WO2017054325A1 - Breast cancer combined diagnosis markers and detection kit - Google Patents

Abstract

Provided are breast cancer combined diagnosis markers and a detection kit. RECQL, RECQL4 and RECQL5 genes can serve as the combined markers, and are used to prepare breast cancer detection and/or therapeutic evaluation reagents.

Description

Breast cancer combined diagnostic marker and test kit Technical field

The invention relates to the fields of biochemistry and molecular biology, in particular to a joint diagnostic marker for breast cancer and a detection kit.

Background technique

The burden of cancer in China is increasing, with 1.6 million people diagnosed with cancer each year and 1.2 million people dying of cancer. Like many other countries, breast cancer is the most common cancer among Chinese women. Worldwide, China accounts for 12.2% of newly diagnosed breast cancer cases and 9.6% of breast cancer deaths.

Breast cancer is a systemic disease, the incidence rate is increasing year by year, and the age of onset is gradually youngening. As a highly heterogeneous tumor, lymphatic or hematogenous metastasis may occur in the early stage, which is one of the common malignant tumors in women.

Although the incidence of breast cancer in China is currently low, since the 1990s, the incidence of breast cancer in China has increased more than twice as much as in the world, especially in urban areas. At present, breast cancer is the highest incidence of cancer among Chinese women, and the cause of cancer death ranks sixth. As of 2008, China had a total of 169,452 new invasive breast cancers and 44,908 deaths from breast cancer, accounting for 12.2% and 9.6% of the world, respectively.

The annual number of breast cancer detected in China is half that of Europe (332,000 cases in 2008 and a total population of 498 million), which is basically equivalent to that of the United States (a total of 182,000 cases in 2008 with a total population of 404 million). . If this trend remains unchanged, by 2021, the number of breast cancer patients in China will reach 2.5 million, and the incidence will increase from less than 60 cases/100,000 women (aged between 55 and 69) to more than 100 cases/ 100,000 women. (Lancet Oncol 2014; 15(7): e279-e289)

At present, there are three main technologies for early warning and early diagnosis of breast cancer in the world:

1. Clinical physical examination: Regular clinical physical examination is one of the effective methods for early detection of breast cancer. Beginning in New York's Health Insurance Program (HIP) in 1963, after 18 years of research, women with regular clinical physical examinations had a 23% lower breast cancer mortality rate than the control group. China has also carried out a series of breast cancer prevention work, but the breast cancer census is costly and the cost/effectiveness ratio is high.

2. Imaging diagnosis: Imaging examination is based on mammography, supplemented by ultrasound and MRI. The mammography method can detect fine calcifications, which can indicate breast cancer if it manifests as sediment-like calcification or fine-grained calcification, or more than 5 in <0.5 mm per 1 cm ² . However, for atypical lesions, especially lesions in the dense breast and lesions near the chest wall are easily missed. Published in the famous British Medical Journal on February 11, 2014, an annual mammography test for women aged 40 to 59 years does not reduce breast cancer mortality, nor is it superior to the diagnosis of general medical examinations. Rate, and 22% of women were over-diagnosed because of mammography (British Medical Journal, 2014, 348.). In addition, the female breast in China is small and dense, which will also reduce the positive rate of patients receiving mammography.

3. Early detection of biological targets: including breast cancer-related gene screening, such as mutation analysis of BRCA1 and BRCA2; detection of nipple discharge specificity and determination of serum tumor markers. The Her-2/neu oncogene is overexpressed in breast cancer and Her-2 protein fragments can be detected in serum. Determination of serum Her-2 levels by enzyme-linked immunosorbent assay can be used as one of the indicators for the diagnosis of breast cancer. However, most of the current molecular diagnostic studies focus on the relationship between genetic polymorphism and breast cancer, which can only give patients a risk of susceptibility, and is not suitable for the diagnosis of clinical diseases.

Therefore, there is an urgent need in the field to establish a new breast cancer screening technology that can diagnose breast cancer in an objective, sensitive and stable manner in the early stage, so as to timely and targeted treatment, improve the quality of life and survival of patients, reduce medical costs, and save community resource.

Summary of the invention

It is an object of the present invention to provide novel breast cancer joint diagnostic markers in view of the aforementioned deficiencies of prior art detection means.

Another object of the present invention is to provide a detection kit prepared based on the above-mentioned markers, and the kit detection method of the invention has the characteristics of small trauma, rapid detection, high throughput, sensitivity and specificity compared with the conventional diagnostic method. Can better diagnose early breast cancer and evaluate the efficacy.

