CN115287350A - Application of exosome miR-106b-3p, miR-10a-3p and the like in lung cancer diagnosis - Google Patents
Application of exosome miR-106b-3p, miR-10a-3p and the like in lung cancer diagnosis Download PDFInfo
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Abstract
The invention discloses an application of exosome miR-106b-3p, miR-10a-3p and the like in lung cancer diagnosis, wherein a kit comprises primers and probes for detecting exosome miRNA markers, and the exosome miRNA markers comprise one or more of let-7a-3p, let-7f-2, miR-106b-3p, miR-10a-5p, miR-125a-5p, miR-1294, miR-19a-3p, miR-22-3p, miR-29a-3p, miR-30e-5p, miR-3158-3p, miR-330-5p, miR-3605-3p, miR-3615, miR-425-3p, miR-450b-5p, miR-4746-5p, miR-483-3p, miR-502-3p, miR-550a-5p, miR-651-775 p, miR-885 p and miR-775 p. The invention provides a noninvasive lung cancer diagnosis method based on exosome, which has high sensitivity and high specificity in lung cancer and provides important values for early diagnosis and recurrence monitoring of lung cancer. It is helpful for preventing and treating lung cancer in our country.
Description
The application is a divisional application, has a divisional application number of 202111059955.0 and an application date of 2020, 05 and 11, and is named as a kit, a device and a method for lung cancer diagnosis.
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
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, a device and a method for non-invasive early lung cancer diagnosis.
The invention provides a kit for lung cancer diagnosis, which comprises primers and probes for detecting exosome miRNA markers, wherein the exosome miRNA markers comprise one or more of let-7a-3p, let-7f-2, miR-106b-3p, miR-10a-5p, miR-125a-5p, miR-1294, miR-19a-3p, miR-22-3p, miR-29a-3p, miR-30e-5p, miR-3158-3p, miR-330-5p, miR-3605-3p, miR-3615, miR-378h, miR-425-3p, miR-450b-5p, miR-4746-5p, miR-483-3p, miR-502-3p, miR-550a-5p, miR-651-775 p, miR-7706 and miR-885 p.
Preferably, the exosome miRNA marker is one or more of miR-3615, miR-502-3p, miR-450b-5p, miR-4746-5p, miR-10a-5p, miR-106b-3p, miR-125a-5p and miR-885-5p.
Preferably, the exosome miRNA marker is a combination of miR-106-3p, miR-125a-5p and miR-3615.
Preferably, the exosome miRNA marker is a combination of miR-106b-3p, miR-3615 and miR-450b-5 p.
Preferably, the exosome miRNA marker is a combination of miR-106b-3p, miR-125a-5p, miR-3615, miR-450b-5p and miR-885-5p.
Preferably, the exosome miRNA marker is a combination of miR-106b-3p, miR-10a-3p, miR-125a-5p, miR-3615 and miR-450b-5 p.
Preferably, the source of exosomes comprises one or more of blood, saliva and sputum.
