WO2019033831A1 - 血清外泌体中肺癌相关microRNA分子标记的应用及其检测试剂盒 - Google Patents

血清外泌体中肺癌相关microRNA分子标记的应用及其检测试剂盒 Download PDF

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WO2019033831A1
WO2019033831A1 PCT/CN2018/090043 CN2018090043W WO2019033831A1 WO 2019033831 A1 WO2019033831 A1 WO 2019033831A1 CN 2018090043 W CN2018090043 W CN 2018090043W WO 2019033831 A1 WO2019033831 A1 WO 2019033831A1
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mir
seq
mirna
pcr
detection
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王弢
渠香云
董肇楠
贾芸莉
马雪情
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江苏为真生物医药技术股份有限公司
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • the invention belongs to the technical field of medical molecular biology, and particularly relates to the application of a molecular marker of IgG of lung cancer exosomes and a kit thereof.
  • Exosome is a membranous vesicle that is widely distributed and distributed in various body fluids and can be secreted by various cells. It is usually between 30 and 120 nm in diameter and contains cell-specific proteins, lipids and Nucleic acids, in addition to being able to carry and transmit important signaling molecules, form a new intercellular information transmission system that changes the functions of other cells, and plays an important role in many physiological and pathological phenomena. Studies have shown that the molecular features of tumor exosome reflect the phenotype of the tumor from which it originates, and the tumor-specific microRNAs and antigens carried can be used as tumor diagnostic markers.
  • exosome can selectively remove certain cellular proteins and transfer many types of molecules between cells, which can induce and enhance the immune response of the body.
  • physiological and pathological processes such as immune surveillance, inflammatory reaction and cancer development, Important features.
  • exosome can be isolated from body fluids such as serum or urine for early clinical diagnosis, and can also be used for clinical risk of tumors. Or efficacy assessment, and prognosis.
  • the exosomes contain a large amount of mRNA and microRNA, which not only protects the in vitro RNA from being degraded, but also acts as an effective carrier to transport RNA into specific target cells and play an important regulatory role. More than 120 microRNAs carried by Exosome have multiple functions. For example, miR-1, miR-17, miR-18, miR-181 and miR-375 are involved in angiogenesis, hematopoiesis, extracellular secretion and tumorigenesis.
  • MicroRNA also known as miRNA or miR
  • miRNA - as the first of the top ten technological breakthroughs in Science in 2002 - is one of the major discoveries in life science research in the 21st century. It plays a very important role in the regulation of biological development and the occurrence of diseases. Important role.
  • miRNAs regulate cell differentiation, proliferation, and apoptosis, thereby promoting or inhibiting the occurrence of tumors.
  • complex regulatory mechanisms are formed to form a regulatory network to promote or inhibit tumorigenesis. Methylation, defects in biological origin, mutations, abnormal transcription, and loss or amplification of genomics all lead to abnormalities in miRNAs in human tumors.
  • miRNAs directly appear as a proto-oncogene or tumor suppressor gene.
  • Carcinogenic and tumor suppressor miRNAs directly or negatively regulate tumor suppressor genes, oncogenes, or genes that control cell cycle progression, differentiation, or apoptosis, directly regulate tumor cell proliferation, differentiation, and apoptosis, and participate in tumor formation, development, and even Invasion and transfer.
  • miRNAs have characteristic expression profiles in tumor cells, cancer tissues, paracancerous tissues, and normal tissues, and there are also characteristic expression levels in hematuria of tumor patients, which provides for the diagnosis of tumors.
  • miRNA may be an important molecular biological marker for tumor diagnosis.
  • Peripheral blood has been the main source of clinical disease marker detection because of its advantages of small trauma, easy access, repeatability, and many detectable indicators.
  • Recent studies have found that endogenous circulating miRNAs exist in peripheral blood, and because of its high stability and specificity, it is expected to become a biological marker for various diseases such as tumors.
  • Some researchers have suggested that circulating miRNAs are mainly found in exosomes and may be a good source of serum miRNAs. Therefore, if combined with the relevant characteristics of exo-miRNA, if the corresponding high-specificity lung cancer diagnostic kit can be developed to be applied to the differential diagnosis and research field of pulmonary nodules and benign and malignant, it will promote lung cancer screening well.
  • the research and the transformation of scientific research results will greatly promote the differentiation and diagnosis of benign and malignant lung tumors.
  • the existing microRNA is used as a molecular marker for detecting lung cancer. Because of its low accuracy and specificity, it requires several markers to be detected at the same time, and the detection cost is high, and there is no kit suitable for commercial production.
  • the two-step detection system based on PCR platform miRNA mainly includes probe miRNA quantitative detection technology and dye detection technology, 1) quantitative detection technology combined with probe, including stem-loop RT-PCR Probe method, key-like method and Ligation Assay.
  • probe miRNA quantitative detection technology and dye detection technology 1) quantitative detection technology combined with probe, including stem-loop RT-PCR Probe method, key-like method and Ligation Assay.
  • SYBR Green Quantitative detection technology based on PCR and fluorescent dyes
  • SYBR Green including poly(A) polymerase tailing method, Stem-loop dye method, primer extension method, multi-multiplexed method (Multiplexed RT) and the like.
  • Most of the SYBR Green technology uses higher sensitivity, generally lower cost, but lower specificity.
  • the method of adding PolyA tail and stem loop structure, the sequence of miRNA pairing is extended, and then normal reverse transcription and subsequent PCR detection are performed.
  • the stem-loop method is only specific for mature miRNAs, and the specificity is relatively high.
  • the tail-tail method can detect mature miRNAs and pre-miRNAs, and the specificity and sensitivity are poor, but the operation and primer design are simple.
  • nucleic acid isothermal amplification technology has flourished. It can amplify specific DNA or RNA at a specific temperature. Compared with traditional PCR technology, the instrument and reaction time are greatly simplified, and it can meet the needs of quick and easy detection. .
  • isothermal amplification techniques to miRNA detection. Summary analysis Based on the temperature amplification technology miRNA one-step detection method: 1. According to the type of amplification can be divided into reactive fluorescence signal linear amplification and exponential amplification (EXPAR). Linear amplification is generally performed by fluorescence spectrophotometer after the end of the reaction, and quantitative analysis is performed according to the fluorescence value of the endpoint Flu.
  • the exponential amplification is exponentially amplified by the EXPAR isothermal amplification technique to reach the standard S. Type amplification curve.
  • the former can be reacted on a common PCR instrument and then collected at the end point of the fluorescence spectrophotometer, which is more conducive to the development of POCT products, but the detection sensitivity and stability are generally poor.
  • the latter can use the POI (similar to the Ct value) on a real-time PCR instrument to achieve accurate quantitative detection of traditional RT-PCR.
  • Isothermal amplification can be divided into probe method and dye method according to the different fluorescent substances used. Similar to traditional RT-PCR, the general probe method has higher specificity and less sensitivity, and the dye method is just the opposite. 3.
  • DSN Duplex-Specific Nuclease
  • DSN Duplex-Specific Nuclease
  • DSN is a thermostable nuclease that does not require a specific recognition site and is capable of selectively degrading DNA in double-stranded DNA and DNA-RNA hybrids, but It has little effect on single-stranded DNA/RNA nucleic acid molecules and double-stranded RNA molecules, and is capable of distinguishing between fully and incompletely matched duplexes.
  • nicking incision enzymes are a special type of nicking enzyme (nicking endonuclease) in restriction endonucleases that recognize specific cleavage sites and cleave only one strand of double-stranded DNA, resulting in a nick that can be directed against DNA.
  • the invention combines the relevant characteristics of exosomes and miRNAs, evaluates the changes of serum exo-miRNA, exo-miRNA expression profiles of lung cancer tissues and the correlation with tumors, and screens out the diagnosis and prediction of lung cancer exosomal microRNAs.
  • the exosomal-associated microRNA molecule comprises at least one upregulated exosomal microRNA molecule, or comprises at least one down-regulated exosomal microRNA, or comprises at least one up-regulated and at least one down-regulated exosomes MicroRNA molecule;
  • the up-regulated exosomal microRNA molecule is at least one of miR-21 or miR-486-5p or miR-205 or miR-126, and the down-regulated microRNA molecule is miR-152 or Let-7a or At least one of miR-148a.
  • the marker is miR-21 in combination with Let-7a, miR-205 in combination with Let-7a, miR-126 in combination with miR-152 or miR-486-5p in combination with miR-148a.
  • diagnosis and prediction are specifically lung cancer screening, auxiliary diagnosis, efficacy evaluation, prognosis evaluation or recurrence monitoring.
  • the molecular markers are derived from body fluids or cells; the body fluids include at least one of blood, sputum, pleural effusion, pleural lavage, urine or saliva.
  • the invention relates to a lung cancer auxiliary diagnosis and detection kit, which is based on a PCR platform miRNA two-step detection kit, and all the two-step detection systems described in the specification are the theoretical basis for constructing the kit.
  • the invention comprises: a microRNA molecular marker specific stem loop structure reverse transcription primer, a PCR upstream primer, a PCR universal downstream primer, and a specific probe for detecting a microRNA molecular marker, wherein the microRNA molecular marker has at least two types. One of them is selected from the up-regulated markers miR-21, miR-486-5p, miR-205 or miR-126; the other is selected from the down-regulated markers miR-152, Let-7a or miR-148a.
  • the loop loop of the neck of the specific stem-loop structure reverse transcription primer is designed to form a key-like structure of the non-contiguous complementary base pair TGCG and CGCA, and the short arm is connected to the microRNA molecule by a ligase during the reverse transcription reaction. .
  • the molecular marker miR-21 reverse transcription primer sequence is SEQ ID NO.
  • the miR-21 PCR upstream primer sequence is SEQ ID NO. 2:
  • the miR-21 PCR universal downstream primer sequence is SEQ ID NO. 3:
  • the miR-21 specific probe sequence is SEQ ID NO. 4:
  • the molecular marker miR-486-5p reverse transcription primer sequence is SEQ ID NO. 5:
  • the miR-486-5p PCR upstream primer sequence is SEQ ID NO. 6:
  • the miR-486-5p PCR universal downstream primer sequence is SEQ ID NO. 3:
  • the miR-486-5p specific probe sequence is as SEQ ID NO. 7:
  • the molecular marker miR-205 reverse transcription primer sequence is SEQ ID NO. 8:
  • the miR-205 PCR upstream primer sequence is as SEQ ID NO. 9:
  • the miR-205 PCR universal downstream primer sequence is SEQ ID NO. 3:
  • the miR-205 specific probe sequence is as SEQ ID NO. 10:
  • the molecular marker miR-126 reverse transcription primer sequence is SEQ ID NO. 11:
  • the miR-126 PCR upstream primer sequence is SEQ ID NO. 12:
  • the miR-126 PCR universal downstream primer sequence is SEQ ID NO. 3:
  • the miR-126 specific probe sequence is SEQ ID NO. 13:
  • the molecular marker let-7a reverse transcription primer sequence is as SEQ ID NO. 14:
  • the let-7a PCR upstream primer sequence is SEQ ID NO. 15:
  • the let-7a PCR universal downstream primer sequence is SEQ ID NO. 3:
  • the let-7a specific probe sequence is SEQ ID NO. 16:
  • the molecular marker miR-152 reverse transcription primer sequence is SEQ ID NO. 17:
  • the miR-152PCR upstream primer sequence is SEQ ID NO. 18:
  • the miR-152PCR universal downstream primer sequence is SEQ ID NO. 3:
  • the miR-152 specific probe sequence is SEQ ID NO. 19:
  • the molecular marker miR-148a reverse transcription primer sequence is SEQ NO. 20:
  • the miR-148a PCR upstream primer sequence is SEQ ID NO. 21:
  • the miR-148aPCR universal downstream primer sequence is SEQ ID NO. 3:
  • the miR-148a specific probe sequence is SEQ ID NO. 22:
  • the miRNA molecular marker calibrator is also included: the miR-21 molecular marker standard is miR-21, the diluted concentration is 10 13 copy/ ⁇ L; the miR-486-5p molecular marker standard is miR-486 -5p, diluted to a concentration of 10 13 copy/ ⁇ L; miR-205 molecular marker standard is miR-205, diluted to a concentration of 10 13 copy / ⁇ L; miR-126 molecular marker standard is miR-126, diluted The post concentration was 10 13 copy/ ⁇ L; the let-7a molecular marker standard was let-7a, the diluted concentration was 10 13 copy/ ⁇ L; the miR-152 molecular marker standard was miR-152, and the diluted concentration was 10 13 copy/ ⁇ L; miR-148a molecular marker standard is miR-148a, diluted to a concentration of 10 13 copy / ⁇ L.
  • the kit further includes a microRNA molecule-specific amplification template, a Vent (exo-) DNA polymerase, a Nicking enzyme, a double-strand specific nuclease, and a molecular hybridization probe.
  • a miRNA diagnostic kit based on isothermal amplification technology the molecular marker miR-21 first amplification template sequence is SEQ ID NO. 23:
  • the second amplification template sequence of miR-21 is SEQ ID NO. 24:
  • the miR-21 hybridization probe sequence is SEQ ID NO. 25:
  • the first amplification template sequence of the molecular marker miR-486-5p is SEQ ID NO. 26:
  • the second amplification template sequence of miR-486-5p is SEQ ID NO.
  • the miR-486-5p hybridization probe sequence is SEQ ID NO. 28:
  • the molecular marker miR-205 first amplification template sequence is SEQ ID NO. 29:
  • the second amplification template sequence of miR-205 is SEQ ID NO. 30:
  • the miR-205 hybridization probe sequence is SEQ ID NO. 31:
  • the molecular marker miR-126 first amplification template sequence is SEQ ID NO. 32:
  • the second amplification template sequence of miR-126 is SEQ ID NO. 33:
  • the miR-126 hybridization probe sequence is SEQ ID NO. 34:
  • the first amplification template sequence of the internal control gene Let-7a is SEQ ID NO. 35:
  • the second amplification template sequence of Let-7a is SEQ ID NO. 36:
  • the Let-7a hybridization probe sequence is SEQ ID NO. 37:
  • the miRNA molecular marker calibrator is also included: the miR-21 molecular marker standard is miR-21, diluted to a concentration of 10 13 copy/ ⁇ L, and diluted to a gradient standard; miR-486-5p molecular marker The standard was miR-486-5p, diluted to a concentration of 10 13 copy/ ⁇ L, and diluted to a gradient standard; miR-205 molecular marker standard was miR-205, diluted to a concentration of 10 13 copy / ⁇ L, And diluted to a gradient standard; miR-126 molecular marker standard is miR-126, diluted to a concentration of 10 13 copy / ⁇ L, and diluted to a gradient standard; Let-7a molecular marker standard is Let-7a, The diluted concentration was 10 13 copy/ ⁇ L and diluted to a gradient standard.
