CN114410794B - Application of exosome miR-106b-3P, ARPC5 in lung cancer diagnosis - Google Patents
Application of exosome miR-106b-3P, ARPC5 in lung cancer diagnosis Download PDFInfo
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Abstract
The invention discloses a kit, a device and a method for diagnosing lung cancer, wherein the kit comprises a primer and a probe for detecting an exosome long RNA marker and a miRNA marker combination, the long RNA marker comprises one or more of ARPC5, MBOAT2 and IL1B, and the miRNA marker comprises one or more of miR-450B-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106B-3p, miR-30e-5p, miR-4746-5p and miR-125a-5p. The exosome-based noninvasive early lung cancer diagnosis method provided by the invention has high sensitivity and high specificity in early lung cancer, and provides important value for early lung cancer diagnosis. Has great help to the prevention and treatment of lung cancer in China.
Description
The application is a divisional application, the application number of the main application is 202111109658.2, the application date is 2020, 05 and 11, and the invention is named as a kit, a device and a method for diagnosing lung cancer.
Technical Field
The invention relates to the field of medical diagnosis, in particular to a diagnostic kit, a device and a method for early lung cancer.
Background
Lung cancer is one of the major cancer species in our country and even worldwide. According to the latest global cancer statistics in 2018, lung cancer incidence and mortality rate are the first among all cancer species. According to 2019 Chinese tumor registration data, 390.2 ten thousand cancer cases are newly increased in China 2015, and about 233.8 ten thousand cancer death cases are obtained, wherein lung cancer is the main cause of Chinese cancer death. Therefore, in order to promote the diagnosis and treatment behavior of lung cancer in China and improve the prognosis of lung cancer patients, early diagnosis becomes an important problem for diagnosis and treatment of lung cancer.
With the application of low-dose helical CT, more and more imaging was found as lung nodules (single lesions <3cm in the lung interstitium, and no associated atelectasis or lymphadenopathy). However, not all lung nodules are malignant, and the identification of benign and malignant lung nodules has been a difficulty in clinical diagnosis and treatment in thoracic surgery. At present, non-invasive detection means such as blood plasma circulating tumor cells, circulating tumor free DNA and the like are also adopted, but the sensitivity of detection in early lung cancer diagnosis is not high; therefore, development of a high-sensitivity noninvasive lung cancer early detection method is needed.
Disclosure of Invention
The invention provides a reagent, a device and a method for noninvasive early lung cancer diagnosis based on exosomes.
In one aspect, the invention provides a kit for lung cancer diagnosis, which comprises a primer and a probe for detecting an exosome long RNA marker and a miRNA marker combination, wherein the long RNA marker comprises one or more of ARPC5, MBOAT2 and IL1B, and the miRNA marker comprises one or more of miR-450B-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106B-3p, miR-30e-5p, miR-4746-5p and miR-125a-5p.
Preferably, the marker is a combination of miR-450b-5p, let-7f-2-3p and ARPC 5.
Preferably, the marker is a combination of miR-106b-3P, miR-30e-5p and MBOAT 2.
Preferably, the marker is a combination of miR-106b-3P, miR-30e-5p, miR-3615, miR-885-5p and ARPC 5.
Preferably, the marker is a combination of miR-106B-3P, miR-125a-5p, miR-3615, miR-450B-5p and IL1B. .
Preferably, the exosome source includes one or more of blood, saliva, and sputum.
