CN103173448A - Early-stage lung adenocarcinoma miRNA (micro ribonucleic acid) specific expression profile and reverse transcription primer and application thereof - Google Patents
Early-stage lung adenocarcinoma miRNA (micro ribonucleic acid) specific expression profile and reverse transcription primer and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological and medicine examination, relates to a tumor biomarker, and in particular relates to an early-stage lung adenocarcinoma miRNA (micro ribonucleic acid) specific expression profile and a reverse transcription primer and an application thereof. The early-stage lung adenocarcinoma miRNA specific expression profile comprises more than one of up-regulated expression miR-103, miR-146a, miR-151, miR-221, miR-222 and miR-223. The invention provides an early-stage lung adenocarcinoma diagnosis model, a reverse transcription primer of specific expression miRNA and a kit or biochip for early-stage lung adenocarcinoma diagnosis based on the specific expression profile. The technical scheme provided by the invention can realize accurate diagnosis of early-stage lung adenocarcinoma by detecting a small amount of miRNA, and provides possibility to the production of accurate, efficient and economical detection products. The established lung adenocarcinoma prediction model can distinguish lung adenocarcinoma from a healthy individual, and has great practical significance in clinic.
Description
Technical field
The invention belongs to biology and field of medical examination, relate to a kind of knubble biological flag thing, be specifically related to a kind of early stage adenocarcinoma of lung miRNA specifically expressing spectrum and reverse transcription primer and purposes.
Background technology
Due to adenocarcinoma of lung morbidity concealment, lack special clinical manifestation, there is no again at present effective method of early diagnosis, although 5 years survival rates of I phase adenocarcinoma of lung postoperative can reach 70%, but only 10% patient the stage (TIN0M0/T2N0M0) is found in early days, 40% patients with lung adenocarcinoma enters middle and advanced stage and loses surgical engine meeting, 5 years survival rate＜15% when making a definite diagnosis.Therefore, the early diagnosis of adenocarcinoma of lung has very important significance to life quality, the extending life that improves patients with lung adenocarcinoma.
The method of the adenocarcinoma of lung early diagnosis that present stage generally acknowledges has: Sputum is learned and is checked, but recall rate only has 20% ~ 30%; Low-dose spiral CT only can detect the peripheral adenocarcinoma of lung, and somewhat expensive; Branchofiberoscope, genus have the inspection of wound property, and the patient should not accept; Increase focus though PET and PET/CT can identify the above metabolism of the interior 0.5mm of lung, the inflammation focus is prone to false positive, and false negative can appear in low adenocarcinoma of lung such as the alveolar cell carcinoma of some metabolic rates.
Along with deepening constantly of biology field research, the new way that relevant early molecule event is expected to become the adenocarcinoma of lung early diagnosis occurs to adenocarcinoma of lung in examination.At present existing Diagnostic Value of Several Serum Tumor Markers is used for the detection of adenocarcinoma of lung, is used for the detection of adenocarcinoma of lung as carcinomebryonic antigen (CEA); Cytokeratin 21-1 segment (CYFRA21-1) is used for the diagnosis of lung squamous cancer; Neuronspecific enolase (NSE) is used for the diagnosis of minicell adenocarcinoma of lung etc., but because of susceptibility and specific problem, is difficult to they rigid indexs as the adenocarcinoma of lung diagnosis.
Small ribonic acid (miRNA, miRNA) be the non-coding strand small ribonucleic acid molecules that a class is about 22 Nucleotide, by to miRNA express spectra in multiple cancerous tissue and healthy tissues comparative analysis, find that miRNA has specific express spectra in different tumours, the miRNA express spectra has significant cancer-related, tissue specificity and expression stability.Therefore determine that early stage adenocarcinoma of lung miRNA specifically expressing spectrum will help the diagnosis of early stage adenocarcinoma of lung.
