CN105671181B

CN105671181B - Gene marker, primer, probe and kit for detecting lung cancer

Info

Publication number: CN105671181B
Application number: CN201610168981.XA
Authority: CN
Inventors: 王义明; 罗国安
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-03-23
Filing date: 2016-03-23
Publication date: 2020-01-14
Anticipated expiration: 2036-03-23
Also published as: CN105671181A

Abstract

The invention discloses a gene marker for detecting lung cancer, which comprises an SFTPB gene, an FGFR1 gene and a FILIP1L gene. Studies find that the expressions of SFTPB, FGFR1 and FILIP1L mRNA in blood mononuclear cells of a lung cancer patient are abnormal, the diagnosis accuracy rate of the integrated lung cancer reaches 98.3 percent, and the diagnosis specificity and sensitivity are far better than those of a single gene. By measuring the expression levels of the three genes SFTPB, FGFR1 and FILIP1L in blood, the diagnosis of lung cancer (especially early lung cancer, namely lung cancer occurs, but the human body has no symptoms basically) is achieved, the detection rate of clinical lung cancer is improved, and the death rate of lung cancer patients is reduced.

Description

Gene marker, primer, probe and kit for detecting lung cancer

Technical Field

The invention relates to the technical field of lung cancer detection, in particular to a gene marker, a primer, a probe and a kit for detecting lung cancer.

Background

Lung cancer occurs in bronchial mucosa epithelium, and the incidence and mortality of lung cancer are always on the rise in recent years, which has become a serious health problem facing the world in the 21 st century. In china, lung cancer patients are expected to reach 100 million, which is the 1 st in the world, by 2025. The research on the occurrence, development, treatment and prognosis of lung cancer has been an important issue facing the medical community. At present, the early diagnosis of the lung cancer comprises chest X-ray and chest CT examination, sputum cell examination, fiber bronchoscopy, percutaneous lung puncture biopsy and other examination methods, and the clinical diagnosis methods have high cost for diagnosing the lung cancer, have instrument specificity and technical limitation, and have the reasons of wound to patients during detection, so that the diagnosis methods have many limitations and cannot be widely applied, and almost 2/3 lung cancer patients are in late stage (stage III or stage IV) during diagnosis and miss the optimal treatment period.

At present, lung cancer markers are also researched more, but free micro RNA mostly concentrated in lung cancer tissues and blood is not developed to a clinical diagnosis stage.

The detection of lung cancer markers in tissues requires surgery or biopsy to obtain cancer tissues, and is not suitable for early detection or multiple detections of lung cancer, because once the biopsy or surgery is required, the lung cancer is in a relatively advanced stage, and diagnosis of the lung cancer markers is not necessary.

Therefore, a new lung cancer detection marker is found and developed into a simple and practical technology for clinical detection, the clinical diagnosis of lung cancer patients is assisted, the diagnosis accuracy of lung cancer is improved, and the method has great significance for early discovery and early treatment of lung cancer.

Disclosure of Invention

The invention aims to solve the technical problems that the detection of the lung cancer marker in the existing tissue needs operation or biopsy, is not suitable for early detection or multiple detections of lung cancer, and increases the pain of patients; the diagnosis technology of blood markers mostly stays in the research stage, the detection effect is yet to be further verified, and the clinical diagnosis stage is not yet developed.

In order to solve the technical problems, the invention provides a gene marker for detecting early lung cancer, which is a combined marker of three genes, and specifically comprises an SFTPB gene, an FGFR1 gene and a FILIP1L gene. By measuring the expression levels of the three genes SFTPB, FGFR1 and FILIP1L in blood, the early diagnosis of lung cancer (lung cancer occurs, but human bodies have no symptoms basically) is achieved, the detection rate of clinical lung cancer is improved in an auxiliary mode, and the death rate of lung cancer patients is reduced.