Preferably, the primers and probes comprise:
primers and probes for detecting reference ACTB: the upstream primer of the RNA is the nucleotide sequence shown in a sequence number 1, the downstream primer is the nucleotide sequence shown in a sequence number 2, and the probe is the nucleotide sequence shown in a sequence number 3;
primers and probes for detection of ARPC 5: the upstream primer of the RNA is the nucleotide sequence shown in a sequence number 4, the downstream primer is the nucleotide sequence shown in a sequence number 5, and the probe is the nucleotide sequence shown in a sequence number 6;
primers and probes for detection of IL 1B: the upstream primer of the RNA is the nucleotide sequence shown in a sequence number 7, the downstream primer is the nucleotide sequence shown in a sequence number 8, and the probe is the nucleotide sequence shown in a sequence number 9;
primers and probes for detection of MBOAT 2: the upstream primer of the RNA is the nucleotide sequence shown as a sequence number 10, the downstream primer is the nucleotide sequence shown as a sequence number 11, and the probe is the nucleotide sequence shown as a sequence number 12;
reverse transcription primers, PCR primers and probes for detection of let-7 f-2: the reverse transcription primer has a nucleotide sequence shown as a sequence number 13, the PCR upstream primer has a nucleotide sequence shown as a sequence number 14, the downstream primer has a nucleotide sequence shown as a sequence number 41, and the probe has a nucleotide sequence shown as a sequence number 15;
reverse transcription primer, PCR primer and probe for detecting miR-106b-3 p: the reverse transcription primer has a nucleotide sequence shown as a sequence number 16, the PCR upstream primer has a nucleotide sequence shown as a sequence number 17, the downstream primer has a nucleotide sequence shown as a sequence number 41, and the probe has a nucleotide sequence shown as a sequence number 18;
reverse transcription primers, PCR primers and probes for detecting miR-125a-5 p: the reverse transcription primer has a nucleotide sequence shown as a sequence number 19, the PCR upstream primer has a nucleotide sequence shown as a sequence number 20, the downstream primer has a nucleotide sequence shown as a sequence number 41, and the probe has a nucleotide sequence shown as a sequence number 21;
reverse transcription primer, PCR primer and probe for detecting miR-30e-5 p: the reverse transcription primer has a nucleotide sequence shown as a sequence number 22, the PCR upstream primer has a nucleotide sequence shown as a sequence number 23, the downstream primer has a nucleotide sequence shown as a sequence number 41, and the probe has a nucleotide sequence shown as a sequence number 24;
reverse transcription primer, PCR primer and probe for detecting miR-3615: the reverse transcription primer has a nucleotide sequence shown as a sequence number 25, the PCR upstream primer has a nucleotide sequence shown as a sequence number 26, the downstream primer has a nucleotide sequence shown as a sequence number 41, and the probe has a nucleotide sequence shown as a sequence number 27;
reverse transcription primers, PCR primers and probes for detecting miR-450b-5 p: the reverse transcription primer has a nucleotide sequence shown as a sequence number 28, the PCR upstream primer has a nucleotide sequence shown as a sequence number 29, the downstream primer has a nucleotide sequence shown as a sequence number 41, and the probe has a nucleotide sequence shown as a sequence number 30;
reverse transcription primers, PCR primers and probes for detecting miR-4746-5 p: the reverse transcription primer has a nucleotide sequence shown as a sequence number 31, the PCR upstream primer has a nucleotide sequence shown as a sequence number 32, the downstream primer has a nucleotide sequence shown as a sequence number 41, and the probe has a nucleotide sequence shown as a sequence number 33;
reverse transcription primer, PCR primer and probe for detecting miR-885-5 p: the reverse transcription primer is a nucleotide sequence shown as a sequence number 34, the PCR upstream primer is a nucleotide sequence shown as a sequence number 35, the downstream primer is a nucleotide sequence shown as a sequence number 41, and the probe is a nucleotide sequence shown as a sequence number 36;
reverse transcription primer, PCR primer and probe for detecting reference U6: the reverse transcription primer of U6 has the nucleotide sequence shown in the sequence No. 39, the PCR upstream primer has the nucleotide sequence shown in the sequence No. 37, the downstream primer has the nucleotide sequence shown in the sequence No. 38, and the probe has the nucleotide sequence shown in the sequence No. 40.