The present invention provides the use of the RECQL, RECQL4 and RECQL5 genes as a combined marker for the preparation of a breast cancer detection and/or therapeutic evaluation reagent.

The human RecQ cleavase is a multifunctional DNA cleavage enzyme that can separate dinucleotide chains by the energy generated by hydrolysis of nucleoside triphosphates (NTPs) and plays an important role in many aspects of DNA metabolism. Participate in various types of DNA repair, including mismatch repair, nucleotide excision repair, and direct repair enzymes.

The RECQL, RECQL4 and RECQL5 genes are all members of the RecQ DNA helicase family. Studies have shown that RECQL gene mutations are closely related to tumor susceptibility, such as breast cancer, osteosarcoma and resectable pancreatic cancer. RECQL4 is widely involved in DNA replication and damage repair. Early studies have confirmed that RECQL4 mutations lead to three syndromes of RTS, BGS and RAPA, in which patients with RTS and BGS have a high tendency to osteosarcoma and lymphoma. In addition, RECQL4 can also prevent multiple tumorigenesis by maintaining gene stabilization of mitochondria and telomeres, and RECQL4 is also thought to be associated with hematologic malignancies. A recent study showed that mutations in the RECQL5 locus are associated with breast cancer (Tumor Biol. 2014; 35: 12201–12204). The above genes are susceptibility genes of tumors. The current research focuses on the relationship between DNA site mutations and diseases, but susceptibility locus mutations are often used to provide the risk of certain diseases, and only detect the sites of susceptible genes. Mutations are not suitable for clinical diagnosis and evaluation of disease. Based on this, we investigated the relationship between the expression levels of the above susceptible genes and breast cancer.

We have found and confirmed by studies that mRNA expression levels of RECQL, RECQL4 and RECQL5 genes in peripheral blood mononuclear cells and urine are significantly changed in patients' body fluids, especially peripheral blood mononuclear cells and urine, and this change The occurrence and treatment of breast cancer are closely related. The mRNA expression of RECQL, RECQL4 and RECQL5 genes can be used as biomarkers for early diagnosis and evaluation of breast cancer.

The invention also provides specific RT-PCR primers and probes for breast cancer detection and/or therapeutic evaluation, including specific amplification primers and probes for RECQL, RECQL4 and RECQL5 genes, and the nucleotide sequence of the probe is 5' a fluorescent reporter group is attached, and a non- 5' end is linked to a fluorescence quenching group, wherein

The nucleotide sequence of the RECQL amplification primer set is shown in SEQ ID NOS: 1 to 2,

The nucleotide sequence of the RECQL-specific probe is shown in SEQ ID NO:3;

The nucleotide sequence of the RECQL4 amplification primer set is shown in SEQ ID NOS: 4 to 5,

The nucleotide sequence of the RECQL4-specific probe is shown in SEQ ID NO: 6;

The nucleotide sequence of the RECQL5 amplification primer set is shown in SEQ ID NOS: 7-8.

The nucleotide sequence of the RECQL5 specific probe is shown in SEQ ID NO: 9.

The above primers and probe sequences screened by the invention can efficiently and specifically amplify the corresponding genes, and only need to take the peripheral blood and morning urine of the person to be tested, and can be widely applied to RECQL of breast cancer patients, The detection of RECQL4 and RECQL5 gene amplification levels, using RT-PCR technology to quantitatively determine three target genes, has the advantages of high specificity and sensitivity, simple operation and easy to large-throughput screening.

Preferably, the specific RT-PCR primers and probes for breast cancer detection and/or therapeutic evaluation include the specific amplification primers and probes of the internal reference gene GAPDH,

The nucleotide sequence of the GAPDH amplification primer set is shown in SEQ ID NOs: 10 to 11,

The nucleotide sequence of the GAPDH-specific probe is shown in SEQ ID NO:12;

The nucleotide sequence of the probe is linked to a fluorescent reporter group at the 5' end and a fluorescence quencher group to the non- 5' end.

The main purpose of GAPDH is to serve as an internal reference to correct detection bias and further improve the detection accuracy of the primer set.

Preferably, the specific RT-PCR primers and probes for any of the above breast cancer detection and/or therapeutic evaluation, the nucleotide sequence of the probe is linked to the fluorescent reporter group FAM at the 5' end and the fluorescence quenching at the 3' end. Kill the group TAMRA. FAM is 6-carboxyfluorescein, and TAMRA is 6-carboxytetramethylrhodamine.

The present invention also provides a kit for detecting and/or evaluating a breast cancer, comprising the specific RT-PCR primers and probes for detecting breast cancer according to any of the above.