Preferably, the primers and probes comprise:
reverse transcription primers, PCR primers and probes for detecting let-7a-3 p: the reverse transcription primer of the let-7a-3p has a nucleotide sequence shown as a sequence number 1, the PCR upstream primer has a nucleotide sequence shown as a sequence number 2, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 3;
reverse transcription primers, PCR primers and probes for detecting let-7 f-2: the reverse transcription primer of let-7f-2 has a nucleotide sequence shown as a sequence number 4, the PCR upstream primer has a nucleotide sequence shown as a sequence number 5, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 6;
reverse transcription primers, PCR primers and probes for detecting miR-106b-3 p: the reverse transcription primer of miR-106b-3p has a nucleotide sequence shown as sequence number 7, the PCR upstream primer has a nucleotide sequence shown as sequence number 8, the downstream primer has a nucleotide sequence shown as sequence number 80, and the probe has a nucleotide sequence shown as sequence number 9;
reverse transcription primers, PCR primers and probes for detecting miR-10a-3 p: the reverse transcription primer of miR-10a-3p has a nucleotide sequence shown as a sequence number 10, the PCR upstream primer has a nucleotide sequence shown as a sequence number 11, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 12;
reverse transcription primers, PCR primers and probes for detecting miR-10a-5 p: the reverse transcription primer of miR-10a-5p 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 80, and the probe has a nucleotide sequence shown as a sequence number 15;
reverse transcription primers, PCR primers and probes for detecting miR-125a-5 p: the reverse transcription primer of miR-125a-5p has a nucleotide sequence shown as sequence number 16, the PCR upstream primer has a nucleotide sequence shown as sequence number 17, the downstream primer has a nucleotide sequence shown as sequence number 80, and the probe has a nucleotide sequence shown as sequence number 18;
reverse transcription primers, PCR primers and probes for detecting miR-1294: the reverse transcription primer of miR-1294 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 80, and the probe has a nucleotide sequence shown as a sequence number 21;
reverse transcription primers, PCR primers and probes for detecting miR-19a-3 p: the reverse transcription primer of miR-19a-3p 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 80, and the probe has a nucleotide sequence shown as a sequence number 24;
reverse transcription primers, PCR primers and probes for detecting miR-22-3 p: the reverse transcription primer of miR-22-3p 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 PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 27;
reverse transcription primers, PCR primers and probes for detecting miR-29a-3 p: the reverse transcription primer of miR-29a-3p 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 PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 30;
reverse transcription primers, PCR primers and probes for detecting miR-30e-5 p: the reverse transcription primer of miR-30e-5p 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 80, and the probe has a nucleotide sequence shown as a sequence number 33;
reverse transcription primers, PCR primers and probes for detecting miR-3158-3 p: the reverse transcription primer of miR-3158-3p has a nucleotide sequence shown as sequence number 34, the PCR upstream primer has a nucleotide sequence shown as sequence number 35, the PCR downstream primer has a nucleotide sequence shown as sequence number 80, and the probe has a nucleotide sequence shown as sequence number 36;
reverse transcription primers, PCR primers and probes for detecting miR-330-5 p: the reverse transcription primer of miR-330-5p has a nucleotide sequence shown as a sequence number 37, the PCR upstream primer has a nucleotide sequence shown as a sequence number 38, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 39;
reverse transcription primers, PCR primers and probes for detecting miR-3605-3 p: the reverse transcription primer of miR-3605-3p has a nucleotide sequence shown as a sequence number 40, the PCR upstream primer has a nucleotide sequence shown as a sequence number 41, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 42;
reverse transcription primers, PCR primers and probes for detecting miR-3615: the reverse transcription primer of miR-3615 has a nucleotide sequence shown as a sequence number 43, the PCR upstream primer has a nucleotide sequence shown as a sequence number 44, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 45;
reverse transcription primers, PCR primers and probes for detecting miR-378 h: the reverse transcription primer of miR-378h has a nucleotide sequence shown as a sequence number 46, the PCR upstream primer has a nucleotide sequence shown as a sequence number 47, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 48;
