CN114959018A - Non-small cell lung cancer miRNA marker and application thereof - Google Patents

Non-small cell lung cancer miRNA marker and application thereof Download PDF

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CN114959018A
CN114959018A CN202210772807.1A CN202210772807A CN114959018A CN 114959018 A CN114959018 A CN 114959018A CN 202210772807 A CN202210772807 A CN 202210772807A CN 114959018 A CN114959018 A CN 114959018A
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mir
lung cancer
small cell
cell lung
mirna
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时广利
陈燕
张鑫桐
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Beijing Chest Hospital
Beijing Tuberculosis and Thoracic Tumor Research Institute
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Beijing Chest Hospital
Beijing Tuberculosis and Thoracic Tumor Research Institute
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The invention provides a non-small cell lung cancer miRNA marker and application thereof, relating to the technical field of biomedicine. The markers comprise at least miR-3149; preferably, the miRNA markers include a combination of miR-3149 and miR-6803-5 p. The miRNA marker provided by the invention has obvious difference in expression quantity between non-small cell lung cancer population and healthy population, and miR-3149 can be used alone or combined with miR-6803-5p as a marker for clinical detection or auxiliary diagnosis of non-small cell lung cancer.

Description

Non-small cell lung cancer miRNA marker and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to a non-small cell lung cancer miRNA marker and application thereof.
Background
In China, the incidence of lung cancer is the first of malignant tumors, most lung cancer patients are found in the late stage, the surgical treatment chance is lost, the 5-year survival rate of the lung cancer patients is only 15-20%, and the 5-year survival rate of the lung cancer patients in the early stage reaches about 40%. Therefore, early detection and early treatment are critical to improve survival in lung cancer patients. The survival of NSCLC patients has not improved significantly over the past few decades due to the lack of effective early diagnostic methods.
At present, the means for detecting and screening lung cancer is mainly diagnostic screening by imaging such as X chest radiography, nuclear magnetic resonance imaging and low-dose spiral CT, however, the specificity and accuracy of the 2 screening means are not ideal.
The micro RNA (microRNA, miRNA) is a non-coding single-stranded small molecule RNA with the length of 18-25 nucleotides, which is highly conserved endogenously, and regulates the translation or expression of a target gene mainly through a post-transcriptional mechanism, so that the micro RNA has different functions of carcinogenesis and cancer suppressor miRNA in the occurrence and development of tumors. miRNA is very stably present in human plasma and serum, can resist degradation of RNA endonuclease, and can be used as a new serum marker index for diagnosing lung cancer.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a non-small cell lung cancer miRNA marker and application thereof, and provides a new way and a new method for specific detection or auxiliary diagnosis of non-small cell lung cancer.
The technical scheme provided by the invention is as follows:
in one aspect, the present invention provides non-small cell lung cancer miRNA markers comprising at least miR-3149; preferably, the miRNA markers include a combination of miR-3149 and miR-6803-5 p. In one embodiment, the miRNA markers consist of miR-3149 and miR-6803-5 p.
The miRNA (miR-3149 and/or miR-6803-5p) sequence is a known sequence, and a corresponding sequence can be obtained from database miRBase.
The present invention finds that the expression of peripheral blood miRNA markers (miR-3149 and/or miR-6803-5p) is significantly upregulated in peripheral blood of patients diagnosed with non-small cell lung cancer, as compared to the expression in control samples; the diagnosis marker miR-3149 is provided for the first time and can be used as a marker for detecting or diagnosing the non-small cell lung cancer in the population, the expression level of the marker is obviously different between the non-small cell lung cancer population and the healthy population, the sensitivity of the marker in the non-small cell lung cancer can reach 79.5 percent, and the reliability is high.
In a preferred embodiment, the miRNA marker is a serum miRNA marker.
In another aspect, the invention provides application of the non-small cell lung cancer miRNA marker in preparation of a kit for detecting and/or diagnosing non-small cell lung cancer. miR-3149 can be used as a marker for detecting or assisting in diagnosis of clinical non-small cell lung cancer alone or in combination with miR-6803-5p, and therefore, can be used for preparing a kit for detecting and/or diagnosing non-small cell lung cancer.
In another aspect, the invention provides an application of the product for detecting the miRNA marker in preparing a kit for detecting and/or diagnosing non-small cell lung cancer. Specifically, the product for detecting the miRNA marker is a product capable of detecting the expression and/or transcription of the miRNA marker.
