CN106929599B - Application of miRNA-6126 as lung cancer diagnosis marker - Google Patents

Abstract

The invention discloses application of miRNA-6126 as a lung cancer diagnosis marker. The invention provides application of a substance for detecting the expression quantity of miRNA-6126 in exosomes of a sample to be detected in preparation of a product for diagnosing or assisting in diagnosing whether the sample to be detected is a lung cancer sample. The invention also provides application of the substance for detecting the miRNA-6126 expression quantity in the exosome of the sample to be detected in preparing a product for screening or assisting in screening whether the sample to be detected is a lung cancer sample. Experiments prove that miRNA-6126 is expected to be a biomarker for clinical lung cancer diagnosis through the verification of the blood exosomes of the lung cancer cell line A549 carcinogenic mice.

Description

Application of miRNA-6126 as lung cancer diagnosis marker

Technical Field

The invention relates to the technical field of biology, in particular to application of miRNA-6126 as a lung cancer diagnosis marker.

Background

The miRNA in the blood circulation is found to have higher value for diagnosing the NSCLC by methods such as a gene chip, a high-throughput sequencing method, a real-time quantitative polymerase chain reaction (qRT-PCR) and the like. Wherein Roth et al found that miR-361-3p and miR-625 in blood contribute to the identification of malignant lung cancer from benign lung lesions. And the up-regulation of miR-21 in blood is a reliable biological marker for diagnosing lung squamous cell carcinoma, and miR-200b-5p, miR-502-5p, miR-629, miR-17 and miR-100 in the blood of patients with lung adenocarcinoma is obviously higher than that of patients with lung granuloma and healthy smokers. In addition, Rodriguez and the like find that miRNA content of miR-122-5p and the like contained in exosome in blood of a lung tumor patient is obviously higher than that of bronchoalveolar lavage fluid. Munagala et al found that the increase of miR-21 and miR-155 in lung cancer exosomes is a potential biological marker for the diagnosis of lung cancer recurrence.

Micro RNA (microRNA, miRNA) is taken as a small non-coding RNA which is abnormally expressed in some tumors, and can be taken as a biomarker for tumor diagnosis and prognosis judgment. However, the method of taking tumor tissue by means of tissue biopsy and analyzing its miRNA is traumatic, thus limiting its further application. Exosomes (exosomes) are subcellular bilayer membrane particles with the diameter of 50-90nm, and contain miRNA, and the exosomes are continuously released to the surrounding environment during the growth process of tumors, so that the detection of body fluid exosomes and exosome RNA is helpful for tumor diagnosis and prognosis judgment. Taylor et al first proposed the diagnosis of ovarian cancer by detecting blood exosome miRNA and further used to determine prognosis.

Disclosure of Invention

The invention aims to provide application of a substance for detecting the expression quantity of miRNA-6126 in exosomes of a sample to be detected.

The invention provides application of a substance for detecting the expression quantity of miRNA-6126 in exosomes of a sample to be detected in preparation of a product for diagnosing or assisting in diagnosing whether the sample to be detected is a lung cancer sample.

The invention also provides application of the substance for detecting the miRNA-6126 expression quantity in the exosome of the sample to be detected in preparing a product for screening or assisting in screening whether the sample to be detected is a lung cancer sample.

The expression quantity is relative expression quantity, and is relative to the expression quantity of an internal reference gene, and the internal reference gene is has-5.8 s.

In the application, the sample to be detected is in vitro serum.

In the application, the substance for detecting the expression quantity of miRNA-6126 in the exosome of the sample to be detected comprises a primer pair for amplifying miRNA-6126.

If the expression of miRNA-6126 in the exosome of the test sample is obviously higher than that in the non-lung cancer sample, the test sample is or is not a lung cancer sample, otherwise, the test sample is or is not a lung cancer sample.

The invention also provides application of the substance for detecting the miRNA-6126 expression quantity in the human serum exosomes to be detected in preparation of products for diagnosing or assisting in diagnosing whether the human to be detected is a lung cancer patient.

The invention also provides application of the substance for detecting the miRNA-6126 expression quantity in the human serum exosomes to be detected in preparation of products for screening or assisting in screening whether the human to be detected is a lung cancer patient.

In the application, the substance for detecting the miRNA-6126 expression quantity in the human serum exosome to be detected comprises a primer pair for amplifying miRNA-6126.

In the application, the primer pair consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.