SFTPB, FGFR1 and FILIP1L were each found to be associated with lung cancer in different studies. Wherein SFTPB is a surfactant protein, proteomics research shows that the plasma level of the SFTPB is related to clinically known independent risk factors of lung cancer, and the SFTPB is a promising blood marker of non-small cell lung cancer. In addition, SFTPB is associated with early stage lung cancer, suggesting whether tumors of potential non-small cell lung cancer are suitable for surgical resection at early stage. FGFR1 is a fibroblast growth factor receptor, whose expression can reduce apoptosis and promote cancer cell survival, and is an independent poor prognostic marker of early-stage squamous cell lung cancer. FILIP1L is a filament protein, and can inhibit invasion and metastasis of cancer cells by inhibiting expression of matrix metalloproteinase, and has FILIP1L protein and mRNA expression level of FILIP1L protein regulated in lung cancer tissue lower than that in normal tissue. The current study only stayed in the correlation of the contents of SFTPB, FGFR1 and FILIP1L proteins with different typing of lung cancer. According to the research, the SFTPB, FGFR1 and FILIP1L gene expressions in blood mononuclear cells are obtained through a large number of early-stage researches through screening and are obviously related to lung cancer, the early-stage lung cancer diagnosis assisting method can be used for assisting in early diagnosis of the lung cancer, after the three gene markers are integrated, the early-stage lung cancer diagnosis accuracy can reach 98.3%, and the early-stage lung cancer diagnosis assisting method is suitable for diagnosis of different types of lung cancer.

The primer and the probe for detecting the lung cancer provided by the invention comprise an SFTPB primer probe group, an FGFR1 primer probe group and an FILIP1L primer probe group, and the nucleotide sequences are as follows:

SFTPB primer probe set:

an upstream primer: 1 (5'-CAGAAACTACAGACAAAGAGAGTGGAA-3') of SEQ ID NO,

a downstream primer: 2 (5'-TTCAGTTGCTTCAGGCCATCT-3') of SEQ ID NO,

and (3) probe: 3 (5'-CAGGCCTCTGAGCCCAAGCTAAGCC-3') SEQ ID NO;

FGFR1 primer probe sets:

an upstream primer: 4 (5'-CACGGGACATTCACCACATC-3') of SEQ ID NO,

a downstream primer: 5 (5'-ACCCCGAAAGACCACACATC-3') in SEQ ID NO,

and (3) probe: 6 (5'-ACTATAAAAAGACAACCAACGGCCGACTGC-3') SEQ ID NO;

FILIP1L primer probe set:

an upstream primer: 7 (5'-CATCCCTGAAACACCTTGATTTTAT-3') in SEQ ID NO,

a downstream primer: 8 (5'-TCAAGGCTTAAAACAACATCCATTT-3') in SEQ ID NO,

and (3) probe: 9 (5'-AAGCCACGCTGTATCTGGACTTCTGATCTG-3') SEQ ID NO;

wherein, the 5 'end of the nucleotide sequence of the probe is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group.

Preferably, the fluorescence reporter is FAM and the fluorescence quencher is TAMRA.

The lung cancer detection kit provided by the invention comprises the primer and the probe for detecting lung cancer. In order to facilitate the fluorescent quantitative PCR detection, the kit preferably also comprises Taqman reaction buffer solution, dNTPs and Mg²⁺And the like.

Preferably, the lung cancer detection kit further comprises a detection primer and a probe of an internal reference gene GAPDH, and the specific nucleotide sequence of the detection primer is an upstream primer: 10, downstream primer SEQ ID NO:11, probe: 12 is SEQ ID NO; the 5 'end of the nucleotide sequence of the probe is marked with a fluorescent reporter group, and the 3' end of the nucleotide sequence of the probe is marked with a fluorescent quenching group. The fluorescence reporter group is preferably FAM, and the fluorescence quencher group is preferably TAMRA.

SEQ ID NO:10(5’-GCATCCTGGGCTACACTGAG-3’)；

SEQ ID NO:11(5’-TCCACCACCCTGTTGCTGTA-3’)；

SEQ ID NO:12(5’-TCCTCTGACTTCAACAGCGACACCC-3’)。

Preferably, the lung cancer detection kit further comprises a positive control, and the positive control comprises a gradient concentration of SFTPB gene, FGFR1 gene, FILIP1L gene and GAPDH gene.

