CN111041092A - Application of reagent for detecting expression level of Fas associated factor family member 2 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 the expression level of a Fas associated factor family member 2 and a kit. More particularly, the application of the reagent for detecting the expression level of FAF2 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 are provided. The detection result of a clinical sample shows that the expression of FAF2 in breast cancer is obviously increased compared with that of a tissue beside the cancer; and the high expression of FAF2 is not beneficial to the 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 the expression level of Fas associated factor family member 2(FAF2) 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 (BC) is the most common gynecological malignancy. The incidence rate in China reaches 25.89/10 ten thousand, and accounts for about 16.83 percent of the incidence rate of all female malignant tumors. Moreover, the proportion of the number of breast cancer patients and the number of newly diagnosed patients in China is increasing year by year. With the increase of the levels of surgical treatment and radiotherapy and chemotherapy, the survival time of breast cancer patients is obviously prolonged, but the overall morbidity and mortality are still the first female tumor. In recent years, with the rapid increase of high-throughput and multigroup data, some novel markers are found to be related to the occurrence and prognosis of breast cancer, and some novel potential targets with anti-breast cancer druggability are suggested, so that the possibility is provided for breast cancer typing, accurate prognosis and breast cancer treatment.
Disclosure of Invention
The invention aims to provide a novel breast cancer prognostic marker Fas associated factor family member 2(FAF2), and further provides application of a reagent for detecting the expression level of FAF2 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.
FAF2, also known as UBXN3B, is a protein-encoding gene, located at 5q35.2, containing 11 exons, GeneID:23197, which encodes a protein that is predominantly located in the endoplasmic reticulum, extracellularly, and lysosomes. At present, no report is found about the role of FAF2 gene in the development of breast cancer. The inventor discovers that the high expression of FAF2 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 FAF2 expression difference on breast Cancer patients through collecting clinical breast Cancer patient samples and follow-up information.
In order to achieve the above object, the first aspect of the present invention provides the use of an agent for detecting the expression level of Fas associated factor family member 2(FAF2) for the preparation of a preparation for use in the auxiliary diagnosis of breast cancer and/or the prognostic judgment of breast cancer patients.
Further, the detecting of the expression level of the FAF2 comprises detecting the expression level of a gene of FAF2 and/or detecting the expression level of a protein of FAF 2.
More specifically, the method for detecting the expression level of FAF2 comprises the following steps: detecting the expression level of FAF2 in breast cancer and para-carcinoma tissues by an RT-qPCR method; detecting the expression quantity of FAF2mRNA in breast cancer and tissues beside the breast cancer by a molecular probe technology; the FAF2 protein expression quantity in breast cancer and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the expression level of FAF2 is an oligonucleotide probe targeting FAF2 coding DNA sequence, a PCR primer or an antibody targeting FAF 2.
According to the present invention, preferably, the reagent for detecting the expression level of FAF2 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.
5’-TGTCGCCATACCTTGGAACA-3’，SEQ ID NO：1；
5’-TCGTGATGGAGGTGGGTTGA-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: and (3) reagents for detecting the expression level of FAF 2.
Further, the reagent for detecting the expression level of FAF2 is an oligonucleotide probe targeting FAF2 coding DNA sequence, a PCR primer or an antibody targeting FAF 2.
Specifically, the reagent for detecting the expression level of FAF2 is a reagent with 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.
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 FAF2 in the breast cancer is obviously increased compared with that in a para-carcinoma tissue (P < 0.001); and high expression of FAF2 is detrimental to overall survival of breast cancer patients (P ═ 0.0062). 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.
Drawings
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.
Fig. 1 shows TCGA high throughput data analysis of FAF2 expression in breast cancer. FAF2 expression was significantly higher in breast cancer tissues than in normal tissues (P < 0.001).
Figure 2 shows TCGA high throughput data analysis of the effect of high expression of FAF2 on survival of breast cancer patients. High expression of FAF2 is detrimental to overall survival of breast cancer patients (P0.006947).
Fig. 3 shows the expression of FAF2 in breast cancer tissues. FAF2 expression was significantly higher in breast cancer tissues than in paracarcinoma tissues (P < 0.001).
Figure 4 shows the effect of high expression of FAF2 in breast cancer tissues on survival of breast cancer patients. High expression of FAF2 is detrimental to overall survival in breast cancer patients (P ═ 0.0062).
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 FAF2 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 "FAF 2", 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 FAF2 was significantly elevated in breast cancer tissues compared to normal tissues (P <0.001), as shown in figure 1.
Example 2
This example illustrates the relationship between high expression of FAF2 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-scenes (RNA Seq V2 RSEM)" was selected, gene input "FAF 2: 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 FAF2 was detrimental to overall survival in breast cancer patients (P0.006947) (fig. 2).