In another aspect, the invention provides a device for lung cancer diagnosis, comprising a primer and a probe for detecting an exosome long RNA marker and a miRNA marker combination, wherein the long RNA marker comprises one or more of ARPC5, MBOAT2 and IL1B, and the miRNA marker comprises one or more of miR-450B-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106B-3p, miR-30e-5p, miR-4746-5p and miR-125a-5p.
In another aspect, the invention provides a method for lung cancer diagnosis, comprising detecting the specificity of an exosome long RNA marker comprising one or more of ARPC5, MBOAT2, IL1B and a combination of miRNA markers comprising one or more of miR-450B-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106B-3p, miR-30e-5p, miR-4746-5p, miR-125a-5p.
The noninvasive early lung cancer diagnosis method based on exosomes provided by the invention has high sensitivity and high specificity in early lung cancer, and provides important value for early lung cancer diagnosis. Has great help to the prevention and treatment of lung cancer in China. Furthermore, the highest sensitivity combination can reach 100% sensitivity, and the highest specificity combination can realize 96.67% specificity, so that the method has better performance.
Drawings
FIG. 1 is a ROC curve of miR-450b-5p+let-7f-2-3p+ARPC5 combination detection of lung cancer.
FIG. 2 is an ROC curve of miR-106b-3P+miR-30e-5p+MBOAT2 combination detection of lung cancer.
FIG. 3 is a ROC curve of miR-106b-3P+miR-30e-5p+miR-450b-5p+miR-885-5p+ARPC5 combination detection of lung cancer.
FIG. 4 is a ROC curve of miR-106b-3P+miR-30e-5p+miR-4746-5p+miR-885-5p+ARPC5 combination detection lung cancer.
FIG. 5 is a ROC curve of miR-106b-3P+miR-30e-5p+miR-3615+miR-885-5p+ARPC5 combination detection of lung cancer.
FIG. 6 is an ROC curve of miR-106b-3P+miR-125a-5p+miR-3615+miR-450b-5p+IL1B combination for detecting lung cancer.
Detailed Description
Extracellular vesicles (Extracellular Vesicles; EVs; hereinafter referred to as extracellular vesicles) are vesicle-like small bodies with a bilayer membrane structure, which are detached from the cell membrane or secreted by the cell, and have diameters varying from 30 to 1000nm, and are mainly composed of microvesicles (MicroVesicles, MVs) and exosomes (exosomes), which are small vesicles detached from the cell membrane after activation or injury of the cell. Because of its unique biological characteristics, extracellular vesicles are of great importance in the diagnosis of disease, particularly the exosomes therein.
The exosome is a membranous vesicle with the particle size of 30-150 nm secreted into the extracellular environment after the fusion of the intracellular multivesicular body and the cell membrane, is an important medium for information transmission among cells, and plays an important role in antigen presentation, apoptosis, inflammatory reaction, tumorigenesis, development and metastasis. It is widely distributed in body fluids, including blood, saliva, urine, milk, hydrothorax and ascites; contains various contents such as DNA, RNA, protein and the like, and can be used as a noninvasive diagnosis marker for various diseases such as tumors and the like. The miRNA is the most abundant nucleic acid component in exosomes, so that the exosome miRNA has potential for early diagnosis of lung cancer.
The kit, the device and the method provided by the invention are used for finding out that the combination of long RNA and miRNA which show obvious differential expression in the exosomes of patients suffering from early lung cancer is used as a marker for diagnosing early lung cancer through experimental study.
In embodiments, a marker combination for diagnosing lung cancer comprises at least one long RNA and one miRNA. Wherein the long RNA comprises: ARPC5, MBOAT2, IL1B. MiRNA includes: miR-450b-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106b-3p, miR-30e-5p, miR-4746-5p and miR-125a-5p.