  • the level of at least one microRNA gene product of the test sample is higher than the level of the corresponding microRNA gene product of the control sample (ie, the expression of the microRNA gene product is "upregulated”).
  • the expression of the microRNA gene product is "upregulated” when the amount of microRNA gene product from the subject sample is greater than the amount of the same gene product of the control sample.
  • the level of at least one microRNA gene product of the test sample is lower than the level of the corresponding microRNA gene product of the control sample (ie, the expression of the microRNA gene product is "down-regulated”).
  • the expression of the microRNA gene is “down-regulated” when the amount of microRNA gene product produced by the microRNA gene from the subject is less than the amount produced from the same gene in the control sample.
  • a preferred embodiment is that at least one of the up-regulated microRNAs in the test sample is combined with at least one down-regulated microRNA to predict the risk of disease.
  • Reverse transcription primers The specific reverse transcription primers of the present invention combine the advantages of the stem-loop RT-PCR method and the key-like method: 1.
  • Stem-loop RT reverse transcription primer (Fig. 1) The Stem base pair is extended, and four pairs of non-contiguous complementary base pairs are designed in the loop loop to enhance their ability to form a key-like structure, thereby facilitating the RT primer to better maintain the stem-loop structure throughout the reverse transcription process, not only Eliminating the mismatching of stem-loop primers and non-target miRNAs increases the specificity, and increases the number of bases of reverse transcripts, which is more conducive to subsequent PCR detection. 2.
  • Stem-loop RT is fully complementary to the miRNA with 5 pairs of bases, and the enzyme ligation step is added before reverse transcription (Fig. 1), which makes the miRNA and Stem-loop RT more strongly and enhances the efficiency of reverse transcription. 3.
  • the present invention utilizes Stem-loop RT primers for miRNA reverse transcription products and can also be used for fluorescent dye PCR detection.
  • PCR upstream and downstream primers specific upstream primers plus Tag to extend the amplification template to increase amplification efficiency, and adjust the downstream primers so that the Tm values of the upstream and downstream primers are basically the same, so that the upstream and downstream primers can simultaneously bind to the template after PCR pre-denaturation. Amplification, annealing and extension are carried out at the same temperature.
  • Hydrolysis probe adopts the design method of TaqMan technology to design a specific hydrolysis probe complementary to the template (Fig. 1) to enhance the specificity of the detection.
  • the miRNA was selected as the internal control gene of the miRNA marker, and the relative quantitative formula (2 - ⁇ Cp ) was used to calculate the fold change of the relative expression of the marker according to the CP value, and the miRNA score was calculated.
  • the Pearson correlation coefficient was used to analyze the correlation between the relative expression of miRNA markers and patients with demographic characteristics, benign lesions and healthy individuals.
  • Clinical pathology diagnosis is used as a reference standard to determine the sensitivity and specificity of miRNA markers.
  • Clinical pathology diagnosis is used as a reference standard to determine the sensitivity and specificity of miRNA markers.
  • ROC characteristic curves and AUC analysis were used to determine the accuracy of the combined detection of miRNAs, and the cut results were used to interpret the sample results.
  • the relative expression amount 2 - ⁇ Cp was calculated using a relative quantitative formula, and the score of each miRNA was calculated.
  • the Pearson correlation coefficient was used to analyze the correlation between the relative expression score of each miRNA marker and the patients with demographic characteristics, benign lesions and healthy individuals. Clinical pathology diagnosis is used as a reference standard to determine the sensitivity and specificity of each miRNA marker miRNA marker.
  • Clinical pathology diagnosis is used as a reference standard to determine the sensitivity and specificity of miRNA markers.
  • Logistic regression models were then used to derive binary logistic regression equations and to select the best diagnostic combination of miRNA markers.
  • ROC characteristic curves and AUC analysis were used to determine the accuracy of the combined detection of miRNAs, and the cut results were used to interpret the sample results.
  • Amplification template The specific amplification template A and B of the present invention are divided into three parts (Fig. 2), the first part is completely complementary to the miRNA, which is favorable for detection specificity; the second part is the corresponding Nicking enzyme recognition and cleavage position. Point, the new amplified single-stranded (tirggers) cyclically cut, replace, release under the action of specific polymerase; the third part is the tirggers complementary strand, which can continuously release tirggers.
  • the miRNA binds to the amplification template A, it triggers the EXPAR 1 reaction.
  • the product tirggers is released from the source and binds to the amplification template B to trigger the EXPAR 2 reaction.
  • the product New tirggers is released by the source and then returned to the amplification template A.
  • EXPAR 1 reaction The sequence of the third part can be designed at will, avoiding the formation of secondary structure by the template itself and reducing the fluorescence background. 2.
  • the amplification templates A and B only align the 1 part and the 3 part, and the A and B are not. Primer dimer will be formed; 3.
  • One-step method, two-cycle amplification is performed by isothermal reaction, and a cyclic chain reaction is formed by two consecutive SDA reactions in series to achieve exponential amplification of EXPAR cycle mode, and expansion can be completed within 30 minutes. Increase the reaction.
  • DSN enzyme Duplex-Specific Nuclease double-strand specific nuclease
  • DSN enzyme Duplex-Specific Nuclease double-strand specific nuclease
  • the invention is based on the linear amplification technique of isothermal signal, and the designed molecular beacon (MB) probe specifically hybridizes with the miRNA, and degrades the DNA probe strand in the DNA-RNA hybrid double strand under the action of the DSN enzyme.
  • the fluorescent signal is released, and the miRNA is again hybridized with the probe into the next cycle, thereby achieving the purpose of fluorescence signal amplification.
  • a microRNA molecular marker in a lung cancer auxiliary diagnostic reagent comprising: 1) up-regulation of at least one of miR-21, miR-486-5p, miR-205 or miR-126; 2) miR-152, Downregulation of at least one of Let-7a or miR-148a; 3) Combination of up-regulated molecular markers and down-regulated molecular markers.
  • the present invention optimizes and improves the existing detection method, and develops a two-step detection kit based on PCR platform miRNA, and can select a corresponding detection and analysis method according to the purpose of detection and the requirements of experimental conditions. Due to the effectiveness of miRNA itself and its correlation with lung cancer tumors, this kit can be used for early and malignant identification of early pulmonary nodules, as well as for prognosis, real-time monitoring of preoperative, postoperative, therapeutic and therapeutic effects.
  • the two-step detection system consists of a uniquely designed specific stem-loop structure RT primer, PCR upstream and downstream primers and Taqman probe primers.
  • the specific primers make the miRNA detection specificity distinguish single base difference, and the sensitivity is at least 1 copy/
  • the detection limit of ⁇ L greatly improves the detection efficiency and accuracy of miRNA.
  • the PCR thermal cycling conditions of different markers can not only detect multiple markers on the same plate in the same batch, but also improve the accuracy of detection of combined markers. Detection efficiency also reduces time and cost.
  • the one-step detection system of the miRNA developed by the isothermal amplification technology of the invention is faster and more convenient, and can greatly improve the detection efficiency and reduce the detection cost.
  • the exo-miRNA has good stability.
  • the exo-miRNA can be effectively extracted after storage for 20 days at 4 °C.
  • the extracted exo-miRNA can be stored at -20 °C for 50 days and stored at -80 °C for a long time.
  • Figure 1 is a schematic diagram of miRNA two-step PCR detection amplification
  • FIG. 2 Schematic diagram of one-step isothermal EXPAR amplification
  • Figure 3 is a schematic diagram of one-step isothermal linear amplification
  • Figure 4 PCR standard curve and detection sensitivity of miRNA markers (A: PCR minimum detection limit of Let-7a; B: PCR standard curve of Let-7a; C: PCR minimum detection limit of miR-21; D: PCR of miR-21 Standard curve; E: lower limit of PCR detection of miR-486-5p; F: PCR standard curve of miR-486-5p; G: lower limit of PCR detection of miR-205; PCR standard curve of H: miR-205; : PCR minimum detection limit of miR-126; J: PCR standard curve of miR-126; K: minimum detection limit of PCR of miR-152; PCR standard curve of L: miR-152; minimum detection of PCR of M: miR-148a Lower limit; N: PCR standard curve of miR-148a).
  • Figure 5 shows the stability results of the two-step miRNA detection system for clinical samples (A: intra-assay difference CV value; B: inter-assay difference CV value).
  • FIG. 6 Results of miR-21 combined with Let-7a in lung cancer clinical samples
  • A lung cancer tissue sample miR-21 combined with Let-7a detection results
  • A-1 miR-21 combined with Let-7a miRNA detection
  • A-2 miR- 21 combined with Let-7a ROC curve
  • B lung cancer serum exosome miR-21 combined with Let-7a test results
  • B-1 lung cancer serum exosome miR-21 combined with Let-7a miRNA detection relative quantitative results
  • B-2 lung cancer serum exosome miR-21 combined with Let-7a detection ROC curve
  • B-3 lung cancer serum exosome miR-21 combined with Let-7a detection logistic regression analysis results
  • B-4 lung cancer serum exosomes miR- 21 combined with Let-7a detection logistic regression ROC curve
  • B-5 lung cancer plasma exosome miR-21 combined with Let-7a miRNA detection results
  • B-6 lung cancer plasma exosome miR-21 combined with Let-7a detection
  • B-8 lung cancer urine exosome
  • Fig.7 Results of miR-205 combined with Let-7a in lung cancer clinical samples
  • A lung cancer tissue sample miR-205 combined with Let-7a detection miRNA results
  • B lung cancer tissue sample miR-205 combined with Let-7a detection ROC curve
  • C lung cancer Serum exosomal miR-205 combined with Let-7a to detect miRNA results
  • D lung cancer serum miR-205 combined with Let-7a ROC curve
  • E lung cancer plasma exosome miR-205 combined with Let-7a detection miRNA results
  • F ROC curve of lung cancer plasma exosome miR-205 combined with Let-7a
  • G lung cancer urine exosome miR-205 combined with Let-7a detection of miRNA results
  • H lung cancer urine exosome miR-205 combined with Let-7a Detect ROC curve
  • Figure 8 Results of miR-126 combined with miR-152 in lung cancer clinical samples (A: lung cancer tissue sample miR-126 combined with miR-152 test results; B: lung cancer tissue sample miR-126 combined with miR-152 detection ROC curve; C: lung cancer serum Exosome miR-126 combined with miR-152 test results; D: lung cancer serum exosome miR-126 combined with miR-152 detection ROC curve; E: lung cancer plasma exosome miR-126 combined with miR-152 test results; F: Detection of ROC curve of lung cancer plasma exosome miR-126 combined with miR-152; G: detection of lung cancer urine exosome miR-126 combined with miR-152; H: detection of lung cancer urine exosome miR-126 combined with miR-152 ROC curve).
  • FIG. 9 Detection results of miR-486-5p combined with miR-148a in lung cancer clinical samples
  • A lung cancer tissue sample miR-486-5p combined with miR-148a test results
  • B lung cancer tissue sample miR-486-5p combined with miR-148a test ROC curve
  • C lung cancer serum exosome miR-486-5p combined with miR-148a
  • D lung cancer serum exosome miR-486-5p combined with miR-148a detection ROC curve
  • E lung cancer plasma exosome miR -486-5p combined with miR-148a test results
  • F lung cancer plasma exosome miR-486-5p combined with miR-148a detection ROC curve
  • G lung cancer urine exosomes miR-486-5p combined with miR-148a test results
  • H lung cancer urine exosome miR-486-5p combined with miR-148a detection ROC curve
  • FIG 10 Preoperative and postoperative exosome miRNA expression levels and their differences
  • A preoperative and postoperative serum exosomal expression levels and differences
  • A-1 miR-21 test results, A-2: miRNA- 205 test results, A-3: miRNA-126 test results, A-4: miRNA-486-5p test results, A-5: Let-7a test results, A-6: miR-152 test results, A-7: miR-148a test results
  • B pre- and post-operative plasma exosome expression levels and their differences
  • C surgery Pre- and post-urinary exosomal expression levels and their differences
  • C-1 miR-21 test results, C-2: miRNA-205 test results, C-3: miRNA-126 test results, C-4: miRNA -4
  • Figure 11 Combined detection of exosome miR-21 and Let-7a for prognosis of lung cancer (A: combined detection of serum exosomes miR-21 and Let-7a for prognosis of lung cancer, A-1, miR-21 and Let-7a Expression level, A-2: ROC curve; A-3: progression-free survival curve; A-4: overall survival rate; B: combined detection of plasma exosomes miR-21 and Let-7a for prognosis of lung cancer, B-1 , miR-21 and Let-7a expression levels, B-2: ROC curve; B-3: progression-free survival curve; B-4: overall survival; C: urine exosome miR-21 and Let-7a combination Prognostic assessment of lung cancer; C-1, miR-21 and Let-7a expression levels, C-2: ROC curve; C-3: overall survival rate).
  • Figure 12 Combined detection of exosome miR-205 and Let-7a for prognosis of lung cancer (A: combined detection of serum exosomes miR-205 and Let-7a for prognosis of lung cancer, A-1, miR-205 and Let-7a Expression level, A-2: ROC curve; A-3: progression-free survival curve; A-4: overall survival rate; B: combined detection of plasma exosomes miR-205 and Let-7a for prognosis of lung cancer, B-1 , miR-205 and Let-7a expression levels, B-2: ROC curve; B-3: progression-free survival curve; B-4: overall survival rate; C: urine exosome miR-205 and Let-7a combination Prognostic assessment of lung cancer, C-1, miR-205 and Let-7a expression levels, C-2: ROC curve; C-3: overall survival rate).
