Biomarkers for breast cancer metastasis and prognostic diagnosis
阅读说明：本技术 乳腺癌转移和预后诊断的生物标志物 (Biomarkers for breast cancer metastasis and prognostic diagnosis ) 是由 李中伟 王典典 白津 郑骏年 侯平甫 李敏乐 于 2019-11-21 设计创作，主要内容包括：本发明涉及一种乳腺癌转移和预后诊断的生物标志物,所述生物标志物为meR342-EZH2,发明人通过一系列实验,发现并证实meR342-EZH2能够促进乳腺癌的EMT进程和乳腺癌细胞的体外侵袭转移和体内远端转移,通过对乳腺癌患者的病例分级、转移和预后等相关分析,发现meR342-EZH2高表达和乳腺癌患者肿瘤变大、分级较高以及更多的远端转移灶点都呈现正相关,meR342-EZH2高表达预示着乳腺癌患者的较低五年生存率。因此可将meR342-EZH2用于制备或者筛选乳腺癌转移诊断试剂和乳腺癌预后诊断试剂,为乳腺癌转移和预后提供了新的诊断及评估方法。(The invention relates to a biomarker for breast cancer metastasis and prognosis diagnosis, wherein the biomarker is meR342-EZH2, the inventor discovers and proves that meR342-EZH2 can promote the EMT process of breast cancer and the in vitro invasion and metastasis and in vivo far-end metastasis of breast cancer cells through a series of experiments, and through the relevant analysis of case grading, metastasis, prognosis and the like of a breast cancer patient, the inventor discovers that meR342-EZH2 high expression and the tumor enlargement, higher grading and more far-end metastasis foci of the breast cancer patient all show positive correlation, and meR342-EZH2 high expression indicates lower five-year survival rate of the breast cancer patient. Therefore, meR342-EZH2 can be used for preparing or screening a breast cancer metastasis diagnostic reagent and a breast cancer prognosis diagnostic reagent, and a novel diagnosis and evaluation method is provided for breast cancer metastasis and prognosis.)
1. A biomarker for breast cancer metastasis and prognostic diagnosis, wherein the biomarker is meR342-EZH 2.
2. Use of the biomarker meR342-EZH2 of claim 1 in the preparation or screening of a diagnostic reagent for breast cancer metastasis.
3. Use of the biomarker meR342-EZH2 of claim 1 in the preparation or screening of a prognostic diagnostic agent for breast cancer.
4. An antibody for detecting the biomarker of claim 1, wherein the amino acid sequence of the antibody is shown as SEQ ID No. 1.
5. The use of the antibody of claim 4 for the preparation of a diagnostic reagent for breast cancer metastasis and a diagnostic reagent for breast cancer prognosis.
Technical Field
The invention relates to a biomarker for breast cancer metastasis and prognosis diagnosis, and belongs to the technical field of biomedicine.
Background
Breast cancer is one of the most common malignant tumors of women, and the incidence rate accounts for 7-10% of various malignant tumors of the whole body, and is the first of the malignant tumors of women. In china, breast cancer is the highest incidence cancer among women, and the rate of incidence increases 2 times the world's average level, with the number of deaths accounting for 9.6% of the world. The deaths of breast cancer patients caused by metastasis account for over 90% of all deaths, and malignant metastasis of breast cancer has become a significant cause of death. Clinical data indicate that a large number of patients with tumor metastases have metastasized when their tumor size in situ is small, but cannot be treated in a timely manner because they are not easily detected. At present, no molecular target and product for predicting breast cancer metastasis and effectively evaluating prognosis exist in the market, so that a new prognostic target for breast cancer metastasis is deeply explored, and the clinical significance is very important.
EZH2(Enhancer of Zeste homo log 2) is an important histone methyltransferase which can catalyze the trimethylation modification of the 27 th lysine of histone H3 of a target gene (H3K27me 3). PRC2 is the result of the transcriptional silencing of downstream target genes by the SETDomain modification of the EZH2 protein to the histone by
The inventor researches and discovers that: the R342 site of EZH2 can be asymmetrically dimethyl-modified by PRMT1 (meR342-EZH2), and meR342-EZH2 can enhance the protein stability of
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a biomarker for breast cancer metastasis and prognosis diagnosis.