In some specific embodiments, the product for detecting the miRNA marker is a reagent suitable for use in a method comprising: reagents for detecting expression levels by using qRT-PCR, digital PCR, fluorescent dye method, blot hybridization, in situ hybridization, array hybridization, gene chip or next generation sequencing. In the present invention, the expression level of miRNA in a sample may be measured using any technique suitable for detecting the expression level of RNA in a biological sample. Suitable techniques for determining the level of RNA expression in a biological sample (cells, tissues, body fluids) are well known to those skilled in the art.
In one embodiment, the product comprises one or more of PCR amplification primers, probes, gene chips; preferably, the product is a reagent for quantifying the expression amount of the miRNA. More preferably, the reagents are suitable for use in reagents for real-time fluorescent quantitative PCR, including amplification primers and/or probes. In the present invention, the expression level is a relative expression level, and is an expression level relative to an internal reference gene.
In one embodiment, the reagents comprise primers and/or probes for amplification of miR-3149 and/or miR-6803-5 p.
In another aspect, the invention provides a non-small cell lung cancer detection kit, which contains primers and/or probes for miR-3149 and/or miR-6803-5p amplification.
In a specific embodiment, the sequence of the primer probe of miR-3149 is shown in SEQ ID No.1, and the sequence of the primer probe of miR-6803-5p is shown in SEQ ID No. 2.
The invention also provides a probe combination of miRNA for detecting non-small cell lung cancer markers, wherein the probe combination is a combination of SEQ ID No.1 and SEQ ID No. 2.
In one embodiment, the kit further comprises amplification primers and/or probes for an internal reference gene; preferably, the reference gene is at least one selected from U6, GAPDH and beta-actin.
In one embodiment, the kit further comprises at least one of an RNA extraction buffer, an RNA reverse transcription reaction, and a qPCR reaction.
For detection or diagnosis of whether a subject has, or is at high risk for having, non-small cell lung cancer, it can be diagnosed by measuring the levels of miR-3149 or miR-3149 and miR-6803-5p in a test sample from said subject, wherein significantly higher expression levels of miR-3149 or miR-3149 and miR-6803-5p in the test sample, as compared to levels in a control sample (a healthy population sample), are indicative of the subject either having, or being at high risk for having, non-small cell lung cancer.
In the present invention, the expression level of the miRNA is any one of concentration, log (concentration), Ct/Cq number, and Ct/Cq power of 2; the sample is a tissue sample or a body fluid sample, preferably a blood sample, optionally a serum sample or a plasma sample.
In the present invention, the non-small cell lung cancer includes one or more of squamous cell lung cancer, adenocarcinoma lung cancer.
In the present invention, the expression level of the miRNA marker may be obtained by a method comprising the steps of:
(a) collecting a fasting venous blood sample, and separating serum;
(b) extracting serum RNA;
(c) synthesizing cDNA by using the extracted RNA as a template;
(d) and detecting the expression quantity of the miRNA molecular marker by a real-time fluorescent quantitative PCR method by using the synthesized cDNA as a template and adopting the specific primer and the probe of the miRNA.
Specifically, simultaneously carrying out RT-qPCR reaction by taking U6 snRNA as an internal reference, and quantifying the content of miRNA in the sample to be detected according to the cycle number when the fluorescence signal reaches a set threshold value; relative expression amount of serum miRNA 2 -ΔΔCt And (4) showing.
Has the advantages that:
the invention provides a biomarker with high diagnostic value for non-small cell lung cancer, wherein the expression levels of miR-3149 and miR-6803-5P in the serum of a non-small cell lung cancer (NSCLC) patient are obviously higher than those of a healthy human group, and the difference is statistically significant (P is less than 0.001). The sensitivity of the serum miR-3149 and miR-6803-5p in the NSCLC reaches 79.5 percent and 48.7 percent respectively. Therefore, the serum miR-3149 and miR-6803-5p can be used as a tumor marker index of the NSCLC; the miR-3149 alone or in combination with miR-6803-5p can be used as a novel biological marker for detecting or diagnosing the non-small cell lung cancer.
In addition, the invention also provides a kit for detecting the miRNA marker, which can more conveniently detect the expression level of the miRNA marker and provide a basis for clinical diagnosis and treatment of the non-small cell lung cancer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a comparison of miR-3149 expression levels in serum of NSCLC patients and healthy persons according to an embodiment of the invention;
FIG. 2 is the result of comparing the expression level of miR-6803-5p in serum of NSCLC patients and healthy human provided by the embodiment of the invention;
