CN106350582B

CN106350582B - Serum miRNA marker related to lung squamous carcinoma auxiliary diagnosis and application thereof

Info

Publication number: CN106350582B
Application number: CN201610723241.8A
Authority: CN
Inventors: 朱伟; 单霞; 尹荣; 朱军; 张澜; 黄泽波; 张获; 朱丹霞; 吴俚蓉; 周鑫; 刘平
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-08-25
Filing date: 2016-08-25
Publication date: 2020-02-14
Anticipated expiration: 2036-08-25
Also published as: CN106350582A

Abstract

The invention discloses a serum miRNA marker related to lung squamous carcinoma auxiliary diagnosis and application thereof, wherein the marker is one or more of miR-106a-5p, miR-20a-5p and miR-93-5 p. The serum miRNA is used as a novel biomarker and has the characteristics of good stability, easy minimally invasive acquisition, and high sensitivity and specificity. The development and utilization of the molecular markers can provide a new direction for the diagnosis and further treatment of various diseases including tumors. The research can obtain the lung squamous carcinoma serum miRNA marker with clinical diagnosis potential more specifically. The research proves the reliability and repeatability of the group of miRNAs as noninvasive markers for diagnosing squamous cell lung carcinoma.

Description

Serum miRNA marker related to lung squamous carcinoma auxiliary diagnosis and application thereof

Technical Field

The invention belongs to the field of genetic engineering and oncology, and relates to a serum miRNA marker related to lung squamous carcinoma auxiliary diagnosis and application thereof.

Background

Lung Cancer (Lung Cancer) is currently one of the most common malignancies worldwide. In China, the incidence and mortality of lung cancer are the top. Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer. Among them, squamous cell lung carcinoma is one of the most common subtypes in NSCLC. Although surgical treatment is the first choice and the most main treatment method for squamous cell lung carcinoma at present and can achieve the aim of curing early squamous cell lung carcinoma patients, the early diagnosis rate of squamous cell lung carcinoma patients in China is still low because the early clinical manifestations of squamous cell lung carcinoma patients have no obvious specificity. Currently, Low-dose CT (Low-dose computed tomogry) is the more recommended screening for lung cancer and pulmonary nodules. But its use is somewhat limited due to reasons such as high false alarm rates, potential over-diagnosis, excessive costs, and possible radiation exposure hazards. The tumor markers currently most widely used in clinical applications, such as squamous cell carcinoma antigen (SCC), carcinoembryonic antigen (CEA), neuronic enzyme (NSE) and Cyfra 21-1, also lack certain sensitivity and specificity. With the development of biotechnology such as genomics, proteomics, and metabolomics, more and more biomarkers have been discovered or studied. Therefore, the discovery of a new marker capable of early diagnosing the squamous cell lung carcinoma indicates that the patient can be waited for, thereby promoting the early intervention and treatment of the squamous cell lung carcinoma and prolonging the life of the patient.

Micro RNA (miRNAs) is a small non-coding RNA molecule with the length of about 22 nucleotides, and is widely involved in various life activity processes including tumor occurrence, invasion, metastasis and the like through posttranscriptional regulation. Researches show that the expression of miRNA has different degrees of up-regulation and down-regulation in tumors, and lays a foundation for the miRNA to be used as a new tumor marker. It has been found that free mirnas can be stably present in peripheral blood and can be used as noninvasive markers for diagnosing tumors. Multiple studies show that circulating miRNA has potential diagnosis value in tumors such as gastric cancer, breast cancer, colorectal cancer and the like. Therefore, the research aims to search serum miRNA with potential diagnostic value for lung squamous carcinoma through the research on lung squamous carcinoma serum of a large sample by utilizing an Exiqon miRNAqPCR panel chip and a qRT-PCR-based relative quantification method. And the expression of the miRNA in lung squamous carcinoma tissues and peripheral serum exosomes is verified so as to further define the relationship between the miRNA and the lung squamous carcinoma. If a diagnosis kit aiming at the squamous cell lung carcinoma is designed according to the miRNA, the diagnosis and treatment level of the squamous cell lung carcinoma in China can be promoted, and a thought is provided for further research on the squamous cell lung carcinoma in the future.

Disclosure of Invention

The invention aims to provide a serum miRNA marker related to lung squamous carcinoma auxiliary diagnosis.

The invention also aims to provide the application of the serum miRNA marker and the primer thereof in preparing an auxiliary diagnosis kit for squamous cell lung carcinoma and in preparing a medicament for treating squamous cell lung carcinoma.

The invention also aims to provide a kit and a medicament for auxiliary diagnosis and treatment of the lung squamous carcinoma.