reverse transcription primers, PCR primers and probes for detection of miR-425-3 p: the reverse transcription primer of miR-425-3p has a nucleotide sequence shown as a sequence number 49, the PCR upstream primer has a nucleotide sequence shown as a sequence number 50, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 51;
reverse transcription primers, PCR primers and probes for detecting miR-450b-5 p: the reverse transcription primer of miR-450b-5p has a nucleotide sequence shown as a sequence number 52, the PCR upstream primer has a nucleotide sequence shown as a sequence number 53, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 54;
reverse transcription primers, PCR primers and probes for detecting miR-4746-5 p: the reverse transcription primer of miR-4746-5p has a nucleotide sequence shown as a sequence number 55, the PCR upstream primer has a nucleotide sequence shown as a sequence number 56, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 57;
reverse transcription primers, PCR primers and probes for detecting miR-483-3 p: the reverse transcription primer of miR-483-3p has a nucleotide sequence shown as a sequence number 58, the PCR upstream primer has a nucleotide sequence shown as a sequence number 59, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 60;
reverse transcription primers, PCR primers and probes for detecting miR-502-3 p: the reverse transcription primer of miR-502-3p has a nucleotide sequence shown as a sequence number 61, the PCR upstream primer has a nucleotide sequence shown as a sequence number 62, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 63;
reverse transcription primers, PCR primers and probes for detecting miR-550a-5 p: the reverse transcription primer of miR-550a-5p has a nucleotide sequence shown as sequence number 64, the PCR upstream primer has a nucleotide sequence shown as sequence number 65, the downstream primer has a nucleotide sequence shown as sequence number 80, and the probe has a nucleotide sequence shown as sequence number 66;
reverse transcription primers, PCR primers and probes for detecting miR-651-5 p: the reverse transcription primer of miR-651-5p has a nucleotide sequence shown as a sequence number 67, the PCR upstream primer has a nucleotide sequence shown as a sequence number 68, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 69;
reverse transcription primers, PCR primers and probes for detecting miR-7706: the reverse transcription primer of miR-7706 is a nucleotide sequence shown as a sequence number 70, the PCR upstream primer is a nucleotide sequence shown as a sequence number 71, the downstream primer is a nucleotide sequence shown as a sequence number 80, and the probe is a nucleotide sequence shown as a sequence number 72;
reverse transcription primers, PCR primers and probes for detecting miR-885-5 p: the reverse transcription primer of miR-885-5p has a nucleotide sequence shown as a sequence number 73, the PCR upstream primer has a nucleotide sequence shown as a sequence number 74, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 75.
In another aspect, the invention provides a device for lung cancer diagnosis, comprising a reagent for detecting exosome miRNA markers, wherein the exosome miRNA markers comprise one or more of let-7a-3p, let-7f-2, miR-106b-3p, miR-10a-5p, miR-125a-5p, miR-1294, miR-19a-3p, miR-22-3p, miR-29a-3p, miR-30e-5p, miR-3158-3p, miR-330-5p, miR-3605-3p, miR-3615, miR-378h, miR-425-3p, miR-450b-5p, miR-4746-5p, miR-483-3p, miR-502-3p, miR-550a-5p, miR-651-5p, miR-7706 and miR-885-5p.
In another aspect, the invention provides a method for lung cancer diagnosis, which comprises detecting the specificity of exosome miRNA markers, wherein the exosome miRNA markers comprise one or more of let-7a-3p, let-7f-2, miR-106b-3p, miR-10a-5p, miR-125a-5p, miR-1294, miR-19a-3p, miR-22-3p, miR-29a-3p, miR-30e-5p, miR-3158-3p, miR-330-5p, miR-3605-3p, miR-3615, miR-378h, miR-425-3p, miR-450b-5p, miR-4746-5p, miR-483-3p, miR-502-3p, miR-550a-5p, miR-651-5p, miR-7706 and miR-5 p.
The invention provides a noninvasive lung cancer diagnosis method based on exosome, which has high sensitivity and high specificity in lung cancer and provides important values for early diagnosis and recurrence monitoring of lung cancer. It is helpful for preventing and treating lung cancer in our country. Furthermore, 5 of the miRNA markers (the combined AUC can reach 0.951 at most, the negative predictive value is 90.32%, the sensitivity is 90.00%, and the specificity is 93.33%), have extremely excellent diagnostic performance.
Drawings
FIG. 1 shows the electron microscope identification results of exosomes.
FIG. 2 is a ROC curve of miR-3615 alone for detecting lung cancer.
FIG. 3 is a ROC curve of miR-502-3p alone for detecting lung cancer.
FIG. 4 is a ROC curve of miR-450b-5p alone for detecting lung cancer.
FIG. 5 is a ROC curve for miR-4746-5p alone to detect lung cancer.
FIG. 6 is a ROC curve for miR-10a-5p alone to detect lung cancer.
FIG. 7 is an ROC curve for combination detection of lung cancer by miR-106-3p + miR-125a-5p + miR-3615.
FIG. 8 is a ROC curve for combination detection of lung cancer by miR-106b-3p + miR-3615+ miR-450b-5 p.
FIG. 9 is an ROC curve for combination detection of lung cancer by miR-106b-3p + miR-125a-5p + miR-3615+ miR-450b-5p + miR-885-5.
