CN110358833B - Application of reagent for detecting expression level of Tudor structural domain protein 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 Tudor structural domain protein 2 and a kit. More particularly, the application of the reagent for detecting the TDRKH expression level 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 TDRKH expression in the breast cancer is obviously increased compared with that of a tissue beside the cancer; and the high expression of TDRKH is not beneficial to the overall survival of the 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 Tudor structural domain protein 2(TDRKH) 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 a malignant lesion of abnormal differentiation of mammary gland epithelial tissues, is the most common malignant tumor of women, and the morbidity and mortality of the breast cancer are ranked first in female cancers. With the progress of research, remarkable progress has been made in the diagnosis and treatment thereof, and it is no longer considered as a disease but a disease having a plurality of heterogeneous subtypes. The proposal of molecular typing enables the individualized treatment of the breast cancer to enter the classified treatment era, and the proposal of prognosis staging enables the individualized treatment of the breast cancer to realize the transition from quality to quality and from macro to micro. With the rapid increase of multigroup high-throughput data, some molecular biological markers are found to be related to the occurrence and prognosis of breast cancer, which makes it possible to diagnose breast cancer more accurately and effectively. At present, the development of new breast cancer treatment targets and prognostic markers is still urgently needed in clinic.
Disclosure of Invention
The invention aims to provide a novel Tudor domain protein 2(TDRKH) as a breast cancer prognostic marker, and further provides application of a reagent for detecting the expression level of the TDRKH 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.
TDRKH, also known as TDRD2, is a protein-coding Gene, located at 1q21.3, containing 16 exons, Gene ID:11022, which encodes a protein predominantly located in the mitochondria and cytoplasm. At present, the TDRKH gene has no reported role in the occurrence and development of breast cancer. The inventor discovers that the high expression of TDRKH is not beneficial to the prognosis of a breast Cancer patient through high-throughput data mining of Cancer Genome Atlas (TCGA), and further verifies the prognostic effect of TDRKH expression difference on the breast Cancer patient 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 a use of an agent for detecting Tudor domain protein 2(TDRKH) expression level in the preparation of a preparation for breast cancer auxiliary diagnosis and/or breast cancer patient prognosis judgment.
Further, the detecting the expression level of TDRKH comprises detecting the gene expression level of TDRKH and/or detecting the protein expression level of TDRKH.
More specifically, the method for detecting the expression level of TDRKH comprises the following steps: detecting the expression quantity of TDRKH in breast cancer and tissues beside the cancer by an RT-qPCR method; detecting the expression quantity of TDRKH mRNA in breast cancer and tissues beside the breast cancer by a molecular probe technology; detecting the expression level of the TDRKH protein in the breast cancer and the tissues beside the cancer by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the TDRKH expression level is an oligonucleotide probe targeting TDRKH coding DNA sequence, a PCR primer or an antibody targeting TDRKH.
According to the present invention, preferably, the reagent for detecting the expression level of TDRKH 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’-CGCAAACAGCCAATCAGTGT-3’，SEQ ID NO：1；
5’-CCTTTGGGTGGAGGAGTCAC-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 TDRKH.
Further, the reagent for detecting the TDRKH expression level is an oligonucleotide probe targeting TDRKH coding DNA sequence, a PCR primer or an antibody targeting TDRKH.
Specifically, the reagent for detecting the TDRKH expression level 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 TDRKH expression in the breast cancer is obviously increased compared with that in a tissue beside the cancer (P < 0.001); and high expression of TDRKH is not beneficial to the overall survival of breast cancer patients (P ═ 0.0222). 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 TDRKH expression in breast cancer. TDRKH expression in breast cancer tissues is significantly higher than that in normal tissues (P < 0.001).
FIG. 2 shows the effect of TCGA high throughput data on TDRKH high expression on survival of breast cancer patients. High TDRKH expression is detrimental to the overall survival of breast cancer patients (P0.0102).
Figure 3 shows the expression of TDRKH in breast cancer tissue. TDRKH expression in breast cancer tissues is remarkably higher than that in paracarcinoma tissues (P < 0.001).
FIG. 4 shows the effect of high TDRKH expression in breast cancer tissues on survival of breast cancer patients. High expression of TDRKH is detrimental to the overall survival of breast cancer patients (P ═ 0.0222).
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 is presented to demonstrate the TCGA high throughput data analysis of the expression changes of TDRKH 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 "Analysis", enter the Gene name "TDRKH ", selecting TCGA dataset" Breast innovative cardio ", searching, clicking" Expression ", and recording 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: the expression of TDRKH in breast cancer tissues is significantly increased compared with normal tissues (P <0.001), as shown in FIG. 1.