The purpose of the invention can be realized by the following technical scheme:

a serum miRNA marker related to lung squamous carcinoma auxiliary diagnosis is one or more of miR-106a-5p (AAAAGUGCUUACAGUGCAGGUAG), miR-20a-5p (UAAAGUGCUUAAUAGUGCAGGUAG) and miR-93-5p (CAAAGUGCUGUUCGGUGCAGGUAG). The serum miRNA marker is preferably a combination of two or more of miR-106a-5p, miR-20a-5p and miR-93-5p, and is further preferably a combination consisting of three miRNAs of miR-106a-5p, miR-20a-5p and miR-93-5 p.

The serum miRNA marker is applied to the auxiliary diagnosis of squamous cell lung carcinoma.

The serum miRNA marker is applied to preparation of a lung squamous carcinoma auxiliary diagnosis kit or a lung squamous carcinoma treatment drug.

A primer of a serum miRNA marker related to lung squamous cancer auxiliary diagnosis, which comprises a primer of one or more miRNAs in miR-106a-5p, miR-20a-5p and miR-93-5 p; preferably primers containing two or more of miR-106a-5p, miR-20a-5p and miR-93-5p in serum miRNA; further preferably, the primer comprises three miRNAs of miR-106a-5p, miR-20a-5p and miR-93-5p in serum miRNAs.

The primer is applied to the auxiliary diagnosis of the squamous cell lung carcinoma or the preparation of an auxiliary diagnosis kit for the squamous cell lung carcinoma.

An auxiliary diagnosis kit for squamous cell lung carcinoma, which is used for detecting one or more miRNAs in serum miR-106a-5p, miR-20a-5p and miR-93-5 p; preferably, the kit is used for detecting two or more miRNAs in serum miR-106a-5p, miR-20a-5p and miR-93-5 p; further preferably, the miRNA is used for detecting three miRNAs of miR-106a-5p, miR-20a-5p and miR-93-5p in serum.

An auxiliary diagnosis kit for squamous cell lung carcinoma, which contains a primer of one or more miRNAs in miR-106a-5p, miR-20a-5p and miR-93-5p in serum miRNAs; preferably primers containing two or more of miR-106a-5p, miR-20a-5p and miR-93-5p in serum miRNA; further preferably primers containing three miRNAs of serum miR-106a-5p, miR-20a-5p and miR-93-5 p.

The kit may also comprise reagents commonly used in PCR technology, such as reverse transcriptase, buffer, dNTPs, MgCl₂DEPC water and Taq enzyme, etc.; standards and/or controls may also be included.

The sequence of each miRNA in the serum miRNA markers miR-106a-5p, miR-20a-5p and miR-93-5p related to lung squamous carcinoma diagnosis is disclosed, but creative labor is required for a person skilled in the art to use each miRNA marker alone or in combination as an auxiliary diagnosis marker of lung squamous carcinoma. The amplification primers of the miRNA markers can be obtained by market purchase, and the primers of the serum miRNA markers used in the embodiment of the invention are specific miRNA stem-loop RT-PCR primers synthesized and produced by Sharpbo, Guangzhou.

Specifically, the technical solution of the present invention to solve the problem includes: (1) establishing a unified specimen library and a database: standard procedures (SOP) were used to collect blood samples meeting the standards and the system collected complete demographic and clinical data. (2) Differential expression profiling of serum mirnas: differential expression of serum miRNAs in squamous cell lung carcinoma and normal control populations was analyzed and further large sample multi-stage validation of differentially expressed miRNAs was performed. (3) The ability of these mirnas to diagnose squamous cell lung carcinoma is clear by multi-stage validation. (4) Development of a serum miRNA diagnosis kit: miRNAs diagnostic kit is developed according to the differential expression of miRNA in the serums of patients with squamous cell lung cancer and normal people, so that noninvasive auxiliary diagnosis of patients with squamous cell lung cancer is realized. (4) The expression conditions of the miRNAs in lung squamous carcinoma tissues and exosomes are analyzed, the relationship between the miRNAs and lung squamous carcinoma is disclosed, and a basis is provided for developing medicaments possibly related to the miRNAs for treating lung squamous carcinoma in the future.

The inventor collects blood samples meeting the standard by a Standard Operation Procedure (SOP), systematically collects complete demographic data and clinical data, and adopts an Exiqon miRNAqPCR panel chip, a qRT-PCR method and the like.

The experimental method of research mainly includes the following parts:

1. study sample selection: patients who are initially treated, have no intervention of operation or radiotherapy and chemotherapy and are pathologically confirmed to be lung squamous carcinoma. The normal control is a normal population for physical examination in a hospital.