Preferably, the lung cancer detection kit further comprises

Universal PCR Master Mix and RNase-free water. Both reagents are commercially available.

The invention also provides a use method of the lung cancer detection kit, which comprises the following steps:

(1) extracting total RNA of a sample, and performing reverse transcription to synthesize cDNA;

(2) performing a fluorescent quantitative PCR reaction using the primer and the probe for detecting lung cancer according to claim 2, respectively, using cDNA as a template, and collecting fluorescent signals to obtain expression levels of FGFR1 gene, SFTPB gene and FILIP1L gene; when the kit contains an internal reference gene, carrying out fluorescent quantitative PCR reaction by using a detection primer and a probe of the internal reference gene GAPDH, normalizing the detection results of the three target genes by using the PCR reaction result of the GAPDH gene, and correcting the Ct value difference caused by the initial sampling amount;

(3) calculating a joint predictor: the expression of the joint predictor is: y is 1.77x1+3.68x2-x3, wherein y represents a combined predictor, x1 represents the relative expression level of the FGFR1 gene of the sample normalized by the reference gene, x2 represents the relative expression level of the SFTPB gene of the sample normalized by the reference gene, and x3 represents the relative expression level of the FILIP1L gene of the sample normalized by the reference gene; when y is more than 34.44, the sample is judged to be positive; when y is less than 34.44, the sample is judged to be negative, namely, a normal sample.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the expression of SFTPB, FGFR1 and FILIP1L mRNA in blood mononuclear cells of a lung cancer patient is found to be abnormal through research, the diagnosis accuracy rate of the integrated lung cancer reaches 98.3%, and the diagnosis specificity and sensitivity are far better than those of a single gene.

Moreover, the detection sample of the kit is mononuclear cells in peripheral blood, so that the kit is convenient to obtain materials, small in wound and safe; the quantitative determination of the three target genes is carried out by adopting the RT-PCR technology, and the method has the advantages of high specificity and sensitivity, simple operation and easy large-flux screening.

The invention can be widely applied to the detection of the amplification levels of the SFTPB, FGFR1 and FILIP1L genes of lung cancer patients, can be used for the early diagnosis and curative effect evaluation of the lung cancer, improves the repeatability and accuracy of pathological detection, avoids excessive tissue biopsy, screens out the lung cancer patients more accurately, intervenes in the early treatment, can greatly reduce the medical cost and expense, reduces the waste of medical resources, prolongs the survival period of part of patients and improves the survival quality of the patients.

Drawings

FIG. 1 shows the change in △ Ct of the three target genes SFTPB, FGFR1 and FILIP1L in normal control and patient samples, Note: P < 0.05 compared to normal.

FIG. 2 shows the results of ROC curve analysis of three target genes SFTPB, FGFR1 and FILIP 1L.

FIG. 3 shows the results of ROC curve analysis after integration of three target genes SFTPB, FGFR1 and FILIP 1L.

Detailed Description

The present invention is further described with reference to specific examples to enable those skilled in the art to better understand the present invention and to practice the same, but the examples are not intended to limit the present invention.

Example 1 preparation and use of a kit for detecting Lung cancer

1. The real-time fluorescent quantitative PCR kit for detecting the expression levels of SFTPB, FGFR1 and FILIP1L genes comprises:

SFTPB gene specific primers (upstream and downstream primer pre-mix), 10. mu.M; specific fluorescent probe, 2 μ M;

FGFR1 gene specific primers (upstream and downstream primer pre-mix), 10 μ M; specific fluorescent probe, 2 μ M;

FILIP1L gene specific primers (pre-mixed upstream and downstream primers), 10. mu.M; specific fluorescent probe, 2 μ M;

GAPDH gene specific primers (upstream and downstream primer pre-mix), 10 μ M; specific fluorescent probe, 2 μ M;

universal PCR Master Mix, purchased from ABI corporation;

RNase-free water, available from Tiangen Biochemical technology (Beijing) Ltd.

