CN114277139A - Kit, device and method for lung cancer diagnosis - Google Patents
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Abstract
The invention discloses a kit, a device and a method for lung cancer diagnosis, wherein the kit comprises one or more of LRRC37A2, TLE4, AZI2, MRPL9, YPEL2, RAB3GAP1, TBC1D23, STK3, IL1B, HMGN1, ERI3, HOPX, CDA, CCBL2, TBK1, RAD54L2, ARPC5, GIMAP5, PLA2G1B, SNHG5 and RPL21 for detecting exosome long RNA markers. The lung cancer detection method based on the exosome long RNA marker has high sensitivity and high specificity in early lung cancer, and provides important value for early diagnosis of lung cancer. It is helpful for preventing and treating lung cancer in our country.
Description
The invention relates to a kit, a device and a method for diagnosing lung cancer, which are divided applications, wherein the application number of a parent application is 202010391309.3, the application date is 2020 and 05 and 11 days.
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 main cancer species in China and even in the world. According to the latest global cancer statistics in 2018, the incidence and mortality of lung cancer are the first in all cancer species. According to the Chinese tumor registration data of 2019, 390.2 ten thousand cancer cases are newly added in 2015 of China, and the death cases of cancer are about 233.8 ten thousand, wherein lung cancer is the main cause of cancer death in China. Therefore, in order to improve the diagnosis and treatment behaviors 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 is shown with lung nodules (single lesions <3cm in the lung interstitium and no associated atelectasis or lymphadenopathy) being discovered. However, not all lung nodules are malignant, and identification of benign and malignant lung nodules has been a difficult point in thoracic surgical clinical diagnosis and treatment. At present, noninvasive detection means such as plasma circulating tumor cells and circulating tumor free DNA are adopted, but the detection sensitivity in early lung cancer diagnosis is not high; therefore, there is a need to develop a highly sensitive method for noninvasive early detection of lung cancer.
Disclosure of Invention
The invention provides an exosome-based reagent, an exosome-based device and an exosome-based method for non-invasive early lung cancer diagnosis.
In one aspect, the present invention provides a kit for lung cancer diagnosis, comprising primers and probes for detecting exosome-long RNA markers including one or more of LRRC37a2, TLE4, az 2, MRPL9, YPEL2, RAB3GAP1, TBC1D23, STK3, IL1B, HMGN1, ERI3, HOPX, CDA, CCBL2, TBK1, RAD54L2, ARPC5, GIMAP5, PLA2G1B, SNHG5, RPL 21.
Preferably, the exosome long RNA marker is a combination of one or more of FHL1, HMGN1, YPEL2, GIMAP5, az 2, CDA, MBOAT2 and RAD54L 2.
Preferably, the long RNA marker of exosome is a combination of FHL1, HMGN1 and YPEL 2.
Preferably, the exosome long RNA marker is a combination of FHL1, GIMAP5 and YPEL 2.
Preferably, the exosome long RNA marker is a combination of AZI2, CDA and FHL 1.
Preferably, the long RNA marker of exosome is a combination of FHL1, MBOAT2 and RAD54L 2.
Preferably, the source of exosomes comprises one or more of blood, saliva and sputum.