Technical scheme
A biomarker for breast cancer metastasis and prognostic diagnosis, wherein the biomarker is meR342-EZH2 (asymmetric dimethyl modification of R342 site of EZH 2).
The inventor combines breast cancer clinical data, explores the relation and molecular mechanism between meR342-EZH2 expression and breast cancer metastasis on the aspects of biochemistry, molecules, cell level, animal models, clinical tissue samples and the like, discovers and proves that meR342-EZH2 can promote the EMT process of breast cancer, promotes in-vitro invasion and metastasis and in-vivo far-end metastasis of breast cancer cells, and discovers that meR342-EZH2 high expression and tumor enlargement, higher grade and more far-end metastasis points of breast cancer patients are positively correlated by analyzing the expression level of meR342-EZH2 and the case grade, metastasis and prognosis of breast cancer patients; finally, statistics is carried out through the relation between the expression level of meR342-EZH2 and the prognosis of the breast cancer patient, and the result shows that the high expression level of meR342-EZH2 is indicative of the lower five-year survival rate of the breast cancer patient.
The biomarker meR342-EZH2 is used for preparing or screening breast cancer metastasis diagnostic reagents.
The biomarker meR342-EZH2 is used for preparing or screening breast cancer prognosis diagnostic reagents.
An antibody for detecting the biomarker meR342-EZH2, wherein the amino acid sequence of the antibody is shown as SEQ ID No.1 (TAERIKTPPKRPGGC).
The meR342-EZH2 antibody is used for preparing a breast cancer metastasis diagnostic reagent and a breast cancer prognosis diagnostic reagent.
The invention has the beneficial effects that: the invention provides a biomarker meR342-EZH2 for breast cancer metastasis and prognosis diagnosis, and a series of experimental studies show that the expression of meR342-EZH2 is related to breast cancer metastasis and prognosis, so meR342-EZH2 can be used for preparing or screening a breast cancer metastasis diagnosis reagent and a breast cancer prognosis diagnosis reagent, and a novel diagnosis and evaluation method is provided for breast cancer metastasis and prognosis, and in addition, the biomarker meR342-EZH2 can also be used for screening medicines for treating breast cancer metastasis and prognosis.
Drawings
FIG. 1 shows the results of the expression levels of meR342-EZH2 in the tissues adjacent to breast cancer, in situ breast cancer and metastatic focus of breast cancer;
FIG. 2 shows mRNA expression at the transcriptional level of E-cadherin, N-cadherin and Vimentin MCF7-Vector, MCF7-Flag-EZH2-WT and MCF7-Flag-EZH2-R342K cells detected by qRT-PCR;
FIG. 3 shows the Western blot detection of the expression of E-cadherin, N-cadherin and Vimentin at the protein level in MCF7-Vector, MCF7-Flag-EZH2-WT and MCF7-Flag-EZH2-R342K cells;
FIG. 4 shows mRNA expression at the transcriptional level of Snail, Twist and ZEB1 detected by qRT-PCR;
FIG. 5 shows the results of Transwell tests on the migratory ability of MCF7-Vector, MCF7-Flag-EZH2-WT and MCF7-Flag-EZH2-R342K cells;
FIG. 6 shows the results of the scratch test for the migration ability of MCF7-Vector, MCF7-Flag-EZH2-WT, and MCF7-Flag-EZH2-R342K cells;
FIG. 7 shows the results of MDA-MB-231-Vector, MDA-MB-231-Flag-EZH2-WT and MDA-MB-231-Flag-EZH2-R342K cells tested for metastasis in the lungs of nude mice;
FIG. 8 is a statistical result of metastatic foci of lung tissues of nude mice after tail vein injection of MDA-MB-231-Vector, MDA-MB-231-Flag-EZH2-WT and MDA-MB-231-Flag-EZH2-R342K cells;
FIG. 9 shows meR342-EZH2 expression profile test results in lung tissues of breast cancer of three groups, MDA-MB-231-Vector, MDA-MB-231-Flag-EZH2-WT and MDA-MB-231-Flag-EZH 2-R342K;
FIG. 10 shows the results of testing the relationship between the expression of meR342-EZH2 and tumor size;