FIG. 10 is ROC curve for combination detection of lung cancer by miR-106b-3p + miR-10a-3p + miR-125a-5p + miR-3615+ miR-450b-5 p.
FIG. 11 is ROC curve (with U6 as reference) for combined detection of lung cancer by miR-106b-3P + miR-10a-5p + miR-125a-5p + miR-3615+ miR-450b-5 p.
Detailed Description
Extracellular vesicles (EVs; hereinafter vesicles are referred to as extracellular vesicles) refer to vesicular bodies with a double-layer membrane structure, which are shed from cell membranes or secreted from cells, and have diameters varying from 30 to 1000nm, and are mainly composed of MicroVesicles (MVs) and exosomes (exosomes), which 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 the intracellular vesicle is fused with a cell membrane, 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 the exosome miRNA has the potential of early diagnosis of lung cancer.
The kit, the device and the method provided by the invention find out through experimental research that one or more of miRNA in exosomes of patients with early lung cancer show significant differential expression is used as a marker for diagnosing early lung cancer.
Significantly differentially expressed mirnas include: let-7a-3p, let-7f-2, miR-106b-3p, miR-10a-5p, miR-125a-5p, miR-1294, miR-19a-3p, miR-22-3p, miR-29a-3p, miR-30e-5p, miR-3158-3p, miR-330-5p, miR-3605-3p, miR-3615, miR-378h, miR-425-3p, miR-450b-5p, miR-4746-5p, miR-483-3p, miR-502-3p, miR-550a-5p, miR-651-5p, miR-7706 and miR-885-5p.
In some preferred embodiments, significantly differentially expressed miRNA molecule markers are used in combination, with the preferred combination being: one or more combinations of miR-3615, miR-502-3p, miR-450b-5p, miR-4746-5p, miR-10a-5p, miR-106b-3p, miR-125a-5p and miR-885-5p. The combination can provide better basis for early diagnosis of lung cancer and indicate the risk of diseases.
In addition, the kit for lung cancer diagnosis of the invention comprises a primer and a probe for detecting the exosome miRNA marker. The primers and the probes for detecting the exosome miRNA markers comprise:
reverse transcription primers, PCR primers and probes for detecting let-7a-3 p: the reverse transcription primer of the let-7a-3p has a nucleotide sequence shown as a sequence number 1, the PCR upstream primer has a nucleotide sequence shown as a sequence number 2, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 3;
reverse transcription primers, PCR primers and probes for detecting let-7 f-2: the reverse transcription primer of let-7f-2 is a nucleotide sequence shown as a sequence number 4, the PCR upstream primer is a nucleotide sequence shown as a sequence number 5, the PCR downstream primer is a nucleotide sequence shown as a sequence number 80, and the probe is a nucleotide sequence shown as a sequence number 6;
reverse transcription primers, PCR primers and probes for detecting miR-106b-3 p: the reverse transcription primer of miR-106b-3p has a nucleotide sequence shown as sequence number 7, the PCR upstream primer has a nucleotide sequence shown as sequence number 8, the downstream primer has a nucleotide sequence shown as sequence number 80, and the probe has a nucleotide sequence shown as sequence number 9;
reverse transcription primers, PCR primers and probes for detecting miR-10a-3 p: the reverse transcription primer of miR-10a-3p has a nucleotide sequence shown as a sequence number 10, the PCR upstream primer has a nucleotide sequence shown as a sequence number 11, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 12;
reverse transcription primers, PCR primers and probes for detecting miR-10a-5 p: the reverse transcription primer of miR-10a-5p 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 PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 15;
reverse transcription primers, PCR primers and probes for detecting miR-125a-5 p: the reverse transcription primer of miR-125a-5p 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 PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 18;
reverse transcription primers, PCR primers and probes for detecting miR-1294: the reverse transcription primer of miR-1294 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 PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 21;
reverse transcription primers, PCR primers and probes for detecting miR-19a-3 p: the reverse transcription primer of miR-19a-3p 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 PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 24;
reverse transcription primers, PCR primers and probes for detecting miR-22-3 p: the reverse transcription primer of miR-22-3p 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 PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 27;
reverse transcription primers, PCR primers and probes for detecting miR-29a-3 p: the reverse transcription primer of miR-29a-3p 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 PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 30;
reverse transcription primers, PCR primers and probes for detecting miR-30e-5 p: the reverse transcription primer of miR-30e-5p 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 PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 33;
reverse transcription primers, PCR primers and probes for detecting miR-3158-3 p: the reverse transcription primer of miR-3158-3p is a nucleotide sequence shown in sequence number 34, the PCR upstream primer is a nucleotide sequence shown in sequence number 35, the PCR downstream primer is a nucleotide sequence shown in sequence number 80, and the probe is a nucleotide sequence shown in sequence number 36;