In some preferred embodiments, wherein the preferred combinations are: miR-450b-5p+let-7f-2-3p+ARPC5, miR-106b-3P+miR-30e-5p+MBOAT2, miR-106b-3P+miR-30e-5p+miR-450b-5p+miR-885-5p+ARPC5miR-106b-3P+miR-30e-5p+miR-4746-5p+miR-885-5p+ARPC5, miR-106b-3P+miR-30e-5p+miR-3615+miR-885-5p+ARPC5 and miR-106b-3P+miR-125a-5p+miR-3615+miR-450b-5p+IL1B. The combination can provide better basis for early diagnosis of lung cancer and indicate disease risk.
In addition, the kit for diagnosing lung cancer comprises a primer and a probe for detecting the exosome long RNA marker. Primers and probes for detecting exosome markers include:
primers and probes for detecting reference ACTB: the upstream primer of the RNA is specifically shown as a nucleotide sequence shown as a sequence number 1, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 2, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 3;
primers and probes for detection of ARPC 5: the upstream primer of the RNA is specifically shown as a nucleotide sequence shown as a sequence number 4, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 5, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 6;
primers and probes for detection of IL 1B: the upstream primer of the RNA is specifically shown as a nucleotide sequence shown as a sequence number 7, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 8, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 9;
primers and probes for detection of MBOAT 2: the upstream primer of the RNA is specifically shown as a nucleotide sequence shown as a sequence number 10, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 11, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 12;
reverse transcription primers, PCR primers and probes for detection of let-7 f-2: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 13, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 14, the downstream primer is specifically shown as a nucleotide sequence number 41, and the probe is specifically shown as a nucleotide sequence number 15;
reverse transcription primer, PCR primer and probe for detecting miR-106b-3 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 16, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 17, the downstream primer is specifically shown as a nucleotide sequence number 41, and the probe is specifically shown as a nucleotide sequence number 18;
reverse transcription primers, PCR primers and probes for detecting miR-125a-5 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 19, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 20, the downstream primer is specifically shown as a nucleotide sequence number 41, and the probe is specifically shown as a nucleotide sequence number 21;
reverse transcription primer, PCR primer and probe for detecting miR-30e-5 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 22, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 23, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 41, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 24;
reverse transcription primer, PCR primer and probe for detecting miR-3615: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 25, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 26, the downstream primer is specifically shown as a nucleotide sequence number 41, and the probe is specifically shown as a nucleotide sequence number 27;
reverse transcription primers, PCR primers and probes for detecting miR-450b-5 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 28, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 29, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 41, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 30;
reverse transcription primers, PCR primers and probes for detecting miR-4746-5 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 31, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 32, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 41, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 33;
reverse transcription primer, PCR primer and probe for detecting miR-885-5 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 34, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 35, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 41, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 36;
reverse transcription primer, PCR primer and probe for detecting reference U6: the reverse transcription primer of U6 is specifically shown as a nucleotide sequence of SEQ ID NO. 39, the PCR upstream primer is specifically shown as a nucleotide sequence of SEQ ID NO. 37, the downstream primer is specifically shown as a nucleotide sequence of SEQ ID NO. 38, and the probe is specifically shown as a nucleotide sequence of SEQ ID NO. 40. The nucleotide sequences of the primers and probes are shown in Table 1.
TABLE 1
Further, the exosome sources include one or more of blood, saliva, and sputum.
The kit, the device and the method are applicable to individuals with high risk of lung cancer, normal individuals and patients after lung cancer operation.
The following description of the embodiments of the present invention will be made in full and clear by reference to the accompanying drawings, in which some, but not all embodiments of the invention are described. Based on the embodiments of the present invention, other embodiments that may be obtained by those of ordinary skill in the art without making any inventive effort are within the scope of the present invention.
In order to screen the exosome markers related to the diagnosis of the colon cancer, 50 cases of early lung cancer patients and 72 cases of control patients respectively, blood is taken to be not less than 10ml and plasma is separated, exosomes in the plasma are separated by a classical ultracentrifugation method, RNA is extracted, and the obtained RNA is subjected to RNA library-building sequencing respectively. The data obtained were analyzed bioinformatically to compare differentially expressed RNAs in early lung cancer patients and controls. These RNA-level markers from exosomes can be used for early diagnosis of lung cancer.