In recent years, the sickness rate of adenocarcinoma of lung is keeping the trend of sustainable growth especially in China, particularly in women and non-smoker's history colony.Therefore, for the NSCLC of this particular type of adenocarcinoma of lung, the research of carrying out the non-invasive early diagnosis is particularly important.
Summary of the invention
Technical problem to be solved by this invention is that susceptibility, the specificity of early stage adenocarcinoma of lung diagnosis is low, in order to overcome the deficiencies in the prior art, provides a kind of early stage adenocarcinoma of lung miRNA specifically expressing spectrum and reverse transcription primer and purposes.
In order to solve the problems of the technologies described above, technical scheme of the present invention is: described early stage adenocarcinoma of lung miRNA specifically expressing is composed more than one in miR-103, miR-146a, miR-151, miR-221, miR-222 and the miR-223 that comprises up-regulated expression.
Further, described early stage adenocarcinoma of lung miRNA specifically expressing spectrum comprises miR-146a, the miR-222 of up-regulated expression and more than one in miR-223.
Further, described early stage adenocarcinoma of lung miRNA specifically expressing spectrum comprises miR-146a, miR-222 and the miR-223 of up-regulated expression.
Further, described miRNA is from the serum of peripheric venous blood.
The present invention also provides the reverse transcription primer of specifically expressing miRNA in above-mentioned early stage adenocarcinoma of lung miRNA specifically expressing spectrum, is specially the probe-miR-222 of the probe-miR-221 of the probe-miR-151 of the probe-miR-146a of the probe-miR-103 of corresponding miR-103, corresponding miR-146a, corresponding miR-151, corresponding miR-221, corresponding miR-222 and the probe-miR-223 of corresponding miR-223.
The present invention also provides the purposes of the reverse transcription primer of above-mentioned specifically expressing miRNA, is used for early stage adenocarcinoma of lung and diagnoses with test kit or biochip.
Further, described test kit or biochip comprise more than one of probe-miR-103, probe-miR-146a, probe-miR-151, probe-miR-221, probe-miR-222 and probe-miR-223.
Further, described test kit or biochip comprise more than one of probe-miR-146a, probe-miR-222 and probe-miR-223.
Further, described test kit or biochip comprise probe-miR-146a, probe-miR-222 and probe-miR-223.
Technical scheme provided by the invention can realize Accurate Diagnosis to early stage adenocarcinoma of lung by the detection to a small amount of miRNA, may for producing that accurate, efficient and economic testing product provides.The adenocarcinoma of lung predictive model of setting up can be distinguished the ability of adenocarcinoma of lung and healthy individuals preferably, has stronger clinical practice meaning.
Description of drawings
Fig. 1 is the statistical data of adenocarcinoma of lung differential expression circulation miRNA in express spectra of the present invention.
Embodiment
The present invention is described in detail below in conjunction with accompanying drawing:
One, the collection of serum specimen: the serum of the peripheric venous blood of the physical examination of healthy population (control) that the serum before patients with lung adenocarcinoma (AD) art in peripheric venous blood and age, sex and experimental group are complementary.
Two, the extracting of the total RNA of serum and purifying:
1. with 350ul serum specimen and equal-volume 2xDenaturing Solution mixed at room temperature, hatch 5min on ice.
2. add acid phenol: chloroform, volume=sample volume+2xDenaturing Solution volume, vibration mixing 30 ~ 60s, the centrifugal 5min of 12000rpm collects water in clean EP pipe.
3. add room temperature 100% ethanol of 1.25 times of volumes in the aqueous phase of collecting, mixing immediately.
4. mixture is added in strainer, maximum 700ul once, if volume greater than 700ul, reusable same strainer.Abandon filtrate after centrifugal 30s, repeat until all mixtures all pass through strainer.
5. with 700ul miRNA Wash Solution I washing, centrifugal 30s abandons filtrate.
6. with 500ul Wash Solution2/3 washed twice, abandon filtrate.
7. empty from 1min, remove debris.