Preferably, the primers and probes comprise:
primers and probes for detection of internal reference ACTB: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 1, the downstream primer is a nucleotide sequence shown as a sequence number 2, and the probe is a nucleotide sequence shown as a sequence number 3;
primers and probes for detection of internal reference GAPDH: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 4, the downstream primer is a nucleotide sequence shown as a sequence number 5, and the probe is a nucleotide sequence shown as a sequence number 6;
primers and probes for detection of RPL 21: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 7, the downstream primer is a nucleotide sequence shown as a sequence number 8, and the probe is a nucleotide sequence shown as a sequence number 9;
primers and probes for detection of SNHG 5: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 10, the downstream primer is a nucleotide sequence shown as a sequence number 11, and the probe is a nucleotide sequence shown as a sequence number 12;
primers and probes for detection of PLA2G 1B: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 13, the downstream primer is shown as a nucleotide sequence in a sequence number 14, and the probe is shown as a nucleotide sequence in a sequence number 15;
primers and probes for detection of HMGN 1: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 16, the downstream primer is shown as a nucleotide sequence in a sequence number 17, and the probe is shown as a nucleotide sequence in a sequence number 18;
primers and probes for detection of TLE 4: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 19, the downstream primer is a nucleotide sequence shown as a sequence number 20, and the probe is a nucleotide sequence shown as a sequence number 21;
primers and probes for detection of LRRC37a 2: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 22, the downstream primer is shown as a nucleotide sequence shown in a sequence number 23, and the probe is shown as a nucleotide sequence shown in a sequence number 24;
primers and probes for detection of FHL 1: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 25, the downstream primer is shown as a nucleotide sequence in a sequence number 26, and the probe is shown as a nucleotide sequence in a sequence number 27;
primers and probes for detection of ARPC 5: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 28, the downstream primer is shown as a nucleotide sequence shown in a sequence number 29, and the probe is shown as a nucleotide sequence shown in a sequence number 30;
primers and probes for detection of AZI 2: the upstream primer of the RNA is shown as a nucleotide sequence of a sequence number 31, the downstream primer is shown as a nucleotide sequence of a sequence number 32, and the probe is shown as a nucleotide sequence of a sequence number 33;
primers and probes for detection of CDA: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 34, the downstream primer is shown as a nucleotide sequence in a sequence number 35, and the probe is shown as a nucleotide sequence in a sequence number 36;
primers and probes for detecting ERI 3: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 37, the downstream primer is shown as a nucleotide sequence in a sequence number 38, and the probe is shown as a nucleotide sequence in a sequence number 39;
primers and probes for detection of GIMAP 5: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 40, the downstream primer is shown as a nucleotide sequence shown in a sequence number 41, and the probe is shown as a nucleotide sequence shown in a sequence number 42;
primers and probes for detection of HOPX: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 43, the downstream primer is shown as a nucleotide sequence in a sequence number 44, and the probe is shown as a nucleotide sequence in a sequence number 45;
primers and probes for detection of IL 1B: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 46, the downstream primer is shown as a nucleotide sequence shown in a sequence number 47, and the probe is shown as a nucleotide sequence shown in a sequence number 48;
primers and probes for detection of KYAT 3: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 49, the downstream primer is shown as a nucleotide sequence in a sequence number 50, and the probe is shown as a nucleotide sequence in a sequence number 51;
primers and probes for detection of MBOAT 2: the upstream primer of the RNA is shown as a nucleotide sequence in sequence number 52, the downstream primer is shown as a nucleotide sequence in sequence number 53, and the probe is shown as a nucleotide sequence in sequence number 54;
primers and probes for detection of RAB3GAP 1: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 55, the downstream primer is shown as a nucleotide sequence in a sequence number 56, and the probe is shown as a nucleotide sequence in a sequence number 57;
primers and probes for detection of RAD54L 2: the upstream primer of the RNA is shown as a nucleotide sequence shown as a sequence number 58, the downstream primer is shown as a nucleotide sequence shown as a sequence number 59, and the probe is shown as a nucleotide sequence shown as a sequence number 60;
primers and probes for detection of STK 3: the upstream primer of the RNA is shown as a nucleotide sequence of a sequence number 61, the downstream primer is shown as a nucleotide sequence of a sequence number 62, and the probe is shown as a nucleotide sequence of a sequence number 63;
primers and probes for detection of MRPL 9: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 64, the downstream primer is shown as a nucleotide sequence shown in a sequence number 65, and the probe is shown as a nucleotide sequence shown in a sequence number 66;
primers and probes for detection of TBC1D 23: the upstream primer of the RNA is shown as a nucleotide sequence of a sequence number 67, the downstream primer is shown as a nucleotide sequence of a sequence number 68, and the probe is shown as a nucleotide sequence of a sequence number 69;
primers and probes for detection of TBK 1: the upstream primer of the RNA is shown as a nucleotide sequence of a sequence number 70, the downstream primer is shown as a nucleotide sequence of a sequence number 71, and the probe is shown as a nucleotide sequence of a sequence number 72;
primers and probes for detection of YPEL 2: the upstream primer of the RNA is shown as a nucleotide sequence in sequence number 73, the downstream primer is shown as a nucleotide sequence in sequence number 74, and the probe is shown as a nucleotide sequence in sequence number 75.