The positive control samples were stored separately and diluted with a concentration gradient during use.

2. The use method of the kit comprises the following steps:

(1) real-time fluorescent quantitative PCR reaction system

TABLE 1 real-time fluorescent quantitative PCR reaction System

Note: the reaction reagent adopts Taqman fluorescent quantitative kit of ABI company in America, and can also be products of the same type of other companies.

The PCR amplification conditions were: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, and amplification at 72 ℃ for 30s, for 45 cycles.

(2) Data processing

The kit adopts a relative quantitative method to analyze data. The Ct values of 3 target genes and 1 reference gene in the sample are detected, and the Ct values are detected by 2^-△△CtThe expression level of SFTPB, FGFR1 and FILIP1L genes is expressed by the difference △ Ct of the amplified Ct value of internal reference gene GAPDH, the size of △ Ct is inversely related to the content of SFTPB, FGFR1 and FILIP1L genes, compared with a normal group, if the △ Ct value of the SFTPB gene of a lung cancer patient is reduced, the expression level of the SFTPB gene is increased in the lung cancer patient group.

Calculation of diagnostic thresholds after integration of SFTPB, FGFR1 and FILIP1L genes: the Ct values of the SFTPB, FGFR1 and FILIP1L gene expression are normalized by using an internal reference gene, and the threshold value is calculated according to the ratio of a target gene to the internal reference gene. And substituting the normalized relative expression quantity of the target gene into an equation y of 1.77x1+3.68x2-x3 for calculation, wherein y represents a combined prediction factor, x1 represents the relative expression quantity of the FGFR1 gene normalized by the reference gene, x2 represents the relative expression quantity of the SFTPB gene normalized by the reference gene, and x3 represents the relative expression quantity of the FILIP1L gene normalized by the reference gene. Normalizing the expression quantity of the target gene measured by a clinical sample, and then substituting the normalized expression quantity into an equation for calculation, wherein if y is more than 34.44, the sample is diagnosed as a lung cancer patient; if y is less than 34.44, the sample is diagnosed as normal.

(3) In-batch and inter-batch repeatability of kit detection system

Randomly taking any sample, repeating the detection for 6 times in one experiment, and inspecting the repeatability in batches; any sample was taken randomly and the reactions were repeated 6 times at different times to investigate batch-to-batch reproducibility with results shown in table 2.

TABLE 2 Intra-and inter-batch Gene repeatability

As can be seen from Table 2, the kit detection method system has an RSD of 0.69% in the absolute amount of genes in batches and an RSD of 1.09% in the absolute amount of genes in batches, which are less than 5%. The data fully show that the established detection method system has good in-batch and batch repeatability, and can ensure the accuracy and reliability of the measurement result.

Example 2 clinical sample testing

1. And (3) collecting clinical samples:

in the study, 50 samples of healthy volunteers and 50 samples of lung cancer patients were collected.

2. Extraction of total RNA from fresh peripheral blood:

(1) centrifuging 1mL of EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood at 5000rpm for 10min, discarding plasma, adding 1mL of erythrocyte lysate, mixing well, reacting on ice for 15min, and centrifuging at 5000rpm for 5 min;

(2) discarding the supernatant, adding 1mL of PBS (Phosphate Buffered Saline), mixing well, centrifuging at 5000rpm for 5 min; repeating the operation for three times until the supernatant is transparent, and discarding the supernatant;

(3) adding 1mL Trizol reagent into the lower layer precipitation cells, fully and uniformly mixing, and standing for 5min at room temperature;

(4) adding chloroform 200 μ L, mixing well for 30s, standing at room temperature for 3min, centrifuging at 10000rpm for 20 min;

(5) taking 500 mu L of supernatant fluid, putting the supernatant fluid into a new PE tube, adding 500 mu L of isopropanol, uniformly mixing, and standing at minus 80 ℃ for overnight; or standing at room temperature for half an hour, and directly performing the centrifugation operation of the step (6).