  • FIG 13 Exogenous miR-126 and miR-152 for prognosis of lung cancer (A: combined detection of serum exosomes miR-126 and miR-152 for prognosis of lung cancer, A-1, miR-126 and miR-152 expression levels , A-2: ROC curve; A-3: overall survival rate; B: combined detection of plasma exosomes miR-126 and miR-152 for lung cancer prognosis, B-1, miR-126 and miR-152 expression levels, B-2: ROC curve; B-3: overall survival rate; C: combined detection of urine exosomes miR-126 and miR-152 for lung cancer prognosis, C-1, miR-126 and miR-152 expression levels, C-2: ROC curve; C-3: overall survival rate).
  • Figure 14 One-step detection kit based on isothermal amplification technology miRNA and its application (A: One-step detection sensitivity and standard curve of Let-7a isothermal exponential amplification; B: Isothermal exponential amplification one-step clinical sample detection C: Isothermal linear amplification one-step Let-7a standard curve; D: isothermal linear amplification one-step miR-21 standard curve; E-1: lung cancer urine exosome miR-21 combined with Let-7a test results; E-2 lung cancer Urine exosome miR-21 combined with Let-7a to detect ROC curve; F-1: lung cancer serum exosome miR-21 combined with Let-7a test results; F-2: lung cancer serum exosome miR-21 combined with Let- 7a detects the ROC curve).
  • A One-step detection sensitivity and standard curve of Let-7a isothermal exponential amplification
  • B Isothermal exponential amplification one-step clinical sample detection
  • C Isothermal linear amplification one-step Let-7a standard curve
  • D isothermal linear
  • Example 1 Two-step detection system kit based on PCR platform miRNA
  • Reagents used to prepare the reverse transcription reaction system include reverse transcription primers (RT-Primer, Shanghai Infineon Synthesis), miRNA standard powder (Shanghai Yingjijieji synthesis), T4 DNA Ligase (T4 DNA Ligase, Supplier: NEB, product number: M0202S, containing 10 ⁇ T4 DNA Ligase Buffer), RNase inhibitor (supplier: Fermentas, trade number: K1622), transcriptase Transcriptase (supplier: Shanghai Yingjie Jieji) Biotechnology Co., Ltd., product number: K1622, containing RNase inhibitor, dNTPs, nuclease-free water), T4 Polynucleotide Kinase (supplier: NEB, trade number: M0201S) and nuclease-free pure water (nuclease-free water, supplier: Shanghai Yingjie Jieji Biological Co., Ltd., product number: K1622).
  • the cDNA was 10-fold diluted and stored at 4 ° C for subsequent PCR amplification.
  • the reagents used to prepare the PCR reaction system include deoxynucleotide deoxynucleotide dNTPs (dCTP, dGTP, dATP, dTTP, dUTP (supplier: Thermo Scientific) formulated as dNTPs.
  • Upstream primer solution (F primer, Shanghai Yingjiejie) Base synthesis), general downstream primer solution (R primer, Shanghai Infineon synthesis), probe (Probe, ABI synthesis), DNA polymerase (HS Taq, supplier: Takara, product number: R007A), uracil -DNA glycosylase (UDG, supplier: NEB, trade number: M0280S) and pure water (H 2 O).
  • the reagents for preparing the PCR reaction system are packaged bottle by bottle, and are used in a certain proportion to prepare a PCR reaction system.
  • the PCR reaction system is 20 ⁇ L/time, and the volume of the package is 50 times, as shown in Table 3.
  • the amplification reaction was then carried out under the conditions of Table 4.
  • the cDNA stock solution was 10 12 copy/ ⁇ L, and 10 ⁇ L of the cDNA stock solution was added to 90 ⁇ L of sterilized purified water and diluted to 10 11 copy/ ⁇ L, and then 10 ⁇ L of 10 11 copy/ ⁇ L dilution solution was added to 90 ⁇ L of sterilized purified water. Dilute to 10 10 copy/ ⁇ L and serially dilute to 1 copy/ ⁇ L of the dilution.
  • miR-21 two-step molecular marker standard configuration:
  • the standard product miR-21 was reverse-transcribed cDNA stock solution was 10 12 copy/ ⁇ L, 10 ⁇ L cDNA stock solution was added to 90 ⁇ L sterile water to dilute to 10 11 copy/ ⁇ L, then 10 ⁇ L 10 11 copy/ ⁇ L dilution was added to 90 ⁇ L sterilization.
  • the purified water was diluted to 10 10 copy/ ⁇ L and serially diluted to 1 copy/ ⁇ L of the dilution.
  • miRNA molecular markers two-step detection system construction and standard configuration configuration reference miR-21, only template, primers, and probes, PCR reaction conditions are the same.
  • Serum Exo-miR-21 combined with miRNA down-regulation marker Exo-Let-7a was used to evaluate the stability of the test results. 4 cases of different clinical serum samples, each sample was divided into 3 batches, 3 batches per batch, verifying the stability of the detection and evaluation system (including Exo-miRNA extraction purification, reverse transcription, PCR on-machine detection). The results are shown in Fig. 5. The difference CV value within the same sample batch can reach 4% or less, and the inter-assay difference CV value can reach 8% or less, indicating that the miRNA two-step detection and evaluation system has good stability.
  • Tissue, serum, plasma and urine samples from a series of lung cancers including different stages, different subtypes, different genders and different age groups), benign lung diseases and healthy people were collected.
  • the miRNAs in the purified tissues and serum were extracted using QIAGEN's commercial product miRNeasy Serum/Plasma Kit (Cat. No. 217184), and the RNA nucleic acid quality was measured by Nano-Drop 2000. The RNA concentration and purity were recorded, and the tissue miRNAs were returned. One treatment.
  • Serum and plasma exosomes were extracted using SBI's commercial ExoQuickTM kit (Cat. EXOQ5A-1).
  • the miRNAs in the purified exosomes were extracted and purified using QIAGEN's commercial product miRNeasy mini kit (Cat. No. 217004), and the RNA nucleic acid quality was measured by Nano-Drop 2000 to record the RNA concentration and purity.
  • Urine exosomes were extracted using SBI's commercial product ExoQuick-TC for Tissue Culture Media and Urine kit (Cat. No. EXOTC10A-1).
  • the miRNAs in the purified exosomes were extracted and purified using QIAGEN's commercial product miRNeasy mini kit (Cat. No. 217004), and the RNA nucleic acid quality was measured by Nano-Drop 2000 to record the RNA concentration and purity.
  • Example 1 A two-step assay system kit based on PCR platform miRNA in Example 1 was used. Exo-miRNA was detected in 56 patients with stage Ia early stage lung cancer, and 76 samples (healthy and benign) were tested for miR-152, Let-7a, miR-148a, miR-21, miR-486- The expression level of 5p, miR-205 or miR-126 is CP value, and the relative expression amount is calculated according to the CP value using a relative quantitative formula.
  • PI 50.979 + 3.462 ⁇ log [copy (miR-21)] - 7.516 ⁇ log [copy (let-7a)] - 1.09 ⁇ CP (let-7a).
  • AUC 0.913, when the Cutoff value is taken as (0.2063), the diagnostic sensitivity is 91%, and the specificity is 92%, which has a significant advantage (B-3 and B-4 in Fig. 6).
  • the results show that plasma exosomal microRNA has a certain degree of discrimination between lung cancer and healthy human controls.
  • Exo-miRNA was detected in 32 urine samples from 22 patients with stage Ia early stage and 22 controls (healthy and benign), and miRNA up-regulated marker miR was detected. -21 and miRNA down-regulate the expression of the marker Let-7a, the CP value, and obtain the score of the relative expression of miRNA.
  • tissue and serum and plasma exosome miRNA markers show that serum exosome miRNA has a significant non-invasive diagnostic effect compared with tumor tissue biopsy and plasma exosome miRNA diagnostics.
  • Exo-miRNA was detected in serum samples from 25 patients with early stage Ia lung cancer and 15 controls (healthy and benign), and miRNA up-regulated markers miR-205 and miRNA were down-regulated.
  • the expression value of the marker Let-7a is CP value.
  • the relative quantitative formula (2 - ⁇ Cp ) is used to calculate the fold change of the relative expression of the joint marker, and then the score of the relative expression of the miRNA is obtained.
  • the detection results were analyzed by SPSS 17.0 for t test analysis (P ⁇ 0.05), indicating that the combined markers were significantly associated with early lung cancer prediction.
  • AUC 0.803 and the Cutoff value was 37.32, the diagnostic sensitivity was 76.1% and the specificity was 83.0%. Has a significant advantage.
  • the miRNA up-regulated marker miR-205 and the miRNA down-regulated marker Let-7a were used to treat plasma exosomal microRNAs in 16 clinical stage Ia lung cancer samples and 3 healthy human control clinical samples. The results show that plasma exosomal microRNA has a certain degree of discrimination between lung cancer and healthy human controls.
  • Exo-miRNA was detected in 25 urine samples from 15 patients with early stage Ia lung cancer, and 15 controls (healthy and benign) were tested for miRNA up-regulation markers miR-205 and miRNA.
  • the CP value of the expression of the marker Let-7a was down-regulated, and the score of the relative expression of the miRNA was obtained based on the CP value.
  • the detection results were analyzed by SPSS 17.0 for t test analysis (P ⁇ 0.05), indicating that the combined markers were significantly associated with early lung cancer prediction.
  • AUC 0.745, when the Cutoff value is 29.348, the diagnostic sensitivity is 83.5%, and the specificity is 65.2%, which has a significant advantage.
  • Exo-miRNA was detected in serum samples from 25 patients with early stage Ia lung cancer and 15 controls (healthy and benign), and miRNA up-regulation marker miR-126 and miRNA were down-regulated.
  • the expression value of the marker miR-152 is CP value.
  • the relative quantitative formula (2 - ⁇ Cp ) is used to calculate the fold change of the relative expression of the combined marker, and then the score of the relative expression of the miRNA is obtained.
  • the detection results were analyzed by SPSS 17.0 for t test analysis (P ⁇ 0.05), indicating that the combined markers were significantly associated with early lung cancer prediction.
  • AUC 0.728, when the Cutoff value is 55.98, the diagnostic sensitivity is 64.0%, and the specificity is 81.0%, which has a significant advantage.
  • miRNA up-regulated marker miR-126 and miRNA down-regulated marker miR-152 were used to control plasma exosomal microRNA in 16 clinical stage Ia lung cancer samples and 3 healthy human control clinical samples. The results show that plasma exosomal microRNA has a certain degree of discrimination between lung cancer and healthy human controls.
  • Exo-miRNA was detected in 25 urine samples from 15 patients with early stage Ia lung cancer and 15 controls (healthy and benign).
  • the miRNA up-regulated markers miR-126 and miRNA were detected.
  • the expression level of the marker miR-152 was down-regulated, and the score of the relative expression of the miRNA was obtained based on the CP value.
  • the detection results were analyzed by SPSS 17.0 for t test analysis (P ⁇ 0.05), indicating that the combined markers were significantly associated with early lung cancer prediction.
  • AUC 0.719, when the Cutoff value is 28.2230, the diagnostic sensitivity is 65.9% and the specificity is 81.4%, which has a significant advantage.
  • miRNA up-regulation marker miR-486-5p combined with miR-148a for clinical sample test results
  • Exo-miRNA was detected in serum samples from 25 patients with early stage Ia lung cancer and 15 controls (healthy and benign), and the miRNA up-regulated marker miR-486-5p was detected.
  • the miRNA down-regulated marker miR-148a expression CP value according to the CP value, using the relative quantitative formula (2 - ⁇ Cp ) value, calculate the fold change of the relative expression of the joint marker, and then obtain the miRNA relative expression score.
  • the detection results were analyzed by SPSS 17.0 for t test analysis (P ⁇ 0.05), indicating that the combined markers were significantly associated with early lung cancer prediction.
  • AUC 0.677, when the Cutoff value is 9.98, the diagnostic sensitivity is 61.5%, and the specificity is 75.0%, which has a significant advantage.
  • the miRNA marker miR-486-5p in combination with miR-148a was used to compare plasma exosomal microRNAs in 16 clinical stage Ia lung cancer samples and 3 healthy human control clinical samples. The results show that plasma exosomal microRNA has a certain degree of discrimination between lung cancer and healthy human controls.
  • Exo-miRNA was detected in 25 urine samples from 15 patients with early stage Ia lung cancer and 15 controls (healthy and benign).
  • the miRNA up-regulated marker miR-486-5p was detected.
  • the miRNA down-regulated marker miR-148a expression amount CP value according to the CP value, the miRNA relative expression amount score.
  • the detection results were analyzed by SPSS 17.0 for t test analysis (P ⁇ 0.05), indicating that the combined markers were significantly associated with early lung cancer prediction.
  • AUC 0.663, when the Cutoff value is 28.9214, the diagnostic sensitivity is 65.2% and the specificity is 78.1%, which has a significant advantage.
  • miRNA up-regulation markers miR-21, miR-205, miR-126, miR-486-5p for clinical sample testing, 10 cases of lung cancer patients without any treatment before surgery (serum, plasma, urine)
  • Exo-miRNA of samples and corresponding postoperative body fluid (serum, plasma, urine) samples were detected, and the expression levels of miRNA up-regulated markers miR-21, miR-205, miR-126, and miR-486-5p were detected.
  • the detection results were analyzed by SPSS 17.0 for t detection analysis P ⁇ 0.05. Results As shown in A of Fig.
  • Exosomal miRNA is used for prognostic evaluation test results
  • Exo-miRNA was detected in 20 samples of IA patients with early stage lung cancer without distant metastasis, no major systemic disease, and curable surgery.
  • the inclusion criteria for the sample were: the ability to complete chemotherapy as scheduled. Body fluids can be collected from before and after chemotherapy, and the samples were divided into 10 treatment effective groups and 10 treatment ineffective groups by pathological diagnosis.