Further directed to the application of the assay wherein the RNA markers are as follows: (1) Collecting a body fluid sample (including blood, sputum and saliva) of an individual to be tested; (2) isolating exosomes in the body fluid; (3) detecting the expression level of the target RNA by a two-step method; (4) Normalizing the expression level of the detection target RNA by using an internal reference gene; (5) Substituting the normalized gene expression level into a judgment model to obtain an output value; (6) And judging whether the individual to be tested is lung cancer or not according to the output value of the model and the judging threshold value.
The kit comprises a PCR primer, a probe, a standard substance and a PCR two-step detection system for detecting the exosome long RNA marker.
The invention comprises the steps of selecting an internal reference beta-Actin (ACTB) to quantify long RNA, and quantifying miRNA by taking U6 as an internal reference. Wherein quantification of target RNA at the time of reference selection is based on the detected Ct value using the quantitative formula 2ΔΔCt to calculate the expression level of the marker. After target RNA expression levels are obtained, ROC characteristic curves and AUCs are used to assess the accuracy of lung cancer detection with single RNA or in combination with multiple RNAs.
Example 1 screening for exosome mRNA and LncRNA markers associated with early lung cancer based on high throughput sequencing
In order to screen the exosome markers related to early lung cancer diagnosis, 50 cases of early lung cancer diagnosis patients 72 and controls were taken, blood was not less than 10ml and plasma was separated, exosomes in plasma were separated by classical ultracentrifugation method and RNA was extracted by qiagen miRNeasy mini kit, and the obtained RNA was subjected to microscale-ribosome-strand-specific RNA pool-building sequencing and small RNA pool-building sequencing. The data obtained were analyzed bioinformatically and compared for long RNAs and mirnas differentially expressed in early lung cancer patients and controls, resulting in significantly different long RNAs as shown in table 2 below for mirnas. These RNA-level markers from exosomes can be used for early diagnosis of lung cancer.
TABLE 2
| RNA type | ID | Pvalue | log2FC | regulated |
| mRNA | MBOAT2 | 4.23E-05 | 1.023772461 | up |
| mRNA | IL1B | 0.000162055 | 1.050636789 | up |
| mRNA | ARPC5 | 0.000871259 | 1.066858402 | up |
| miRNA | hsa-let-7f-2-3p | 0.00027664 | -0.446890593 | down |
| miRNA | hsa-miR-450b-5p | 0.000293888 | -0.402238286 | down |
| miRNA | hsa-miR-106b-3p | 0.001429894 | 0.189007463 | up |
| miRNA | hsa-miR-885-5p | 0.001455856 | -0.589409694 | down |
| miRNA | hsa-miR-30e-5p | 0.001592304 | -0.329476279 | down |
| miRNA | hsa-miR-3615 | 0.00283095 | 0.419677035 | up |
| miRNA | hsa-miR-4746-5p | 0.003524579 | 0.315578167 | up |
| miRNA | hsa-miR-125a-5p | 0.004298578 | -0.492462437 | down |
Example 2 miRNA detection System based on fluorescent quantitative PCR platform
1. miRNA reverse transcription reaction system
miRNA reverse transcription reagents, enzymes and oligo dT are purchased from TAKARA, standards are synthesized from Shanghai Yingfei WeiJiuji, and specific reverse primers are synthesized from Suzhou Hongxun. A20 ul reverse transcription system was used, as shown in Table 3 below.
TABLE 3 Table 3
2. PCR reaction system
The PCR reaction mixture is purchased from TAKARA, and the upstream primer and the probe, namely the universal downstream primer, are synthesized by Suzhou hong, and the fluorescent quantitative PCR instrument is ABI 7500. The PCR reaction system is shown in Table 4 below:
TABLE 4 Table 4
The PCR procedure was performed at 95℃for 10min, 15 cycles of (95℃15s;55℃30 s) not collecting fluorescence, and 35 cycles of (95℃15s;55℃30 s) collecting fluorescence.