8. preheating Elution is Solution to 95 ℃, and the Elution Solution50ul of preheating is added the filter membrane center, standing 2 ~ 3min, and centrifugal 30s again adds filtrate in strainer and repeats wash-out once again, collects elutriant, is stored in-80 ℃.
Three, reverse transcription reaction:
The reverse transcription primer sequence of the miRNA of 6 up-regulated expressions is provided by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd as shown in Table 1.
The miRNA standard substance are bought the sharp rich bio tech ltd in the Guangzhou.
Table one
| The miRNA title | Probe | The reverse transcription primer |
| miR-151a-3p | probe--miR-151a-3p | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCTCAA |
| miR-103a-3p | probe-miR-103a-3p | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCATAG |
| miR-23a-3p | probe-miR-23a-3p | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGAAAT |
| miR-425-5p | probe--miR-425-5p | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACG |
| miR-146a-5p | probe-miR-146a-5p | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACCCA |
| miR-185-5p | probe-miR-185-5p | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGGA |
| miR-222-3p | probe-miR-222-3p | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACCCAG |
| miR-223-3p | probe-miR-223-3p | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGGGGT |
| miR-221 | probe-miR-221 | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAAACC |
| miR-92a | probe-miR-92a | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGGC |
| miR-21 | probe-miR-21 | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA |
7 μ LRNA utilize PrimeScript RT Enzyme System to specifications step carry out reverse transcription reaction (DRR037A, TaKaRa, Japan).
Four, PCR reaction:
Use SYBR Green I PCR Master Mix (DRR081A, TaKaRa, Japan) to run on Roche480 real-time fluorescence quantitative PCR system (Roche).
20 μ L reaction systems comprise: 2 μ LcDNA, 3 μ L forward direction primers, 1.4 μ L universal primer and 10 μ LPower SYBR Green I PCR Master Mix, forward direction primer and universal primer sequence provide by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd as shown in Table 2.
Reaction conditions is: the 95 ℃ of 5min that anneal, 95 ℃ of 15s of 40 circulations anneal and extend 1min. for 60 ℃
Set up the typical curve of 1fmol/L to 1000pmol/L by standard substance, the Ct value that obtains is converted to the concentration of each miRNA by standard substance.
Table two
| Title | The forward direction primer | Universal primer |
| hsa-miR-151a-3p | TCGGGCTAGACTGAAGCTCC | GTGCAGGGTCCGAGGT |
| hsa-miR-103a-3p | GCCCGAGCAGCATTGTACAG | GTGCAGGGTCCGAGGT |
| hsa-miR-23a-3p | GCCTGATCACATTGCCAGGG | GTGCAGGGTCCGAGGT |
| hsa-miR-425-5p | GCCCGAATGACACGATCACT | GTGCAGGGTCCGAGGT |
| hsa-miR-146a-5p | CGCCGCTGAGAACTGAATTCC | GTGCAGGGTCCGAGGT |
| hsa-miR-185-5p | TCGGGTGGAGAGAAAGGCAG | GTGCAGGGTCCGAGGT |
| hsa-miR-222-3p | GCCCGAGCTACATCTGGCTA | GTGCAGGGTCCGAGGT |
| hsa-miR-223-3p | CGGCGGTGTCAGTTTGTCAAA | GTGCAGGGTCCGAGGT |
| hsa-miR-221 | GCCCGAGCTACATTGTCTGC | GTGCAGGGTCCGAGGT |
| hsa-miR-92a | CGCGCTATTGCACTTGTCCC | GTGCAGGGTCCGAGGT |
| hsa-miR-21 | GCCCGCTAGCTTATCAGACTGATG | GTGCAGGGTCCGAGGT |
As shown in Figure 1, miR-103, miR-146a, miR-151, miR-221, miR-222 and miR-223 have significant up-regulated expression (p＜0.0005) and have significant difference.