  • the expression levels of miR-21, miRNA-205, miRNA-126, miRNA-486-5p, Let-7a, miR-152, and miR-148a were examined.
  • Exo-miRNA samples from 10 samples of body fluids with significant differences in prognosis were tested for miRNA up-regulation markers miR-21 and miR-205, and down-regulated markers
  • miRNA up-regulation markers miR-21 and miR-205 were tested for miRNA up-regulation markers miR-21 and miR-205, and down-regulated markers
  • Exosomal miRNA is used for recurrence monitoring test results
  • the pathological stage with the same pathological stage and the prognosis survival in 2015-2016 was 10 cases, all of which were the first diagnosis of the primary lesion, 5 cases were still healthy after 3 years, and 5 cases were within 2 years after treatment. Recurrence or lymph node metastasis or liver metastasis, died within 2 years.
  • the expression of exosome miRNA was detected by body fluids before and after operation in 10 samples. The samples were taken every three months after surgery, and the expression of exosome miRNA was predicted according to the patient's exogenous miRNA expression to judge recurrence or metastasis. Statistical analysis was performed to evaluate the correlation between exosome miRNA expression levels and imaging findings. Statistical analysis of the relationship between exosome miRNA expression levels and patient survival.
  • the evaluation rate was 100%, indicating that the discovery of body fluid exosomes miRNAs earlier than clinical signs and symptoms can be used to predict lung cancer recurrence or metastasis.
  • Kaplan-Meier survival and relapse analysis the results are shown in Figures 11, 12, and 13, exosomes miR-21 or miR-486-5p or miR-205 or miR-126 or miR-152 or Let-7a or miR-
  • the gene expression level of 148a is significantly correlated with survival and can be used for recurrence risk assessment.
  • the one-step isothermal exponential amplification method was used to detect the Let-7a standard.
  • the POI values of the samples fell between 10 7 and 10 10 copies of the standard linear range, indicating that the isothermal amplification EXPAR one-step method can be used for sample detection.
  • the detection limit is shown in comparison with other similar EXPAR methods (document: Guo-lei Wang and Chun-yang Zhang, Sensitive Detection of MicroRNAs with Hairpin Probe-Based Circular Exponential Amplification Assay. Anal. Chem.
  • the collection of the fluorescent signal of the reaction tube in the instrument used should be set, and the selected fluorescent detection channel is identical to the fluorescent reporter group labeled by the probe.
  • the specific setting method varies depending on the instrument, and should refer to the instruction manual of the instrument.
  • Urine samples from a series of lung cancer diagnosed by the hospital including different stages, different subtypes, different genders and different age groups), benign lung diseases and healthy people were collected.
  • Urine exosomes were extracted using SBI's commercial product ExoQuick-TC for Tissue Culture Media and Urine kit (Cat. No. EXOTC10A-1).
  • the miRNAs in the purified exosomes were extracted and purified using QIAGEN's commercial product miRNeasy mini kit (Cat. No. 217004), and the RNA nucleic acid quality was measured by Nano-Drop 2000 to record the RNA concentration and purity.
  • the one-step detection system kit based on the isothermal amplification technique miRNA in Example 2 was used. Exo-miRNA was detected in 50 patients with early stage Ia lung cancer and 50 controls (healthy and benign). The expression of miR-152 and Let-7a was detected. According to CP value, relative quantification was used. The formula calculates the relative expression.
  • Exo-miRNA was detected in 32 urine samples from 22 patients with early stage Ia and 22 controls (healthy and benign), and miRNA up-regulated markers miR-21 and miRNA down-regulated markers were detected.
  • the expression level of Let-7a is CP value, and the score of relative expression of miRNA is obtained.
  • the detection results were analyzed by SPSS 17.0 for t test analysis (P ⁇ 0.05), indicating that the combined markers were significantly associated with early lung cancer prediction.
  • AUC 0.844, when the Cutoff value was 19.878, the diagnostic sensitivity was 87.5% and the specificity was 81.8%. Has a significant advantage.
  • Exo-miRNA was detected in serum samples from 56 patients with early stage Ia lung cancer and 76 controls (healthy and benign), and miRNA up-regulated markers miR-21 and miRNA down-regulated markers were detected.
  • the expression level of Let-7a is CP, and the score of relative expression of miRNA is obtained based on the CP value.

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Abstract

提供了外泌体相关microRNA在制备诊断和预示肺癌标志物中的应用及试剂盒，开发出基于PCR平台miRNA的两步法检测试剂盒，通过上调分子标志物和下调分子标志物联合使用，在肺癌的辅助诊断方面具有突出优势，灵敏度最低可达到1copy/μL，极大提高了检测的准确度。另外，开发出等温扩增技术miRNA的一步法检测体系更快速、便捷，可大大提高检测效率，降低检测成本。

Description

血清外泌体中肺癌相关microRNA分子标记的应用及其检测试剂盒 技术领域
本发明属于医学分子生物学技术领域，具体涉及肺癌外泌体miRNA分子标记的应用及其试剂盒。
背景技术
外泌体(exosome)是一种广泛存在并分布于各种体液中可由多种细胞分泌的膜性小囊泡，直径一般介于30～120nm之间，包含有细胞特异的蛋白、脂质和核酸，除了能作携带和传递重要的信号分子，形成一种全新的细胞间信息传递系统从而改变其他细胞的功能外，在很多生理病理上起着重要的作用。研究表明，肿瘤exosome的分子特征部分反映其来源肿瘤的表型，所携带的肿瘤特异性microRNA和抗原可以作为肿瘤诊断标志物。此外exosome可以选择性的将某些细胞蛋白移出，在细胞之间传递很多种类型的分子，可以诱发与增强机体免疫反应，在免疫监视、炎症反应及癌症发生发展等许多生理和病理过程中有重要的功能。在包括膀胱癌、脑瘤、结直肠癌和黑色素瘤在内的多种肿瘤临床病例中，均能从患者血清或尿液等体液中分离exosome用于早期临床诊断，也可用于肿瘤的临床风险或疗效评估，以及预后判定。
外泌体中含有大量mRNA和microRNA，不仅保护体外RNA稳定存在免遭降解，还能够作为有效的载体将RNA转运到特定的靶细胞中，发挥重要的调控作用。Exosome承载的120多种microRNA具有多种功能。如miR-1、miR-17、miR-18、miR-181和miR-375与血管生成、造血、细胞外分泌和肿瘤的发生有关。
microRNA(又称miRNA或miR)——作为Science 2002年十大科技突破的第一名——是21世纪生命科学研究重大发现之一，它在生物的发育时序调控和疾病的发生中起到非常重要的作用。miRNA通过调节癌基因及抑癌基因的表达，调控细胞的分化、增殖、凋亡，从而促进或抑制肿瘤的发生，期间有着复杂的调节机制，形成调控网络，共同促进或抑制肿瘤的发生。甲基化、生物起源上的缺陷、变异、转录的异常以及基因组的丢失或扩增等均导致miRNA在人类肿瘤中的异常，许多miRNA直接表现为一种原癌基因或抑癌基因的作用，致癌与抑癌miRNA通过正向或负向调控肿瘤抑制基因、癌基因或控制细胞周期进程、分化或凋亡的基因，直接调控肿瘤细胞的增殖、分化和凋亡，参与肿瘤的生成、发展甚至侵袭转移。大量的研究显示，miRNA在肿瘤细胞、癌组织、癌旁组织、正常组织中都有特征性的表达谱改变，在肿瘤患者的血尿中也有特征性的表达水平改变，这就为肿瘤的诊断提供了新的思路，也提示miRNA可能可以成为肿瘤诊断的重要的分子生物学标志。
外周血因其具有创伤小、易获取、可重复、可检测指标众多等优点，一直是临床疾病标志物检测的主要标本来源。近年研究发现，外周血中存在内源性循环miRNA，且因其具有较高的稳定性和特异性，有望成为诸如肿瘤等多种疾病的生物学标志物。有研究者提出，循环miRNA主要存在于外泌体中，有可能成为较好的检测血清miRNA的来源。因此，若结合exo-miRNA的相关特点，如果能研发出相应的高特异性肺癌诊断试剂盒使之能够应用于肺部结节良恶性辅助的鉴别诊断及科研领域，将很好地促进肺癌筛查研究及科研成果的转化，对于良恶性肺部肿瘤的区分、诊疗必将起到巨大的推动作用。
目前，已有的microRNA做为检测肺癌分子标志物的应用，因其准确性、特异性不高或需要几个标志物同时检测，检测成本高等缺陷，并没有适合商业化生产的试剂盒。
此外，基于PCR平台miRNA的两步法检测体系主要包括探针法miRNA定量检测技术和染料法检测技术，1)、结合探针的定量检测技术，包括茎环引物(Stem-loop RT-PCR)探针法，key-like法和酶连法(Ligation Assay)。这三种方法需要使用miRNA特异的探针，这类方法的突出优点是特异性强，常常能区分同一miRNA家族的不同变体。但存在miRNA与Stem-loop RT结合不牢固，茎环引物与非目标miRNA的错配现象。2)、基于PCR与荧光染料如SYBR Green的定量检测技术，包括poly(A)聚合酶加尾法、Stem-loop染料法、引物延伸法、多通路法(Multiplexed RT)等。使用SYBR Green技术大多灵敏度较高，费用普遍较低，但特异性较低。相对而言，加PolyA尾及茎环结构的方法，将miRNA配对的序列延长，然后进行正常的反转录及后续的PCR检测。茎环法只针对成熟miRNA，特异性相对较高；加尾法可以检测到成熟miRNA及pre-miRNA，特异性和灵敏度都差一些，但操作及引物设计简单。