The serum is in vitro serum.

It is another object of the present invention to provide a product.

The product provided by the invention is the substance for detecting the expression quantity of miRNA-6126 in the exosome of the sample to be detected or the substance for detecting the expression quantity of miRNA-6126 in the exosome of human serum to be detected.

The product is any one of the following 1) to 4):

1) products for diagnosing or assisting in diagnosing whether the sample to be tested is a lung cancer sample;

2) screening or assisting in screening whether the sample to be tested is a product of the lung cancer sample;

3) products for diagnosing or assisting in diagnosing whether the human to be tested is a lung cancer patient;

4) screening or assisting in screening whether the human to be tested is a product of a lung cancer patient.

The application of miRNA-6126 as a lung cancer diagnostic marker is also within the scope of the invention.

The invention aims to establish a method for diagnosing and prognosing lung cancer by using exosome, which adopts immortalized human bronchial epithelial cells BEP2D and α particle radiation to induce the cell line BERP35T44-1 of the cells which become cancerous, separates exosome, obtains miRNA with obviously increased expression in the exosome of the cells BERP35T44-1 of the cancerous cell line by using miRNA chip technology, detects in serum of 31 cases of clinical lung cancer patients, obviously increases the expression of the miRNA-6126 compared with normal people, lays a foundation for clinical lung cancer diagnosis, and can be used for preparing a diagnosis kit.

Drawings

FIG. 1 is a graph showing real-time amplification and product lysis curves of hsa-5.8s gene.

FIG. 2 is a real-time amplification curve and a product dissolution curve of miR-4530 gene.

FIG. 3 shows that miRNA-6126 is highly expressed in human lung cancer serum.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 discovery of miRNA associated with Lung cancer cells

Immortalized human bronchial epithelial cells BEP2D are described in Sedum indicum, Nexatron, Wudchang 238Pu α particles in the first study of transformation of human bronchial epithelial cells BEP2D, J.CHINA Lung cancer, 2000, 3 (6): 428-431.2;

the tumor cell BERP35T44-1 has high serum resistance, strong anchoring independent growth capacity, high proliferation speed and certain migration capacity, and is recorded in the following documents, namely, the biological characteristic research of malignant transformation of human bronchial epithelial cells into tumor cells induced by α particle radiation, Huiyingchun, military science institute, Master thesis of national liberty military science institute in 2003.

1. Cell culture: BEP2D cells and BERP35T44-1 cells were cultured in LHC-8 serum-free medium and subcultured in a 37C, 5% CO incubator.

2. Extraction of exosomes: harvesting 10⁷The culture supernatant of BEP2D and BERP35T44-1 cells with good growth state is separated by adopting a multi-step differential centrifugation method: cells were removed by centrifugation at 300g for 10 min at 4 ℃; centrifuging at 4 deg.C for 20 min at 2000g and at 4 deg.C for 30 min at 10000g to remove cell debris; finally, the precipitate obtained by ultracentrifugation at 100000g for 60 minutes at 4 ℃ is the exosome.

3. Electron microscopy analysis of exosomes: adding 1ml of PBS into the exosome precipitate, blowing, dissolving, then taking 20 mu l of dissolved exosome, dropwise adding the dissolved exosome precipitate onto a sample-carrying copper net, standing for 1 minute at room temperature, sucking the liquid from the side by using filter paper, dropwise adding 30 mu l of 2% phosphotungstic acid solution (PH6.8) onto the copper net, and carrying out negative staining for 1 minute at room temperature. The negative staining solution was blotted by filter paper, baked for 2 minutes under an incandescent lamp, and photographed under a transmission electron microscope.

4. Extraction of exosome RNA: to the exosome pellet was added 500. mu.l of Trizol reagent (Invitrogen, USA) to extract total RNA, thereby obtaining BEP2D extracellular exosome RNA and BERP35T44-1 extracellular exosome RNA.

Total RNA concentration was determined using a ultramicro-spectrophotometer. Purity was assessed as the A260nm/A280nm ratio. In order to determine the existence of miRNA in the extracted total RNA, RNU6B is selected as an internal reference, and the miRNA in the total RNA is detected by a real-time fluorescent quantitative PCR method.

miRNA chip experiment: 2 total RNA samples of exosomes of two cells BEP2D and BERP35T44-1 were subjected to miRNA chip experiments. The miRNA chip experiment is completed by an LC Sciences company by adopting a human LC Sciences microRNAMicroarray-Single gene expression profiling chip.