Preferably, the above breast cancer detection and/or therapeutic evaluation kit further comprises a positive template, and the positive template comprises purified: RECQL gene amplification product, RECQL4 gene amplification product, RECQL5 gene amplification product and GAPDH gene amplification. product.

The positive template of the four gene amplification products is mainly used for the drawing of the standard curve, which is convenient for quantitative analysis of the detection results.

Preferably, the above-mentioned breast cancer detection and/or therapeutic evaluation kit is divided into a real-time fluorescent quantitative PCR reaction system of four genes RECQL, RECQL4, RECQL5 and GAPDH and a positive template system of the four genes, wherein

19.0 μL of real-time PCR system for each gene, including 10 μL of PCR reaction buffer, 0.8 μL of the gene amplification primer at a concentration of 10 μM, 1.0 μL of the gene-specific probe at a concentration of 2 μM, and RNase-free water of 7.2 μL. ;

The positive template system for each gene included a purified amplification product of the gene at a concentration of 1 x 10 ⁹ copies/μL.

The PCR reaction buffer contains at least DNA polymerase, dNTPs, ^Mg2+, etc., and can be directly purchased from commercially available products, such as ABI products, or can be prepared by itself.

The present invention also provides a method for using the above-described breast cancer detection and/or therapeutic evaluation kit, comprising the following steps:

The positive templates of the four genes RECQL, RECQL4, RECQL5 and GAPDH were serially diluted and added to a real-time fluorescent quantitative PCR reaction system for fluorescence quantitative PCR reaction. The quantitative standard of each gene was drawn according to the diluted copy number and the amplified Ct value. curve;

After the total RNA is extracted, the sample is reverse-transcribed into cDNA, and the cDNA is used as a fluorescent quantitative PCR template. After adding a real-time fluorescent quantitative PCR reaction system, a quantitative PCR reaction is performed, and the obtained amplified Ct value is brought into a quantitative standard curve of the corresponding gene. Obtaining an initial expression amount of the gene in the sample;

The measurement result of the sample to be tested is normalized by the result of the internal reference gene GAPDH of the sample, and the combined prediction of the three marker genes RECQL, RECQL4 and RECQL5 is calculated according to the equation y=x1+42.016×x2+7.87×x3. Factor, where y represents the joint predictor, x1 represents the expression level of the sample RECQL gene, x2 represents the expression level of the sample RECQL4 gene, and x3 represents the expression level of the sample RECQL5 gene;

When the y is less than 0.006, the sample to be tested is positive, and when y is greater than 0.006, the sample to be tested is negative.

Preferably, in the above method for using the breast cancer detection and/or therapeutic evaluation kit, the fluorescence quantitative PCR reaction conditions are: pre-denaturation at 95 ° C for 10 min; denaturation at 95 ° C for 10 s, annealing at 60 ° C for 30 s, expansion at 72 ° C for 30 s, and performing 45 Cycles.

Preferably, in the above method for using the breast cancer detecting and/or therapeutic evaluation kit, the sample to be detected is peripheral blood and/or urine.

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

The present invention finds that the combination of RECQL, RECQL4 and RECQL5 genes can be used as a breast cancer marker to detect diseases with high accuracy, and the provided primers and the kit consisting of the primers are used for measuring peripheral blood and urine. Convenient, minimally invasive or non-invasive, safe; RT-PCR technology for quantitative determination of three target genes, with specificity and sensitivity, easy to operate and easy to large-throughput screening.

The invention can be widely applied to the amplification of RECQL, RECQL4 and RECQL5 genes in breast cancer patients Horizontal detection can be used for early diagnosis and evaluation of breast cancer, improve the repeatability and accuracy of pathological examination, avoid excessive tissue biopsy, and more accurately screen breast cancer patients. Early treatment intervention can greatly reduce medical treatment. Costs and expenses, reduce the waste of medical resources, prolong the survival of some patients, and improve the quality of life of patients.

DRAWINGS

Figure 1 is a standard curve of three target genes and one housekeeping gene;

Figure 2 is a box plot showing the expression of a target gene in a normal control and patient samples before and after treatment;

Figure 3 is a result of ROC curve analysis of three target genes;

Figure 4 is the result of ROC curve analysis after integration of three target genes;

Figure 5 is a result of ROC curve analysis of target gene expression of a patient sample before and after treatment;

Figure 6 Box plot of RECQL and RECQL5 in urine expression in breast cancer patients;

Figure 7 is a box plot of expression of RECQL4 in the urine of normal controls and breast cancer patients.

detailed description

The present invention will be further described in conjunction with the specific embodiments, which are to be understood by those skilled in the art.