Preliminary screening of the Exiqon miRNAqPCR panel chip: and (3) carrying out RNA extraction on the serum mixed sample by using a TRIZOL-LS reagent, and carrying out qRT-PCR operation to obtain a primary screening result.

3. Training set and verification set: RNA extraction is carried out on each serum sample by using an AM1556 kit (ABI company), a cDNA sample is obtained through reverse transcription reaction, and a PCR primer and SYBR Green fluorescent dye are added for PCR reaction. 4. The RNA in lung squamous carcinoma and paracancerous tissues is extracted by a TRIZOL-LS reagent, the RNA in exosomes is extracted by an ExoQuick kit (SBI company) and an AM1556 kit (ABI company), and the expression difference of miRNA in tissues and exosomes is detected by a qRT-PCR method.

5. Statistical analysis: exercise chi²Tests, paired t tests, and non-parametric rank-sum tests compare the differences in miRNA expression levels among different study groups. The diagnostic value of serum miRNA is proved by calculating ROC curve analysis.

At present, by carrying out systematic expression analysis on miRNA in peripheral serum of a squamous cell lung carcinoma patient, the research group of the invention has found a group of 3 lung squamous cell carcinoma serum microRNA markers (miR-106a-5p, miR-20a-5p and miR-93-5p) with clinical diagnosis potential.

The invention has the beneficial effects that:

1. compared with the traditional tumor marker, the serum miRNA is used as a novel biomarker, and has the characteristics of good stability, easy minimally invasive acquisition, high sensitivity and high specificity. The development and utilization of the molecular markers can provide a new direction for the diagnosis and further treatment of various diseases including tumors.

2. Researchers performed rigorous and multistage verification and evaluation of differentially expressed mirnas in the serum of lung squamous carcinoma and normal control population through an Exiqon miRNAqPCR panel chip and an absolute quantitative method based on qRT-PCR. The reliability and repeatability of the group of miRNAs as noninvasive markers for diagnosing squamous cell lung carcinoma are proved.

3. Researchers find that the expression of miR-106a-5p, miR-20a-5p and miR-93-5p in lung squamous carcinoma patients is obviously higher than that of a normal control group. Meanwhile, the expressions of miR-106a-5p, miR-20a-5p and miR-93-5p in the tissue and serum exosome of the lung squamous carcinoma patient are higher than those of a normal control. These results will provide new ideas for future research on the mechanism of these mirnas for lung squamous carcinoma and for the treatment of lung squamous carcinoma by these mirnas.

Drawings

FIG. 1: flow chart of experiment

FIG. 2: high expression of 3 miRNAs in lung squamous carcinoma serum P <0.001

FIG. 3: ROC curve analysis of the obtained miRNA

a: a training set, a test set and a verification set; b: training a set; c: testing the set; and d, verifying the set.

FIG. 4: expression of 3 miRNAs in lung squamous carcinoma tissue P <0.05, P <0.001

FIG. 5: expression of 3 miRNAs in serum exosomes of patients with lung squamous carcinoma P <0.001

Detailed Description

The inventor collects a large number of vein serum samples of squamous cell lung carcinoma patients and normal physical examination people from the first subsidiary hospital of Nanjing medical university in 2012 to 2015, and selects 100 samples of squamous cell lung carcinoma and 108 samples of normal control as experimental samples for initial screening and subsequent series of qRT-PCR verification of an Exiqon miRNA qPCR panel chip by sorting sample data. At the same time, 32 lung squamous carcinoma tissues and 36 paracarcinoma tissues are reserved. Selected patient serum samples were obtained from patients who were initially treated, were not operated, and had undergone chemo-and chemo-radiotherapy intervention and were pathologically confirmed as squamous cell lung carcinoma. And the system collects the demographic data and clinical data of the samples.

Referring to the flowchart (fig. 1), 30 lung squamous carcinoma samples and 10 normal controls were randomly selected from lung squamous carcinoma and normal control serum samples, and mixed into 3 lung squamous carcinoma serum mixed samples and 1 normal mixed sample (one mixed sample is formed by mixing 10 200ul serum samples to form a 2ml sample). The 4 mixed samples were subjected to preliminary screening and analysis of the Exiqon miRNA qPCR panel chip, and the specific steps refer to the specifications of the Exiqon miRNA qPCR panel chip:

1. serum extraction

Serum samples were removed and centrifuged at 3000x g for 5min after thawing to remove some debris and some insoluble components. Transfer 250ul of supernatant to a new 1.5ml tube, add 750ul TRIZOL-LS, and shake vigorously for 5 s.