reverse transcription primers, PCR primers and probes for detecting miR-330-5 p: the reverse transcription primer of miR-330-5p has a nucleotide sequence shown as a sequence number 37, the PCR upstream primer has a nucleotide sequence shown as a sequence number 38, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 39;
reverse transcription primers, PCR primers and probes for detecting miR-3605-3 p: the reverse transcription primer of miR-3605-3p has a nucleotide sequence shown as a sequence number 40, the PCR upstream primer has a nucleotide sequence shown as a sequence number 41, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 42;
reverse transcription primers, PCR primers and probes for detecting miR-3615: the reverse transcription primer of miR-3615 has a nucleotide sequence shown as a sequence number 43, the PCR upstream primer has a nucleotide sequence shown as a sequence number 44, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 45;
reverse transcription primers, PCR primers and probes for detecting miR-378 h: the reverse transcription primer of miR-378h has a nucleotide sequence shown as a sequence number 46, the PCR upstream primer has a nucleotide sequence shown as a sequence number 47, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 48;
reverse transcription primers, PCR primers and probes for detection of miR-425-3 p: the reverse transcription primer of miR-425-3p has a nucleotide sequence shown as a sequence number 49, the PCR upstream primer has a nucleotide sequence shown as a sequence number 50, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 51;
reverse transcription primers, PCR primers and probes for detecting miR-450b-5 p: the reverse transcription primer of miR-450b-5p has a nucleotide sequence shown as sequence number 52, the PCR upstream primer has a nucleotide sequence shown as sequence number 53, the downstream primer has a nucleotide sequence shown as sequence number 80, and the probe has a nucleotide sequence shown as sequence number 54;
reverse transcription primers, PCR primers and probes for detecting miR-4746-5 p: the reverse transcription primer of miR-4746-5p has a nucleotide sequence shown as sequence number 55, the PCR upstream primer has a nucleotide sequence shown as sequence number 56, the downstream primer has a nucleotide sequence shown as sequence number 80, and the probe has a nucleotide sequence shown as sequence number 57;
reverse transcription primers, PCR primers and probes for detecting miR-483-3 p: the reverse transcription primer of miR-483-3p has a nucleotide sequence shown as a sequence number 58, the PCR upstream primer has a nucleotide sequence shown as a sequence number 59, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 60;
reverse transcription primers, PCR primers and probes for detecting miR-502-3 p: the reverse transcription primer of miR-502-3p has a nucleotide sequence shown as a sequence number 61, the PCR upstream primer has a nucleotide sequence shown as a sequence number 62, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 63;
reverse transcription primers, PCR primers and probes for detecting miR-550a-5 p: the reverse transcription primer of miR-550a-5p has a nucleotide sequence shown as sequence number 64, the PCR upstream primer has a nucleotide sequence shown as sequence number 65, the PCR downstream primer has a nucleotide sequence shown as sequence number 80, and the probe has a nucleotide sequence shown as sequence number 66;
reverse transcription primers, PCR primers and probes for detecting miR-651-5 p: the reverse transcription primer of miR-651-5p has a nucleotide sequence shown as a sequence number 67, the PCR upstream primer has a nucleotide sequence shown as a sequence number 68, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 69;
reverse transcription primers, PCR primers and probes for detecting miR-7706: the reverse transcription primer of miR-7706 is a nucleotide sequence shown as a sequence number 70, the PCR upstream primer is a nucleotide sequence shown as a sequence number 71, the PCR downstream primer is a nucleotide sequence shown as a sequence number 80, and the probe is a nucleotide sequence shown as a sequence number 72;
reverse transcription primers, PCR primers and probes for detecting miR-885-5 p: the reverse transcription primer of miR-885-5p has a nucleotide sequence shown as a sequence number 73, the PCR upstream primer has a nucleotide sequence shown as a sequence number 74, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 75;
reverse transcription primers, PCR primers and probes for detection of internal reference U6: the reverse transcription primer of U6 has a nucleotide sequence shown as a sequence number 78, the PCR upstream primer has a nucleotide sequence shown as a sequence number 76, the PCR downstream primer has a nucleotide sequence shown as a sequence number 78, and the probe has a nucleotide sequence shown as a sequence number 79. 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 solutions of the present invention will be described more fully and clearly below with reference to the embodiments, which are a part of the embodiments of the present invention, rather than the whole embodiments. 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.