Example 1 Breast Cancer Detection and/or Efficacy Evaluation Kit

Kit 1

Including 8 systems, real-time PCR system for the four genes RECQL, RECQL4, RECQL5 and GAPDH, and a positive template system for these four genes, wherein

19.0 μL of real-time PCR system for each gene, including 10 μL of PCR reaction buffer, 0.8 μL of the gene amplification primer at a concentration of 10 μM, 1.0 μL of the gene-specific probe at a concentration of 2 μM, and RNase-free water of 7.2 μL. ;

The positive template system for each gene included a purified amplification product of the gene at a concentration of 1 x 10 ⁹ copies/μL.

RECQL upstream primer: 5'-ACAAAGGGCAATCAGGAATCA-3';

RECQL downstream primer: 5'-CATTGGCTGACCATTTTCTATGAAC-3';

RECQL probe sequence: 5'-AATTCATGCAGGTGCTTACCATGCCAA-3';

RECQL4 upstream primer: 5'-TCTCTCCCCTGCTGTCACTCA-3';

RECQL4 downstream primer: 5'-GACAGATTCCCGTTGCTTCCT-3';

RECQL4 probe sequence: 5'-CTGGCCTGCCACCGTGTCTCAAG-3';

RECQL5 upstream primer: 5'-AGAAGGTCCCTGTAATTGTTGCA-3';

RECQL5 downstream primer: 5'-GCCATAGACTTGGCAATATTCCA-3';

RECQL5 probe sequence: 5'-AAGCCAATGTCAGGTTTGTCGCCCA-3';

GAPDH upstream primer: 5'-GCATCCTGGGCTACACTGAG-3';

GAPDH upstream primer: 5'-TCCACCACCCTGTTGCTGTA-3';

GAPDH probe sequence: 5'-TCCTCTGACTTCAACAGCGACACCC-3'.

The 5' end of the RECQL, RECQL4, RECQL5 and GAPDH-specific probes binds to the fluorescent generating group FAM, and the 3' end binds to the fluorescent quenching group TAMRA.

The PCR reaction buffer is Taqman reaction solution (trade name)

Universal PCR Master Mix).

When the kit is detected, the cDNA of the sample to be detected is added.

Kit 2

Including the following systems:

1RECQL gene-specific primer (upstream and downstream primer premix), 10 μM; specific fluorescent probe, 2 μM;

2RECQL4 gene-specific primer (premixed with upstream and downstream primers), 10 μM; specific fluorescent probe, 2 μM;

3RECQL5 gene-specific primer (premixed with upstream and downstream primers), 10 μM; specific fluorescent probe, 2 μM;

4GAPDH gene-specific primer (upstream and downstream primer premix), 10 μM; specific fluorescent probe, 2 μM;

5, 6, 7, and 8 are quantitative positive templates: the amplified products of purified RECQL, RECQL4, RECQL5 and GAPDH genes, respectively, at a concentration of ¹⁰⁹ copies/μL.

Universal PCR Master Mix, purchased from ABI;

RNase-free water, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

Example 2 Method of Using the Kit of the Present Invention

(1) Real-time fluorescent quantitative PCR reaction

The kit of the present invention may also include primers and probes of only four genes, and positive template of amplification products of four genes, and then assist the existing real-time fluorescent quantitative PCR kit for detection together.

Single gene real-time fluorescent quantitative PCR reaction system

Reaction reagent	Volume (μL)
PCR reaction buffer	10
Primer Primer F/R (10μM)	0.8
Probe (2μM)	1.0
cDNA	1.0
RNase-free water	7.2
total capacity	20

Note: The above reagents are obtained by American ABI Taqman Fluorescence Quantitation Kit, and other products of the same type can be used.

The PCR amplification conditions were: 95 □ predenaturation for 10 min, 95 □ denaturation for 10 s, 60 □ annealing for 30 s, 72 □ amplification for 30 s, and 45 cycles.

(2) Drawing a standard curve

The quantitative positive template of known concentration provided in the kit is diluted to a concentration gradient of 10 ³ to 10 ⁷ copies/μL by a 10-fold dilution method, and the reaction is carried out according to the above reaction system and amplification conditions, and after completion, According to the diluted copy number and the amplified Ct value, the standard curve y=A×log x+B, R ^{2 is obtained} , where x represents the starting template copy number, y represents the Ct value, A and B are specific values, and the R ² value is required. To be greater than 0.99 (R ² represents the correlation coefficient of the established equation, the closer to 1, the better the established equation).