近年来，核酸等温扩增技术蓬勃发展起来，它能在某一特定的温度下扩增特定的DNA或者RNA，与传统PCR技术相比仪器、反应时间大大简化，更能满足快捷简便的检测需求。已有大量的研究将等温扩增技术应用到miRNA检测方面。总结分析基于温扩增技术miRNA一步法检测方法：1.根据扩增类型可分为反应荧光信号线性扩增和指数型扩增(EXPAR)。线性扩增一般是在反应结束后利用荧光分光光度计采集荧光信号，根据终点Flu荧光值大小进行定量分析，指数型扩增是利用EXPAR等温扩增技术将检测信号呈指数型放大，达到标准S型扩增曲线。前者可在普通PCR仪上进行反应然后在荧光分光光度计采集终点信号，更有利于POCT产品开发，但一般检测灵敏度和稳定性较差。后者可在实时荧光定量PCR仪上利用POI(类似于Ct值)达到传统RT-PCR的精确定量检测检测结果。2.根据所用荧光物质不同也可将等温扩增分为探针法和染料法，同传统RT-PCR类似，一般探针法检测特异性较高而灵敏度稍差，染料法正好与之相反。3.DSN(Duplex-Specific Nuclease双链特异性核酸酶)，是一种热稳定核酸酶，不需要特异的识别位点，能够选择性降解双链DNA和DNA-RNA杂交体中的DNA,但对单链DNA/RNA核酸分子和双链RNA分子几乎没有作用，能够区分完全和不完全匹配的双链体。nicking切口酶是限制性内切酶中有一类特殊的切口酶(nicking enzyme，或者nicking endonuclease)，识别特异性的切割位点，只切割双链DNA中的一条链，造成一个切口，可对DNA分子定点切割，一些研究是基于nicking切口酶或DSN双链特异性核酸酶等特异性切割酶的作用设计出快速简便的一步法等温miRNA检测技术。但普遍的缺点是荧光背景较高，检测下限达不到要求，通过降低反应温度等方法提高了灵敏度，所需反应时间又过长。本发明通过结合不同的荧光信号类型、扩增技术及所用的特异性试剂，极大地拓展了设计思路，大大缩短了检测时间并提高了检测的灵敏度和特异性，达到了一步法miRNA临床检测的要求。
发明内容：
本发明结合外泌体(exosome)和miRNA的相关特点，对肺癌血清exo-miRNA、肺癌组织exo-miRNA表达图谱的变化及与肿瘤的相关性进行评价，筛选出诊断和预示肺癌外泌体microRNA分子标志物：
优选的，所述外泌体相关microRNA分子包括至少一种上调的外泌体microRNA分子，或包 含至少一种下调的外泌体microRNA，或包含至少一种上调和至少一种下调的外泌体microRNA分子；
优选的，所述上调的外泌体microRNA分子为miR-21或miR-486-5p或miR-205或miR-126中至少一种，所述下调的microRNA分子为miR-152或Let-7a或miR-148a中至少一种。
更优选的，在一种microRNA分子标志物在肺癌诊断和预示的应用的实施方案中，其中，上调组中至少一种或几种microRNA与下调组中至少一种或几种microRNA联合检测，较优选的，所述标志物为miR-21与Let-7a联合，miR-205与Let-7a联合，miR-126与miR-152联合或miR-486-5p与miR-148a联合。
更优选的，所述诊断和预示具体为肺癌筛查、辅助诊断、疗效评价、预后评估或复发监控。
microRNA分子标志物在肺癌诊断和预示的应用中,分子标志物来源于体液或细胞；所述体液包括血液、痰液、胸腔积液、胸腔灌洗液、尿液或唾液中的至少一种。
一种肺癌辅助诊断检测试剂盒，该试剂盒是基于PCR平台miRNA两步法检测试剂盒，说明书中所述的所有两步法检测体系为构建该试剂盒的理论基础。包含：microRNA分子标志物特异性茎环结构逆转录引物、PCR上游引物、PCR通用下游引物，用于检测microRNA分子标记物的特异探针，其中，所述的microRNA分子标志物至少为两种，其中一种选自上调标志物miR-21、miR-486-5p、miR-205或miR-126；另一种选自下调标志物miR-152、Let-7a或miR-148a。
优选的，所述特异性茎环结构逆转录引物的颈部的loop环部设计非连续互补碱基对TGCG和CGCA构成钥匙状结构，逆转录反应时通过连接酶将短臂与microRNA分子相连接。
更优选的，所述的分子标志物miR-21逆转录引物序列如SEQ ID NO.1：
5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCATCAACAT-3'；
miR-21 PCR上游引物序列如SEQ ID NO.2：
5'-CTCCGTCAGGGTAGCTTATCAGACTG-3'；
miR-21 PCR通用下游引物序列如SEQ ID NO.3：
5'－CTCAAGTGTCGTGGAGTCGGC-3'；
miR-21特异探针序列如SEQ ID NO.4：
5'-FAM-TTTCCTCATCATCAACAT-MGB-3'
所述的分子标志物miR-486-5p逆转录引物序列如SEQ ID NO.5：
5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCACTCGGGG-3'；
miR-486-5p PCR上游引物序列如SEQ ID NO.6：
5'-CTCCGTCAGGGTCCTGTACTGAGCTG-3'；
miR-486-5p PCR通用下游引物序列如SEQ ID NO.3：
5'－CTCAAGTGTCGTGGAGTCGGC-3'；
miR-486-5p特异探针序列如SEQ ID NO.7：
5'-FAM-TTTCCTCATCACTCGGGG-MGB-3'
所述的分子标志物miR-205逆转录引物序列如SEQ ID NO.8：
5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCACAGACTC-3'；
miR-205 PCR上游引物序列如SEQ ID NO.9：
5'-CTCCGTCAGGGTCCTTCATTCCACCG-3'；
miR-205 PCR通用下游引物序列如SEQ ID NO.3：
5'－CTCAAGTGTCGTGGAGTCGGC-3'；
miR-205特异探针序列如SEQ ID NO.10:
5'-FAM-TTTCCTCATCACAGACTC-MGB-3'；
所述的分子标志物miR-126逆转录引物序列如SEQ ID NO.11：
5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCACGCATTA-3'；
miR-126 PCR上游引物序列如SEQ ID NO.12：
5'-CTCCGTCAGGGTCGTACCGTGAGTAA-3'；
miR-126 PCR通用下游引物序列如SEQ ID NO.3：
5'－CTCAAGTGTCGTGGAGTCGGC-3'；
miR-126特异探针序列如SEQ ID NO.13：
5'-FAM-TTTCCTCATCACGCATTA-MGB-3'
所述的分子标志物let-7a逆转录引物序列如SEQ ID NO.14：
5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCAACTATAC-3'；
let-7a PCR上游引物序列如SEQ ID NO.15：
5'-CTCCGTCAGGGTGAGGTAGTAGGTT-3'；
let-7a PCR通用下游引物序列如SEQ ID NO.3：
5'－CTCAAGTGTCGTGGAGTCGGC-3'；
let-7a特异探针序列如SEQ ID NO.16：
5'-FAM-TTTCCTCATCAACTATAC-MGB-3'
所述的分子标志物miR-152逆转录引物序列如SEQ ID NO.17：
5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCAAGTCGGAG-3'；
miR-152PCR上游引物序列如SEQ ID NO.18：
5'-CTCCGTCAGGGAGGTTCTGTGATACA-3'；
miR-152PCR通用下游引物序列如SEQ ID NO.3：
5'－CTCAAGTGTCGTGGAGTCGGC-3'；
miR-152特异探针序列如SEQ ID NO.19：
5'-FAM-TTTCCTCATCAAGTCGGAG-MGB-3'；
所述的分子标志物miR-148a逆转录引物序列如SEQ NO.20：
5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCAAGTCGGAG-3'；
miR-148a PCR上游引物序列如SEQ ID NO.21：
5'-CTCCGTCAGGGAAAGTTCTGAGACA-3'；
miR-148aPCR通用下游引物序列如SEQ ID NO.3：
5'－CTCAAGTGTCGTGGAGTCGGC-3'；
miR-148a特异探针序列如SEQ ID NO.22：
5'-FAM-TTTCCTCATCAAGTCGGAG-MGB-3'；
更优选的，还包括miRNA分子标志物校准品：miR-21分子标志物标准品为miR-21，稀 释后浓度为10 13copy/μL；miR-486-5p分子标志物标准品为miR-486-5p，稀释后浓度为10 13copy/μL；miR-205分子标志物标准品为miR-205，稀释后浓度为10 13copy/μL；miR-126分子标志物标准品为miR-126，稀释后浓度为10 13copy/μL；let-7a分子标志物标准品为let-7a，稀释后浓度为10 13copy/μL；miR-152分子标志物标准品为miR-152，稀释后浓度为10 13copy/μL；miR-148a分子标志物标准品为miR-148a，稀释后浓度为10 13copy/μL。
所述试剂盒还包括microRNA分子特异性扩增模板、Vent(exo-)DNA聚合酶、Nicking酶、双链特异性核酸酶和分子杂交探针。
一种基于等温扩增技术miRNA诊断试剂盒，所述的分子标志物miR-21第一条扩增模板序列如SEQ ID NO.23：
5'-GTCATCGCAGACAACCTCATCTAGACTCATCAACATCAGTCTGATAAGCTAA-NH 2-3'
miR-21第二条扩增模板序列如SEQ ID NO.24：
5'-ATCAACATCAGTCTGATAAGCTAATCTAGACTCGTCATCGCAGACAACCTCA-NH 2-3'
miR-21杂交探针序列如SEQ ID NO.25：
5'-FAM-AGCCTATCAACATCAGTCTGATAAGCTAATAGGCTGCATC-Tamra-3'
所述的分子标志物miR-486-5p第一条扩增模板序列如SEQ ID NO.26：
5'-GTCATCGCAGTGTTCCTCAACAGACTCTCTCGGGGCAGCTCAGTACAGGAA-NH 2-3'
miR-486-5p第二条扩增模板序列如SEQ ID NO.27：
5'-CTCGGGGCAGCTCAGTACAGGAAAACAGACTCAGTCATCGCAGTGTTCCTCA-N H 2-3'
miR-486-5p杂交探针序列如SEQ ID NO.28：
5'-FAM-AGCCTAACTCGGGGCAGCTCAGTACAGGAATAGGCTGCATC-Tamra-3'
所述的分子标志物miR-205第一条扩增模板序列如SEQ ID NO.29：
5'-GTCATCGCAGTGTTCCTCAACAGACTCTCAGACTCCGGTGGAATGAAGGAA-NH 2-3'
miR-205第二条扩增模板序列如SEQ ID NO.30：
5'-CAGACTCCGGTGGAATGAAGGAAACAGACTCAGTCATCGCAGTGTTCCTCA-NH 2-3'
miR-205杂交探针序列如SEQ ID NO.31：
5'-FAM-AGCCTAACAGACTCCGGTGGAATGAAGGAATAGGCTGCATC-Tamra-3'
所述的分子标志物miR-126第一条扩增模板序列如SEQ ID NO.32：
5'-GTCATCGCAGTGTTCCTCAACAGACTCTCGCATTATTACTCACGGTACGAA-NH 2-3'
miR-126第二条扩增模板序列如SEQ ID NO.33：
5'-CGCATTATTACTCACGGTACGAAACAGACTCAGTCATCGCAGTGTTCCTCA-NH 2-3'
miR-126杂交探针序列如SEQ ID NO.34：
5'-FAM-AGCCTAACGCATTATTACTCACGGTACGAATAGGCTGCATC-Tamra-3'
所述的内控基因Let-7a第一条扩增模板序列如SEQ ID NO.35：
5'-GTC ATC GCAGTGTTCCTCAACAGACTCTAACTATACAACCTACTACCTCA-NH 2-3'
Let-7a第二条扩增模板序列如SEQ ID NO.36：
5'-AACTATACAACCTACTACCTCAAACAGACTCAGTCATCGCAGTGTTCCTCA-NH 2-3'
Let-7a杂交探针序列如SEQ ID NO.37：
5'-FAM-AGCCTAAACTATACAACCTACTACCTCAATAGGCTGCATC-Tamra-3'
更优选的，还包括miRNA分子标志物校准品：miR-21分子标志物标准品为miR-21，稀释后浓度为10 13copy/μL，并稀释为梯度标准品；miR-486-5p分子标志物标准品为miR-486-5p，稀释后浓度为10 13copy/μL，并稀释为梯度标准品；miR-205分子标志物标准品为miR-205，稀释后浓度为10 13copy/μL，并稀释为梯度标准品；miR-126分子标志物标准品为miR-126，稀释后浓度为10 13copy/μL，并稀释为梯度标准品；Let-7a分子标志物标准品为Let-7a，稀释后浓度为10 13copy/μL，并稀释为梯度标准品。
在一个实施方案中，测试样品的至少一种microRNA基因产物的水平高于对照样品的对应microRNA基因产物的水平(即，microRNA基因产物的表达被"上调")。当来自受试者样品的microRNA基因产物的量高于对照样品的相同基因产物的量时，microRNA基因产物的表达被“上调”。
在另一个实施方案中，测试样品的至少一种microRNA基因产物的水平低于对照样品的对应microRNA基因产物的水平(即，microRNA基因产物的表达被"下调")。当来自受试者的microRNA基因产生的microRNA基因产物的量低于对照样品中从相同基因产生的量时，microRNA基因的表达被“下调”。
优选的实施方案为，测试样品中至少一种上调microRNA联合至少一种下调microRNA，进而预示患病风险。
基于PCR平台miRNA的两步法检测试剂盒
逆转录引物：本发明特异性的逆转录引物结合了茎环引物(Stem-loop RT-PCR)法和key-like法的设计优点：1.Stem-loop RT逆转录引物(图1)颈部Stem碱基对延长，并且在loop环部设计了4对非连续互补碱基对加强其形成key-like结构的能力，从而促使RT引物在整个逆转录过程能更好地保持茎环结构，不仅杜绝了茎环引物与非目标miRNA的错配提高特异性，而且使逆转录产物碱基数增加，更有利于后续的PCR检测。2.Stem-loop RT与miRNA有5对碱基完全互补，并且在逆转录前增加了酶连接步骤(图1)，使miRNA与Stem-loop RT结合更牢固，增强了逆转录的效率。3.本发明利用Stem-loop RT引物对miRNA逆转录产物也可用于荧光染料法PCR检测。
PCR上下游引物：特异性上游引物加上Tag标签从而延长扩增模板增加扩增效率，并调整下游引物使上下游引物Tm值基本相同，使得在PCR预变性后上下游引物可同时与模板结合并进行扩增，退火和延伸同一温度下进行。
水解探针：本发明采用TaqMan技术的设计方法，设计一条与模版互补的特异性水解探针(图1)，增强检测的特异性。
对一种miRNA标志物定量检测，选取该miRNA作为miRNA标志物的内控基因，根据CP值，使用相对定量公式(2 -ΔΔCp)计算标志物相对表达量的倍数变化，算出miRNA的得分。并利用皮尔逊相关系数(Pearson correlation coefficient)分析miRNA标志物的相对表达量与具有人口统计学特征的患者、良性病变和健康个体三者之间的相关性。临床病理诊断用作参 考标准以决定miRNA标志物的敏感性和特异性。临床病理诊断用作参考标准确定miRNA标志物的敏感性和特异性。使用ROC特征曲线和AUC分析来确定miRNA联合检测的准确度，以cut off值对样本结果进行判读。