The experimental procedures in the following examples are all conventional ones unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
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 are respectively screened, blood is taken to be not less than 10ml, plasma is separated, exosome in the plasma is separated and RNA is extracted by a classical ultracentrifugation method, 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 mRNA 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) extracting exosome RNA from spkin exogenous cel-miR-39; (4) detecting the expression level of the target RNA by using a two-step method; (5) Normalizing the expression level of the target RNA to be detected by using the external reference gene; (6) Substituting the normalized gene expression level into a judgment model to obtain an output value; (7) 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 exosome RmiNA marker and a two-step detection system of reverse-plus-PCR.
Comprises selecting an external reference Cel-miR-39 or an internal reference U6 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 miRNA markers associated with early lung cancer based on high throughput sequencing
In order to screen the exosome markers relevant to early lung cancer diagnosis, 50 cases of early lung cancer diagnosis patients 72 and controls are respectively taken, blood is not less than 10ml, plasma is separated, exosome in the plasma is separated by a classical ultracentrifugation method, RNA is extracted by a qiagen miRNeasy mini kit, and the obtained RNA is subjected to small RNA library sequencing. The obtained data were analyzed by bioinformatics, and mirnas differentially expressed in early lung cancer patients and controls were compared to obtain significantly different mirnas as shown in table 2 below. These exosome-derived RNA-level markers can be used for early diagnosis of lung cancer.
TABLE 2
Example 2 fluorescent quantitation PCR platform based miRNA detection system
1. miRNA reverse transcription reaction system
miRNA reverse transcription reagents, enzymes and oligdT were purchased from TAKARA, standards were synthesized from Shanghai Yinxie substrate, and primers with reversed specificity were synthesized by Suzhou Hongyu. A20 ul reverse transcription system was used, as shown in Table 3 below.
TABLE 3
2. PCR reaction system
The PCR reaction mixture was purchased from TAKARA, the upstream primer and probe, i.e., the universal downstream primer, were synthesized by hong, suzhou, and the fluorescent quantitative PCR instrument was ABI 7500. The PCR reaction system is shown in Table 4 below.
TABLE 4
The PCR procedure was 95 ℃ for 10min, (95 ℃ 15s, 55 ℃ 30 s) 15 cycles without fluorescence acquisition, (95 ℃ 15s, 55 ℃ 30 s) 35 cycles with fluorescence acquisition.
Example 3 evaluation of early diagnosis and detection effects of lung cancer by taking Ex-seng Cel-miR-39 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 Exosucur of Echobiotech (Beijing Enzekangtai) (the exosome electron microscope identification result is shown in figure 1), miRNA in exosome is extracted from the separated exosome by a Qiagen MIReasy mini kit, RNA concentration and quality are detected by Agilent 2100, and the RNA concentration is recorded.
3. RNA two-step detection system
The two-step method detection system based on the PCR platform miRNA in the embodiment 1 is adopted to detect plasma exosome miRNA of 30 cases and 30 cases of control samples (healthy persons and benign nodules) of early lung cancer patients, detect Ct value of target miRNA, and calculate relative expression according to the Ct value and a relative quantitative formula.
