Application of CircEGFR as molecular target and prognosis biomarker in preparation of triple negative breast cancer treatment product

Abstract

The invention relates to an application of circEGFR (Epidermal Growth Factor Receptor) as a molecular target and a prognosis biomarker in preparation of a triple negative breast cancer treatment product. The circBase ID of the circEGFR is hsacirc0080220. According to the application disclosed by the invention, a lentivirus overexpression vector and a lentivirus silencing vector of the circEGFR are constructed, and the influence of overexpression or silencing of the lentivirus overexpression vector and the lentivirus silencing vector on MDA-MB-231 cell proliferation, migration, invasion and EMT is evaluated, so that the triple negative breast cancer is treated by utilizing the silenced circEGFR. The circEGFR expression quantity in tissues and plasma exosomes of a triple negative breast cancer patient is detected through RT-qPCR, and the circEGFR is found to have the effect of predicting prognosis of the patient. Therefore, the invention provides a prognostic marker and a promising therapeutic target for the TNBC.

Description

Technical Field

[0001] The present invention belongs to the technical field of breast cancer markers, and specifically relates to an application of circEGFR as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product. Background Art

[0002] According to the latest global data released by the World Health Organization's International Agency for Research on Cancer, there will be approximately 2.26 million new cases of breast cancer in 2020, making it the most commonly diagnosed cancer. It is the cancer with the highest mortality rate in developing countries and the cancer with the highest malignant tumor mortality rate in women. Breast cancer mainly includes luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) overexpression and triple-negative breast cancer (TNBC). The purpose of breast cancer classification is to determine the best treatment plan for patients based on the known association between the underlying tumor molecular biology and treatment sensitivity. Breast cancer has become a global health problem, of which the TNBC subtype accounts for about 15% to 20%. It is characterized by strong invasiveness, high mortality and poor prognosis. The age of onset is trending towards younger age. It is a highly heterogeneous cancer. Its molecular characteristics are that estrogen receptor (ER), progesterone receptor (PR) and HER2 expression are all negative. There is still a lack of standardized TNBC treatment options. Therefore, the development of new TNBC treatment strategies has become an urgent clinical need.

[0003] Neoadjuvant chemotherapy based on anthracyclines is still one of the standard treatments for TNBC. Pirarubicin (THP, also known as pyranorubicin) is an anthracycline antitumor drug that can non-specifically embed between double-stranded DNA bases to form a relatively stable anthracycline-DNA complex, inhibiting DNA replication and mRNA synthesis; inhibiting DNA polymerase and DNA topoisomerase II activity, interfering with DNA synthesis; and generating oxygen free radicals to destroy cell membrane function. However, chemotherapy has not yet formed a particularly effective targeted treatment strategy for TNBC, so there is an urgent need to find new drug intervention targets to provide a new theoretical basis for the occurrence, development and treatment of TNBC, so as to guide individualized treatment and improve the prognosis of cancer patients. THP is widely used in neoadjuvant treatment of TNBC, but its mechanism of action in treating TNBC by intervening in circRNAs has not yet been clarified.

[0004] Circular RNA (circRNA) belongs to non-coding RNA (ncRNA) and has become an emerging research hotspot in recent years. CircRNA is a type of RNA molecule that does not have a 5' cap and a 3' poly A tail structure and forms a circular molecular structure with covalent bonds. CircRNA is mainly produced by reverse splicing, which is significantly different from the classic 5'-3' pattern of linear RNA. Due to the special circular structure of circRNA, it has the characteristics of longer half-life and greater stability. A large number of circRNAs are believed to be stable and exist at high levels in body fluids, including plasma, serum, exosomes and urine. Studies have revealed that plasma exosome circRNA can be used as a biomarker for cancer diagnosis and prognosis. An important function of circRNA is to compete as a competitive endogenous RNA (Competitive endogenous RNA, ceRNA) to sponge adsorb microRNA (microRNA, miRNA) to affect the expression of miRNA target genes. At present, the research on circRNA not only focuses on the mechanism of the occurrence and development of TNBC, but also includes circRNA as a biomarker for the diagnosis, treatment and prognosis of TNBC.