2. Two-phase separation

After homogenization the sample is incubated for 5 minutes at 15 to 30 ℃. 0.2ml of chloroform was added to 1ml of the sample homogenized with TRIZOL-LS reagent, and the cap was closed. After manually shaking the tube vigorously for 15 seconds, the tube is incubated at 15 to 30 ℃ for 2 to 3 minutes. Centrifuge at 13,000g for 15 minutes at 4 ℃.

RNA precipitation

The aqueous phase was transferred to a fresh centrifuge tube. The aqueous phase was mixed with isopropanol to precipitate the RNA therein, the amount of isopropanol added was: to 1ml of TRIZOL-LS reagent homogenate was added 0.5ml of isopropanol and 5ul of glycogen. Standing at 4 ℃ for half an hour to separate out RNA as much as possible. Centrifuge at 13,000g for 15 minutes at 4 ℃.

RNA washing

The supernatant was removed and at least 1ml of 75% (v/v) ethanol was added to each 1ml of the TRIZOL-LS reagent homogenate sample to wash the RNA pellet. The mixture was allowed to stand for 10 minutes and then centrifuged at 10000g at 4 ℃ for 5 minutes.

5. Re-solubilization of RNA pellets

The ethanol solution was removed, the RNA pellet was air-dried for 5-10 minutes, repeatedly blown several times with a gun by adding RNase-free water, and then incubated at 55 to 60 ℃ for 10 minutes.

6. And (3) measuring the concentration:

typically, 5. mu.g RNA/50ml serum is obtained.

cDNA Synthesis

(1) Diluting template RNA: 20-25 ng template RNA was diluted to 14ul (final concentration 1.492-1.786 ng/. mu.l) using DEPC water.

(2) Preparing a reaction solution: the 5 × Reaction Buffer and DEPC water were dissolved on ice and shaken well. The Enzyme mix was placed in an ice box at-20 ℃ and gently mixed before use and then placed on ice. All reagents were used after centrifugation.

(3) Preparing a reaction solution: the reaction solution in the following table was prepared

(4) Mix and centrifuge reagents: and shaking or pumping the reaction solution uniformly and then centrifuging to ensure that all the solutions are thoroughly and uniformly mixed.

(5) Reverse transcription and heat inactivation: after incubating the reaction solution at 42 ℃ for 60 minutes, the reverse transcriptase was inactivated by incubating at 95 ℃ for 5 minutes.

8.Real-Time PCR

Reagent:

Nuclease free water(Exiqon)

SYBR^TMGreen master mix(Exiqon)

cDNA template

ROX(Invitrogen)

miRNA PCR ARRAY(Exiqon)

The instrument comprises the following steps:

ABI PRISM7900 system(Applied Biosystems)

(1) preparation of Real-time PCR reagents: subjecting the prepared cDNA template, DEPC Water and SYBR^TMGreen mastermix was dissolved on ice for 15-20 minutes.

(2) Diluting the cDNA template: the cDNA template obtained from the RT reaction was diluted 110-fold with nucleoease free water (for example, 2180. mu.l of nucleoease free water was added to 20. mu.l of the reaction solution).

(3) Mixing all reaction reagents:

A. after simple centrifugation of the PCR plate, the membrane was removed.

B. The 110-fold diluted cDNA template was mixed with 2 × SYBR Green master mix as described in 1: mixing at a volume ratio of 1.

C. Inverting and mixing the reaction solution and centrifuging

D. Adding the mixed reaction solution to each well in the plate

E. Resealing the PCR plate

(4) Subjecting the PCR plate to simple low-temperature centrifugation

(5) Real-time PCR amplification: real-time PCR amplification and dissolution curve analysis were performed according to the reaction conditions in the following table.

Real-time PCR cycling conditions are as follows:

and (3) data analysis: using the Delta Ct method

Preliminary data analysis was performed using software attached to the PCR instrument to obtain the original Cq value (Cp or Ct, which may vary from instrument to instrument).

We propose to use GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis) for standard and in-depth data analysis.

a. The Δ Ct for each pathway-associated gene in each treatment group was calculated.

ΔCt(group 1)＝average Ct–average of HK genes’Ct for group 1 array

ΔCt(group 2)＝average Ct–average of HK genes’Ct for group 2 array

b. The Δ Δ Ct for each gene in 2 PCR arrays (or two groups) was calculated.

