CN110358832B - Application of reagent for detecting expression level of PH domain family A member 6 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 PH domain family A member 6 and a kit. More particularly, relates to the application of a reagent for detecting the expression level of PLEKHA6 in the preparation of a preparation for the auxiliary diagnosis of breast cancer and/or the prognosis of a breast cancer patient and a kit for the auxiliary diagnosis of breast cancer and/or the prognosis of the breast cancer patient. The detection result of a clinical sample shows that the expression of PLEKHA6 in the breast cancer is obviously increased compared with that of a tissue beside the cancer; and the high expression of PLEKHA6 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 PH domain family A member 6(PLEKHA6) 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 gynecological malignancy. Global tumor epidemiological data show that 1677000 new cases of breast cancer are second to lung cancer in total 1677000 worldwide in 2012, and are the highest malignant tumors in women with highest morbidity and mortality. The incidence rate of breast cancer in China is up to 25.89/10 ten thousand, and accounts for about 16.83 percent of the incidence rate of malignant tumors of all women. Moreover, the proportion of the number of breast cancer patients and the number of newly diagnosed patients in China is increasing year by year, and the patients show a trend of younger onset, which seriously affects the physical and mental health of women and even endangers life. Therefore, the study of pathogenesis and prognosis of breast cancer is of far-reaching importance to breast cancer patients. The breast cancer shows great complexity and heterogeneity in the aspects of cell origin, histological morphology, disease grading, clinical representation, treatment response, metastatic potential and the like, and the application of the breast cancer prognosis marker is limited. Therefore, there is an urgent need to develop more targeted breast cancer prognostic markers to meet clinical needs.
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.
Disclosure of Invention
The invention aims to provide a novel breast cancer prognosis marker PH domain family A member 6(PLEKHA6), and further provides application of a reagent for detecting the expression level of PLEKHA6 in preparing 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.
PLEKHA6, also known as PEPP3, is a protein-encoding Gene, located at 1q32.1, containing 36 exons, Gene ID:22874, which encodes a protein that is predominantly localized in the cytoplasm and nucleus. At present, no report is found about the role of the PLEKHA6 gene in the occurrence and development of breast cancer. The inventor discovers that the high expression of PLEKHA6 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 PLEKHA6 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 a reagent for detecting the expression level of PH domain family a member 6(PLEKHA6) in the preparation of a preparation for use in the auxiliary diagnosis of breast cancer and/or the prognostic judgment of breast cancer patients.
Further, said detecting the expression level of PLEKHA6 comprises detecting the gene expression level of PLEKHA6 and/or detecting the protein expression level of PLEKHA 6.
More specifically, the method for detecting the expression level of the PLEKHA6 comprises the following steps: detecting the expression level of PLEKHA6 in breast cancer and paracarcinoma tissues by an RT-qPCR method; detecting the expression quantity of PLEKHA6mRNA in breast cancer and tissues beside the breast cancer by a molecular probe technology; the expression level of the PLEKHA6 protein in breast cancer and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the expression level of the PLEKHA6 is an oligonucleotide probe targeting a PLEKHA6 coding DNA sequence, a PCR primer or an antibody targeting PLEKHA 6.
According to the invention, preferably, the reagent for detecting the expression level of PLEKHA6 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.
5’-AGGCAAGAGGTAGAGGCAGA-3’，SEQ ID NO：1；
5’-GGAGAGTCCATTGGTGAGCC-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 PLEKHA 6.
Further, the reagent for detecting the expression level of the PLEKHA6 is an oligonucleotide probe targeting a PLEKHA6 coding DNA sequence, a PCR primer or an antibody targeting PLEKHA 6.
Specifically, the reagent for detecting the expression level of the PLEKHA6 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 the PLEKHA6 in the breast cancer is obviously increased compared with that in a para-carcinoma tissue (P < 0.001); and the high expression of PLEKHA6 is not beneficial to the overall survival of breast cancer patients (P ═ 0.0285). 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.
Fig. 1 shows TCGA high-throughput data analysis of the expression of PLEKHA6 in breast cancer. The expression of PLEKHA6 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 PLEKHA6 on survival of breast cancer patients. High expression of PLEKHA6 was detrimental to overall survival of breast cancer patients (P ═ 0.0059).