[0005] CircEGFR (circBase ID: hsa_circ_0080220) is located in the genome chr7: 55209978-55214433. It is formed by reverse splicing of exons 2, 3 and 4 of its parent gene EGFR, and is 471bp in length. Studies have reported that circEGFR can regulate the malignant progression of colorectal cancer through the miR-106a-5p / DDX5 axis. Although studies have reported that circEGFR plays an important role in colorectal cancer, the mechanism by which circEGFR plays a role in TNBC is still unclear. Therefore, how to use circEGFR to treat TNBC is an urgent problem to be solved. Summary of the invention

[0006] The purpose of the present invention is to make up for the defects of the existing technology and provide an application of circEGFR as a molecular target and prognostic biomarker in the preparation of triple-negative breast cancer treatment products. The present invention constructs a lentiviral overexpression vector and a lentiviral silencing vector of circEGFR, evaluates the effects of overexpression or silencing on MDA-MB-231 cell proliferation, migration, invasion and EMT, and realizes the use of silencing circEGFR to treat triple-negative breast cancer. The expression of circEGFR in tissues and plasma exosomes of patients with triple-negative breast cancer was detected by RT-qPCR, and it was found that circEGFR has the effect of predicting patient prognosis.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] A use of circEGFR as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product, wherein the circBase ID of the circEGFR is: hsa_circ_0080220.

[0009] Preferably, the use of circEGFR as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product comprises the following steps:

[0010] (1) shRNA was cloned into a lentiviral silencing vector to obtain a lentiviral vector that stably silenced circEGFR;

[0011] (2) transfecting MDA-MB-231 cells with the stably silenced circEGFR lentiviral vector obtained in step (1);

[0012] (3) RT-qPCR was used to detect the expression level of circEGFR in tissue and plasma exosomes of patients with triple-negative breast cancer.

[0013] Preferably, the lentiviral silencing vector in step (1) is PLent-U6-GFP-Puro.

[0014] Preferably, the shRNA sequence in step (1) is any one or more of the following three pairs of complementary chain shRNAs designed for the cleavage site of circEGFR;

[0015] CircEGFR shRNA-1BamHI F: 5'-gatccCCTGGGCAGCTTTTGCCAAGGC TCGAGCCTTGGCAAAAGCTGCCCAGGTTTTTa-3';

[0016] CircEGFR shRNA-1Mlul R: 5'-cgcgtAAAAACCTGGGCAGCTTTTGCCAAGGCTCGAGCCTTGGCAAAAGCTGCCCAGGg-3';

[0017] CircEGFR shRNA-2BamHI F: 5'-gatccTGGGCAGCTTTTGCCAAGGCAC TCGAGTGCCTTGGCAAAAGCTGCCCATTTTTa-3';

[0018] CircEGFR shRNA-2Mlul R: 5'-cgcgtAAAAATGGGCAGCTTTTGCCAAGGCACTCGAGTGCCTTGGCAAAAGCTGCCCAg-3';

[0019] CircEGFR shRNA-3BamHI F: 5'-gatccGCAGCTTTGCCAAGGCACGAC TCGAGTCGTGCCTTGGCAAAAGCTGCTTTTTa-3';

[0020] CircEGFR shRNA-3Mlul R: 5'-cgcgtAAAAAGCAGCTTTTGCCAAGGCACGACTCGAGTCGTGCCTTGGCAAAAGCTGCg-3'.

[0021] Preferably, the steps of constructing the shRNA cloned in the lentiviral silencing vector in step (1) are as follows:

[0022] (1) Preparation of shRNA complementary strand;

[0023] (2) Double restriction digestion of lentiviral silencing vector;

[0024] (3) Recovering the enzyme digestion product to obtain the lentiviral silencing vector after enzyme digestion;

[0025] (4) connecting the complementary strand of the target fragment shRNA to the vector obtained in step (3) to obtain a connection product;

[0026] (5) transformation of the ligation product obtained in step (4);

[0027] (6) Sequencing and identifying the converted ligation product obtained in step (5).