FIG. 3 is a schematic diagram of a subject operating curve (ROC) of miR-3149 expression levels in serum of NSCLC patients and healthy persons provided by an embodiment of the invention;
FIG. 4 is a schematic diagram of the operating curve (ROC) of the subjects of the miR-6803-5p expression level in the serum of NSCLC patients and healthy people provided by the embodiment of the invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. 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.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Examples
1. Study object
1.1 case group: 78 patients with NSCLC were admitted to the department of thoracic, beijing, affiliated with the university of capital medical science, of which 58 men and 20 women had an average age of 65.1 years; 38 cases of adenocarcinoma and 40 cases of squamous cell carcinoma; 20 patients in the early stage (stage I + II) and 58 patients in the late stage (stage III + IV). All NSCLC patients are diagnosed for the first time and are pathologically diagnosed as NSCLC, and no surgery, chemotherapy or radiotherapy is performed before.
1.2 healthy controls: 44 Beijing department hospital physical examination health workers, wherein 21 men and 23 women have an average age of 38.2 years and normal indexes such as blood pressure, blood fat, blood sugar, liver and kidney functions, blood routine and the like.
2. Specimen collection
4mL of fasting venous blood samples of NSCLC patients and healthy people are collected, serum is centrifugally separated within 2 hours, then the blood serum is stored in an ultra-low temperature refrigerator at minus 80 ℃, and finally RNA extraction is intensively carried out.
3. Extraction of serum RNA
Total RNA was extracted from Serum using the MiRNeasy Serum/Plasma kit (Qiagen, Germany).
4. Real-time fluorescent quantitative PCR (qRT-PCR) detection
And (3) detecting the expression quantities of miR-3149, miR-572, miR-638, miR-6803-5p and miR-7704 in the blood serum of 78 NSCLC patients and 44 healthy people by using a qRT-PCR method.
The qRT-PCR experiment was performed using the synthesized cDNA as template and U6 as internal reference, with a qRT-PCR reaction system of 20. mu.L.
PCR cycling conditions: 40 cycles of 95 ℃ for 5 minutes, 95 ℃ for 30 seconds, and 60 ℃ for 1 minute; then 95 ℃ for 15 seconds, 60 ℃ for 1 minute, 95 ℃ for 15 seconds, 1 cycle. The qRT-PCR experimental data are analyzed by ABI7500PCR instrument software, the expression quantity detection of each cDNA sample is repeated for 3 times, and the relative expression quantity of serum miRNA is 2 -ΔΔCt And (4) showing.
The primer sequences of the miRNA fluorescent quantitative PCR are as follows:
Figure BDA0003724836370000071
5. results
5.1 the expression levels of miR-572, miR-638 and miR-7704 in the serum of NSCLC patients have no statistical difference compared with healthy people (P is more than 0.05). The expression levels of miR-3149 and miR-6803-5P in the blood serum of the NSCLC patient are obviously higher than that of a healthy human control, and the difference is statistically significant (P is less than 0.001) (see table 1, figure 1 and figure 2).
TABLE 1 results of detection of serum miR-3149 and miR-6803-5p M (QR)
Figure BDA0003724836370000072
Note: NSCL (non-Small-cell-disease)C, comparing the standard sample with a healthy human control, a,b P＜0.001。
5.2 As shown in Table 2, serum miR-3149 has 79.5% sensitivity and 75.0% specificity for NSCLC detection; the sensitivity and the specificity of the serum miR-6803-5p to the NSCLC detection are 48.7% and 75.0%.
TABLE 2 sensitivity, specificity of serum miR-3149 and miR-6803-5p in NSCLC
Figure BDA0003724836370000073
Example 3 ROC of miRNA markers miR-3149 and miR-6803-5p
According to the invention, the relative expression amounts of miR-3149 and miR-6803-5p are taken as the basis, and the respective diagnostic effects are analyzed through an ROC curve (figure 3 and figure 4). The method comprises the following specific steps:
and (4) drawing an ROC curve of the marker, and judging the diagnosis effect of the marker non-small cell lung cancer according to the area under the curve.
The results show that the AUC for a single miR-3149 is 0.831; sensitivity was 79.5%, specificity was 75%.
The results show that the AUC of a single miR-6803-5p is 0.765; sensitivity 48.7%, specificity 75%.
The invention discovers that the serum miR-3149 and miR-6803-5p can be used as tumor markers of NSCLC and can be used as early diagnosis indexes of the NSCLC. The 2 markers provided by the invention stably exist in human serum, and the 2 markers and primers thereof can be used for preparing an early-stage NSCLC diagnosis kit and an auxiliary diagnosis reference index for early-stage NSCLC.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> the university of capital medical department affiliated to the Beijing thoracic hospital; research institute of tuberculosis and breast tumor in Beijing
<120> non-small cell lung cancer miRNA marker and application thereof
<130> PA22002180
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> Has-miR-3149 qPCR primer
<400> 1
tttgtatgga tatgtgtgtg tat 23
<210> 2
<211> 22
<212> DNA
<213> Has-miR-6803-5p qPCR primer
<400> 2
ctgggggtgg ggggctgggc gt 22

