CN111041099B - Application of reagent for detecting expression level of G protein-coupled receptor 137B and kit - Google Patents
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Abstract
The invention belongs to the technical field of biology, and relates to application of a reagent for detecting GPR137B expression level and a kit. More particularly, the invention relates to the application of a reagent for detecting the expression level of GPR137B in the preparation of a preparation for breast cancer auxiliary diagnosis and/or breast cancer patient prognosis and a kit for breast cancer auxiliary diagnosis and/or breast cancer patient prognosis. The detection result of a clinical sample shows that the expression of GPR137B in the breast cancer is obviously increased compared with that in a paracancerous tissue; and high expression of GPR137B is detrimental to overall survival of breast cancer patients. Therefore, the reagent for detecting the change of the gene expression can be used for the prognosis or diagnosis and treatment of the breast cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a reagent for detecting an expression level of a G protein-coupled receptor 137B (GPR137B) in preparation of a preparation for auxiliary diagnosis of breast cancer and/or prognosis of a breast cancer patient, and a kit for auxiliary diagnosis of breast cancer and/or prognosis of a breast cancer patient.
Background
Breast cancer is the most common malignancy in women, with up to 170 million breast cancer patients newly diagnosed worldwide each year, with approximately 522000 deaths from breast cancer, second only to lung cancer, on 2012 epidemiological statistics in developed countries. Breast cancer accounts for 25% of all female tumors, and 15% of all tumor-associated deaths. In China, 278800 new breast cancer cases are detected in 2013, the morbidity is 42.02/100000 according to the standard of Chinese population, the estimated death number is 64600, and the mortality is 9.74/100000. The incidence and mortality of breast cancer are increasing in China with the change of social and economic factors. With the understanding of the biological mechanism of the occurrence and development of breast cancer, tumor targeted therapy, endocrine therapy and the like play an important role in the treatment of breast cancer. With the development of molecular typing and precise medicine, the research and discovery of new key target genes of breast cancer have important significance for the individualized diagnosis and treatment of breast cancer.
Disclosure of Invention
The invention aims to provide a novel breast cancer prognosis marker G protein-coupled receptor 137B (GPR137B), and further provides application of a reagent for detecting the expression level of GPR137B in preparation of a preparation for auxiliary diagnosis of breast cancer and/or prognosis of a breast cancer patient and a kit for auxiliary diagnosis of breast cancer and/or prognosis of the breast cancer patient.
GPR137B is a protein-encoding Gene, located at 1q42.3, containing 7 exons, Gene ID:7107, which encodes a protein that is localized to cell membranes and lysosomes. At present, no report is found about the role of GPR137B gene in the development of breast cancer. The inventor discovers that the high expression of GPR137B is not beneficial to the prognosis of breast Cancer patients through high-throughput data mining of Cancer and tumor gene maps (TCGA), and further verifies the prognostic effect of GPR137B expression difference on breast Cancer patients through collecting clinical breast Cancer patient samples and follow-up information.
In order to achieve the above objects, the first aspect of the present invention provides the use of an agent for detecting the expression level of G protein-coupled receptor 137B (GPR137B) for the preparation of a preparation for use in the auxiliary diagnosis of breast cancer and/or the prognosis of a breast cancer patient.
Further, detecting the expression level of GPR137B comprises detecting the gene expression level of GPR137B and/or detecting the protein expression level of GPR 137B.
More specifically, the method for detecting the expression level of GPR137B comprises the following steps: detecting the expression quantity of GPR137B in breast cancer and paracarcinoma tissues by an RT-qPCR method; detecting the expression quantity of GPR137B mRNA in breast cancer and paracarcinoma tissues by a molecular probe technology; the expression level of GPR137B protein in breast cancer and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the expression level of GPR137B is an oligonucleotide probe targeting GPR137B coding DNA sequence, a PCR primer or an antibody targeting GPR 137B.
According to the invention, preferably, the reagent for detecting the expression level of GPR137B is a reagent with the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
5’-ACAAAGGACCTTACCAACCCT-3’，SEQ ID NO：1
5’-CTGGAGCAAAACCTCCCTGA-3’，SEQ ID NO：2
The second aspect of the present invention provides a kit for breast cancer auxiliary diagnosis and/or breast cancer patient prognosis, which comprises: reagents for detecting the expression level of GPR 137B.
Further, the reagent for detecting the expression level of GPR137B is an oligonucleotide probe targeting GPR137B coding DNA sequence, a PCR primer or an antibody targeting GPR 137B.