Set up miR-103, miR-146a, miR-151, miR-221, miR-222 and miR-223 ROC curve separately, and calculate its area under curve (AUC), data as shown in Table 3:
Table three
| miRNA | AUC | 95%CI | Susceptibility | Specificity |
| miR-103 | 0.7824 | (0.7293-0.8355) | 72.26% | 73.48% |
| miR-146a | 0.9186 | (0.884-0.95311) | 87.10% | 90.34% |
| miR-151-3p | 0.8648 | (0.82235-0.90724) | 81.01% | 84.38% |
| miR-221 | 0.8256 | (0.77869-0.87252) | 73.51% | 76.19% |
| miR-222 | 0.8785 | (0.83551-0.92144) | 83.22% | 83.97% |
| miR-223 | 0.9180 | (0.88652-0.94947) | 83.44% | 88.19% |
AUC to each miRNA carries out LOGIS TIC recurrence, miR-146a, miR-222 and miR-223 have significance to Diagnosis of pulmonary gland cancer in regression equation, finally form the associating diagnostic model of 3 miRNA concentration: [In (P/ (1-P))=-2.06102+0.0508526 * X
1-0.1463608 * X
2+ 0.0226559 * X
3], wherein, X
1Be the concentration of miR-146a, X
2Be the concentration of miR-222, X
2Concentration for miR-223.
Utilize this diagnostic model to analyze discovery for the patient that suffers from of stages I, II and III to the IASLC adenocarcinoma of lung by stages, the ROC area under curve of its Diagnosis of pulmonary gland cancer is 0.9418,0.9678 and 0.9538, illustrate that this model has good diagnosis capability to I phase adenocarcinoma of lung, can distinguish patients with lung adenocarcinoma and healthy population.
When the P value was p=0.5015, the susceptibility of this diagnostic model was 90.83%, and specificity is 84.35%, and model is 87.27% to the differentiation accuracy rate of adenocarcinoma of lung and normal individual, far away higher than other types adenocarcinoma of lung mark.
Claims (8)
1. an early stage gland adenocarcinoma of lung miRNA specifically expressing spectrum, is characterized in that, comprises more than one in miR-103, miR-146a, miR-151, miR-221, miR-222 and the miR-223 of up-regulated expression.
2. a kind of early stage adenocarcinoma of lung miRNA specifically expressing spectrum according to claim 1, is characterized in that, described early stage adenocarcinoma of lung miRNA specifically expressing spectrum comprises miR-146a, the miR-222 of up-regulated expression and more than one in miR-223.
3. a kind of early stage adenocarcinoma of lung miRNA specifically expressing is composed according to claim 2, it is characterized in that, described early stage adenocarcinoma of lung miRNA specifically expressing spectrum comprises miR-146a, miR-222 and the miR-223 of up-regulated expression.
4. a kind of early stage adenocarcinoma of lung miRNA specifically expressing is composed according to claim 1, it is characterized in that, described miRNA is from the serum of peripheric venous blood.
5. the reverse transcription primer of specifically expressing miRNA during the described early stage adenocarcinoma of lung miRNA specifically expressing of claim 1 is composed, it is characterized in that, be respectively the probe-miR-222 of the probe-miR-221 of the probe-miR-151 of the probe-miR-146a of the probe-miR-103 of corresponding miR-103, corresponding miR-146a, corresponding miR-151, corresponding miR-221, corresponding miR-222 and the probe-miR-223 of corresponding miR-223.
6. diagnose with test kit or biochip for early stage adenocarcinoma of lung for one kind, it is characterized in that, described test kit or biochip comprise more than one of probe-miR-103, probe-miR-146a, probe-miR-151, probe-miR-221, probe-miR-222 and probe-miR-223.
According to claim 6 a kind of for early stage adenocarcinoma of lung diagnosis with test kit or biochip, it is characterized in that, described test kit or biochip comprise more than one of probe-miR-146a, probe-miR-222 and probe-miR-223.
According to claim 7 a kind of for early stage adenocarcinoma of lung diagnosis with test kit or biochip, it is characterized in that, described test kit or biochip comprise probe-miR-146a, probe-miR-222 and probe-miR-223.
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