The RNA of BEP2D cell exosome and the RNA of BERP35T44-1 cell exosome are detected by using a human LC Sciences microRNA Microarray-Single gene expression profiling chip (provided by Hangzhou Union bioinformatics technologies, Inc.), and after detection, the RNA quality is found to be intact, and a labeling hybridization test is carried out. The hybridization chip is scanned by a scanner, data signal extraction, LOWESS filtration for normalization processing, differential expression analysis and the like. Obtaining an exosome gene expression profile of human bronchial epithelial cells BEP2D and an exosome gene expression profile of tumor cells BERP35T 44-1.

Comparing the two gene expression profiles, the result shows that compared with the gene expression profile of BEP2D of human bronchial epithelial cells, the gene expression profile of BERP35T44-1 of tumor cells expresses 140 miRNAs with up-regulated expression, and the expression profile of miRNA is 169. The data show that the up-regulation fold of miRNA-6126 is 8.78 fold.

Example 2 application of expression level of miRNA-6126 in diagnosis of patients with lung cancer

First, RNA extraction

Serum sources of lung cancer patients: the serum of the lung cancer patient is provided by the general hospital of the liberation army, the lung cancer is the first diagnosis, and the patient has not received any treatment including operation, radiotherapy, chemotherapy and the like. The diagnosis is clear and the pathology is clear.

Normal control serum source: the normal control serum is provided by the general hospital examination center of the liberation military and is obtained by the conventional physical examination of healthy people.

Respectively extracting the RNA of the lung cancer patient serum exosome and the RNA of the normal control serum exosome.

1. The sample information is as follows:

TABLE 1

Second, primer design and verification

DNAMAN was used to design the reverse transcription primer of the neck loop and the upstream primer of qPCR, the downstream primer was the universal primer, the length of the target fragment was about 60bp, and the annealing temperature was 60 deg.C (see Table 2 for sequence). Primer verification uses the same conditions as quantitative PCR, a cDNA mixed sample is used as a template, the result melting curves are all single peaks, agarose gel electrophoresis detection is a single band, and PCR amplification specificity is good. The quantitative results are shown in FIGS. 1 and 2.

TABLE 2miR-4530 sequences and qPCR primer sequences

Third, RT-PCR method (reverse transcription to synthesize cDNA)

The extracted RNA was removed from a freezer at-80 ℃ and dissolved in ice, and first strand cDNA was synthesized using FastQuant RT Kit (with gDNAse) (TIANGEN).

And then, respectively taking the cDNA as a template, and carrying out relative quantitative PCR by using an upstream primer and a downstream primer.

Relative quantitative PCR system as table 3:

TABLE 3

Relative quantitative PCR reaction Using Roche

Carrying out quantitative PCR by using a 96 fluorescent quantitative PCR instrument:

TABLE 4

The expression level of the target gene in the sample of the experimental group is 2 in comparison with that of the control group (has-5.8s)^-ΔΔCtThe results are shown in Table 5.

TABLE 5 expression level of the target gene in each sample (relative expression level to has-5.8s)

Statistical processing was performed using Gram Pad software the data in table 5 and the results are shown in figure 3.

The data of Table 5 were statistically processed using the software Sas, and the results are shown in tables 6-7 below:

table 6 shows the results of miRNA-6126 in 31 lung cancer patients

Table 7 shows the results of 5 normal control groups of miRNA-6126

Not satisfying normality, this example uses a rank-sum test to design the quantification in groups: z-3.4307 and P-0.0006, the two groups of differences have statistical significance.

Sequence listing

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Claims (2)

1. The application of a substance for detecting the expression quantity of miRNA-6126 in a human serum exosome to be detected in the preparation of a product for diagnosing or assisting in diagnosing whether a human to be detected is a lung cancer patient;

the substance for detecting the miRNA-6126 expression quantity in the human serum exosomes to be detected comprises a primer pair for amplifying miRNA-6126;

the primer pair consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.

2. The application of a substance for detecting the expression quantity of miRNA-6126 in a human serum exosome to be detected in the preparation of a product for screening or assisting in screening whether a human to be detected is a lung cancer patient;

the substance for detecting the miRNA-6126 expression quantity in the human serum exosomes to be detected comprises a primer pair for amplifying miRNA-6126;

the primer pair consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.

Images