Δ Δ Ct ═ Δ Ct (group 2) - Δ Ct (group 1)

Remarking: typically group 1 is the control and group 2 is the experimental group.

c. The expression difference of the corresponding genes of the group 2 and the group 1 is calculated through 2-delta. Ct.

After the initial screening of the chip, 38 differentially expressed mirnas (more than 1.5-fold difference in all of the 3 lung squamous carcinoma serum mixed samples compared to the normal sample) were obtained as shown in the following table.

Verifying 38 differential expression miRNAs obtained by primary screening by a relative quantitative method based on qRT-PCR through a training set, a testing set and a verification set, wherein the specific steps are as follows:

1. serum RNA extraction: serum RNA extraction kit (AM1556) of ABI company is selected, 200ul of RNA is extracted from each sample according to the kit instruction, and finally 100ul of DEPC water is used for dissolving.

Preparation of cDNA:

1) reverse transcription experiment was performed using a 50. mu.L reaction system

The above reaction system was mixed well and after instantaneous centrifugation, the reaction was carried out according to the following procedure:

2) the following reactants are added into the reaction system after the reaction

3.qPCR

1) Using a 5. mu.L reaction system, the following ratio was used for the test

The reaction system is mixed evenly, placed in a real-time quantitative PCR instrument after instantaneous centrifugation, and reacted according to the following procedures:

the dissolution profile was added after the reaction was complete.

And (3) data analysis: the relative concentration of miRNA in each sample can be obtained from the Ct value. Statistical analysis is carried out by using SPSS 16.0 software, and a group of miRNA (micro ribonucleic acid) which are highly expressed in the serum of the lung squamous carcinoma are obtained in a training set, a testing set and a verification set: miR-106a-5P, miR-20a-5P and miR-93-5P (P values in training set, test set and validation set are all less than 0.05, FIG. 2). From these 3 mirnas, the ROC curve for each sample can be calculated. As shown in fig. 3, the molecular marker composed of the 3 mirnas can well distinguish the patients with squamous cell lung carcinoma from the normal population.

The expression of the 3 miRNAs in exosomes in lung squamous carcinoma tissues and serum is further detected after a research group, TRIZOL is used for extracting RNA from the lung squamous carcinoma tissues, and an exosome extraction kit is an ExoQuick kit (SBI company). Exosome RNA extracted from 200ul serum was resuspended in 200ul DEPC water, and exosome RNA was extracted using AM1556 kit (ABI) in the same procedure as the serum RNA extraction.

By nonparametric test analysis, the expression of miR-106a-5p, miR-20a-5p and miR-93-5p in lung squamous carcinoma tissues is higher than that in paracarcinoma tissues (figure 4). The expressions of miR-106a-5p, miR-20a-5p and miR-93-5p in lung squamous carcinoma serum exosome are also obviously higher than those of normal people (figure 5).

The kit comprises a batch of serum miRNA qRT-PCR primers, and can also comprise common reagents required by corresponding PCR technologies, such as: reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, fluorescent probes, RNase inhibitors, Taq enzyme and the like can be selected according to the specific experimental method, the common reagents are well known to those skilled in the art, and in addition, standard substances and controls (such as quantitative standard normal human samples and the like) can be provided. The kit has the value that only serum is needed, other tissue samples are not needed, and the expression content of miRNA in the serum sample is detected through the simplest fluorescence method, so as to assist in diagnosing the possibility of lung squamous cell carcinoma of a patient from which the sample is derived. The serum miRNA is convenient to detect, accurate in quantification and capable of greatly improving the sensitivity and specificity of disease diagnosis, so that the kit can help to guide diagnosis and further individualized treatment when put into practice.

Claims

1. A serum miRNA marker related to lung squamous carcinoma auxiliary diagnosis is characterized in that the serum miRNA marker is a combination of miR-106a-5p, miR-20a-5p and miR-93-5 p.

2. A primer of a serum miRNA marker related to lung squamous carcinoma auxiliary diagnosis is characterized in that the primer consists of primers of miR-106a-5p, miR-20a-5p and miR-93-5p in serum miRNA.

3. The use of the primer of claim 2 in the preparation of a lung squamous carcinoma auxiliary diagnostic kit.

4. An auxiliary diagnosis kit for squamous cell lung carcinoma, which is characterized in that the kit is used for detecting three miRNAs of miR-106a-5p, miR-20a-5p and miR-93-5p in serum.

5. An auxiliary diagnosis kit for squamous cell lung carcinoma is characterized in that the kit contains primers of miR-106a-5p, miR-20a-5p and miR-93-5p in serum miRNA.

6. The diagnostic kit according to claim 4 or 5, wherein the kit further comprises reagents commonly used in PCR technology.