Figure 3 shows the expression of PLEKHA6 in breast cancer tissue. The expression of PLEKHA6 was significantly higher in breast cancer tissues than in paracarcinoma tissues (P < 0.001).
Figure 4 shows the effect of high expression of PLEKHA6 in breast cancer tissue on survival of breast cancer patients. High expression of PLEKHA6 was detrimental to overall survival in breast cancer patients (P ═ 0.0285).
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 the expression changes of PLEKHA6 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 "Analyzis ", enter the gene name" PLEKHA6 ", select TCGA dataset" Breast innovative carcinoma ", search, click" 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: the expression of PLEKHA6 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 TCGA high throughput data analysis and the prognosis of PLEKHA6 high expression in breast cancer.
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 "PLEKHA 6: 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 PLEKHA6 was detrimental to overall survival (P ═ 0.0059) in breast cancer patients (fig. 2).
Example 3
This example illustrates the preparation of reagents for detecting the expression level of PLEKHA6 for preparing 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;
12.10.0 μ M PLEKHA6 real-time fluorescence 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 PLEKHA6 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-ΔΔCTThe relative expression level of PLEKHA6 is calculated as follows: the experiment detects the relative expression change of the PLEKHA6 in 57 breast cancer tissues and 18 paracarcinoma tissues. ACTB as an internal reference gene, and a qPCR-determined target gene PLEKHA6CTC 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 the relative expression of PLEKHA6 in each group was calculated by using the Power function in Excell table. Differential expression of PLEKHA6 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 PLEKHA6 in patients with breast cancer: 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 PLEKHA6 was high, which was 2 times higher than the average expression level of para-carcinoma tissues, in total 13 cases, while the expression level of PLEKHA6 was low, in total 44 cases. 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 the PLEKHA6 in the breast cancer is obviously increased (P <0.001) compared with that in a para-carcinoma tissue, as shown in a figure 3; and high expression of PLEKHA6 was detrimental to overall survival of breast cancer patients (P ═ 0.0285), 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. Use of an agent that detects the level of expression of PH domain family A member 6(PLEKHA6) in the manufacture of a formulation for the prognostic determination of a breast cancer patient.
2. The use of claim 1, wherein said detecting the expression level of PLEKHA6 comprises detecting the gene expression level of PLEKHA6 and/or detecting the protein expression level of PLEKHA 6.
3. The use of claim 1, wherein the method of detecting the expression level of PLEKHA6 comprises: detecting the expression level of PLEKHA6 in breast cancer and paracarcinoma tissues by an RT-qPCR method; detecting the expression quantity of PLEKHA6mRNA in breast cancer and paracarcinoma tissues by a molecular probe technology; the expression level of the PLEKHA6 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 PLEKHA6 is an oligonucleotide probe targeting a PLEKHA 6-encoding DNA sequence, a PCR primer, or an antibody targeting PLEKHA 6.
5. The use of claim 4, wherein the reagent for detecting the expression level of PLEKHA6 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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Effective date of registration: 20220523 Address after: 2081, building a, 88 Jianghai West Road, Liangxi District, Wuxi City, Jiangsu Province, 214000 Patentee after: Wuxi Xiangyuan Information Technology Co.,Ltd. Address before: 224008 no.283, Jiefang South Road, Yancheng City, Jiangsu Province Patentee before: JIANGSU VOCATIONAL College OF MEDICINE Effective date of registration: 20220523 Address after: No.1 Huanghai Road, Shuiji sub district office, Laixi City, Qingdao City, Shandong Province 266600 Patentee after: Qingdao Yanding Biomedical Technology Co.,Ltd. Address before: 2081, building a, 88 Jianghai West Road, Liangxi District, Wuxi City, Jiangsu Province, 214000 Patentee before: Wuxi Xiangyuan Information Technology Co.,Ltd. |
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Denomination of invention: Application of reagents and kits for detecting the expression level of member 6 of Pleckstrin homology domain family A Effective date of registration: 20230721 Granted publication date: 20210115 Pledgee: Weihai commercial bank Limited by Share Ltd. Qingdao branch Pledgor: Qingdao Yanding Biomedical Technology Co.,Ltd. Registration number: Y2023980049237 |