4. Exosome miRNA diagnosis early lung cancer performance assessment
(1) Evaluation of independent detection performance of miR-3615
As shown in FIG. 2, ct values of miR-3615 were detected for plasma exosomes of 30 patients with early lung cancer and 30 control samples (healthy people and benign lesions), and the copy number of miRNA was obtained according to the Ct values with the external reference Cel-miR-39 as a reference. 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 (3) performing t detection analysis on the detection result by adopting an R language, wherein pvalue =0.00058< =0.05, and the exosome miR-3615 is remarkably related to early-stage lung cancer. The AUC of miR-3615 for diagnosing early lung cancer alone is 0.785, the negative predictive value is 85%, the sensitivity is 90%, the specificity is 56.57%, and the potential of a diagnostic marker is realized.
(2) Evaluation of independent detection performance of miR-502-3p
As shown in FIG. 3, ct values of miR-502-3p were detected for plasma exosomes of 30 patients with early lung cancer and 30 control samples (healthy people and benign lesions), and the copy number of miRNA was obtained according to the Ct values with the external reference Cel-miR-39 as a reference. 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 (3) performing t-detection analysis on the detection result by adopting R language, wherein pvalue =0.00139< =0.05, and the exosome miR-502-3p is remarkably related to early lung cancer. The AUC of the miR-502-3p for singly diagnosing early lung cancer is 0.756, the negative predictive value is 68.42%, the sensitivity is 60%, the specificity is 86.67%, and the miR-502-3p has the potential of a diagnostic marker.
(3) Evaluation of independent detection performance of miR-450b-5p
As shown in FIG. 4, ct values of miR-450b-5p were detected for plasma exosomes of 30 early lung cancer patients and 30 control samples (healthy people and benign lesions), and the copy number of miRNA was obtained from Ct values with the external reference Cel-miR-39 as a reference. 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 (3) performing t-detection analysis on the detection result by adopting R language, wherein pvalue =0.00199< =0.05, and the exosome miR-450b-5p is remarkably related to early lung cancer. The AUC of the miR-450b-5p for independently diagnosing early lung cancer is 0.744, the negative predictive value is 80%, the sensitivity is 86.67%, the specificity is 53.33%, and the potential of a diagnostic marker is realized.
(4) Evaluation of independent detection performance of miR-4746-5p
As shown in FIG. 5, ct values of miR-4746-5p were detected for plasma exosomes of 30 patients with early lung cancer and 30 control samples (healthy people and benign lesions), and the copy number of miRNA was obtained from the Ct values with the external reference Cel-miR-39 as a reference. 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. T-detection analysis is carried out on the detection result by adopting R language, and pvalue =0.00183< =0.05, which indicates that the exosome miR-4746-5p is significantly related to early-stage lung cancer. The AUC of miR-4746-5p for diagnosing early lung cancer alone is 0.743, the negative predictive value is 66.67%, the sensitivity is 60%, the specificity is 80%, and the potential of a diagnostic marker is realized.
(5) Evaluation of independent detection performance of miR-10a-5p
As shown in FIG. 6, ct values of miR-10a-5p were detected for plasma exosomes of 30 patients with early lung cancer and 30 control samples (healthy people and benign lesions), and the copy number of miRNA was obtained according to the Ct values with the external reference Cel-miR-39 as a reference. 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 (3) performing t-detection analysis on the detection result by adopting R language, wherein pvalue =0.00183< =0.05, and the exosome miR-10a-5p is remarkably related to early lung cancer. The AUC of the miR-10a-5p for diagnosing early lung cancer alone is 0.73, the negative predictive value is 77.27%, the sensitivity is 83.33%, the specificity is 56.67%, and the potential of a diagnostic marker is realized.
(6) Assessment of the Performance of other markers significantly associated with early Lung cancer
Other significantly related miRNA performance assessments are shown in table 5 below.
TABLE 5
From the data presented in table 5 it can be seen that the mirnas described in the table all have the potential for diagnostic markers.