[0028] Preferably, the specific steps of transfecting MDA-MB-231 cells with the stably silencing circEGFR lentiviral vector in step (2) are as follows:

[0029] a. Transfection of MDA-MB-231 cells with lentiviral vector:

[0030] The day before transfection, MDA-MB-231 cells were passaged and transferred into 24-well plates to a confluency of 70% to 80%. 200 μL of lentiviral silencing vector was added to each well and cultured for 24 h before switching to complete culture medium.

[0031] b. Detection of killing curve:

[0032] (1) The MDA-MB-231 cells obtained in step a were prepared into a cell suspension using a serum-free medium without dual antibodies, inoculated into a 96-well plate, and cultured for 24 hours;

[0033] (2) adding different concentrations of puromycin: the concentrations were 0 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.2 μg / mL, 1.4 μg / mL, 1.6 μg / mL, 1.8 μg / mL, and 2 μg / mL;

[0034] (3) The cells were cultured for 7 days, during which the cell culture medium was changed once. On the 7th day, it was found that the cells with a puromycin concentration of 0 to 1 μg / mL survived, while the cells with a puromycin concentration of 1 to 2 μg / mL partially died.

[0035] (4) Select 1 μg / mL of puromycin as the concentration for stable transfectant screening;

[0036] c. Screening of stable transgenic strains:

[0037] (1) MDA-MB-231 cells were infected with a stably silenced circEGFR lentiviral vector and cultured for 48 h. Complete medium containing 1 μg / mL puromycin was added and cultured for 7 days, during which the medium was changed once;

[0038] (2) Observe and mark individual cell populations on the culture plate under a microscope;

[0039] (3) Digest the cell population with trypsin and inoculate it into a new 24-well plate for culture;

[0040] (4) Continue subclone screening with puromycin for 7 days;

[0041] (5) Observe under a microscope and select the normally growing cell population for subculture, and use 1 μg / mL puromycin to maintain stable culture for 30 days.

[0042] Preferably, the specific sequence information of the amplification primers for circEGFR is as follows:

[0043] Upstream primer: 5′-CATGTCGATGGACTTCCAGA-3′;

[0044] Downstream primer: 5′-AAATTCCCAAGGACCACCTC-3′.

[0045] A use of a substance for overexpressing or silencing circEGFR in preparing a product for treating triple-negative breast cancer, wherein the circBase ID of the circEGFR is: hsa_circ_0080220.

[0046] Preferably, the substance for overexpressing or silencing circEGFR includes amplification primers of circEGFR, and the specific sequence information thereof is as follows:

[0047] Upstream primer: 5′-CATGTCGATGGACTTCCAGA-3′;

[0048] Downstream primer: 5′-AAATTCCCAAGGACCACCTC-3′.

[0049] Preferably, the substance for silencing circEGFR further comprises a shRNA sequence cloned in a lentiviral silencing vector, wherein the shRNA sequence is any one or more of the following three pairs of complementary chain shRNAs designed for the cleavage site of circEGFR;

[0050] CircEGFR shRNA-1BamHI F: 5'-gatccCCTGGGCAGCTTTTGCCAAGGC TCGAGCCTTGGCAAAAGCTGCCCAGGTTTTTa-3';

[0051] CircEGFR shRNA-1Mlul R: 5'-cgcgtAAAAACCTGGGCAGCTTTTGCCAAGGCTCGAGCCTTGGCAAAAGCTGCCCAGGg-3';

[0052] CircEGFR shRNA-2BamHI F: 5'-gatccTGGGCAGCTTTTGCCAAGGCAC TCGAGTGCCTTGGCAAAAGCTGCCCATTTTTa-3';

[0053] CircEGFR shRNA-2Mlul R: 5'-cgcgtAAAAATGGGCAGCTTTTGCCAAGGCACTCGAGTGCCTTGGCAAAAGCTGCCCAg-3';

[0054] CircEGFR shRNA-3BamHI F: 5'-gatccGCAGCTTTGCCAAGGCACGAC TCGAGTCGTGCCTTGGCAAAAGCTGCTTTTTa-3';

[0055] CircEGFR shRNA-3Mlul R: 5'-cgcgtAAAAAGCAGCTTTTGCCAAGGCACGACTCGAGTCGTGCCTTGGCAAAAGCTGCg-3'.