Example 2
This example illustrates the relationship between high TDRKH expression 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 /), the tumor dataset "Breast Invasive Carcinoma (TCGA Provisional)", the omics dataset "mRNA Expression z-scenes (RNA Seq V2 RSEM)", the gene input "TDRKH: 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 TDRKH was detrimental to overall survival in breast cancer patients (P0.0102) (fig. 2).
Example 3
This example illustrates the preparation of reagents for measuring the expression level of TDRKH 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;
8.10.0mM 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;
12.10.0 μ M TDRKH 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 TDRKH 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 TDRKH by the method: the experiment detects the relative expression change of TDRKH in 57 breast cancer tissues and 18 paracarcinoma tissues. ACTB as reference gene, and the target gene TDRKH 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 amount of TDRKH in each group by using a Power function in an Excell table. The difference of TDRKH expression between breast cancer and paracarcinoma was analyzed by using GraphPad Prism 6.0 mapping software and T test<A difference of 0.05 is statistically significant.
High expression of TDRKH 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 total 15 cases of TDRKH with the relative expression amount 2 times higher than that of the para-carcinoma tissues show high expression, and the total 42 cases of TDRKH with the other low expression. 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 TDRKH expression in the breast cancer is obviously increased (P <0.001) compared with that in a tissue beside the cancer, as shown in figure 3; and high expression of TDRKH is detrimental to overall survival of breast cancer patients (P ═ 0.0222), 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 (5)
1. Application of the reagent for detecting the TDRKH expression level in preparing a preparation for breast cancer auxiliary diagnosis and/or breast cancer patient prognosis judgment.
2. Use according to claim 1, wherein the detection of the expression level of TDRKH comprises detection of the gene expression level of TDRKH and/or detection of the protein expression level of TDRKH.
3. The use according to claim 1, wherein the method for detecting the expression level of TDRKH comprises: detecting the expression quantity of TDRKH in breast cancer and tissues beside the cancer by an RT-qPCR method; detecting the expression quantity of TDRKH mRNA in breast cancer and tissues beside the breast cancer by a molecular probe technology; detecting the expression level of the TDRKH protein in the breast cancer and the tissues beside the cancer by immunohistochemistry or Western-Blot.
4. Use according to claim 1, wherein the agent for detecting the expression level of TDRKH is an oligonucleotide probe targeting the DNA sequence of TDRKH, a PCR primer, or an antibody targeting TDRKH.
5. The use according to claim 4, wherein the reagent for detecting the expression level of TDRKH is 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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Citations (2)
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| CN101273144A (en) * | 2005-07-27 | 2008-09-24 | 肿瘤疗法科学股份有限公司 | Method of diagnosing esophageal cancer |
| KR20160087245A (en) * | 2015-01-13 | 2016-07-21 | 오동엽 | Single nucleotide polymorphism marker in TDRKH gene for diagnosis of meat quality in Hanwoo and method for diagnosis of meat quality in Hanwoo using same marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101273144A (en) * | 2005-07-27 | 2008-09-24 | 肿瘤疗法科学股份有限公司 | Method of diagnosing esophageal cancer |
| KR20160087245A (en) * | 2015-01-13 | 2016-07-21 | 오동엽 | Single nucleotide polymorphism marker in TDRKH gene for diagnosis of meat quality in Hanwoo and method for diagnosis of meat quality in Hanwoo using same marker |
Non-Patent Citations (2)
| Title |
|---|
| 1、MiR-34c/CTNND1信号通路是化疗药物抑制肝癌进展的重要靶点 2、RNA结合蛋白DCAF13/TDRKH在肝癌中的异常表达及其临床意义;曹建中;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20180115(第1期);第66页二、实验方法 * |
| RNA sequencing identifies specific PIWI-interacting small non-coding RNA expression patterns in breast cancer;Hashim Adnan等;《Oncotarget》;20140916;第5卷(第20期);第9901-9910页 * |
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Effective date of registration: 20220518 Address after: 266000 No. 1, Huanghai Road, Shuiji sub district office, Laixi City, Qingdao City, Shandong Province Patentee after: Qingdao Yanding Biomedical Technology Co.,Ltd. Address before: 224008 no.283, Jiefang South Road, Yancheng City, Jiangsu Province Patentee before: JIANGSU VOCATIONAL College OF MEDICINE |