(3) Sample testing

Total RNA was extracted from 1 mL of fresh EDTA anticoagulated peripheral blood of the patient using the Trizol method (Invitrogen, USA), and then the total RNA was inverted using the RevertAid First Strand cDNA Synthesis Kit of Thermo Scientific, USA. Recorded as cDNA, the cDNA is used as a template for real-time quantitative PCR, and the reaction can be carried out according to the above reaction system and amplification conditions, and the reaction result is taken into the above standard curve for calculation, and the initial copy value of the target gene in the sample can be obtained.

(4) Judgment of results

The measurement result of the sample to be tested is normalized by the result of the internal reference gene of the sample, and the combined predictive factors of the three target genes are calculated according to the equation y=x1+42.016×x2+7.87×x3, wherein y represents joint prediction. The factor, x1 represents the expression level of the sample RECQL gene, x2 represents the expression level of the sample RECQL4 gene, and x3 represents the expression level of the sample RECQL5 gene.

When y is less than 0.006, it is positive, and when it is greater than 0.006, it is negative.

Example 3 Establishment of three target gene standard curves and method repeatability:

(1) Preparation of standard curve

Template: A quantitative standard curve was prepared using the quantitative positive template of the kit of the present invention, and the assay repeatability of the kit was tested.

Instrument: American ABI 7300 real-time PCR instrument.

METHODS: Three target genes (RECQL, RECQL4, RECQL5) and a positive model of a housekeeping gene (GAPDH) were diluted 10 times, and sequentially diluted to 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ and 10 ³ copies . /μL. 1 μL of each was taken for real-time fluorescent quantitative PCR reaction.

The standard curve is shown in Figure 1. The RECQL standard curve equation is: y=-3.8833x+41.323, the correlation coefficient R ² =0.9969; the RECQL4 standard curve equation is: y=-4.036x+37.778, correlation coefficient R ² =0.9990; RECQL5 The standard curve equation is: y=-3.95x+38.905, correlation coefficient R ² =0.9978; GAPDH standard curve equation is: y=-3.7331x+43.275, correlation coefficient R ² =0.9979.

As can be seen from Figure 1, the standard curve and Ct values made with the positive template have a good linear relationship with the initial copy number.

Example 4 Intra- and Inter-assay Reproducibility of the Kit Detection System

Randomly take any sample, repeat 6 simultaneous tests in one experiment, and examine the repeatability within the batch; randomly take any sample and repeat 6 reactions at different times to examine the repeatability between batches. The results are shown in Table 2.

Table 2 Intra- and inter-assay gene repeatability

As can be seen from Table 2, the RSD of the absolute amount of the intra-assay gene of the kit detection method system was 0.54%, and the RSD of the absolute amount of the inter-assay gene was 1.37%, both of which were less than 5%. The above data fully explain the established inspection The measurement method system has good intra-batch and batch repeatability, which can ensure the accuracy and reliability of the measurement results.

Example 5 Clinical Blood Sample Testing

(1) Clinical sample collection:

A total of 50 healthy volunteer samples, 49 breast cancer patients (stage II, preoperative) and 22 post-treatment patient samples (stage II, postoperative) were collected.

(2) Extraction of total RNA from fresh peripheral blood:

1) 1 mL of EDTA (ethylenediaminetetraacetic acid) anticoagulated whole blood, centrifuged at 5000 rpm for 10 min, discard the plasma, add 1 mL of red blood cell lysate, mix well, place on ice for 15 min, centrifuge at 5000 rpm for 5 min;

2) Discard the supernatant, add 1 mL of PBS (Phosphate Buffered Saline, phosphate buffer), mix well, centrifuge at 5000 rpm for 5 min; repeat the operation three times, until the supernatant is clear, discard the supernatant;

3) Add 1 mL of Trizol reagent to the lower layer of precipitated cells, mix well, and let stand at room temperature for 5 min;

4) Add 200 μL of chloroform, mix well for 30 s, place at room temperature for 3 min, centrifuge at 10,000 rpm for 20 min;

5) Take 500 μL of the supernatant into a new PE tube, add 500 μL of isopropanol, mix and place at -80 □ overnight;

6) Remove the PE tube from -80 □, dissolve at room temperature, centrifuge at 10,000 rpm for 30 min, discard the supernatant, add 1 mL, 75% ethanol-DEPC water (V/V, DEPC is diethylpyrocarbonate), and mix thoroughly. , centrifuged at 7500 rpm for 10 min;

7) Discard the supernatant, precipitate at room temperature, and add 22 μL of DEPC water to dissolve to obtain total RNA solution, which is ready for use or -80 □.