Example 4 evaluation of the Effect of early diagnosis and detection of Lung cancer by combination of multiple markers with exogenous control Cel-miR-39 as reference
1. Three marker combination performance evaluation
The relative expression level of each miRNA was calculated according to the method in example 3, and the three marker combinations were trained using logistic regression, and the combinations with AUC of 0.85 or more of the three marker combinations were obtained as shown in table 6 below. Wherein the combination performances of miR-106-3p + miR-125a-5p + miR-3615, miR-106b-3p + miR-3615+ miR-450b-5p and the like are optimal, the AUC is respectively 0.887 and 0.881, and the AUC curves are respectively shown in figure 7 and figure 8.
TABLE 6
2. Five marker combination performance evaluation
The relative expression level of each miRNA was calculated according to the method in example 3, and five marker combinations were trained by logistic regression, and the combinations having AUC of the five marker combinations of 0.93 or more were obtained as shown in table 7 below. Wherein the combination performance of miR-106b-3p + miR-125a-5p + miR-3615+ miR-450b-5p + miR-885-5p, miR-106b-3p + miR-10a-3p + miR-125a-5p + miR-3615+ miR-450b-5p is optimal, the AUC is 0.951 and 0.948 respectively, and the AUC curves are respectively shown in a graph 9 and a graph 10.
TABLE 7
Example 5 evaluation of the Effect of the Multi-marker combination for early diagnosis and detection of Lung cancer Using internal reference U6 as reference
The relative expression level of each miRNA with U6 as a reference was calculated according to the method in example 3, and five marker combinations were trained by logistic regression, and the resulting combinations of AUC of 85 or more for the five marker combinations are shown in table 8 below. Wherein the combination property of mmiR-106b-3P + miR-10a-5p + miR-125a-5p + miR-3615+ miR-450b-5p is optimal, the AUC is 0.864, and the AUC curves are respectively shown in figure 11.
TABLE 8
The data show that the lung cancer detection method based on the exosome miRNA marker can realize noninvasive diagnosis of lung cancer, provides important values for early diagnosis and recurrence monitoring of lung cancer, and is of great help to prevention and treatment of lung cancer in China. Among them, 5 miRNA markers (the combined AUC can reach up to 0.951, the negative predictive value is 90.32%, the sensitivity is 90.00%, and the specificity is 93.33%) have extremely superior diagnostic performance.
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.
Sequence listing
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Claims (3)
1. The application of primers and probes for detecting plasma exosome miRNA markers in preparation of a lung cancer diagnosis kit is characterized in that the exosome miRNA markers are a combination of miR-106b-3p, miR-10a-3p, miR-125a-5p, miR-22-3p and miR-3615.
2. The use of claim 1, wherein the primers and probes comprise:
reverse transcription primers, PCR primers and probes for detecting miR-106b-3 p: the reverse transcription primer of miR-106b-3p has a nucleotide sequence shown as a sequence number 7, the PCR upstream primer has a nucleotide sequence shown as a sequence number 8, the PCR downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 9;
reverse transcription primers, PCR primers and probes for detecting miR-10a-3 p: the reverse transcription primer of miR-10a-3p has a nucleotide sequence shown as a sequence number 10, the PCR upstream primer has a nucleotide sequence shown as a sequence number 11, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 12;
reverse transcription primers, PCR primers and probes for detecting miR-125a-5 p: the reverse transcription primer of miR-125a-5p 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 80, and the probe has a nucleotide sequence shown as a sequence number 18;
reverse transcription primers, PCR primers and probes for detecting miR-22-3 p: the reverse transcription primer of miR-22-3p 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 80, and the probe has a nucleotide sequence shown as a sequence number 27;
reverse transcription primers, PCR primers and probes for detecting miR-3615: the reverse transcription primer of miR-3615 has a nucleotide sequence shown as a sequence number 43, the PCR upstream primer has a nucleotide sequence shown as a sequence number 44, the downstream primer has a nucleotide sequence shown as a sequence number 80, and the probe has a nucleotide sequence shown as a sequence number 45.
3. The application of primers and probes for detecting plasma exosome miRNA markers in preparation of a device for lung cancer diagnosis is characterized in that the exosome miRNA markers are a combination of miR-106b-3p, miR-10a-3p, miR-125a-5p, miR-22-3p and miR-3615.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
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