Example 3 fluorescent quantitative PCR platform based mRNA and LncRNA detection System
1. Reverse transcription of mRNA and LncRNA
PrimeScript by Takara Corp TM RT reagent Kit (Perfect Real Time) and PremixEx Taq TM The (Probe qPCR) kit performs reverse transcription and qPCR detection.
Preparing a reverse transcription reaction system (the reaction solution is prepared on ice), then placing the reaction system into a PCR instrument to react under the conditions of 37 ℃ for 60min,85 ℃ for 5s and 12 ℃ for infinity, and adding 50ul DEPC-H after the reverse transcription is finished 2 O was diluted, and 3ul of the template was used for PCR. The reverse transcription reaction system is shown in Table 5 below.
TABLE 5
2. mRNA and LncRNA qPCR
A qPCR reaction system was prepared as follows (reaction solution preparation was performed on ice), and a template-free control was set as a negative control. Then, the sample was put into a real-time fluorescent PCR instrument (ABI 7500) to carry out amplification detection under the following reaction conditions. The qPCR reaction system is shown in Table 6 below, and the qPCR reaction conditions are shown in Table 7 below.
TABLE 6
TABLE 7
Example 4 evaluation of early diagnosis detection Effect of Single marker Lung cancer
1. Sample collection
The blood 10ml of control samples of early stage (stage I and stage II) lung nodule lung cancer patients, benign lung nodule patients, healthy people and the like diagnosed by hospitals is collected and separated into plasma.
2. Exosome RNA extraction
Plasma exosomes were isolated using ultracentrifugation or echo (exoupur of beijing enrozemotai), and the isolated exosomes were extracted for long RNAs in the exosomes using Qiagen miReasy mini kit kit and the RNA concentration and quality were detected using Agilent 2100 and the RNA concentration was recorded.
3. RNA two-step detection system
The two-step detection system based on the PCR platform mRNA in the embodiment 1 is adopted to detect plasma exosome mRNA and LncRNA of 30 cases and 30 cases of control samples (healthy people and benign nodules) of early lung cancer patients, the Ct value of target long RNA is detected, and the relative expression quantity is calculated according to the Ct value and a relative quantitative formula.
4. Performance assessment of exosome RNA single marker diagnosis for early lung cancer
As shown in the following table, for plasma exosomes of 30 and 30 control samples (healthy people and benign lesions) of early lung cancer patients, ct values of target genes were detected, wherein long RNAs were referenced to an internal reference β -action, and mirnas were referenced to an internal reference U6; based on the Ct values, the relative expression levels of RNA were derived. And calculating the fold change of the relative expression quantity of the combined marker by using a relative quantitative formula value, so as to obtain the relative expression quantity of RNA. And carrying out statistical analysis on the detection result by adopting R language. The performance of the single markers was evaluated as shown in Table 8 below, where miR-450b-5p and miR-3615 had better sensitivity and let-7f-2-3p had good specificity.
TABLE 8
Example 5 evaluation of early diagnosis Effect of Long RNA and miRNA combined lung cancer
1. Three marker combination performance assessment
The relative expression level of each long RNA was calculated as in example 3, and the three marker combinations were trained using logistic regression, and the combinations with AUC of 0.76 or more for the three marker combinations were obtained as shown in Table 9 below. The combination performance of miR-450b-5p+let-7f-2-3p+ARPC5 is optimal, the accuracy is 76.67%, and the AUC curves are respectively shown in figure 1; miR-106b-3P+miR-30e-5p+MBOAT2 has good specificity (80%), and an AUC curve is shown in the following figure 1.