Example 3
This example illustrates the preparation of reagents for detecting the expression level of FAF2 for the preparation of a kit (50 reactions) for the prognosis of breast cancer patients.
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 FAF2 real-time fluorescent quantitative PCR specific primer 30.0 μ l, the primer sequence is shown in Table 1;
13.10.0 μ M ACTB real-time fluorescent quantitation PCR specific primers 30.0 μ l, the primer sequences are shown in Table 1.
TABLE 1 fluorescent quantitative RT-PCR primer sequences
Example 4
This example illustrates the detection of FAF2 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 level of FAF 2: the experiment detects the relative expression change of FAF2 in 57 breast cancer tissues and 18 paracarcinoma tissues. ACTB as reference gene, and the target gene FAF2C 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), the relative expression of FAF2 in each group was calculated by using the Power function in the Excell table. Differential expression of FAF2 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 FAF2 with prognosis for 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 relative expression level of FAF2 was high, 2-fold higher than the mean relative expression level of para-carcinoma tissues, for a total of 7 cases, while the other cases were low, for a total of 50 cases, FAF 2. 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 FAF2 in the breast cancer is obviously increased compared with that in a para-carcinoma tissue (P <0.001), as shown in figure 3; and high expression of FAF2 was detrimental to overall survival in breast cancer patients (P ═ 0.0062), 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
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Claims (9)
1. Use of a reagent for detecting the expression level of Fas associated factor family member 2(FAF2) in the preparation of a formulation for use in the auxiliary diagnosis of breast cancer and/or the prognostic judgment of breast cancer patients.
2. The use according to claim 1, wherein the detecting of the expression level of FAF2 comprises detecting the gene expression level of FAF2 and/or detecting the protein expression level of FAF 2.
3. The use according to claim 1, wherein the method for detecting the expression level of FAF2 comprises: detecting the expression level of FAF2 in breast cancer and para-carcinoma tissues by an RT-qPCR method; detecting the expression level of FAF2mRNA in breast cancer and tissues beside the breast cancer by a molecular probe technology; the FAF2 protein expression quantity in breast cancer and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
4. The use of claim 1, wherein the reagent for detecting the expression level of FAF2 is an oligonucleotide probe targeting a DNA sequence encoding FAF2, a PCR primer, or an antibody targeting FAF 2.
5. The use of claim 4, wherein the reagent for detecting the expression level of FAF2 is a reagent with 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.
6. A kit for use in breast cancer assisted diagnosis and/or prognosis of a breast cancer patient, the kit comprising: and (3) reagents for detecting the expression level of FAF 2.
7. The kit according to claim 6, wherein the reagent for detecting the expression level of FAF2 is an oligonucleotide probe targeting a DNA sequence encoding FAF2, a PCR primer, or an antibody targeting FAF 2.
8. The kit according to claim 7, wherein the reagent for detecting the expression level of FAF2 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.
9. The kit of claim 6, wherein 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.
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| CN112410346A (en) * | 2020-11-30 | 2021-02-26 | 中国计量大学 | Method for improving insect killing toxicity of biocontrol fungi |
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|---|---|---|---|---|
| WO2006135886A2 (en) * | 2005-06-13 | 2006-12-21 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
| CN101801419A (en) * | 2007-06-08 | 2010-08-11 | 米尔纳疗法公司 | Gene and path as the miR-34 regulation and control for the treatment of the target of intervening |
| US20190161809A1 (en) * | 2016-06-20 | 2019-05-30 | Istituto Europeo Di Oncologia (Ieo) | Methods and kits comprising gene signatures for stratifying breast cancer patients |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006135886A2 (en) * | 2005-06-13 | 2006-12-21 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
| CN101801419A (en) * | 2007-06-08 | 2010-08-11 | 米尔纳疗法公司 | Gene and path as the miR-34 regulation and control for the treatment of the target of intervening |
| US20190161809A1 (en) * | 2016-06-20 | 2019-05-30 | Istituto Europeo Di Oncologia (Ieo) | Methods and kits comprising gene signatures for stratifying breast cancer patients |
Non-Patent Citations (1)
| Title |
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| TREPTE P等: "Homo sapiens Fas associated factor family member 2（FAF2），mRNA", 《NCBI GENEBANK：NM_014613.3》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112410346A (en) * | 2020-11-30 | 2021-02-26 | 中国计量大学 | Method for improving insect killing toxicity of biocontrol fungi |
| CN112410346B (en) * | 2020-11-30 | 2023-07-25 | 中国计量大学 | Method for improving biocontrol fungus insecticidal toxicity |
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