CN113862355A - Group of miRNA biomarkers related to lung cancer and application thereof - Google Patents

Abstract

The invention relates to the field of molecular diagnosis, and particularly discloses a group of miRNA biomarkers related to lung cancer and application thereof. The marker is a combination of hsa-miR-3714, hsa-miR-550a-3-5p and hsa-miR-4706. According to the invention, three miRNAs which are differentially expressed are screened out by collecting serum of a patient and a healthy person, and expression differences of the miRNAs are researched, wherein the miRNAs are expressed by hsa-miR-3714, hsa-miR-550a-3-5P and hsa-miR-4706, and compared with the normal person, the total expression levels of hsa-miR-3714, hsa-miR-550a-3-5P and hsa-miR-4706 in the plasma of 18 lung cancer patients are respectively increased by 2.14, 2.56 and 2.06 times (P is less than 0.05). Therefore, the miRNA marker combination related to lung cancer provided by the invention has the characteristics of simple operation, high specificity and high sensitivity for early screening of lung cancer.

Description

Group of miRNA biomarkers related to lung cancer and application thereof

Technical Field

The invention relates to the field of molecular diagnosis, and particularly discloses a group of miRNA biomarkers related to lung cancer and application thereof.

Background

In 2020, 457 more than ten thousand newly-increased cases of cancer and 301 more than ten thousand newly-increased death cases of cancer account for 23.7 percent and 30.2 percent of the total number of cancer and death worldwide, the cancer is the first to live in the world in 2020, the medical cost of Chinese malignant tumor exceeds 2200 billion yuan, and the family and the society of patients are heavily burdened. Lung cancer is currently the most common malignancy, with incidence and mortality leading among all cancers. The early diagnosis of lung cancer has important social value and economic significance for improving the life quality of patients, prolonging the life and reducing the social medical expenditure.

The human blood is rich in metabolites of human organs and cells, and a series of biomolecules are also introduced into the blood during the process of cancer occurrence and development. Wherein, microRNA (miRNA) as a tumor marker has good early warning effect on early onset and diagnosis and treatment of various malignant tumors (cancers), so that the search for one or a group of high-sensitivity and high-specificity miRNA detection has important medical significance.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a group of miRNA markers and a kit related to lung cancer. According to the invention, three miRNAs which are differentially expressed are screened out by collecting blood serum of a patient and blood serum of a healthy person, and expression difference of the miRNAs is researched, and the miRNAs are used as a group of miRNA marker combinations related to lung cancer, so that a lung cancer auxiliary diagnosis kit is developed, and data support is provided for early diagnosis of the lung cancer.

The invention comprises the following technical scheme:

a group of miRNA markers related to lung cancer is a combination of hsa-miR-3714, hsa-miR-550a-3-5p and hsa-miR-4706.

Further, the group of miRNA markers related to lung cancer is derived from human serum.

Further, a set of detection primers for detecting the set of miRNA markers associated with lung cancer, the detection primers comprising:

the forward detection primer sequence of hsa-miR-3714:

SEQ ID NO.1= 5’-CGCAGGGGCCGGGTGCTCACCG-3’；

the forward detection primer sequence of hsa-miR-550a-3-5p is as follows:

SEQ ID NO.2=5’-TGCCTAGGCTGAGACTGCAGTG-3’；

the forward detection primer sequence of hsa-miR-4706:

SEQ ID NO.3=5’-GGTCCAGAGGGGAGATAGGTTC-3’；

universal reverse detection primer sequence:

SEQ ID NO.4=5’-GCGAGCACAGAATTAATACGAC-3’。

furthermore, the application of the detection primers in preparing a reagent for early diagnosis of lung cancer.

Further, a lung cancer early diagnosis kit comprises the group of detection primers.

Further, the lung cancer early diagnosis kit comprises an enzyme and a reagent for PCR reaction.

Furthermore, the lung cancer early diagnosis kit comprises a total RNA extraction reagent, a total RNA plus poly (A) reagent, an RT-PCR reagent and a quantitative PCR reagent.