In another aspect, the present invention provides a device for lung cancer diagnosis, comprising a reagent for detecting an exosome-length RNA marker comprising one or more of LRRC37a2, TLE4, az 2, MRPL9, YPEL2, RAB3GAP1, TBC1D23, STK3, IL1B, HMGN1, ERI3, HOPX, CDA, CCBL2, TBK1, RAD54L2, ARPC5, GIMAP5, PLA2G1B, hg sn 5, RPL 21.
In another aspect, the present invention provides a method for lung cancer diagnosis, comprising detecting the specificity of exosome-long RNA markers comprising one or more of LRRC37a2, TLE4, az 2, MRPL9, YPEL2, RAB3GAP1, TBC1D23, STK3, IL1B, HMGN1, ERI3, HOPX, CDA, CCBL2, TBK1, RAD54L2, ARPC5, GIMAP5, PLA2G1B, hg sn 5, RPL 21.
The invention provides a noninvasive early lung cancer diagnosis method based on exosome, which has high sensitivity and high specificity in early lung cancer and provides important value for early diagnosis of lung cancer. It is helpful for preventing and treating lung cancer in our country. Furthermore, the combined negative predictive value of 3 long RNA markers is 88.24%, the sensitivity is 93.33%, the specificity is 50%, and the sensitivity is higher.
Drawings
FIG. 1 is a ROC curve for FHL1 alone for lung cancer.
FIG. 2 is a ROC curve of MBOAT2 alone for lung cancer.
FIG. 3 is a ROC curve for the combination FHL1+ HMGN1+ YPEL2 for lung cancer detection.
FIG. 4 is a ROC curve for the combination FHL1+ GIMAP5+ YPEL2 for lung cancer.
FIG. 5 is a ROC curve for the combination AZI2+ CDA + FHL1 for lung cancer.
FIG. 6 is a ROC curve for the combination FHL1+ MBOAT2+ RAD54L2 for lung cancer.
FIG. 7 is a ROC curve for the combination ARPC5+ AZI2+ CDA + HMGN1+ MBOAT2 for lung cancer.
FIG. 8 is a ROC curve for the combination of ARPC5+ HMGN1+ IL1B + MBOAT2+ RAB3GAP1 for lung cancer detection.
Detailed Description
The Extracellular Vesicles (EVs; hereinafter, Vesicles are all referred to as Extracellular Vesicles) refer to vesicular bodies with a double-layer membrane structure, which are shed from cell membranes or secreted by cells, and have diameters of 30-1000nm, and mainly comprise Micro Vesicles (MVs) and exosomes (exosomes), and the Micro Vesicles are small Vesicles shed from cell membranes after cells are activated or damaged. Extracellular vesicles are of great interest in disease diagnosis, particularly exosomes, due to their unique biological characteristics.
The exosome is a membrane vesicle with the particle size of 30-150 nm secreted into the extracellular environment after an intracellular multivesicular body and a cell membrane are fused, is an important medium for intercellular information transfer, and plays an important role in antigen presentation, apoptosis, inflammatory reaction, tumorigenesis development and metastasis processes. It is widely distributed in body fluid, including blood, saliva, urine, milk, hydrothorax and ascites, etc.; contains various inclusion substances such as DNA, RNA, protein and the like, and can be used as noninvasive diagnosis markers of various diseases such as tumors and the like. And miRNA is the most abundant nucleic acid component in exosome, so exosome miRNA has the potential of being used for early diagnosis of lung cancer.
The kit, the device and the method provided by the invention use mRNA and LncRNA (collectively referred to as long RNA) which are found to be remarkably and differentially expressed in exosome of a patient with early lung cancer through experimental research as markers for diagnosing early lung cancer.
Significantly differentially expressed long RNA markers include: one or more of LRRC37a2, TLE4, az 2, MRPL9, YPEL2, RAB3GAP1, TBC1D23, STK3, IL1B, HMGN1, ERI3, HOPX, CDA, CCBL2, TBK1, RAD54L2, ARPC5, GIMAP5, PLA2G1B, SNHG5, RPL 21.