Claims (10)

1. A non-small cell lung cancer miRNA marker, wherein the miRNA marker at least comprises miR-3149; preferably, the miRNA markers include a combination of miR-3149 and miR-6803-5 p.
2. Use of the non-small cell lung cancer miRNA marker according to claim 1 for the preparation of a kit for the detection and/or diagnosis of non-small cell lung cancer.
3. Use of a product for detecting the miRNA marker of claim 1 for the preparation of a kit for detecting and/or diagnosing non-small cell lung cancer.
4. The use of claim 3, wherein the product for detecting the miRNA marker comprises one or more of a PCR amplification primer, a probe, and a gene chip.
5. The use as claimed in claim 3 wherein the product for detecting the miRNA marker is a reagent for quantifying the expression level of the miRNA.
6. The use according to claim 5, wherein the reagents comprise primers and/or probes for amplification of miR-3149 and/or miR-6803-5 p.
7. A non-small cell lung cancer detection kit is characterized by comprising primers and/or probes for miR-3149 and/or miR-6803-5p amplification.
8. The kit according to claim 7, wherein the probe sequence of miR-3149 is shown in SEQ ID No.1, and the probe sequence of miR-6803-5p is shown in SEQ ID No. 2.
9. The kit of claim 7, further comprising amplification primers and/or probes for an internal reference gene; preferably, the reference gene is at least one selected from U6, GAPDH and beta-actin.
10. The kit of any one of claims 7-9, wherein the kit further comprises at least one of an RNA extraction buffer, an RNA reverse transcription reaction, and a qPCR reaction.
CN202210772807.1A 2022-06-30 2022-06-30 Non-small cell lung cancer miRNA marker and application thereof Pending CN114959018A (en)

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