Specifically, the reagent for detecting the expression level of GPR137B is a reagent with the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
According to the present invention, the kit may further contain other conventional reagents for real-time fluorescence quantification, and preferably, the kit further comprises at least one of the following components: trizol, isopropanol, chloroform, absolute ethanol, RNase-free water, random primers, 5 XM-MLV buffer, dNTPs, RNase inhibitor, M-MLV reverse transcriptase, a primer having the sequence of SEQ ID NO: 3 and SEQ ID NO: 4, and (4) ACTB real-time fluorescence quantitative PCR specific primers of the nucleotide sequence shown in the specification.
5’-GGCACCCAGCACAATGAAGA-3’，SEQ ID NO：3
5’-ACTCCTGCTTGCTGATCCAC-3’，SEQ ID NO：4
The detection result of a clinical sample shows that the expression of GPR137B in the breast cancer is obviously increased compared with that in a paracancerous tissue (P < 0.001); and high expression of GPR137B is detrimental to overall survival of breast cancer patients (P ═ 0.0377). Therefore, the reagent for detecting the change of the gene expression can be used for the prognosis or diagnosis and treatment of the breast cancer.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
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The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
Figure 1 shows TCGA high throughput data analysis of GPR137B expression in breast cancer. GPR137B expression was significantly higher in breast cancer tissues than in normal tissues (P < 0.001).
FIG. 2 shows the effect of TCGA high-throughput data analysis of the high expression of GPR137B on survival of breast cancer patients. High expression of GPR137B is detrimental to overall survival in breast cancer patients (P < 0.001).
Figure 3 shows the expression of GPR137B in breast cancer tissues. GPR137B expression was significantly higher in breast cancer tissues than in paracarcinoma tissues (P < 0.001).
Figure 4 shows the effect of high expression of GPR137B in breast cancer tissues on survival of breast cancer patients. High expression of GPR137B is detrimental to overall survival in breast cancer patients (P ═ 0.0377).
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
This example demonstrates TCGA high throughput data analysis of changes in expression of GPR137B in breast cancer.
TCGA high throughput data analysis process:
log-in TCGA Portal website UALCAN ((TM))http://ualcan.path.uab.edu/index.html) First page, click on "Analysis", enter the gene name "GPR 137B", select TCGA dataset "Breast innovative carcinoma", search, click on "Expression", record the result. And (3) mapping by using GraphPad 12.0 software, wherein the statistical method is T test, and the difference is statistically significant when P is less than 0.05.
2. As a result: expression of GPR137B was significantly elevated in breast cancer tissues compared to normal tissues (P <0.001) as shown in fig. 1.
Example 2
This example illustrates the relationship between high expression of GPR137B and breast cancer prognosis in TCGA high-throughput data analysis.
TCGA high throughput data analysis process:
the TCGA Portal site cBioPortal (http:// www.cbioportal.org /), tumor dataset "Breast Invasive Carcinoma (TCGA Provisional)" was selected, omics dataset "mRNA Expression z-Scores (RNA Seq V2 RSEM)" was selected, and gene input "GPR 137B: and EXP > 2 ', searching, clicking ' survivval ' in a pop-up page, and recording a result. The Kaplan-Meier method draws a survival curve, and log-rank tests the difference of the survival curve, wherein the difference has statistical significance when P is less than 0.05.
2. As a result: high expression of GPR137B was detrimental to overall survival (P <0.001) in breast cancer patients (FIG. 2).
Example 3
This example illustrates the preparation of reagents for measuring the expression level of GPR137B for the preparation of a kit for the prognosis of breast cancer patients (50 reactions).
1.Trizol 50.0ml；
2. 50.0ml of isopropanol;
3. 50.0ml of chloroform;
4. 50.0ml of absolute ethyl alcohol;
5. RNase-free water 5.0ml
6.1.0. mu.M Random primers (Random primers) 50.0. mu.l;
7.5 XM-MLV buffer 2.0 ml;
8.10.0 mM deoxynucleotide triphosphate (dNTPs) 100.0. mu.l;
9.40U/. mu.l RNase inhibitor 50.0. mu.l;
10.200U/. mu. l M-MLV reverse transcriptase 50.0. mu.l;
11.ABI 2×PCR Mix 2.0ml；
12.10.0 μ M GPR137B real-time fluorescent quantitative PCR specific primer 30.0 μ l, the primer sequence is shown in Table 1;
13.10.0 mu M ACTB real-time fluorescent quantitative PCR specific primer 30.0 mu l, the primer sequence is shown in Table 1;
TABLE 1 fluorescent quantitative RT-PCR primer sequences
Example 4
This example illustrates the testing of GPR137B in a clinical breast cancer tissue sample.
1. The study was performed with patient informed consent. Clinical information from 57 breast cancer patients was obtained from patient visit records. Breast cancer specimens are divided into two parts: one fraction was immediately frozen in liquid nitrogen and stored at-80 ℃ until RNA extraction, and the other fraction was used for histopathological evaluation.