TABLE 9
2. Five marker combination performance assessment
The relative expression level of each long RNA was calculated as in example 3, and five marker combinations were trained using logistic regression, and the combinations with AUC of 0.85 or more for the five marker combinations were obtained as shown in Table 10 below. The two combinations of miR-106b-3P+miR-30e-5p+miR-450b-5p+miR-885-5p+ARPC5 and miR-106b-3P+miR-30e-5p+miR-4746-5p+miR-885-5p+ARPC5 have good sensitivity, namely 93.33% and 100% respectively, and the AUC curves are shown in figures 3 and 4; miR-106b-3P+miR-30e-5p+miR-3615+miR-885-5p+ARPC5 and miR-106b-3P+miR-125a-5p+miR-3615+miR-450b-5p+IL1B have good specificity of 90% and 96.67%, respectively, and AUC curves are shown in figures 5 and 6.
Table 10
The data show that the exosome-based noninvasive early lung cancer diagnosis method has high sensitivity and high specificity in early lung cancer, and provides important value for early lung cancer diagnosis. Has great help to the prevention and treatment of lung cancer in China. The highest sensitivity combination can reach 100% of sensitivity, the highest specificity combination can realize 96.67% of specificity, and the method has good performance.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
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cgaggtggac tcctgcct 18
<210> 5
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ctgcccggtc cttcactg 18
<210> 6
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
actcttggtg ttgatagggg ggttc 25
<210> 7
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gccctaaaca gatgaagtgc tc 22
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
tctgccctct ggatggcggc at 22
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
tctgctgatt tttcaggccc a 21
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
<210> 12
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
tggcttgcga aattcatgat gggatgt 27
<210> 13
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacggaaag 50
<210> 14
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
agcgcctata cagtctactg t 21
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
tcgcactgga tacgacggaa agac 24
<210> 16
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgcagca 50
<210> 17
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
accgcactgt gggtact 17
<210> 18
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
tcgcactgga tacgacgcag ca 22
<210> 19
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactcacag 50
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
<210> 21
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
tcgcactgga tacgactcac aggt 24
<210> 22
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccttcca 50
<210> 23
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
accgctgtaa acatccttga c 21
<210> 24
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
tcgcactgga tacgaccttc ca 22
<210> 25
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgagccg 50
<210> 26
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
cctctctcgg ctcctcg 17
<210> 27
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
tcgcactgga tacgacgagc cg 22
<210> 28
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactattca 50
<210> 29
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
tccgcttttg caatatgttc c 21
<210> 30
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
ttcgcactgg atacgactat tcagga 26
<210> 31
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactctgca 50
<210> 32
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
ccggtcccag gagaacc 17
<210> 33
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
tcgcactgga tacgactctg ca 22
<210> 34
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacagaggc 50
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
<210> 36
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
tcgcactgga tacgacagag gc 22
<210> 37
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
ctcgcttcgg cagcaca 17
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
<210> 40
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
agaagattag catggcccct gcgca 25
<210> 41
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
gtgcagggtc cgaggt 16
Claims (4)
1. The application of a primer and a probe for detecting an exosome long RNA marker and a miRNA marker in preparation of a lung cancer diagnosis kit is characterized in that the markers are a combination of miR-106b-3P, miR-30e-5p, miR-450b-5p, miR-885-5p and ARPC 5.