FIG. 11 is a graph showing the results of testing the relationship between meR342-EZH2 high expression and pathological grade of breast cancer patients;
FIG. 12 shows the results of a test of meR342-EZH2 methylation-high expression and breast cancer metastasis relationship;
FIG. 13 shows the results of a test on the relationship between the expression level of meR342-EZH2 and the number of lymph node metastases in breast cancer patients;
FIG. 14 is a graph showing the results of testing the relationship between the expression level of meR342-EZH2 and the overall survival rate of breast cancer patients.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
In the examples below, the material sources used are as follows:
plasmids used in the experiments:
GST, GST-PRMT1, GST-EZH2(1-522AA), GST-EZH2(523-746AA) plasmids of pGEX-4T-1 vector; plasmid shEZH2-3' UTR of pLKO.1 vector; the 3xFlag-EZH2-WT plasmids of the pCDH-CMV-MCS-EF1-Puro vector were all from Xuzhou university of medicine;
the construction process of the 3xFlag-EZH2-R342K plasmid of pCDH-CMV-MCS-EF1-Puro vector is as follows:
(1) the following point mutation primers for R342K were designed and synthesized:
an upstream primer: 5'-ACCGCTGAGAAGATAAAGACC-3' the flow of the air in the air conditioner,
a downstream primer: 5'-GGTCTTTATCTTCTCAGCGGT-3' are provided.
(2) The 3xFlag-EZH2-WT plasmid was used as a template, the upstream and downstream primers for point mutation of R342K were used as primers, and PCR was carried out using the purchased Rapid site-directed mutagenesis kit (KM101) from Tiangen Biotech company according to the kit instructions to obtain a mutated plasmid, which was transformed into competent cells.
(3) And after the bacterial colony grows out, selecting bacteria, shaking the bacteria to extract plasmids, and carrying out sequencing verification mutation, wherein the sequencing result shows that the mutation sequence result is correct. Thus, 3xFlag-EZH2-R342K plasmid of pCDH-CMV-MCS-EF1-Puro vector was constructed.
Packaging plasmids for PAX and PMD-2G: all purchased from addge, catalog number: PAX 12260; PMD-2G 12259.
Antibodies used in the experiments:
a first antibody: anti-Flag M2 affinity gel from Bimake (cat # B23101); murine Flag and GST-tagged antibodies were from SUNGENE BIOTECH (cat # KM8004 and # KM 8005); murine EZH2 antibody was from CST (cat # 3147); the murine GAPDH antibody is from Proteitech (Cat #60004-1), and the rabbit Ubiquitin antibody is from CST (Cat # 3933); rabbit-derived E-cadherin, N-cadherin and Vimentin antibodies were obtained from CST (Cathayen #3195, #13116 and #5741 in that order); murine IgG antibodies were obtained from CST (cat # 5415).
Secondary antibody: the goat anti-mouse IgG secondary antibody and the goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase were purchased from Kyowa Kingchi Biotech Co., Ltd. (Cat # ZB-2305 and # ZB-2301).
Cells and media used in the experiment:
HEK293T cells, MCF7 breast cancer cells and MDA-MB-231 breast cancer cells were all purchased from ATCC (american model culture collection stock) (cargo numbers # CRL1573, # HTB22, # HTB129 in order); MDA-MB-231-luc breast cancer cell line was purchased from Shanghai national academy of sciences cell Bank (cat # CBP 30106L); fetal bovine serum was purchased from Beijing Quanjin Biotechnology Ltd (cat # FS 201-02); the cell culture medium used for cell culture was obtained from Sigma (Cat. No. DMEM medium # D7777; RPMI-1640 medium # D7755; L15 medium # L4386).
Tissue samples used in the experiment:
the clinical breast cancer tissue samples used were 367 samples collected at the affiliated hospital of xu medical university; there are also 100 breast cancer tissue chips (cat # TMA-BR1008a) available from west analina.
Other reagent consumables:
use of qRT-PCR
Other materials were commercially available unless otherwise specified, and the experimental procedures used in the examples described below were all conventional.