(3) Synthesis of cDNA:

1) Take 2 μL of total RNA solution, 50-fold dilution, measure OD (absorbance) by ultraviolet spectrophotometer, calculate the concentration of total RNA extracted above, and determine OD260/280≥1.8 to determine purity;

2) Take 0.2 μg of total RNA and reverse transcription reagent, and perform the reaction according to the cDNA synthesis reaction system of Table 3; the reagent is a RevertAid First Strand cDNA Synthesis Kit of Thermo Scientific, USA, and the same type of products can be used by other companies;

Table 3: Reaction system for cDNA synthesis

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

(4) Sample quantitative detection

Using the sample cDNA as a template, the reaction was carried out in an ABI Prism 7300 real-time fluorescent PCR instrument using Taqman reaction method. The reaction system and reaction conditions are shown in Table 1. The PCR amplification conditions were: 95 □ predenaturation for 10 min, 95 □ denaturation for 10 s, 60 □ annealing for 30 s, 72 □ amplification for 30 s, and 45 cycles.

The expression levels of RECQL, RECQL4 and RECQL5 were detected by absolute quantitative method using standard curve. It was found that the expression levels of target genes RECQL, RECQL4 and RECQL5 in blood samples of breast cancer patients (normalized with internal reference) were significantly lower than normal samples. After surgery and chemoradiotherapy, the expression level can be restored to some extent. The results are shown in Figure 2.

It can be seen from Fig. 2 that compared with the normal control group, the expression of RECQL gene was significantly decreased in pre- and post-treatment patients (P<0.05); RECQL4 and RECQL5 genes also showed similar expression trends, and the expression differences were significant. (P<0.05). At the same time, it was found that the expression of three target genes increased in the samples of patients after surgery and chemoradiotherapy, and the expression of RECQL gene was significantly increased in patients after treatment (P<0.05). In summary, the expression of RECQL, RECQL4 and RECQL5 genes in peripheral blood mononuclear cells can be used as potential biomarkers for the diagnosis and evaluation of early breast cancer.

For the ability of the above three genes to evaluate the ability and efficacy of breast cancer patients, this study used the receiver operating characteristic (ROC) curve to evaluate. The ROC curve is an important tool for evaluating the accuracy of markers. The two main indicators that can be obtained include: sensitivity, or true positive rate, which is evaluated for performance in selecting a particular disease patient. High sensitivity is generally required in screening tests to rule out people without disease; specificity, or true negative rate, indicating their ability to correctly select a person without a disease. High specificity is generally required in the diagnosis to obtain a lower false positive rate. The ROC curve analysis was first performed on the three target genes, and the results are shown in Fig. 3. Among them, the abscissa is 100-specific, that is, the false positive rate, and the ordinate is the sensitivity, that is, the true positive rate.

As can be seen from Figure 3, the diagnostic accuracy of the three target genes in the normal control (50 cases) and breast cancer patients (49 cases) can reach more than 80%, RECQL and RECQL4 genes have higher specificity, RECQL5 gene It has a higher diagnostic sensitivity. Further research found that the integration of the three target genes in the kit significantly improved the diagnostic ability compared to a single gene. Firstly, by establishing a logistic regression model, the joint predictor is obtained. The expression of the predictor is: y=x1+42.016×x2+7.87×x3, where y represents the joint predictor, x1 represents the expression level of the sample RECQL gene, and x2 represents the sample. The expression level of the RECQL4 gene, x3 indicates the expression level of the sample RECQL5 gene. The results of the expression of the three target genes in each sample were taken into the predictor equation, and the predictors of each sample were obtained, and the ROC curve analysis of the double normal model was performed using the sample as the analysis index. The result is shown in 4, passing 3 The measured values of the genes were calculated as positive when the combined predictor y was less than 0.006, and diagnosed as breast cancer; when y was greater than 0.006, the sample was a normal sample.

It can be seen from Fig. 4 that the diagnostic sensitivity and specificity of the three target genes are improved after integration, and the sensitivity and specificity of the integrated predictive factors for breast cancer diagnosis are 80.00% and 95.83%, respectively, and under the ROC curve. The area was 0.941 (P<0.0001), indicating that the diagnosis of breast cancer was achieved after the integration of the three target genes with a diagnostic accuracy of 94.1%, which has clinical diagnostic value.

The ability of the three target genes determined for the kit to evaluate the efficacy of breast cancer was also analyzed using the ROC curve. The ROC curve analysis of the expression of three target genes in 49 pre-treatment breast cancer patients and 22 post-treatment breast cancer patients showed that the RECQL gene was the best for breast cancer patients (P<0.0001, significant). Sex), AUC reached 0.816, better than RECQL4 and RECQL5 genes, can be used as an auxiliary indicator for clinical efficacy evaluation. The results of the ROC curve analysis are shown in Figure 5.