2. The use according to claim 1, wherein the exosome source comprises one or more of blood, saliva and sputum.
3. The use according to claim 1, wherein the primers and probes comprise:
primers and probes for detection of ARPC 5: the upstream primer of the RNA is the nucleotide sequence shown in a sequence number 4, the downstream primer is the nucleotide sequence shown in a sequence number 5, and the probe is the nucleotide sequence shown in a sequence number 6;
reverse transcription primer, PCR primer and probe for detecting miR-106b-3 p: the reverse transcription primer has a nucleotide sequence shown as a sequence number 16, the PCR upstream primer has a nucleotide sequence shown as a sequence number 17, the downstream primer has a nucleotide sequence shown as a sequence number 41, and the probe has a nucleotide sequence shown as a sequence number 18;
reverse transcription primer, PCR primer and probe for detecting miR-30e-5 p: the reverse transcription primer has a nucleotide sequence shown as a sequence number 22, the PCR upstream primer has a nucleotide sequence shown as a sequence number 23, the downstream primer has a nucleotide sequence shown as a sequence number 41, and the probe has a nucleotide sequence shown as a sequence number 24;
reverse transcription primers, PCR primers and probes for detecting miR-450b-5 p: the reverse transcription primer has a nucleotide sequence shown as a sequence number 28, the PCR upstream primer has a nucleotide sequence shown as a sequence number 29, the downstream primer has a nucleotide sequence shown as a sequence number 41, and the probe has a nucleotide sequence shown as a sequence number 30;
reverse transcription primer, PCR primer and probe for detecting miR-885-5 p: the reverse transcription primer is shown as a nucleotide sequence shown as a sequence number 34, the PCR upstream primer is shown as a nucleotide sequence number 35, the downstream primer is shown as a nucleotide sequence number 41, and the probe is shown as a nucleotide sequence number 36.
4. The application of a primer and a probe for detecting an exosome long RNA marker and a miRNA marker in preparing a lung cancer diagnosis device is characterized in that the markers are a combination of miR-106b-3P, miR-30e-5p, miR-450b-5p, miR-885-5p and ARPC 5.
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| CN202210144431.XA Active CN114214420B (en) | 2020-03-30 | 2020-05-11 | Application of exosome miR-106B-3P, IL B and the like in lung cancer diagnosis |
| CN202210144442.8A Active CN114231638B (en) | 2020-03-30 | 2020-05-11 | Application of exosome-7 f-2-3p, ARPC5 and the like in lung cancer diagnosis |
| CN202210144440.9A Active CN114214421B (en) | 2020-03-30 | 2020-05-11 | Application of exosome miR-3615, ARPC5 and the like in lung cancer diagnosis |
| CN202111108621.8A Active CN113930505B (en) | 2020-03-30 | 2020-05-11 | Kit and device for lung cancer diagnosis |
| CN202210144616.0A Active CN114214422B (en) | 2020-03-30 | 2020-05-11 | Application of exosome miR-106b-3P, ARPC5 in lung cancer diagnosis |
| CN202111109657.8A Active CN113801936B (en) | 2020-03-30 | 2020-05-11 | Kit, device and method for lung cancer diagnosis |
| CN202010391533.2A Active CN111455057B (en) | 2020-03-30 | 2020-05-11 | Kit, device and method for lung cancer diagnosis |
| CN202210144425.4A Active CN114214419B (en) | 2020-03-30 | 2020-05-11 | Application of exosome miR-3615, MBOAT2 and the like in lung cancer diagnosis |
| CN202210144490.7A Active CN114410794B (en) | 2020-03-30 | 2020-05-11 | Application of exosome miR-106b-3P, ARPC5 in lung cancer diagnosis |
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| CN202210144442.8A Active CN114231638B (en) | 2020-03-30 | 2020-05-11 | Application of exosome-7 f-2-3p, ARPC5 and the like in lung cancer diagnosis |
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| CN114410794A (en) | 2022-04-29 |
| CN114231638B (en) | 2023-06-27 |
| CN113774138A (en) | 2021-12-10 |
| CN113801936B (en) | 2022-04-19 |
| CN114214419A (en) | 2022-03-22 |
| CN114214420B (en) | 2023-06-23 |
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| CN111455057B (en) | 2021-10-26 |
| CN111455057A (en) | 2020-07-28 |
| CN114214423A (en) | 2022-03-22 |
| CN113774138B (en) | 2022-03-18 |
| CN114231638A (en) | 2022-03-25 |
| CN114214419B (en) | 2023-06-27 |
| CN113930505B (en) | 2022-04-19 |
| CN114214420A (en) | 2022-03-22 |
| CN114214422A (en) | 2022-03-22 |
| CN114214423B (en) | 2023-06-27 |
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