In some preferred embodiments, the significantly differentially expressed long RNA marker is a combination of one or more of FHL1, HMGN1, YPEL2, GIMAP5, az 2, CDA, MBOAT2, and RAD54L 2. More preferably, the combination of FHL1, HMGN1 and YPEL2, FHL1, GIMAP5 and YPEL2, AZI2, CDA and FHL1, or FHL1, MBOAT2 and RAD54L 2.
In addition, the kit for lung cancer diagnosis of the present invention includes primers and probes for detecting the above exosome-long RNA marker. The primers and probes for detecting the long RNA markers of the exosomes comprise:
primers and probes for detection of internal reference ACTB: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 1, the downstream primer is a nucleotide sequence shown as a sequence number 2, and the probe is a nucleotide sequence shown as a sequence number 3;
primers and probes for detection of internal reference GAPDH: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 4, the downstream primer is a nucleotide sequence shown as a sequence number 5, and the probe is a nucleotide sequence shown as a sequence number 6;
primers and probes for detection of RPL 21: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 7, the downstream primer is a nucleotide sequence shown as a sequence number 8, and the probe is a nucleotide sequence shown as a sequence number 9;
primers and probes for detection of SNHG 5: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 10, the downstream primer is a nucleotide sequence shown as a sequence number 11, and the probe is a nucleotide sequence shown as a sequence number 12;
primers and probes for detection of PLA2G 1B: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 13, the downstream primer is shown as a nucleotide sequence in a sequence number 14, and the probe is shown as a nucleotide sequence in a sequence number 15;
primers and probes for detection of HMGN 1: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 16, the downstream primer is shown as a nucleotide sequence in a sequence number 17, and the probe is shown as a nucleotide sequence in a sequence number 18;
primers and probes for detection of TLE 4: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 19, the downstream primer is a nucleotide sequence shown as a sequence number 20, and the probe is a nucleotide sequence shown as a sequence number 21;
primers and probes for detection of LRRC37a 2: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 22, the downstream primer is shown as a nucleotide sequence shown in a sequence number 23, and the probe is shown as a nucleotide sequence shown in a sequence number 24;
primers and probes for detection of FHL 1: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 25, the downstream primer is shown as a nucleotide sequence in a sequence number 26, and the probe is shown as a nucleotide sequence in a sequence number 27;
primers and probes for detection of ARPC 5: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 28, the downstream primer is shown as a nucleotide sequence shown in a sequence number 29, and the probe is shown as a nucleotide sequence shown in a sequence number 30;
primers and probes for detection of AZI 2: the upstream primer of the RNA is shown as a nucleotide sequence of a sequence number 31, the downstream primer is shown as a nucleotide sequence of a sequence number 32, and the probe is shown as a nucleotide sequence of a sequence number 33;
primers and probes for detection of CDA: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 34, the downstream primer is shown as a nucleotide sequence in a sequence number 35, and the probe is shown as a nucleotide sequence in a sequence number 36;
primers and probes for detecting ERI 3: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 37, the downstream primer is shown as a nucleotide sequence in a sequence number 38, and the probe is shown as a nucleotide sequence in a sequence number 39;
primers and probes for detection of GIMAP 5: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 40, the downstream primer is shown as a nucleotide sequence shown in a sequence number 41, and the probe is shown as a nucleotide sequence shown in a sequence number 42;
primers and probes for detection of HOPX: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 43, the downstream primer is shown as a nucleotide sequence in a sequence number 44, and the probe is shown as a nucleotide sequence in a sequence number 45;
primers and probes for detection of IL 1B: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 46, the downstream primer is shown as a nucleotide sequence shown in a sequence number 47, and the probe is shown as a nucleotide sequence shown in a sequence number 48;
primers and probes for detection of KYAT 3: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 49, the downstream primer is shown as a nucleotide sequence in a sequence number 50, and the probe is shown as a nucleotide sequence in a sequence number 51;