In summary, RECQL, RECQL4 and RECQL5 can be used as biomarkers for early diagnosis of breast cancer, and the combined diagnostic accuracy is as high as 94.1%. However, the correlation between RECQL gene expression and curative effect in the evaluation of breast cancer patients' efficacy Preferably, the accuracy of the evaluation can reach 81.6%, especially the determination of the effectiveness of the treatment, which can reach an accuracy rate of 90.91%.

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

Example 6 Clinical urine sample test

(1) Clinical sample collection:

A total of 30 urine samples from healthy volunteers and 30 urine samples from breast cancer patients were collected in this study. The urine samples were all morning urine, and free RNA was extracted immediately after collection.

(2) Pretreatment of urine:

1) Immediately after collecting fresh urine, 12000 g, 4 ° C, and centrifugation for 10 minutes, the supernatant was collected.

2) The supernatant was again 12,000 g, centrifuged at 4 ° C for 10 minutes, and the supernatant was collected to completely remove the cellular components.

(3) Extraction of total RNA from urine supernatant:

Use the miRNeasy Serum/Plasma Kit (QIAGEN, other brands of the same type of products) kit to extract free RNA from the urine supernatant. The main steps are as follows:

1) After taking 350 μL of supernatant, add 5 volumes of QIAzol lysis reagent, vortex for 3-5 s, and lyse for 5 min at room temperature (15-25 ° C).

2) Add 3.5 μL of miRNeasy serum/plasma internal reference (concentration: 1.6×10 ⁸ copies/μL), mix, add 350 μL of chloroform, vortex for 3-5 s, and let stand for 3 min at room temperature.

3) 1200g, centrifuge at 15 °C for 15min, carefully pipet the supernatant into a new PV tube and add 1.5 times the volume of absolute ethanol, then mix.

4) Pipette 700 μL of the sample into a purification column, 8500 g, centrifuge at 4 ° C for 15 s, discard the centrate, and repeat the above steps for the remaining sample.

5) Add 700 μL of Buff RWT to the centrifugal purification cartridge, wash the cartridge, 8500 g, centrifuge at 4 ° C for 15 s, and discard the centrate.

6) Add 500 μL of 80% ethanol, 8500 g, and centrifuge at 2 ° C for 2 min in the purification cartridge. Place the purification cartridge in a new PV tube, open the cap of the cartridge, and centrifuge at full speed for 5 min to dry the cartridge.

7) Transfer the purification cartridge to a new PV tube, add 14 μL of RNase-free water to the center of the small membrane, gently cover and centrifuge at full speed for 1 min to elute RNA.

8) Store the collected RNA at -80 ° C and set aside.

(3) Method for synthesizing cDNA The method for synthesizing cDNA from blood is extracted.

(4) Sample quantitative detection

Using the sample cDNA as a template, the reaction was carried out in an ABI Prism 7300 real-time fluorescent PCR instrument using Taqman reaction method. The reaction system and reaction conditions are shown in Table 1. The amplification conditions of the PCR were: pre-denaturation at 95 ° C for 10 min, denaturation at 95 ° C for 10 s, annealing at 60 ° C for 30 s, and amplification at 72 ° C for 30 s for 45 cycles.

RECQL, RECQL4 and RECQL5 gene expression using absolute quantitative method for establishing a standard curve At the level of detection, it was found that only RECQL4 was detectable in normal human urine free nucleotides, and neither RECQL nor RECQL5 was detected, which was negative expression. In the urine samples of breast cancer patients, the target genes RECQL and RECQL5 were detected positively, and the expression results are shown in Figure 6. The content of RECQL4 was significantly lower in breast cancer patients than in normal controls (Figure 7). This result is consistent with the measurement of the gene in the blood of the patient. The results indicate that RECQL, RECQL4 and RECQL5 can be used as urine markers for the diagnosis of breast cancer, providing patients with a non-invasive method for the diagnosis of cancer.

The embodiments described above are merely preferred embodiments for the purpose of fully illustrating the invention, and the scope of the invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of the present invention. The scope of the invention is defined by the claims.