(6) Taking out the PE tube from-80 deg.C, melting at room temperature, centrifuging at 10000rpm for 30min, removing supernatant, adding 1mL of 75% ethanol-DEPC water (V/V, DEPC is diethyl pyrocarbonate), dissolving, mixing, and centrifuging at 7500rpm for 10 min;

(7) discarding supernatant, air drying the precipitate at room temperature, adding 22 μ L DEPC water to dissolve to obtain total RNA solution, or storing at-80 deg.C.

3. Synthesis of cDNA:

(1) taking 2 mu L of total RNA solution, diluting by 50 times, measuring OD (absorbance) by an ultraviolet spectrophotometer, calculating the concentration of the total RNA obtained by extraction, and detecting that OD260/280 is more than or equal to 1.8 to determine the purity;

(2) taking 0.2 mu g of total RNA and a reverse transcription reagent, synthesizing a reaction system according to the cDNA shown in the table 3, and adding samples for reaction; the reagent adopts RevertAId First Strand cDNA Synthesis Kit of Thermo Scientific company in the United states, and can also be products of the same type of other companies.

Table 3: reaction system for cDNA synthesis

Reaction reagent	Volume (μ L)
		10×Buffer RT	2
dNTP Mix(2.5mM each dNTP)	2
		Oligo-dT Primer(10μm)	2
Quant Reverse Transcriptase	1
		RNasin-free water	12
Sample Total RNA (0.2. mu.g)	1
		Total volume	20

(3) The reaction system is incubated at 37 ℃ for 60min, and the cDNA product is stored at-20 ℃.

4. Quantitative detection of samples

The sample cDNA is taken as a template, a Taqman fluorescence quantitative PCR reaction method is adopted, the reaction is carried out in an ABI Prism7300 real-time fluorescence PCR amplification instrument, and the reaction system and the reaction conditions are shown in Table 1. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, and amplification at 72 ℃ for 30s, for 45 cycles.

By using 2^-△△CtThe method calculates the relative content of the target gene in the sample. The calculation results are shown in table 4.

TABLE 4 relative amounts of SFTPB, FGFR1, FILIP1L mRNA

Note: 2^-△△CtThe value is the ratio of the relative content of the target gene to the normal group after normalization.

FIG. 1 is a graph of △ Ct showing that the Ct value of a target gene in a sample is changed, △ Ct is inversely proportional to the content of the target gene in the sample, and △ Ct is increased, which indicates that the content of the target gene in the sample is decreased, and thus it is known that the expression levels of SFTPB and FILIP1L mRNA are increased and the expression level of FGFR1 mRNA is decreased in a sample of a lung cancer patient.

In conclusion, the expression of SFTPB, FGFR1 and FILIP1L genes in peripheral blood mononuclear cells can be used as a potential biomarker for lung cancer diagnosis.

For the diagnostic ability and efficacy evaluation ability of the three genes on lung cancer patients, the study was evaluated by using Receiver Operating Characteristic (ROC) curve. ROC curves are an important tool to evaluate marker accuracy. Two main metrics that can be derived include: sensitivity (sensitivity), or true positive rate, assessed for its performance in selecting patients for a particular disease. High sensitivity is generally required in screening assays to exclude disease-free persons; specificity (specificity), or true negative rate, indicates its ability to correctly select a person without a disease. High specificity is generally required in diagnostics to obtain a low false positive rate. First, ROC curve analysis was performed on three target genes, and the results are shown in FIG. 2. Wherein the abscissa is 100-specificity, i.e., false positive rate, and the ordinate is sensitivity, i.e., true positive rate.

As shown in FIG. 2, when the three target genes are used for distinguishing the normal control (50 cases) from the lung cancer patients (50 cases), the diagnosis accuracy can reach more than 90%, the FGFR1 and FILIP1L genes have higher sensitivity, and the SFTPB gene has higher diagnosis specificity. In-depth research finds that the diagnostic capability of integrating three target genes in the kit can be improved compared with that of a single gene. Firstly, establishing a logistic regression model to obtain a joint prediction factor, wherein the expression of the prediction factor is as follows: y is 1.77x1+3.68x2-x3, wherein y represents a combined predictor, x1 represents the expression level of the FGFR1 gene of the sample, x2 represents the expression level of the SFTPB gene of the sample, and x3 represents the expression level of the FILIP1L gene of the sample. The results of the expression amount measurements of the three target genes in each sample are brought into a predictor equation to obtain the predictors of each sample, and the predictors are used as analysis indexes to perform the ROC curve analysis of the binormal model, wherein the results are shown in 3.