primers and probes for detection of MBOAT 2: the upstream primer of the RNA is shown as a nucleotide sequence in sequence number 52, the downstream primer is shown as a nucleotide sequence in sequence number 53, and the probe is shown as a nucleotide sequence in sequence number 54;
primers and probes for detection of RAB3GAP 1: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 55, the downstream primer is shown as a nucleotide sequence in a sequence number 56, and the probe is shown as a nucleotide sequence in a sequence number 57;
primers and probes for detection of RAD54L 2: the upstream primer of the RNA is shown as a nucleotide sequence shown as a sequence number 58, the downstream primer is shown as a nucleotide sequence shown as a sequence number 59, and the probe is shown as a nucleotide sequence shown as a sequence number 60;
primers and probes for detection of STK 3: the upstream primer of the RNA is shown as a nucleotide sequence of a sequence number 61, the downstream primer is shown as a nucleotide sequence of a sequence number 62, and the probe is shown as a nucleotide sequence of a sequence number 63;
primers and probes for detection of MRPL 9: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 64, the downstream primer is shown as a nucleotide sequence shown in a sequence number 65, and the probe is shown as a nucleotide sequence shown in a sequence number 66;
primers and probes for detection of TBC1D 23: the upstream primer of the RNA is shown as a nucleotide sequence of a sequence number 67, the downstream primer is shown as a nucleotide sequence of a sequence number 68, and the probe is shown as a nucleotide sequence of a sequence number 69;
primers and probes for detection of TBK 1: the upstream primer of the RNA is shown as a nucleotide sequence of a sequence number 70, the downstream primer is shown as a nucleotide sequence of a sequence number 71, and the probe is shown as a nucleotide sequence of a sequence number 72;
primers and probes for detection of YPEL 2: the upstream primer of the RNA is shown as a nucleotide sequence in sequence number 73, the downstream primer is shown as a nucleotide sequence in sequence number 74, and the probe is shown as a nucleotide sequence in sequence number 75. The nucleotide sequences of the primers and probes are shown in Table 1.
TABLE 1
Further, the source of exosomes includes one or more of blood, saliva, and sputum.
The kit, the device and the method are suitable for individuals, such as people at high risk of lung cancer, normal individuals and patients after lung cancer operation.
The technical solution of the present invention will be described in detail with reference to the following embodiments, which are a part of the embodiments of the present invention, but not all of them. Other embodiments, which can be derived by one of ordinary skill in the art from the embodiments of the present invention without creative efforts, are within the protection scope of the present invention.
In order to screen an exosome marker related to diagnosis of colon lung cancer, 50 cases of early lung cancer patients and 72 cases of controls respectively take blood of not less than 10ml and separate plasma, the blood is used for separating exosomes in the plasma by a classical ultracentrifugation method and extracting RNA, and the obtained RNA is respectively subjected to RNA library construction and sequencing. The data obtained were analyzed bioinformatically to compare differentially expressed RNA in early lung cancer patients and controls. These exosome-derived RNA-level markers can be used for early diagnosis of lung cancer.
Further directed to the method wherein the RNA marker is further analyzed by the following steps: (1) collecting body fluid samples (including blood, sputum and saliva) of individuals to be detected; (2) isolating exosomes in the body fluid; (3) detecting the expression level of target RNA by using a two-step method; (4) normalizing the expression level of the detection target RNA by using the 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 detected is lung cancer or not according to the output value of the model and the judgment threshold value.
The kit comprises a PCR primer, a probe and a standard substance for detecting the long RNA marker of the exosome and a two-step detection system of PCR.
Comprises selecting internal reference beta-Actin (ACTB) to carry out the quantification of target RNA. Wherein the expression level of the marker is calculated by using a quantitative formula 2 delta Ct according to the detection Ct value by using the quantification of the target RNA when the reference is selected. Having obtained the target RNA expression level, the ROC characteristic curve and AUC were used to assess the accuracy of lung cancer detection by single RNA or by combining multiple RNAs.