Claims (10)

1. The use of the RECQL, RECQL4 and RECQL5 genes as a combined marker in the preparation of a breast cancer detection and/or therapeutic evaluation reagent.
2. Specific RT-PCR primers and probes for breast cancer detection and/or therapeutic evaluation, characterized by specific amplification primers and probes including RECQL, RECQL4 and RECQL5 genes, and the nucleotide sequence of the probe 5' a fluorescent reporter group is attached, and a non- 5' end is linked to a fluorescence quenching group, wherein

   The nucleotide sequence of the RECQL amplification primer set is shown in SEQ ID NOS: 1 to 2,

   The nucleotide sequence of the RECQL-specific probe is shown in SEQ ID NO:3;

   The nucleotide sequence of the RECQL4 amplification primer set is shown in SEQ ID NOS: 4 to 5,

   The nucleotide sequence of the RECQL4-specific probe is shown in SEQ ID NO: 6;

   The nucleotide sequence of the RECQL5 amplification primer set is shown in SEQ ID NOS: 7-8.

   The nucleotide sequence of the RECQL5 specific probe is shown in SEQ ID NO: 9.
3. The specific RT-PCR primers and probes for breast cancer detection and/or therapeutic evaluation according to claim 2, further comprising specific amplification primers and probes for the internal reference gene GAPDH,

   The nucleotide sequence of the GAPDH amplification primer set is shown in SEQ ID NOs: 10 to 11,

   The nucleotide sequence of the GAPDH-specific probe is shown in SEQ ID NO:12;

   The nucleotide sequence of the probe is linked to a fluorescent reporter group at the 5' end and a fluorescence quencher group to the non- 5' end.
4. The specific RT-PCR primer and probe for breast cancer detection and/or therapeutic evaluation according to claim 2 or 3, wherein the nucleotide sequence of the probe is linked to the fluorescent reporter group FAM at the 5' end. A fluorescent quenching group TAMRA is attached to the 3' end.
5. A kit for detecting and/or evaluating a breast cancer, comprising the specific RT-PCR primer and probe for detecting breast cancer according to any one of claims 2 to 4.
6. The kit for detecting and/or evaluating a breast cancer according to claim 5, further comprising a positive template, wherein the positive template comprises purified: RECQL gene amplification product, RECQL4 gene amplification product, and RECQL5 gene amplification Product and GAPDH gene amplification products.
7. The kit for detecting and/or evaluating a breast cancer according to claim 6, wherein the real-time fluorescent quantitative PCR reaction system of the four genes RECQL, RECQL4, RECQL5 and GAPDH and the positive template of the four genes are used. System, where

   19.0 μL of real-time PCR system for each gene, including 10 μL of PCR reaction buffer, 0.8 μL of the gene amplification primer at a concentration of 10 μM, and a specific probe of 2 μM at a concentration of 2 μM 1.0 μL and RNase-free water 7.2 μL;

   The positive template system for each gene included a purified amplification product of the gene at a concentration of 1 x 10 ⁹ copies/μL.
8. A method of using a breast cancer detection and/or therapeutic evaluation kit according to claim 6 or 7, comprising the steps of:

   The positive templates of the four genes RECQL, RECQL4, RECQL5 and GAPDH were serially diluted and added to a real-time fluorescent quantitative PCR reaction system for fluorescence quantitative PCR reaction. The quantitative standard of each gene was drawn according to the diluted copy number and the amplified Ct value. curve;

   After the total RNA is extracted, the sample is reverse-transcribed into cDNA, and the cDNA is used as a fluorescent quantitative PCR template. After adding a real-time fluorescent quantitative PCR reaction system, a quantitative PCR reaction is performed, and the obtained amplified Ct value is brought into a quantitative standard curve of the corresponding gene. Obtaining an initial expression amount of the gene in the sample;

   The measurement result of the sample to be tested is normalized by the result of the internal reference gene GAPDH of the sample, and the combined prediction of the three marker genes RECQL, RECQL4 and RECQL5 is calculated according to the equation y=x1+42.016×x2+7.87×x3. Factor, where y represents the joint predictor, x1 represents the expression level of the sample RECQL gene, x2 represents the expression level of the sample RECQL4 gene, and x3 represents the expression level of the sample RECQL5 gene;

   When the y is less than 0.006, the sample to be tested is positive, and when y is greater than 0.006, the sample to be tested is negative.
9. The method for detecting a breast cancer detecting and/or therapeutic effect evaluation kit according to claim 8, wherein the fluorescent quantitative PCR reaction conditions are: pre-denaturation at 95 ° C for 10 min; denaturation at 95 ° C for 10 s, annealing at 60 ° C for 30 s, 72 ° C Amplification was carried out for 30 s for 45 cycles.
10. A method of using a breast cancer detection and/or therapeutic evaluation kit according to claim 8 or 9, wherein the sample to be detected is peripheral blood and/or urine.

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