Further, in the kit for early diagnosis of lung cancer, the RT-PCR reagent comprises a universal RT-primer and a primer of an internal reference U6;

the sequence of the universal RT-primer is

SEQ ID NO.5 =5’-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3’；

The sequence of the RT-primer of internal reference U6 is SEQ ID No.6= 5'-GGAACGCTTCACGAATTTG-3';

the forward primer sequence of internal reference U6 SEQ ID No.7= 5'-ATTGGAACGATACAGAGAAGATT-3';

the reverse primer sequence of internal reference U6 is SEQ ID No.8= 5'-GGAACGCTTCACGAATTTG-3'.

The invention has the following beneficial effects:

according to the invention, three miRNAs which are differentially expressed are screened out by collecting serum of a patient and a healthy person, and expression differences of the miRNAs are researched, wherein the miRNAs are expressed by hsa-miR-3714, hsa-miR-550a-3-5P and hsa-miR-4706, and compared with the normal person, the total expression levels of hsa-miR-3714, hsa-miR-550a-3-5P and hsa-miR-4706 in the plasma of 18 lung cancer patients are respectively increased by 2.14, 2.56 and 2.06 times (P is less than 0.05). Therefore, the miRNA marker combination related to lung cancer provided by the invention has the characteristics of simple operation, high specificity and high sensitivity for early screening of lung cancer.

Drawings

FIG. 1 expression levels of hsa-miR-3714 in plasma of 18 patients and 18 normal persons;

FIG. 2 ROC curve of hsa-miR-3714;

FIG. 3 expression levels of hsa-miR-550a-3-5p in plasma of 18 cancer patients and 18 normal persons;

FIG. 4 shows the ROC curve of hsa-miR-550a-3-5 p;

FIG. 5 expression levels of hsa-miR-4706 in plasma of 18 cancer patients and 18 normal persons;

FIG. 6 shows the ROC curve of hsa-miR-4706;

FIG. 7 is a combined ROC curve of three miRNAs of hsa-miR-3714, hsa-miR-550a-3-5p and hsa-miR-4706.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The proper amount of the compound is determined by the ordinary technicians in the field according to the national technical specifications and the actual production conditions. The starting materials described in the present invention are all commercially available unless otherwise specified.

Example 1

Three miRNAs (miRNAs) of hsa-miR-3714, hsa-miR-550a-3-5p and hsa-miR-4706 in serum are detected, serum samples of 18 patients and 18 normal persons are respectively detected, and the method is carried out according to the following steps:

1. a200. mu.l serum sample was mixed with 1ml QIAzol lysis reagent and allowed to stand at room temperature for 5 minutes.

2. Adding 1ml chloroform, mixing by vortex for 30s, and standing for 5 min at room temperature.

Centrifuge at 10000 g for 15 min at 3.4 ℃.

4. The supernatant was aspirated to about 500uL, 750 uL of 100% ethanol was added, and the mixture was mixed well.

5. Taking no more than 700 μ l of sample each time, transferring to a centrifugal column, centrifuging at room temperature of 8000 g for 15s, and discarding waste liquid. Repeat this operation for the remaining samples

6. Add 700. mu.l of buffer RWT to the centrifuge tube, centrifuge at 8000 g for 15s at room temperature, and discard the waste.

7. Add 500. mu.l of buffered RPE to the centrifuge tube, centrifuge at 8000 g for 15s at room temperature, and discard the waste.

8. Add 500. mu.l of buffered 80% ethanol to the centrifuge tube, centrifuge at 8000 g for 2 minutes at room temperature, and discard the waste.

9. And placing the centrifuge tube in a collecting tube, centrifuging at full speed for 5 minutes, and drying by spinning.

10. The tube was placed in a fresh collection tube, 15ul of rnase-free water was added to the center of the spin column membrane, centrifuged at full speed for 1 minute, and the RNA solution was collected. Storing at-20 deg.C.

11. The miRNA is subjected to poly (A) Tailing by using Poly (A) labeling Kit (# AM1350, Ambion, Austin, TX) according to the operating manual.

12. Reverse transcription of poly (A) -added miRNA was performed using M-MuLV Reverse Transcriptase (# E6300S, NEB, Hitchin, UK) and universal RT-primers according to the instruction manual:

wherein the RT-PCR reagent comprises a universal RT-primer and a primer of an internal reference U6;

the sequence of the universal RT-primer is

SEQ ID NO.5 =5’-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3’；

The sequence of the RT-primer of internal reference U6 is SEQ ID No.6= 5'-GGAACGCTTCACGAATTTG-3';

the forward primer sequence of internal reference U6 SEQ ID No.7= 5'-ATTGGAACGATACAGAGAAGATT-3';

the reverse primer sequence of internal reference U6 is SEQ ID No.8= 5'-GGAACGCTTCACGAATTTG-3'.