Example 1 screening of exosome mRNA and LncRNA markers associated with early lung cancer based on high throughput sequencing
In order to screen an exosome marker related to early lung cancer diagnosis, 50 cases of early lung cancer diagnosis patients 72 and controls respectively take blood of not less than 10ml and separate plasma, the blood is used for separating exosomes in the plasma by a classical ultracentrifugation method and extracting RNA by using a qiagen miRNeasy mini kit, and the obtained RNA is subjected to micro-enucleated ribosome strand specific RNA library-building sequencing. The obtained data were analyzed by bioinformatics to compare mRNA and LncRNA (collectively referred to as long RNA) differentially expressed in early lung cancer patients and controls, and the significantly different long RNAs were obtained as shown in table 2 below. These long RNA-level markers from exosomes can be used for early diagnosis of lung cancer.
TABLE 2
Example 2 fluorescent quantitation-based PCR platform mRNA and LncRNA detection system
1. Reverse transcription of mRNA and LncRNA
PrimeScript from takara was usedTMRT reagent Kit (Perfect Real Time) and Premix Ex TaqTM(Probe qPCR) kit for reverse transcription and qPCR detection.
Preparing a reverse transcription reaction system (reaction liquid is prepared on ice), then putting the reaction system into a PCR instrument for reaction under the reaction conditions of 37 ℃ for 60min, 85 ℃ for 5s and 12 ℃ for infinity, and adding 50ul DEPC-H after the reverse transcription is finished2Diluting with O, taking 3ul as a template, and carrying out PCR reaction. The reverse transcription reaction system is shown in Table 3 below.
TABLE 3
2. mRNA and LncRNAqPCR
The qPCR reaction system was prepared as follows (reaction solution was prepared on ice) and a no template control was set as a negative control. Then, the mixture was put into a real-time fluorescence PCR apparatus (ABI7500) to perform amplification detection under the following reaction conditions. The qPCR reaction system is shown in table 4 below and the qPCR reaction conditions are shown in table 5 below.
TABLE 4
TABLE 5 qPCR reaction conditions
Example 3 evaluation of early diagnosis and detection Effect of Lung cancer Using Ex-Shen beta-Actin (ACTB) as reference Single marker
1. Sample collection
10ml of blood of control samples of early stage (stage I and stage II) lung nodule lung cancer patients, benign lung nodule patients, healthy persons, etc. diagnosed in hospitals were collected and separated into plasma.
2. Exosome RNA extraction
Plasma exosome separation is carried out by ultracentrifugation or Exosuurur of Echobiotech (Beijing Enzekangtai), long RNA in exosome is extracted from the separated exosome by a Qiagen MiReasy mini kit, RNA concentration and quality are detected by Agilent2100, and the RNA concentration is recorded.
3. RNA two-step detection system
A two-step method detection system based on mRNA of a PCR platform in example 1 is adopted to detect plasma exosome mRNA and LncRNA of 30 patients with early lung cancer and 30 control samples (healthy people and benign nodules), the Ct value of target long RNA is detected, and the relative expression amount is calculated according to the Ct value and a relative quantitative formula.
4. Evaluation of exosome long-RNA diagnosis early lung cancer performance
(1) FHL1 independent detection performance evaluation
As shown in FIG. 1, Ct values of FHL1 were measured for plasma exosomes of 30 patients with early lung cancer and 30 control samples (healthy people and benign lesions), and RNA copy number was obtained from Ct values with the reference of ginseng beta-Actin. 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 the RNA. And performing statistical analysis on the detection result by adopting an R language. The AUC of FHL1 for diagnosing early lung cancer alone is 0.653, the negative predictive value is 70%, the sensitivity is 80% and the specificity is 46.67%.
(2) MBOAT2 test Performance evaluation alone
As shown in FIG. 2, the Ct value of MBOAT2 was measured for plasma exosomes of 30 patients with early lung cancer and 30 control samples (healthy and benign lesions), and the RNA copy number was obtained from the Ct value with the reference of the external reference β -Actin. 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 the RNA. And performing statistical analysis on the detection result by adopting an R language. The AUC of MBOAT2 for diagnosing early lung cancer alone is 0.595, the negative predictive value is 76.5%, the sensitivity is 86.7%, the specificity is 43.33%, and the sensitivity is better.
(3) Evaluation of Performance of other Individual markers significantly associated with early Lung cancer
Other significantly related long RNA performance evaluations are shown in table 6 below.
TABLE 6
From the data shown in table 6, it can be seen that the RNAs shown in the table all have the potential of diagnostic markers.
