CN111041095B - Application of reagent for detecting expression level of chromosome 8 open reading frame 73 and kit - Google Patents
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention belongs to the technical field of biology, and relates to an application of a reagent for detecting the expression level of an open reading frame 73 of a chromosome 8 and a kit. More particularly, the application of the reagent for detecting the expression level of C8ORF73 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 C8ORF73 in the breast cancer is obviously increased compared with that of a tissue beside the cancer; and the high expression of C8ORF73 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 an open reading frame 73(C8ORF73) of a chromosome 8 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 breast cancer prognosis marker chromosome 8 open reading frame 73(C8ORF73), and further provides application of a reagent for detecting the expression level of C8ORF73 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.
C8ORF73 is a protein-encoding Gene, located at 8q24.3, comprising 15 exons, Gene ID:642475, which encodes a protein as long as it is located in the mitochondria. At present, no report is found about the role of the C8ORF73 gene in the development of breast cancer. The inventor discovers that the high expression of C8ORF73 is not beneficial to the prognosis of breast Cancer patients through high-throughput data mining of Cancer Genome Atlas (TCGA), and further verifies the prognostic effect of the expression difference of C8ORF73 on the 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 a use of a reagent for detecting an expression level of chromosome 8 open reading frame 73(C8ORF73) in the preparation of a preparation for breast cancer auxiliary diagnosis and/or breast cancer patient prognosis.
Further, the detecting the expression level of the C8ORF73 comprises detecting the gene expression level of the C8ORF73 and/or detecting the protein expression level of the C8ORF 73.
More specifically, the method for detecting the expression level of C8ORF73 comprises the following steps: detecting the expression level of C8ORF73 in breast cancer and para-carcinoma tissues by an RT-qPCR method; detecting the expression level of C8ORF73mRNA in breast cancer and tissues beside the cancer by a molecular probe technology; the expression level of C8ORF73 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 C8ORF73 is an oligonucleotide probe targeting a C8ORF73 encoding DNA sequence, a PCR primer or an antibody targeting C8ORF 73.
According to the present invention, preferably, the reagent for detecting the expression level of C8ORF73 is a reagent having the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
5’-CGGCTATGGCCTTCTTCACA-3’，SEQ ID NO：1；
5’-CACCTTCCTGCGATTCAGCG-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 C8ORF 73.
Further, the reagent for detecting the expression level of the C8ORF73 is an oligonucleotide probe targeting a C8ORF73 encoding DNA sequence, a PCR primer or an antibody targeting C8ORF 73.
Specifically, the reagent for detecting the expression level of C8ORF73 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 C8ORF73 in the breast cancer is obviously increased compared with that in a para-carcinoma tissue (P < 0.001); and high expression of C8ORF73 is detrimental to overall survival of breast cancer patients (P ═ 0.0081). 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 high throughput data of TCGA for the analysis of C8ORF73 expression in breast cancer. C8ORF73 expression was significantly higher in breast cancer tissues than in normal tissues (P < 0.001).
FIG. 2 shows a TCGA high throughput data analysis of the effect of high expression of C8ORF73 on survival in breast cancer patients. High expression of C8ORF73 is detrimental to overall survival in breast cancer patients (P ═ 0.0149).
FIG. 3 shows the expression of C8ORF73 in breast cancer tissues. C8ORF73 expression was significantly higher in breast cancer tissues than in paracarcinoma tissues (P < 0.001).
FIG. 4 shows the effect of high expression of C8ORF73 in breast cancer tissues on survival of breast cancer patients. High expression of C8ORF73 is detrimental to overall survival of breast cancer patients (P ═ 0.0081).
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 serves to illustrate the high throughput data analysis of TCGA for the expression change of C8ORF73 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 "C8 ORF 73", 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 C8ORF73 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 C8ORF73 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)" was selected, the omics dataset "mRNA Expression z-scenes (RNA Seq V2 RSEM)" was selected, the gene input "C8 ORF 73: 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 C8ORF73 was detrimental to overall survival in breast cancer patients (P ═ 0.0149) (fig. 2).
Example 3
This example illustrates the preparation of reagents for detecting the expression level of C8ORF73 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 mu M C8ORF73 real-time fluorescent quantitative PCR specific primer 30.0 mu 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 C8ORF73 in clinical breast cancer tissue samples.
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 method calculates the relative expression amount of C8ORF 73: the experiment detects the relative expression change of C8ORF73 in 57 breast cancer tissues and 18 paracarcinoma tissues. ACTB as an internal reference gene, and a target gene C8ORF73C 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 C8ORF73 in each group by using the Power function in the Excell table. Differential expression of C8ORF73 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 C8ORF73 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 C8ORF73 was high when it was 2 times higher than the average relative expression level of the para-carcinoma tissues, 8 cases in total, and the other cases were low in C8ORF73, 49 cases in 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 C8ORF73 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 C8ORF73 is detrimental to overall survival of breast cancer patients (P ═ 0.0081), 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
Application of reagent and kit for detecting expression level of chromosome 8 open reading frame 73
<130> BJI1900778JSYY
<160> 4
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<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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Claims (4)
1. The application of the reagent for detecting the expression level of C8ORF73 in preparing a preparation for prognosis judgment of a breast cancer patient, wherein the expression level of C8ORF73 is the gene expression level of C8ORF 73.
2. The use of claim 1, wherein the method for detecting the expression level of C8ORF73 comprises: the expression level of C8ORF73 in breast cancer and paracarcinoma tissues is detected by RT-qPCR method.
3. The use of claim 1, wherein the reagent for detecting the expression level of C8ORF73 is an oligonucleotide probe targeting a C8ORF73 encoding DNA sequence, or a PCR primer.
4. The use of claim 3, wherein the reagent for detecting the expression level of C8ORF73 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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| WO2014014518A1 (en) * | 2012-07-18 | 2014-01-23 | Dana-Farber Cancer Institute, Inc. | Methods for treating, preventing and predicting risk of developing breast cancer |
| CN104334191A (en) * | 2012-03-29 | 2015-02-04 | 纽约市哥伦比亚大学托管会 | Methods for treating hair loss disorders |
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| WO2012142330A1 (en) * | 2011-04-15 | 2012-10-18 | Brown University | Micro rnas as diagnostic biomarkers and therapeutics for ovarian cancer and metastatic tumors that disseminate within the peritoneal cavity |
| CN104334191A (en) * | 2012-03-29 | 2015-02-04 | 纽约市哥伦比亚大学托管会 | Methods for treating hair loss disorders |
| WO2014014518A1 (en) * | 2012-07-18 | 2014-01-23 | Dana-Farber Cancer Institute, Inc. | Methods for treating, preventing and predicting risk of developing breast cancer |
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Effective date of registration: 20221220 Address after: No.1-4, 5 / F, building B22, Wuhan hi tech medical instrument Park, 818 Gaoxin Avenue, Donghu New Technology Development Zone, Wuhan City, Hubei Province, 430000 Patentee after: Sinopharm (Wuhan) medical laboratory Co.,Ltd. Address before: 224008 no.283, Jiefang South Road, Yancheng City, Jiangsu Province Patentee before: JIANGSU VOCATIONAL College OF MEDICINE |