[0056] The beneficial effects of the present invention are:

[0057] The present invention discloses an application of circEGFR as a molecular target and prognostic biomarker in the preparation of a product for the treatment of triple-negative breast cancer. By constructing a stable silencing circEGFR lentiviral vector, the proliferation, migration, invasion and EMT of MDA-MB-231 cells are interfered with, which provides an important supplement to the biological function of circEGFR, provides an effective treatment for triple-negative breast cancer, and also provides a theoretical basis for the application of circEGFR as a molecular target and prognostic biomarker in triple-negative breast cancer.

[0058] The present invention uses a circEGFR as a molecular target and a prognostic biomarker in the preparation of a triple-negative breast cancer treatment product. By constructing a lentiviral overexpression vector and a lentiviral silencing vector of circEGFR, the effects of overexpression or silencing on MDA-MB-231 cell proliferation, migration, invasion and EMT are evaluated, and the use of silencing circEGFR to treat triple-negative breast cancer is achieved. The expression of circEGFR in tissues and plasma exosomes of patients with triple-negative breast cancer was detected by RT-qPCR, and it was found that circEGFR has the effect of predicting the prognosis of patients. Therefore, the present invention provides a prognostic marker and a promising therapeutic target for TNBC. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0060] Figure 1 This is the sh-circEGFR-1 sequence alignment result diagram; Query is the sequencing result; Sbject is the sh-circEGFR-1 sequence.

[0061] Figure 2 This is the result of sh-circEGFR-2 sequence alignment; Query is the sequencing result; Sbject is the sh-circEGFR-2 sequence.

[0062] Figure 3 This is the sh-circEGFR-3 sequence alignment result diagram; Query is the sequencing result; Sbject is the sh-circEGFR-3 sequence.

[0063] Figure 4 This is a diagram of the transfection efficiency of CircEGFR and sh-circEGFR.

[0064] Figure 5The figure shows the effect of overexpression or silencing of circEGFR on the proliferation ability of MDA-MB-231 cells; A is the cell proliferation detected by CCK-8 at 0, 24, 48, 72, and 96 hours; B is the cell proliferation detected by EdU at 24 hours; **p<0.01vs.MOCK, ##p<0.01vs.sh-NC.

[0065] Figure 6 The figure shows the effect of overexpression or silencing of circEGFR on the migration ability of MDA-MB-231 cells; A is a scratch assay to detect cell migration ability (40×; scale: 50μm); B is a Transwell vertical migration assay to detect cell migration ability; **p<0.01vs.MOCK, ##p<0.01vs.sh-NC.

[0066] Figure 7 The figure shows the effect of overexpression or silencing of circEGFR on the migration ability of MDA-MB-231 cells; **p<0.01vs.MOCK, ##p<0.01vs.sh-NC.

[0067] Figure 8 The figure shows the effect of overexpression or silencing of circEGFR on EMT-related proteins in MDA-MB-231 cells; **p<0.01vs.MOCK, ##p<0.01vs.sh-NC. DETAILED DESCRIPTION

[0068] The inventive concept of the present invention is that breast cancer can be treated with surgical treatment, radiotherapy, chemotherapy, endocrine therapy and targeted therapy. The diversity of such treatment options also reflects the complexity of breast cancer treatment today. Triple-negative breast cancer (TNBC) is a special subtype of breast cancer, accounting for about 15% to 25% of all breast cancers. It is characterized by negative estrogen receptors, progesterone receptors and HER-2, high malignancy, strong invasiveness, high metastatic potential, easy recurrence and poor prognosis. There is still a lack of standardized TNBC treatment options. Therefore, the development of new TNBC treatment strategies has become an urgent clinical need. A large number of studies have revealed that plasma exosome circRNA can be used as a cancer diagnosis and prognosis biomarker. CircRNAs have been shown to play an important role in the occurrence and development of TNBC, but whether the screened target circRNAs can become TNBC treatment targets and prognosis biomarkers still needs to be explored. Find independent prognostic markers and promising therapeutic targets for TNBC; explore the molecular targets that THP may act on; study the intrinsic functions and mechanisms of molecular targets in the occurrence, development and drug treatment of TNBC, and provide theoretical support and basis for the occurrence, development and treatment of TNBC and the anti-TNBC effect of pirarubicin from the perspective of circRNA. Based on this, the present invention provides an application of circEGFR as a molecular target and prognostic biomarker in the preparation of triple-negative breast cancer treatment products. The present invention constructs a lentiviral overexpression vector and a lentiviral silencing vector of circEGFR, evaluates the effects of its overexpression or silencing on the proliferation, migration, invasion and EMT of MDA-MB-231 cells, and realizes the use of silencing circEGFR to treat triple-negative breast cancer. The expression of circEGFR in tissues and plasma exosomes of patients with triple-negative breast cancer was detected by RT-qPCR, and it was found that circEGFR has the effect of predicting the prognosis of patients.