Example 4 evaluation of the Effect of the Multi-marker combination for early diagnosis and detection of Lung cancer Using Ex-reference ACTB as a reference
1. Three marker combination performance evaluation
The relative expression of each long RNA was calculated according to the method described in example 3, and the three marker combinations were trained using logistic regression, and the combinations obtained with the accuracy of the three marker combinations above 0.7 are shown in Table 7 below.
TABLE 7
The data in table 7 show that the combination of FHL1+ HMGN1+ YPEL2, FHL1+ GIMAP5+ YPEL2, az 2+ CDA + FHL1, FHL1+ MBOAT2+ RAD54L2 is the most excellent, and the accuracy is 71.67%. The AUC curves of the above four combinations are shown in fig. 3, 4, 5, and 6, respectively. The negative predictive value of the FHL1+ MBOAT2+ RAD54L2 combination is 88.24%, the sensitivity is 93.33%, the specificity is 50%, and the combination has higher sensitivity.
2. Five marker combination performance evaluation
The relative expression of each long RNA was calculated according to the method of example 3, and five marker combinations were trained using logistic regression, and the combinations having an accuracy of 70% or more of the five marker combinations were obtained as shown in table 8 below.
TABLE 8
From the data in Table 8, it can be seen that the two combinations of ARPC5+ AZI2+ CDA + HMGN1+ MBOAT2 and ARPC5+ HMGN1+ IL1B + MBOAT2+ RAB3GAP1 have the best performance, and the accuracy is 75.0% and 73.3% respectively. The AUC curves of the above five combinations are shown in fig. 7 and fig. 8, respectively.
The data show that the lung cancer detection method based on the exosome long RNA marker has high sensitivity and high specificity in early lung cancer, and provides important value for early diagnosis of the lung cancer. It is helpful for preventing and treating lung cancer in our country. The combined negative predictive value of 3 long RNA markers is 88.24%, the sensitivity is 93.33%, the specificity is 50%, and the sensitivity is higher.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments 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 utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
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Claims (5)
1. A kit for lung cancer diagnosis, which comprises primers and probes for detecting an exosome-long RNA marker, wherein the exosome-long RNA marker is a combination of ARPC5, HMGN1, IL1B, MBOAT2 and SNHG 5.
2. The kit of claim 1, wherein the source of exosomes comprises one or more of blood, saliva, and sputum.
3. The kit of claim 1, wherein the primers and probes comprise:
primers and probes for detection of SNHG 5: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 10, the downstream primer is a nucleotide sequence shown as a sequence number 11, and the probe is a nucleotide sequence shown as a sequence number 12;
primers and probes for detection of HMGN 1: the upstream primer of the RNA is shown as a nucleotide sequence in a sequence number 16, the downstream primer is shown as a nucleotide sequence in a sequence number 17, and the probe is shown as a nucleotide sequence in a sequence number 18;
primers and probes for detection of ARPC 5: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 28, the downstream primer is shown as a nucleotide sequence shown in a sequence number 29, and the probe is shown as a nucleotide sequence shown in a sequence number 30;
primers and probes for detection of IL 1B: the upstream primer of the RNA is shown as a nucleotide sequence shown in a sequence number 46, the downstream primer is shown as a nucleotide sequence shown in a sequence number 47, and the probe is shown as a nucleotide sequence shown in a sequence number 48;
primers and probes for detection of MBOAT 2: the upstream primer of the RNA is shown as a nucleotide sequence in sequence number 52, the downstream primer is shown as a nucleotide sequence in sequence number 53, and the probe is shown as a nucleotide sequence in sequence number 54.
4. A device for lung cancer diagnosis comprising a reagent that detects an exosome-long RNA marker, which is a combination of ARPC5, HMGN1, IL1B, MBOAT2 and SNHG 5.
5. A method for lung cancer diagnosis comprising detecting the specificity of an exosome-long RNA marker, which is a combination of ARPC5, HMGN1, IL1B, MBOAT2 and SNHG 5.
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| CN112433051B (en) * | 2020-11-23 | 2022-07-08 | 四川大学华西医院 | Application of platelet activating factor acetylhydrolase detection reagent in preparation of lung cancer screening kit |
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