13. Quantitative analysis of miRNA was performed using real-time fluorescent quantitative PCR SsoFastTM EvaGreen Supermix (BIO-RAD, Hercules, CA). Quantitative reaction system: SsoFast EvaGreen Supermix 5. mu.L, 0.5. mu.M forward primer 1. mu.L, 0.5. mu.M universal reverse primer 1. mu.L, template cDNA 1. mu.L, increasing RNase-free water to 10. mu.L. Reaction conditions are as follows: 2min at 95 ℃; denaturation 95 ℃ 20s, annealing/extension 58 ℃ 40s, for 40 cycles:

the upstream primer, namely the detection primer comprises:

the forward detection primer sequence of hsa-miR-3714:

SEQ ID NO.1= 5’-CGCAGGGGCCGGGTGCTCACCG-3’；

the forward detection primer sequence of hsa-miR-550a-3-5p is as follows:

SEQ ID NO.2=5’-TGCCTAGGCTGAGACTGCAGTG-3’；

the forward detection primer sequence of hsa-miR-4706:

SEQ ID NO.3=5’-GGTCCAGAGGGGAGATAGGTTC-3’；

universal reverse detection primer sequence:

SEQ ID NO.4=5’-GCGAGCACAGAATTAATACGAC-3’。

14. as shown in the attached figures 1-6, the total expression levels of hsa-miR-3714, hsa-miR-550a-3-5P and hsa-miR-4706 in the plasma of 18 lung cancer patients are respectively increased by 2.14, 2.56 and 2.06 times (P < 0.05) compared with the normal human. Meanwhile, the merged ROC curve of three miRNAs of hsa-miR-3714, hsa-miR-550a-3-5p and hsa-miR-4706 is shown in figure 7.

The above examples illustrate that the miRNA marker combination related to lung cancer provided by the present invention has the characteristics of simple operation, high specificity and high sensitivity for early screening of lung cancer.

The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.

SEQUENCE LISTING

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Claims (8)

1. A group of miRNA markers related to lung cancer is characterized in that the markers are a combination of hsa-miR-3714, hsa-miR-550a-3-5p and hsa-miR-4706.

2. The panel of claim 1, wherein the miRNA markers are derived from human serum.

3. A set of detection primers for detecting the set of lung cancer-associated miRNA markers of claim 1, wherein the detection primers comprise:

the forward detection primer sequence of hsa-miR-3714:

SEQ ID NO.1= 5’-CGCAGGGGCCGGGTGCTCACCG-3’；

the forward detection primer sequence of hsa-miR-550a-3-5p is as follows:

SEQ ID NO.2=5’-TGCCTAGGCTGAGACTGCAGTG-3’；

the forward detection primer sequence of hsa-miR-4706:

SEQ ID NO.3=5’-GGTCCAGAGGGGAGATAGGTTC-3’；

universal reverse detection primer sequence:

SEQ ID NO.4=5’-GCGAGCACAGAATTAATACGAC-3’。

4. use of a set of detection primers according to claim 3 for the preparation of a reagent for the early diagnosis of lung cancer.

5. A kit for early diagnosis of lung cancer, comprising the detection primer according to claim 3.

6. The kit for early diagnosis of lung cancer according to claim 5, wherein the diagnostic kit comprises enzymes and reagents for PCR reaction.

7. The kit for early diagnosis of lung cancer according to claim 5, wherein the kit comprises a total RNA extraction reagent, a total RNA plus poly (A) reagent, an RT-PCR reagent, and a quantitative PCR reagent.

8. The early lung cancer diagnosis kit of claim 7, wherein the RT-PCR reagent comprises a universal RT-primer and a primer of an internal reference U6;

the sequence of the universal RT-primer is

SEQ ID NO.=5’-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3’；

The sequence of the RT-primer of internal reference U6 is SEQ ID No.6= 5'-GGAACGCTTCACGAATTTG-3';

the forward primer sequence of internal reference U6 SEQ ID No.7= 5'-ATTGGAACGATACAGAGAAGATT-3';

the reverse primer sequence of internal reference U6 is SEQ ID No.8= 5'-GGAACGCTTCACGAATTTG-3'.

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