis Software with own Software of the instrument, wherein an internal parameter is U6, and a detection index is a Ct value.
7. And (3) establishing a training set, namely randomly dividing all samples into 2 groups with equal number, wherein the number of the training set and the testing set is 58 respectively, and the training set and the testing set respectively comprise 28 lung cancers and 30 healthy people.
a. The fate of the patient is used to compare the expression difference of the miRNA in the patients with lung cancer and the healthy people, i.e. fateLung cancer-∆CtHealth care。
b. Specific Δ CtHealth care=
；∆CtLung cancer=
。
c. The expression difference of each miRNA in lung cancer population and healthy population = 2-∆∆Ct。
Statistical analysis is carried out by using SPSS 22.0 software, and the training set data is analyzed by adopting an Fisher discriminant method to obtain a Fisher linear discriminant formula of Y =1.991 +0.771 Δ CtmiR 545 +0.439 Δ CtmiR 514b +0.852 Δ CtmiR 590 miR-1.277 Δ CtmiR 627+0.497 CtmiR 653.
8. And (4) detecting the test set, wherein the number of the test sets is 58, 28 lung cancers and 30 healthy people are detected. And obtaining the Δ Ct values of the 7 miRNAs in each sample of the test set by adopting the same detection operation as the training set. Judging each data of the test set by using the Fisher linear discriminant formula, wherein the obtained detection result data are as follows:
according to the detection results of the test set, the true positive rate (sensitivity) of the serum miRNA lung cancer diagnosis marker combination and the detection kit thereof on lung cancer is 93.1%, namely the missed diagnosis rate is 6.9%; the true negative rate (specificity) is 96.6 percent, namely the misdiagnosis rate is 3.4 percent; the total accuracy of all 58 samples in the test set was 94.8%.
And (3) drawing an ROC curve, and evaluating the diagnosis efficiency of the 5 miRNA markers on the lung cancer by using the area AUC under the curve, which is shown in figure 2.
The maximum diagnostic potency of AUC is 1.00, the independent diagnostic potency of the 5 miRNA markers is 0.67-0.82, and the combined diagnostic potency is as high as 0.91, as shown in the following table.
The detection results of all 116 samples in the training set and the test set are summarized, and it can be seen that the 5 miRNAs in the patent have significant expression difference between lung cancer population and healthy population, the expression amount of each miRNA in the serum of the two populations is shown in figure 3, and the specific data is shown in the following table.
| miRNA | ∆CtHealth care | ∆CtLung cancer | ∆∆Ct | Difference in expression | PValue of |
| hsa-miR-545-5p | -0.52 | -2.48 | -1.96 | 3.89 | ＜0.001 |
| hsa-miR-514b-5p | 1.79 | -0.31 | -2.1 | 4.29 | ＜0.001 |
| hsa-miR-590-5p | -1.07 | -3.11 | -2.04 | 4.11 | ＜0.001 |
| hsa-miR-627-5p | -1.59 | -2.44 | -0.85 | 1.80 | ＜0.004 |
| hsa-miR-653-5p | -2.30 | -3.71 | -1.41 | 2.66 | ＜0.002 |
The invention establishes a set of complete operation flow, and can better distinguish lung cancer patients from healthy people by the serum miRNA lung cancer diagnosis marker combination and the detection kit thereof from serum samples.
Sequence listing
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Claims (7)
1. A serum miRNA lung cancer diagnostic marker combination is characterized by consisting of 1 or more of free hsa-miR-545-5p, hsa-miR-514b-5p, hsa-miR-590-5p, hsa-miR-627 and hsa-miR-653-5p in human serum.
2. The combination of serum miRNA lung cancer diagnostic markers according to claim 1, wherein the serum miRNA markers are a combination of two or more of hsa-miR-545-5p, hsa-miR-514b-5p, hsa-miR-590-5p, hsa-miR-627 and hsa-miR-653-5 p.
3. The serum miRNA lung cancer diagnostic marker combination of claim 1, wherein: the miRNA marker is combined with the application in the diagnosis of lung cancer.
4. The detection kit for the serum miRNA lung cancer diagnostic marker combination according to claim 1, wherein: the use of the serum miRNA lung cancer diagnostic marker combination of claim 1 in the preparation of a detection kit.
5. The detection kit for the serum miRNA lung cancer diagnostic marker combination according to claim 4, wherein: the qPCR amplification primer sequence of the lung cancer diagnostic marker combination of claim 1.
6. The detection kit for the serum miRNA lung cancer diagnostic marker combination according to claim 4, wherein: the reagent is poly-A tailing reverse transcription reagent and qPCR detection reagent.
7. The detection kit for the serum miRNA lung diagnosis marker combination according to claim 4, wherein: positive quality control material, negative quality control material, poly-A tailing enzyme, reverse transcriptase, dNTPs, reverse transcription buffer solution, RNA enzyme-free water, qPCR buffer solution, magnesium chloride, DNA polymerase and SYBR Green fluorescent dye.
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