As can be seen from FIG. 3, after the three target genes are integrated, the diagnosis sensitivity and specificity are improved, the sensitivity and specificity of the integrated predictor for lung cancer diagnosis respectively reach 100%, 90% and 91%, and the area under the ROC curve is 0.983(P <0.0001), which indicates that the integrated three target genes have 98.3% diagnosis accuracy for lung cancer diagnosis, have clinical diagnosis application value, and can be used as an auxiliary molecular index for clinical lung cancer diagnosis.

In the sample, the relative expression levels of the SFTPB, FGFR1 and FILIP1L genes after normalization are measured and are substituted into a way joint predictor (y value) which is calculated by the equation y of 1.77x1+3.68x2-x 3. y is more than 34.44, and the sample is judged as a disease sample; y < 34.44, and the sample is judged to be a normal sample.

In addition, the integrated gene marker group can also be considered as a treatment target and an evaluation index for future drug development, and drug screening and new drug development are carried out.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

SEQUENCE LISTING

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Claims

The application of the combination of SFTPB gene, FGFR1 gene and FILIP1L gene as markers in the preparation of lung cancer detection reagents.
2. The primers and probes for detecting lung cancer are characterized by comprising an SFTPB primer probe group, an FGFR1 primer probe group and a FILIP1L primer probe group, and the nucleotide sequences are as follows:

SFTPB primer set:

an upstream primer: 1 (5'-CAGAAACTACAGACAAAGAGAGTGGAA-3') of SEQ ID NO,

a downstream primer: 2 (5'-TTCAGTTGCTTCAGGCCATCT-3') of SEQ ID NO,

and (3) probe: 3 (5'-CAGGCCTCTGAGCCCAAGCTAAGCC-3') SEQ ID NO;

FGFR1 primer set:

an upstream primer: 4 (5'-CACGGGACATTCACCACATC-3') of SEQ ID NO,

a downstream primer: 5 (5'-ACCCCGAAAGACCACACATC-3') in SEQ ID NO,

and (3) probe: 6 (5'-ACTATAAAAAGACAACCAACGGCCGACTGC-3') SEQ ID NO;

FILIP1L primer set:

an upstream primer: 7 (5'-GGCCTGGTGCAAGCAAAGT-3') in SEQ ID NO,

a downstream primer: 8 (5'-GGAATGCGGGTGGGTGTAG-3') in SEQ ID NO,

and (3) probe: 9 (5'-CGAGCACTATCACCATAACACCGGTCACA-3') SEQ ID NO;

wherein, the 5 'end of the nucleotide sequence of the probe is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group.
3. The primers and probe for detecting lung cancer according to claim 2, wherein the fluorescence reporter group is FAM and the fluorescence quencher group is TAMRA.
4. A lung cancer detection kit comprising the primer and the probe for detecting lung cancer according to claim 2 or 3.
5. The lung cancer detection kit according to claim 4, further comprising a detection primer and a probe of a reference gene GAPDH, wherein the specific nucleotide sequence of the detection primer is an upstream primer: 10, downstream primer SEQ ID NO:11, probe: 12 is SEQ ID NO; the 5 'end of the nucleotide sequence of the probe is marked with a fluorescent reporter group, and the 3' end of the nucleotide sequence of the probe is marked with a fluorescent quenching group.
6. The lung cancer detection kit of claim 5, further comprising a positive control, wherein the positive control comprises a gradient concentration of SFTPB gene, FGFR1 gene, FILIP1L gene and GAPDH gene.
7. The lung cancer detection kit according to claim 5, further comprising
Universal PCR Master Mix and RNase-free water.