对两种或两种以上miRNA标志物联合检测，选取1)miR-21、miR-486-5p、miR-205或miR-126的至少一种的上调；2)miR-152、Let-7a或miR-148a的至少一种的下调；3)上调分子标志物和下调分子标志物联合使用。根据CP值，使用相对定量公式计算相对表达量2 -ΔΔCp，算出每种miRNA的得分。并利用皮尔逊相关系数(Pearson correlation coefficient)分析每种miRNA标志物的相对表达量得分与具有人口统计学特征的患者、良性病变和健康个体三者之间的相关性。临床病理诊断用作参考标准以决定每种miRNA标志物miRNA标志物的敏感性和特异性。临床病理诊断用作参考标准确定miRNA标志物的敏感性和特异性。然后用逻辑回归模型(Logistic regression models)得出二元logistic回归方程，并选择miRNA标志物的最佳诊断组合。使用ROC特征曲线和AUC分析来确定miRNA联合检测的准确度，以cut off值对样本结果进行判读。
基于等温扩增技术miRNA的一步法检测体系
等温扩增EXPAR一步法检测体系
扩增模板：本发明特异性的扩增模板A和B分3个部分(图2)，第1部分与miRNA完全互补结合，有利于检测特异性；第2部分为对应的Nicking酶识别切割位点，在特异性聚合酶的作用下对新扩增单链(tirggers)进行循环切割、替换、释放；第3部分为tirggers互补链，可源源不断释放tirggers。miRNA一旦与扩增模板A结合就会触发EXPAR 1反应，产物tirggers被源源释放后会与扩增模板B结合从而触发EXPAR 2反应，产物New tirggers被源源释放后再返回与扩增模板A结合进入EXPAR 1反应。特点：1、第3部分序列基本可随意设计，避免扩增模板自身形成二级结构，降低荧光背景；2、扩增模板A和B只是把1部分和3部分对调，A和B之间不会形成引物二聚体；3、一步法，等温实现2次循环扩增，通过二个连续SDA反应串联构成了一个循环链反应，从而达到指数型扩增EXPAR循环模式，30min内即可完成扩增反应。
等温线性扩增一步法检测体系
DSN酶(Duplex-Specific Nuclease双链特异性核酸酶)，能够选择性降解双链DNA和DNA-RNA杂交体中的DNA链,但对单链DNA/RNA核酸分子和双链RNA分子中的RNA链几乎没有作用。本发明基于等温信号线性扩增技术，设计的分子信标(Molecular beacon，MB)探针与miRNA特异性杂交，在DSN酶的作用下对DNA-RNA杂交双链中的DNA探针链进行降解并释放荧光信号，miRNA再次与探针杂交进入下一个循环，从而达到荧光信号放大的目的。可利用普通荧光分光光度计采集终点信号，有利于POCT产品开发(图3)。特点：1.采用分子信标探针，环部与miRNA完全互补，特异性强能区分单碱基；2.颈部有5对碱基互补，既能保证探针在自由状态时保持茎环结构，减少荧光背景信号，又能在与miRNA杂交时迅速形成刚性链状模板，提高结合效率；3.无PCR目的产物扩增，污染少；4.DSN酶无需特异性识别位点，可适用于所有miRNA检测；5.操作简单、试剂耗材少，成本低。
本发明的有益效果：
(1)microRNA分子标志物在肺癌辅助诊断试剂中的应用，其包含1)miR-21、miR-486-5p、miR-205或miR-126的至少一种的上调；2)miR-152、Let-7a或miR-148a的至少一种的下调； 3)上调分子标志物和下调分子标志物联合使用。经过大数据临床验证实验，在肺癌的辅助诊断指标方面具有突出优势，极大提高了进行miRNA检测时相对定量的准确度。
(2)本发明对现有检测方法进行了优化改进，开发出基于PCR平台miRNA的两步法检测试剂盒，可根据检测的目的及实验条件的需求选择相应的检测和分析方法。由于miRNA自身的实效性及与肺癌肿瘤的相关性，本试剂盒既可用于早期肺部结节良恶性鉴别，也可用于预后、对术前术后、治疗、疗效等起到实时监控作用。其中两步法检测体系包含独立自主设计的特异性的茎环结构RT引物、PCR上下游引物及Taqman探针引物，特异性引物使得miRNA检测特异性可区分单个碱基差异，灵敏度最低可达到1copy/μL的检测下限，大大提高了miRNA的检测效率和精确度，此外，不同标志物PCR热循环条件一样，不仅可同批次同板检测多种标志物，提高了联合标志物检测的准确性和检测效率，也降低时间、成本。
(3)本发明开发出等温扩增技术miRNA的一步法检测体系更快速、便捷，可大大提高检测效率，减少检测费用。
(4)采用血清外泌体exo-miRNA作为标志物联合检测，比血浆外泌体-miRNA或血清-miRNA、血浆-miRNA直接检测效果更好。exo-miRNA具有很好的稳定性，血清在4℃存放20天仍可有效提取exo-miRNA，提取的exo-miRNA可-20℃冻存50天，-80℃长期保存。
附图说明
图1miRNA两步法PCR检测扩增原理图；
图2一步法等温EXPAR扩增原理图；
图3一步法等温线性扩增原理图；
图4miRNA标志物PCR标准曲线及检测灵敏度(A：Let-7a的PCR最低检测下限；B：Let-7a的PCR标准曲线；C：miR-21的PCR最低检测下限；D：miR-21的PCR标准曲线；E：miR-486-5p的PCR最低检测下限；F：miR-486-5p的PCR标准曲线；G：miR-205的PCR最低检测下限；H：miR-205的PCR标准曲线；I：miR-126的PCR最低检测下限；J：miR-126的PCR标准曲线；K：miR-152的PCR最低检测下限；L：miR-152的PCR标准曲线；M：miR-148a的PCR最低检测下限；N：miR-148a的PCR标准曲线)。
图5两步法miRNA检测体系对临床样本检测稳定性结果(A：批内差异CV值；B：批间差异CV值)。
图6肺癌临床样本miR-21联合Let-7a检测结果(A：肺癌组织样本miR-21联合Let-7a检测结果，A-1：miR-21联合Let-7a miRNA检测；A-2：miR-21联合Let-7a ROC曲线；B：肺癌血清外泌体miR-21联合Let-7a检测结果；B-1：肺癌血清外泌体miR-21联合Let-7a miRNA检测相对定量结果；B-2：肺癌血清外泌体miR-21联合Let-7a检测ROC曲线；B-3：肺癌血清外泌体miR-21联合Let-7a检测逻辑回归分析结果；B-4：肺癌血清外泌体miR-21联合Let-7a检测逻辑回归ROC曲线；B-5：肺癌血浆外泌体miR-21联合Let-7a miRNA检测结果；B-6：肺癌血浆外泌体miR-21联合Let-7a检测ROC曲线；B-7：肺癌尿液外泌体miR-21联合Let-7a miRNA检测；B-8：肺癌尿液外泌体miR-21联合Let-7a检测ROC曲线)。
图7肺癌临床样本miR-205联合Let-7a检测结果(A：肺癌组织样本miR-205联合Let-7a检测miRNA结果；B；肺癌组织样本miR-205联合Let-7a检测ROC曲线；C：肺癌血清外泌体miR-205联合Let-7a检测miRNA结果；D：肺癌血清外miR-205联合Let-7a ROC曲线；E：肺癌血浆外泌体miR-205联合Let-7a检测miRNA结果；F：肺癌血浆外泌体miR-205联 合Let-7a检测ROC曲线；G：肺癌尿液外泌体miR-205联合Let-7a检测miRNA结果；H：肺癌尿液外泌体miR-205联合Let-7a检测ROC曲线)。
图8肺癌临床样本miR-126联合miR-152检测结果(A：肺癌组织样本miR-126联合miR-152检测结果；B：肺癌组织样本miR-126联合miR-152检测ROC曲线；C：肺癌血清外泌体miR-126联合miR-152检测结果；D：肺癌血清外泌体miR-126联合miR-152检测ROC曲线；E：肺癌血浆外泌体miR-126联合miR-152检测结果；F：肺癌血浆外泌体miR-126联合miR-152检测ROC曲线；G：肺癌尿液外泌体miR-126联合miR-152检测结果；H：肺癌尿液外泌体miR-126联合miR-152检测ROC曲线)。
图9肺癌临床样本miR-486-5p联合miR-148a的检测结果(A：肺癌组织样本miR-486-5p联合miR-148a检测结果；B：肺癌组织样本miR-486-5p联合miR-148a检测ROC曲线；C：肺癌血清外泌体miR-486-5p联合miR-148a检测结果；D：肺癌血清外泌体miR-486-5p联合miR-148a检测ROC曲线；E：肺癌血浆外泌体miR-486-5p联合miR-148a检测结果；F：肺癌血浆外泌体miR-486-5p联合miR-148a检测ROC曲线；G：肺癌尿液外泌体miR-486-5p联合miR-148a检测结果；H：肺癌尿液外泌体miR-486-5p联合miR-148a检测ROC曲线)。
图10术前和术后外泌体miRNA表达水平及其差异(A：术前和术后血清外泌体表达水平及其差异(A-1：miR-21检测结果、A-2：miRNA-205检测结果、A-3：miRNA-126检测结果、A-4：miRNA-486-5p检测结果、A-5：Let-7a检测结果、A-6：miR-152检测结果、A-7：miR-148a检测结果)；B：术前和术后血浆外泌体表达水平及其差异(B-1：miR-21检测结果、B-2：miRNA-205检测结果、B-3：miRNA-126检测结果、B-4：miRNA-486-5p检测结果、B-5：Let-7a检测结果、B-6：miR-152检测结果、B-7：miR-148a检测结果)；C：术前和术后尿液外泌体表达水平及其差异(C-1：miR-21检测结果、C-2：miRNA-205检测结果、C-3：miRNA-126检测结果、C-4：miRNA-486-5p检测结果、C-5：Let-7a检测结果、C-6：miR-152检测结果、C-7：miR-148a检测结果))。
图11外泌体miR-21和Let-7a联合检测对肺癌预后评估(A：血清外泌体miR-21和Let-7a联合检测对肺癌预后评估，A-1，miR-21和Let-7a表达水平，A-2：ROC曲线；A-3：无进展生存曲线；A-4：总体生存率；B：血浆外泌体miR-21和Let-7a联合检测对肺癌预后评估，B-1，miR-21和Let-7a表达水平，B-2：ROC曲线；B-3：无进展生存曲线；B-4：总体生存率；C：尿液外泌体miR-21和Let-7a联合检测对肺癌预后评估；C-1，miR-21和Let-7a表达水平，C-2：ROC曲线；C-3：总体生存率)。
图12外泌体miR-205和Let-7a联合检测对肺癌预后评估(A：血清外泌体miR-205和Let-7a联合检测对肺癌预后评估,A-1，miR-205和Let-7a表达水平，A-2：ROC曲线；A-3：无进展生存曲线；A-4：总体生存率；B：血浆外泌体miR-205和Let-7a联合检测对肺癌预后评估，B-1，miR-205和Let-7a表达水平，B-2：ROC曲线；B-3：无进展生存曲线；B-4：总体生存率；C：尿液外泌体miR-205和Let-7a联合检测对肺癌预后评估，C-1，miR-205和Let-7a表达水平，C-2：ROC曲线；C-3：总体生存率)。
图13外泌体miR-126和miR-152对肺癌预后评估(A：血清外泌体miR-126和miR-152联合检测对肺癌预后评估，A-1，miR-126和miR-152表达水平，A-2：ROC曲线；A-3：总体生存率；B：血浆外泌体miR-126和miR-152联合检测对肺癌预后评，B-1，miR-126和miR-152表达水平，B-2：ROC曲线；B-3：总体生存率；C：尿液外泌体miR-126和miR-152联合检 测对肺癌预后评估，C-1，miR-126和miR-152表达水平，C-2：ROC曲线；C-3：总体生存率)。
图14基于等温扩增技术miRNA的一步法检测试剂盒及其应用(A：Let-7a等温指数型扩增一步法检测灵敏度及标准曲线；B：等温指数型扩增一步法临床样本检测C：等温线性扩增一步法Let-7a标准曲线；D：等温线性扩增一步法miR-21标准曲线；E-1：肺癌尿液外泌体miR-21联合Let-7a检测结果；E-2肺癌尿液外泌体miR-21联合Let-7a检测ROC曲线；F-1：肺癌血清外泌体miR-21联合Let-7a检测结果；F-2：肺癌血清外泌体miR-21联合Let-7a检测ROC曲线)。
具体实施方式
下面将结合附图，对本发明的优选实施例进行详细的描述。优选实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件进行。
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1、基于PCR平台miRNA的两步法检测体系试剂盒
1)本实施例使用仪器如下：
4℃低温离心机(Thermo Fisher Fresco17)、LightCycler 480实时荧光定量PCR仪(罗氏公司)、超净工作台(SW-CJ-1D，龙扬科学仪器)、常规PCR仪(A100，杭州朗基科学仪器有限公司)。
2)RNA逆转录反应体系：
试剂：用于配制逆转录反应体系的试剂包括逆转录引物(RT-Primer，上海英潍捷基合成)，miRNA标准品粉末(上海英潍捷基合成)、T4 DNA连接酶(T4 DNA Ligase，供应商：NEB，商品号：M0202S，包含10×T4 DNA Ligase Buffer)、RNA酶抑制剂(RNase inhibitor，供应商：Fermentas，商品号：K1622)、转录酶Transcriptase(供应商：上海英潍捷基生物技术有限公司，商品号：K1622，包含RNase inhibitor、dNTPs、nuclease-free water)、T4多聚核苷酸激酶(T4 Polynucleotide Kinase，供应商：NEB，商品号：M0201S)和无核酸酶纯水(nuclease-free water，供应商：上海英潍捷基生物有限公司，商品号：K1622)。用于配制逆转录反应体系的试剂逐瓶封装，使用时按一定的比例制成逆转录体系，逆转录反应体系为20μL/次，分装的体积是50次的用量，如表1所示。
表1 通用逆转录反应体系组分表
组分 终浓度 体积/μL
逆转录RT-Primer 5μmol/L 5
T4 DNALigaseBuffer 10× 2
dNTPs(含dUTP) 1mmol/L 2
RNase inhibitor 20UμL -1 1
Transcriptase 200UμL -1 1
T4 DNA Ligase 10UμL -1 0.5
T4 Polynucleotide Kinase 2.5UμL -1 0.25
模板   5
nuclease-free water   加水至20μL
按表2条件进行逆转录。
表2 通用RNA逆转录条件
Figure PCTCN2018090043-appb-000001
cDNA 10倍稀释后4℃保存用于后续PCR扩增。
3)PCR反应体系：
试剂：用于配制PCR反应体系的试剂包括三磷酸脱氧核苷酸dNTPs(dCTP、dGTP、dATP、dTTP、dUTP(供应商：Thermo Scientific)配制成dNTPs。上游引物液(F primer，上海英潍捷基合成)、通用下游引物液(R primer，上海英潍捷基合成)、探针(Probe，ABI合成)、DNA聚合酶(HS Taq，供应商：Takara公司，商品号：R007A)、尿嘧啶-DNA糖基化酶(UDG，供应商：NEB，商品号：M0280S)和纯水(H 2O)。
用于配制PCR反应体系的试剂逐瓶封装，使用时按一定比例配制成PCR反应体系，PCR反应体系为20μL/次，分装的体积是50次的用量，如表3所示。
表3 优化后的PCR反应体系
组分 终浓度 体积μL
10×Buffer 10× 2
MgCl 2 25mmol/L 3
dNTPs 1mmol/L 0.8
上游F 0.5μmol/L 0.5
下游R 0.5μmol/L 0.5
Probe 0.2mol/L 0.2
HS Tag 1UμL -1 0.2
UDG 0.5UμL -1 0.1
模板   2
ddH 2O   加水至20μL
然后按表4条件进行扩增反应。
表4 PCR热循环条件
Figure PCTCN2018090043-appb-000002
4)miRNA两步法分子标志物标准品配置：
标准品miRNA经逆转录后cDNA原液为10 12copy/μL，取10μL cDNA原液加入90μL灭菌纯化水稀释到10 11copy/μL，再取10μL 10 11copy/μL稀释液加入90μL灭菌纯化水稀释到10 10copy/μL，依次逐级稀释到1copy/μL的稀释液。