2. Extraction of total RNA from tissues: the experiment was performed in an ice bath. Placing 30-50 mg of tissue (fresh or tissue preserved at-70 ℃ in liquid nitrogen) into a 1.5ml centrifuge tube, adding 1ml Trizol, homogenizing fully, and standing at room temperature for 5 min; adding 200 μ l chloroform into each tube, mixing vigorously for 30sec, standing for 15min, and centrifuging at 4 deg.C and 12000rpm for 15 min; gently sucking 400 μ l of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 12000rpm at 4 ℃ for 10 min; discarding the supernatant, adding 1ml of 75% alcohol to wash the precipitate, and centrifuging at 4 ℃ and 12000rpm for 10 min; the supernatant was discarded as much as possible, air-dried at room temperature for 10min, and dissolved by adding 10. mu.l of RNase-free water to each tube (10-15 min. lysis-promoting at 65 ℃). OD260 was measured and the RNA concentration was calculated.
RNA(mg/ml)＝40×OD260X dilution multiple (n)/1000
3. Reverse transcription: each 25. mu.l reverse transcription system contained 100pmol of random primer, 2. mu.g of total RNA, 1. mu.l of M-MLV reverse transcriptase, 0.625. mu.l of RNase inhibitor, 1.25. mu.l of dNTPs (10mM), 5. mu.l of 5 XM-MLV buffer, and a 25. mu.l of RNase-free water. The reaction conditions are as follows: 1h at 37 ℃ and 5min at 95 ℃.
4. Quantitative PCR: each 20. mu.l reaction system contained 2 XPCR Mix 10. mu.l, upstream and downstream primers 0.4. mu.l each, cDNA 1. mu.l, ddH2O8.2. mu.l. The reaction conditions are as follows: 94 ℃ for 2min, 94 ℃ for 15s, 60 ℃ for 40s, 40 cycles.
5.2-ΔΔCTCalculating the relative expression quantity of GPR 137B: the experiment examined the relative expression level changes of GPR137B in 57 breast cancer tissues and 18 paracarcinoma tissues. ACTB as reference gene, the target gene GPR137B C measured by qPCRTC of reference gene ACTB with the same value as that of the tissue sourceTSubtracting the values to obtain Δ CTThen, Δ C is addedTAnd control group Δ CTSubtracting to obtain Δ Δ CT(taking a paracarcinoma sample Δ CTHas an average value of Δ CTControl), and calculating the relative expression of GPR137B in each group by using the Power function in an Excell table. Differential expression of GPR137B between breast and paracarcinoma, P, was analyzed using a software GraphPad Prism 6.0 mapping, T-test<A difference of 0.05 is statistically significant.
High expression of GPR137B and prognosis of breast cancer patients: the follow-up time of the patients is 1-32 months, and the number of successfully received follow-up patients is 57. The expression level of GPR137B was high, 2 times higher than that of the paracarcinoma tissues, and 9 cases were total, while the expression level of GPR137B was low, and 48 cases were total. The Kaplan-Meier method draws a survival curve, and log-rank tests the difference of the survival curve, wherein the difference has statistical significance when P is less than 0.05.
7. As a result:
the detection result of a clinical sample shows that the expression of GPR137B in the breast cancer is obviously increased compared with that in a paracancerous tissue (P <0.001), as shown in a figure 3; and high expression of GPR137B was detrimental to overall survival in breast cancer patients (P ═ 0.0377), as shown in fig. 4.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
<110> Jiangsu medical profession college
<120> application of reagent for detecting expression level of G protein-coupled receptor 137B and kit
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Claims (5)
1. Use of an agent for detecting the expression level of G protein-coupled receptor 137B (GPR137B) in the preparation of a formulation for the prognostic determination of breast cancer patients.
2. The use of claim 1, wherein the detecting the expression level of GPR137B comprises detecting the gene expression level of GPR137B and/or detecting the protein expression level of GPR 137B.
3. The use of claim 1, wherein the method for detecting the expression level of GPR137B comprises: detecting the expression quantity of GPR137B in breast cancer and paracarcinoma tissues by an RT-qPCR method; detecting the expression quantity of GPR137B mRNA in breast cancer and paracarcinoma tissues by a molecular probe technology; the expression level of GPR137B protein in breast cancer and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
4. The use of claim 1, wherein the agent for detecting the expression level of GPR137B is an oligonucleotide probe targeting GPR137B encoding DNA sequence, a PCR primer, or an antibody targeting GPR 137B.
5. The use of claim 4, wherein the agent for detecting the expression level of GPR137B is a reagent having the sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
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