5)miRNA两步法检测体系灵敏度：
采用上述基于PCR平台miRNA的两步法检测体系试剂盒，检测miR-152、Let-7a、miR-148a、miR-21、miR-486-5p、miR-205或miR-126的标准品，得出检测下限及扩增效率。检测原理如图1所示。
以miR-21为例，miR-21两步法分子标志物标准品配置：
标准品miR-21经逆转录后cDNA原液为10 12copy/μL，取10μL cDNA原液加入90μL灭菌纯化水稀释到10 11copy/μL，再取10μL 10 11copy/μL稀释液加入90μL灭菌纯化水稀释到10 10copy/μL，依次逐级稀释到1copy/μL的稀释液。
其他miRNA分子标志物两步法检测体系构建及标准品配置参考miR-21，仅模板、引物、及探针不同，PCR反应条件相同。
两步法检测体系miRNA标准品检测结果，如表5所示。
表5 miRNA标准品检测结果
Figure PCTCN2018090043-appb-000003
6)两步法miRNA检测体系对临床样本检测稳定性评价
肺癌临床样本血清Exo-miR-21联合miRNA下调标志物Exo-Let-7a对检测结果稳定性进行评价。4例不同临床血清样本，每例样本分3个批次检测，每个批次3个重复，验证检测评价体系(包括Exo-miRNA提取纯化、逆转录、PCR上机检测)稳定性。结果如图5所示，同一样本批内差异CV值可达到4％以内，批间差异CV值可达到8％以内，说明miRNA两步法检测评价体系具有良好的稳定性。
7)miRNA的两步法检测试剂盒肺癌早期诊断及肺部结节良恶性鉴别效果评价
(1)样本采集
收集经医院检查确诊的肺癌(包括不同分期、不同亚型、不同性别及不同年龄段)、肺部疾病良性病变和健康人等系列人群的组织、血清、血浆、尿液样本。
(2)组织miRNA提取纯化
采用QIAGEN公司商业化产品miRNeasy Serum/Plasma Kit试剂盒(货号217184)，提取纯化组织和血清中的miRNA，并利用Nano-Drop 2000测RNA核酸质量，记录RNA浓度及纯度，并对组织miRNA进行归一化处理。
(3)血清外泌体miRNA、血浆外泌体miRNA提取纯化
采用SBI公司商业化产品ExoQuickTM试剂盒(货号EXOQ5A-1)提取血清和血浆外泌体。采用QIAGEN公司商业化产品miRNeasy mini kit试剂盒(货号217004)，提取纯化外泌体中的miRNA，并利用Nano-Drop 2000测RNA核酸质量，记录RNA浓度及纯度。
(4)尿液外泌体miRNA提取纯化
采用SBI公司商业化产品ExoQuick-TC for Tissue Culture Media and Urine试剂盒(货号EXOTC10A-1)提取尿液外泌体。采用QIAGEN公司商业化产品miRNeasy mini kit试剂盒(货号217004)，提取纯化外泌体中的miRNA，并利用Nano-Drop 2000测RNA核酸质量，记录RNA浓度及纯度。
(5)miRNA两步法检测体系
采用实施例1中基于PCR平台miRNA的两步法检测体系试剂盒。对Ⅰa期早期肺癌病人56例，76例对照(健康人和良性病变)的血清样本的Exo-miRNA进行检测，检测miR-152、Let-7a、miR-148a、miR-21、miR-486-5p、miR-205或miR-126的表达量CP值，根据CP值，使用相对定量公式计算相对表达量。
(6)肺部相关疾病miRNA检测结果
(1)肺癌临床样本miR-21联合Let-7a检测结果
如图6中A所示，对肺癌病人22例包括癌和癌旁的组织样本，miRNA进行检测，检测miRNA上调标志物miR-21和miRNA下调标志物Let-7a表达量CP值，根据CP值，使用相对定量公式(2 -ΔΔCp)值，计算联合标志物相对表达量的倍数变化，进而得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关，AUC＝1.0，具有显著优势。
如图6中B所示，对Ⅰa期早期肺癌病人56例，76例对照(健康人和良性病变)的血清样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-21和miRNA下调标志物Let-7a的表达量CP值，根据CP值，使用相对定量公式(2 -ΔΔCp)值，计算联合标志物相对表达量的倍数变化，进而得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.897，Cutoff值取20.42时，诊断灵敏度82.2％，特异性92.7％。具有显著优势(图6中B-1和B-2)。对标记物联合诊断得到的CP(CT)值、拷贝数进行逻辑回归分析，建立诊断模型：通过以下公式计算P值：
P＝EXP(PI)/(1+EXP(PI))，其中PI计算如下：
PI＝50.979+3.462×log[copy(miR-21)]-7.516×log[copy(let-7a)]-1.09×CP(let-7a)。AUC＝0.913，Cutoff值取(0.2063)时，诊断灵敏度91％，特异性92％，具有显著优势(图6中B-3和B-4)。
如图6中B-5和B-6，miRNA上调标志物miR-21和miRNA下调标志物Let-7a对16例Ⅰa期肺癌样本和3例健康人对照临床样本血浆外泌体microRNA。结果显示血浆外泌体microRNA对肺癌和健康人对照具有一定区分度。
如图6中B-7和B-8所示，对Ⅰa期早期肺癌病人32例，22例对照(健康人和良性病变)的尿液样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-21和miRNA下调标志物Let-7a的表达量CP值，得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.823，Cutoff值取23.6645时，诊断灵敏度87.5％，特异性78.3％，具有显著优势。
通过组织及血清、血浆外泌体miRNA标志物诊断效果显示，与肿瘤组织穿刺活检、血浆外泌体miRNA诊断效果相比，血清外泌体miRNA有着无创诊断效果显著的优点。
(2)肺癌临床样本miR-205联合Let-7a检测结果
如图7中A和B所示，对肺癌病人14例包括癌和癌旁的组织样本，miRNA进行检测，检测miRNA上调标志物miR-205和miRNA下调标志物Let-7a表达量CP值，根据CP值，使用相对定量公式(2 -ΔΔCp)值，计算联合标志物相对表达量的倍数变化，进而得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.901，具有显著优势。
如图7中C和D所示，对Ⅰa期早期肺癌病人25例，15例对照(健康人和良性病变)的血清样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-205和miRNA下调标志物Let-7a的表达量CP值，根据CP值，使用相对定量公式(2 -ΔΔCp)值，计算联合标志物相对表达量的倍数变化，进而得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.803，Cutoff值取37.32时，诊断灵敏度76.1％，特异性83.0％。具有显著优势。
如图7中E和F所示，miRNA上调标志物miR-205和miRNA下调标志物Let-7a对16例Ⅰa期肺癌样本和3例健康人对照临床样本血浆外泌体microRNA。结果显示血浆外泌体microRNA对肺癌和健康人对照具有一定区分度。
如图7中G和H所示，对Ⅰa期早期肺癌病人25例，15例对照(健康人和良性病变)的尿液样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-205和miRNA下调标志物Let-7a的表达量CP值，根据CP值，得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.745，Cutoff值取29.348时，诊断灵敏度83.5％，特异性65.2％，具有显著优势。
(3)肺癌临床样本miR-126联合miR-152检测结果
如图8中A和B所示，对肺癌病人14例包括癌和癌旁的组织样本，miRNA进行检测，检测miRNA上调标志物miR-205和miRNA下调标志物Let-7a表达量CP值，根据CP值，使用相对定量公式(2 -ΔΔCp)值，计算联合标志物相对表达量的倍数变化，进而得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.821，具有显著优势。
如图8中C和D所示，对Ⅰa期早期肺癌病人25例，15例对照(健康人和良性病变)的血清样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-126和miRNA下调标志物miR-152的表达量CP值，根据CP值，使用相对定量公式(2 -ΔΔCp)值，计算联合标志物相对表达量的倍数变化，进而得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.728，Cutoff值取55.98时，诊断灵敏度64.0％，特异性81.0％，具有显著优势。
如图8中E和F所示，miRNA上调标志物miR-126和miRNA下调标志物miR-152对16例Ⅰa期肺癌样本和3例健康人对照临床样本血浆外泌体microRNA。结果显示血浆外泌体microRNA对肺癌和健康人对照具有一定区分度。
如图8中G和H所示，对Ⅰa期早期肺癌病人25例，15例对照(健康人和良性病变)的尿液样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-126和miRNA下调标志 物miR-152的表达量CP值，根据CP值，得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.719，Cutoff值取28.2230时，诊断灵敏度65.9％，特异性81.4％，具有显著优势。
(4)miRNA上调标志物miR-486-5p联合miR-148a进行临床样本检测结果
如图9中A和B所示，对肺癌病人14例包括癌和癌旁的组织样本，miRNA进行检测，检测miRNA上调标志物miR-205和miRNA下调标志物Let-7a表达量CP值，根据CP值，使用相对定量公式(2 -ΔΔCp)值，计算联合标志物相对表达量的倍数变化，进而得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关，AUC＝0.837，具有显著优势。
如图9中C和D所示，对Ⅰa期早期肺癌病人25例，15例对照(健康人和良性病变)的血清样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-486-5p和miRNA下调标志物miR-148a表达量CP值，根据CP值，使用相对定量公式(2 -ΔΔCp)值，计算联合标志物相对表达量的倍数变化，进而得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.677，Cutoff值取9.98时，诊断灵敏度61.5％，特异性75.0％，具有显著优势。
如图9中E和F所示，miRNA标志物miR-486-5p联合miR-148a对16例Ⅰa期肺癌样本和3例健康人对照临床样本血浆外泌体microRNA。结果显示血浆外泌体microRNA对肺癌和健康人对照具有一定区分度。
如图9中G和H所示，对Ⅰa期早期肺癌病人25例，15例对照(健康人和良性病变)的尿液样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-486-5p和miRNA下调标志物miR-148a表达量CP值，根据CP值，得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.663，Cutoff值取28.9214时，诊断灵敏度65.2％，特异性78.1％，具有显著优势。
8)肺癌术前和术后体液外泌体miRNA表达水平及其差异
(1)收集经医院检查确诊的肺癌(包括不同分期、不同亚型、不同性别及不同年龄段)、收集未经任何治疗的10例肺癌患者术前体液(血清、血浆、尿液)样本和对应的术后体液(血清、血浆、尿液)样本，检测miR-21、miRNA-205、miRNA-126、miRNA-486-5p、Let-7a、miR-152、miR-148a的表达水平。
(2)miRNA上调标志物miR-21，miR-205，miR-126，miR-486-5p进行临床样本检测，对未经任何治疗的10例肺癌患者术前体液(血清、血浆、尿液)样本和对应的术后体液(血清、血浆、尿液)样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-21，miR-205，miR-126，miR-486-5p的表达量。对检测结果采用SPSS17.0进行t检测分析P<0.05。结果如图10中A所示，血清外泌体miR-21，miR-205，miR-126，miR-486-5p术前和术后1周之间比较差异有统计学意义；如图10中B所示血浆外泌体miR-21，miR-205，miR-126，miR-486-5p术前和术后1周之间比较差异有统计学意义；如图10中C所示尿液外泌体miR-21，miR-205，miR-126，miR-486-5p术前和术后1周之间比较差异有统计学意义。说明体液外泌体miR-21，miR-205，miR-126，miR-486-5p有可能成为肺癌术后检测的生化标志物。
9)外泌体miRNA用于预后评估检测结果
(1)对无远处转移、无全身重大疾病、可手术根治的ⅠA期早期肺癌患者共20例样本的 Exo-miRNA进行检测。样本纳入标准为：能够按预定方案完成化疗。可从化疗前后收集体液，通过病理诊断将样本分为10例治疗有效组，10例治疗无效组。检测miR-21、miRNA-205、miRNA-126、miRNA-486-5p、Let-7a、miR-152、miR-148a的表达水平。采用相对定量法，计算基因的相对表达量F＝2 -△△cp。对预后生存明显差异的体液样本10例(其中五例2年内复发或转移，导致死亡)的体液样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-21和miR-205，下调标志物Let-7a的表达量，基因的表达量F＝2 —△△ct。
(2)外泌体miR-21和Let-7a联合检测对肺癌预后评估
如图11中A所示，10例治疗有效样本和10例治疗无效样本，血清外泌体miR-21和Let-7a联合检测结果，AUC＝0.840，与疗效显著性相关；Kaplan-Meier曲线显示，血清Exo-miR-21联合Let-7a表达水平与患者PFS密切相关，F>cutoff组的患者PFS更长(P<0.05)。
如图11中B所示，10例治疗有效样本和10例治疗无效样本，血浆外泌体miR-21和Let-7a联合检测结果，AUC＝0.810，与疗效显著性相关；Kaplan-Meier曲线显示，血浆Exo-miR-21联合Let-7a表达水平与患者PFS密切相关，F>cutoff组的患者PFS更长(P<0.05)。
如图11中C所示，10例治疗有效样本和10例治疗无效样本，尿液外泌体miR-21和Let-7a联合检测结果，AUC＝0.750，与疗效显著性相关。Kaplan-Meier曲线显示，尿液Exo-miR-21联合Let-7a表达水平与患者PFS密切相关，F>cutoff组的患者PFS更长(P<0.05)。
(3)外泌体miR-205和Let-7a联合检测对肺癌预后评估
如图12中A所示，10例治疗有效样本和10例治疗无效样本，血清外泌体miR-205和Let-7a联合检测结果，AUC＝0.750，与疗效显著性相关；Kaplan-Meier曲线显示，血清Exo-miR-205联合Let-7a表达水平与患者PFS密切相关，F>cutoff组的患者PFS更长(P<0.05)。
如图12中B所示，10例治疗有效样本和10例治疗无效样本，血浆外泌体miR-205和Let-7a联合检测结果，AUC＝0.780，与疗效显著性相关；Kaplan-Meier曲线显示，血浆Exo-miR-205联合Let-7a表达水平与患者PFS密切相关，F>cutoff组的患者PFS更长(P<0.05)。
如图12中C所示，10例治疗有效样本和10例治疗无效样本，尿液外泌体miR-205和Let-7a联合检测结果，AUC＝0.780。Kaplan-Meier曲线显示，尿液Exo-miR-205联合Let-7a表达水平与患者PFS密切相关，F>cutoff组的患者PFS更长(P<0.05)。
(4)外泌体miR-126和miR-152联合检测对肺癌预后评估
如图13中A所示，10例治疗有效样本和10例治疗无效样本，血清外泌体miR-126和miR-152联合检测结果，AUC＝0.760，与疗效显著性相关；Kaplan-Meier曲线显示，血清Exo-miR-126联合miR-152表达水平与患者PFS密切相关，F>cutoff组的患者PFS更长(P<0.05)。
如图13中B所示，10例治疗有效样本和10例治疗无效样本，血浆外泌体miR-126和miR-152联合检测结果，AUC＝0.750，与疗效显著性相关；Kaplan-Meier曲线显示，血浆Exo-miR-126联合miR-152表达水平与患者PFS密切相关，F>cutoff组的患者PFS更长(P<0.05)。
如图13中C所示，10例治疗有效样本和10例治疗无效样本，尿液外泌体miR-126和miR-152联合检测结果，AUC＝0.770，与疗效显著性相关；Kaplan-Meier曲线显示，尿液Exo-miR-126联合miR-152表达水平与患者PFS密切相关，F>cutoff组的患者PFS更长(P<0.05)。
10)外泌体miRNA用于复发监控检测结果
(1)对2015-2016年收治的病理分期相同，预后生存明显差异的体液样本各10例，均为原发病灶的首次诊查，五例术后3年仍健在，五例治疗后2年内复发或淋巴结转移或肝转移， 2年内死亡。10例样本采集术前术后体液检测外泌体miRNA表达水平，术后每隔三个月进行随访取样检测，根据患者外泌体miRNA表达情况进行预测，判断复发或转移。统计分析评价外泌体miRNA表达水平与影像学检测相关性。统计学分析外泌体miRNA表达水平与患者生存期的关系。
(2)基因的表达量F＝2 -△△cp,5例复发样本根据P＝疗程结束后不同时间的F/化疗后F,P>1.5判断为复发，P≤1.5判断为未复发，将P值判断结果与五例患者临床评价结果进行比较。(3)对miRNA标志物进行分析。结果如表6～10所示，在5例复发患者中外泌体miR-21或miR-486-5p或miR-205或miR-126或miR-152或Let-7a或miR-148a的基因表达量评价符合率为100％，说明体液外泌体miRNA早于临床症状和体征的发现，可用于预测肺癌复发或转移。Kaplan-Meier生存及复发分析，结果如图11,12,13所示，外泌体miR-21或miR-486-5p或miR-205或miR-126或miR-152或Let-7a或miR-148a的基因表达量与生存期显著性相关，可用于复发风险评估。
表6、1号患者复发患者体液外泌体miRNA及临床检测结果
Figure PCTCN2018090043-appb-000004
Figure PCTCN2018090043-appb-000005
表7、2号患者复发患者体液外泌体miRNA及临床检测结果
Figure PCTCN2018090043-appb-000006
表8、3号患者复发患者体液外泌体miRNA及临床检测结果
Figure PCTCN2018090043-appb-000007
Figure PCTCN2018090043-appb-000008
表9、4号患者复发患者体液外泌体miRNA及临床检测结果
Figure PCTCN2018090043-appb-000009
Figure PCTCN2018090043-appb-000010
表10、5号患者复发患者体液外泌体miRNA及临床检测结果
Figure PCTCN2018090043-appb-000011
Figure PCTCN2018090043-appb-000012
实施例2
一、基于等温扩增技术miRNA的一步法检测试剂盒
1、等温扩增EXPAR一步法检测体系，扩增原理如图2所示
1)在等温条件下，通过EXPAR技术，无需逆转录，达到指数型PCR扩增目的，miRNA分子标志物PCR扩增体系如表11所示：
表11、miRNA分子标志物PCR扩增体系
Figure PCTCN2018090043-appb-000013
扩增条件如表12所示：
表12、PCR反应热循环条件
扩增 灭活 冷却
55℃,30sec signal 60cycles 85℃,5min 50℃,30sec
2)Let-7a等温扩增EXPAR一步法检测结果，扩增原理如图3所示
如图14中A所示，采用等温指数型扩增一步法检测Let-7a标准品，标准品线性方程为：POI＝-17.9319-3.61353Log C(m)，R 2>0.99，检测下限达到10 5copy/μL，线性范围为5。如图14中B所示，10例临床样本检测，样本POI值均落在标准品线性范围10 7--10 10拷贝之间，说明等温扩增EXPAR一步法可以用于样本检测。
3)本发明的优势
利用特异的扩增引物和酶试剂，实现miRNA一步法检测的目的，标准品线性方程为：POI＝-17.9319-3.61353Log C(m)，R 2>0.99，检测下限达到10 5copy/μL，线性范围为5；30sec采集一次荧光信号，30min内即可结束反应。相对于其他类似EXPAR法(文献：Guo-lei Wang and  Chun-yang Zhang，Sensitive Detection of MicroRNAs with Hairpin Probe-Based Circular Exponential Amplification Assay.Anal.Chem.2012,84,7037-7042)所示，检测下限为10 6copy/μL，线性范围为4，反应体系操作较为复杂，需配制A、B、C3个体系在反应过程逐个加入，反应时间较长约100min。本发明线性范围更广，反应时间更快，检测下限更好，扩增效率更好，等温扩增更适宜于POCT领域。
2、等温线性扩增一步法检测体系
表13、优化后的PCR反应体系
Figure PCTCN2018090043-appb-000014
表14、PCR反应热循环条件
Figure PCTCN2018090043-appb-000015
注：a.在荧光PCR反应体积不同时，各试剂应按比例调整,
b.使用的仪器不同，应将反应参数作适当调整,
c.仪器检测通道的选择：
在进行荧光PCR反应时，应对所用仪器中反应管荧光信号的收集进行设置，选择的荧光检测通道与探针所标记的荧光报告基团一致。具体设置方法因仪器而异，应参照仪器使用说明书。
等温反应结束后，利用荧光分光光度计测定荧光值，做标准曲线。结果如图14中C所示，Let-7a标准品线性方程为：Fluorescence(a.u.)＝-339.22+307.44Log C(m)，R 2>0.99，检测下限可达到10 7copy/μL；如图14中D所示，miR-21标准品线性方程为：Fluorescence(a.u.)＝-44.509+125.55Log C(m)，R 2>0.99，检测下限可达到10 7copy/μL，线性范围为4。说明等温线性扩增一步法体系可以用于miRNA检测。
表11 Let-7a荧光值
Figure PCTCN2018090043-appb-000016
表12 miR-21荧光值
Figure PCTCN2018090043-appb-000017
二、基于等温扩增技术miRNA的一步法检测试剂盒临床效果评价
1)样本采集
收集经医院检查确诊的肺癌(包括不同分期、不同亚型、不同性别及不同年龄段)、肺部疾病良性病变和健康人等系列人群的尿液样本。
2)尿液外泌体miRNA提取纯化
采用SBI公司商业化产品ExoQuick-TC for Tissue Culture Media and Urine试剂盒(货号EXOTC10A-1)提取尿液外泌体。采用QIAGEN公司商业化产品miRNeasy mini kit试剂盒(货号217004)，提取纯化外泌体中的miRNA，并利用Nano-Drop 2000测RNA核酸质量，记录RNA浓度及纯度。
3)miRNA一步法检测体系
采用实施例2中基于等温扩增技术miRNA的一步法检测体系试剂盒。对Ⅰa期早期肺癌病人50例，50例对照(健康人和良性病变)的血清样本的Exo-miRNA进行检测，检测miR-152、Let-7a的表达量CP值，根据CP值，使用相对定量公式计算相对表达量。
4)肺癌临床样本miR-21联合Let-7a检测结果
如图14中E所示，对Ⅰa期早期肺癌病人32例，22例对照(健康人和良性病变)的尿液样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-21和miRNA下调标志物Let-7a的表达量CP值，得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.844，Cutoff值取19.878时，诊断灵敏度87.5％，特异性81.8％。具有显著优势。
如图14中F所示，对Ⅰa期早期肺癌病人56例，76例对照(健康人和良性病变)的血清样本的Exo-miRNA进行检测，检测miRNA上调标志物miR-21和miRNA下调标志物Let-7a的表达量CP值，根据CP值，得出miRNA相对表达量的得分。对检测结果采用SPSS17.0进行t检测分析P<0.05，说明联合标志物与早期肺癌预测显著相关。AUC＝0.879，Cutoff值取21.02时，诊断灵敏度81.3％，特异性81.8％。具有显著优势。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。

Claims (14)

  1. 外泌体相关microRNA分子在制备诊断和预示肺癌标志物中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述外泌体相关microRNA分子包括至少一种上调的外泌体microRNA分子，或包含至少一种下调的外泌体microRNA，或包含至少一种上调和至少一种下调的外泌体microRNA分子。
  3. 根据权利要求2所述的应用，其特征在于：所述上调的外泌体microRNA分子为miR-21或miR-486-5p或miR-205或miR-126中至少一种，所述下调的microRNA分子为miR-152或Let-7a或miR-148a中至少一种。
  4. 根据权利要求1所述的应用，其特征在于：所述标志物为miR-21与Let-7a联合，miR-205与Let-7a联合，miR-126与miR-152联合或miR-486-5p与miR-148a联合。
  5. 根据权利要求1所述的应用，其特征在于：所述诊断和预示具体为肺癌筛查、辅助诊断、疗效评价、预后评估或复发监控。
  6. 根据权利要求1所述的应用，其特征在于：所述外泌体来源为体液或细胞。
  7. 根据权利要求6所述的应用，其特征在于：所述体液包括血液、痰液、胸腔积液、胸腔灌洗液、尿液或唾液中的至少一种。
  8. 检测权利要求1～7任一项所述外泌体相关microRNA分子的试剂盒，其特征在于：所述试剂盒为基于PCR平台miRNA两步法检测试剂盒或基于等温扩增技术miRNA的一步法检测试剂盒。
  9. 根据权利要求8所述的试剂盒，其特征在于：所述试剂盒为基于PCR平台miRNA两步法检测试剂盒，含microRNA分子标志物特异性茎环结构逆转录引物、PCR上游引物、PCR通用下游引物，用于检测microRNA分子标记物的特异探针，系列梯度稀释浓度的microRNA标准品。
  10. 根据权利要求9所述的试剂盒，其特征在于：所述特异性茎环结构逆转录引物的颈部的loop环部设计非连续互补碱基对TGCG和CGCA构成钥匙状结构，逆转录反应时通过连接酶将短臂与microRNA分子相连接。
  11. 根据权利要求9中所述的试剂盒，其特征在于：所述microRNA分子标志物为miR-21、miR-486-5p、miR-205、miR-126、miR-152、Let-7a或miR-148a中的至少一种，所述miR-21的逆转录引物序列如SEQ ID NO.1所示，PCR上游引物如SEQ ID NO.2所示，PCR通用下游引物如SEQ ID NO.3所示，特异探针核苷酸序列如SEQ ID NO.4；所述miR-486-5p的逆转录引物序列如SEQ ID NO.5所示，PCR上游引物如SEQ ID NO.6所示，PCR通用下游引物如SEQ ID NO.3所示，特异探针核苷酸序列如SEQ ID NO.7；所述miR-205的逆转录引 物序列如SEQ ID NO.8所示，PCR上游引物如SEQ ID NO.9所示，PCR通用下游引物如SEQ ID NO.3所示，特异探针核苷酸序列如SEQ ID NO.10；所述miR-126的逆转录引物序列如SEQ ID NO.11所示，PCR上游引物如SEQ ID NO.12所示，PCR通用下游引物如SEQ ID NO.3所示，特异探针核苷酸序列如SEQ ID NO.13；所述let-7a的逆转录引物序列如SEQ ID NO.14所示，PCR上游引物如SEQ ID NO.15所示，PCR通用下游引物如SEQ ID NO.3所示，特异探针核苷酸序列如SEQ ID NO.16；所述miR-152的逆转录引物序列如SEQ ID NO.17所示，PCR上游引物如SEQ ID NO.18所示，PCR通用下游引物如SEQ ID NO.3所示，特异探针核苷酸序列如SEQ ID NO.19；所述miR-148a的逆转录引物序列如SEQ ID NO.20所示，PCR上游引物如SEQ ID NO.21所示，PCR通用下游引物如SEQ ID NO.3所示，特异探针核苷酸序列如SEQ ID NO.22。
  12. 根据权利要求9中所述的试剂盒，其特征在于，还包括miRNA分子标志物标准品：miR-21分子标志物标准品为miR-21，稀释后浓度为10 13copy/μL，并梯度稀释为系列标准品；miR-486-5p分子标志物标准品为miR-486-5p，稀释后浓度为10 13copy/μL；miR-205分子标志物标准品为miR-205，稀释后浓度为10 13copy/μL；miR-126分子标志物标准品为miR-126，稀释后浓度为10 13copy/μL；let-7a分子标志物标准品为let-7a，稀释后浓度为10 13copy/μL；miR-152分子标志物标准品为miR-152，稀释后浓度为10 13copy/μL；miR-148a分子标志物标准品为miR-148a，稀释后浓度为10 13copy/μL。
  13. 根据权利要求8所述的试剂盒，其特征在于：所述试剂盒为基于等温扩增技术miRNA的一步法检测试剂盒，包括microRNA分子特异性扩增模板、Vent(exo-)DNA聚合酶、Nicking酶、双链特异性核酸酶和分子杂交探针。
  14. 根据权利要求13所述的检测试剂盒，其特征在于：miR-21第一条特异性扩增模板核苷酸如SEQ ID NO.23所示，miR-21第二条扩增模板核苷酸序列如SEQ ID NO.24所示，miR-21杂交探针序列如SEQ ID NO.25；miR-486-5p第一条特异性扩增模板核苷酸如SEQ ID NO.26所示，miR-486-5p第二条扩增模板核苷酸序列如SEQ ID NO.27所示，miR-486-5p杂交探针序列如SEQ ID NO.28；miR-205第一条特异性扩增模板核苷酸如SEQ ID NO.29所示，miR-205第二条扩增模板核苷酸序列如SEQ ID NO.30所示，miR-205杂交探针序列如SEQ ID NO.31；miR-126第一条特异性扩增模板核苷酸如SEQ ID NO.32所示，miR-126第二条扩增模板核苷酸序列如SEQ ID NO.33所示，miR-126杂交探针序列如SEQ ID NO.34；Let-7a第一条特异性扩增模板核苷酸如SEQ ID NO.35所示，Let-7a第二条扩增模板核苷酸序列如SEQ ID NO.36所示，Let-7a杂交探针序列如SEQ ID NO.37。
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