[0069] The present invention provides an application of circEGFR as a molecular target and a prognostic biomarker in the preparation of a triple-negative breast cancer treatment product, wherein the circBase ID of the circEGFR is: hsa_circ_0080220.

[0070] The use of circEGFR of the present invention as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product specifically comprises the following steps:

[0071] (1) shRNA was cloned into a lentiviral silencing vector to obtain a lentiviral vector that stably silenced circEGFR; the lentiviral silencing vector was PLent-U6-GFP-Puro;

[0072] (2) transfecting MDA-MB-231 cells with the stably silenced circEGFR lentiviral vector obtained in step (1);

[0073] (3) RT-qPCR was used to detect the expression level of circEGFR in tissue and plasma exosomes of patients with triple-negative breast cancer.

[0074] The sh-RNA sequence is any one or more of the following three pairs of complementary chain shRNAs designed for the cleavage site of circEGFR;

[0075] CircEGFR shRNA-1BamHI F: 5'-gatccCCTGGGCAGCTTTTGCCAAGGC TCGAGCCTTGGCAAAAGCTGCCCAGGTTTTTa-3';

[0076] CircEGFR shRNA-1Mlul R: 5'-cgcgtAAAAACCTGGGCAGCTTTTGCCAAGGCTCGAGCCTTGGCAAAAGCTGCCCAGGg-3';

[0077] CircEGFR shRNA-2BamHI F: 5'-gatccTGGGCAGCTTTTGCCAAGGCAC TCGAGTGCCTTGGCAAAAGCTGCCCATTTTTa-3';

[0078] CircEGFR shRNA-2Mlul R: 5'-cgcgtAAAAATGGGCAGCTTTTGCCAAGGCACTCGAGTGCCTTGGCAAAAGCTGCCCAg-3';

[0079] CircEGFR shRNA-3BamHI F: 5'-gatccGCAGCTTTGCCAAGGCACGAC TCGAGTCGTGCCTTGGCAAAAGCTGCTTTTTa-3';

[0080] CircEGFR shRNA-3Mlul R: 5'-cgcgtAAAAAGCAGCTTTTGCCAAGGCACGACTCGAGTCGTGCCTTGGCAAAAGCTGCg-3'.

[0081] In the above sh-RNA sequence, the capital letters are the main bases that play a role, and the lowercase letters are the bases added to increase the stability of the sequence.

[0082] Further preferably, the specific steps for constructing a lentiviral vector for stably silencing circEGFR are as follows:

[0083] (1) Preparation of shRNA complementary strand;

[0084] (2) Double restriction digestion of lentiviral silencing vector;

[0085] (3) Recovering the enzyme digestion product to obtain the lentiviral silencing vector after enzyme digestion;

[0086] (4) connecting the complementary strand of the target fragment shRNA to the vector obtained in step (3) to obtain a connection product;

[0087] (5) transformation of the ligation product obtained in step (4);

[0088] (6) Sequencing and identifying the converted ligation product obtained in step (5).

[0089] Further preferably, the specific information of the circEGFR amplification primer sequence is as follows:

[0090] Upstream primer: 5′-CATGTCGATGGACTTCCAGA-3′;

[0091] Downstream primer: 5′-AAATTCCCAAGGACCACCTC-3′.

[0092] Further preferably, the specific steps of transfecting MDA-MB-231 cells with a lentiviral vector for stably silencing circEGFR are as follows:

[0093] a. Transfection of MDA-MB-231 cells with lentiviral vector:

[0094] The day before transfection, MDA-MB-231 cells were passaged and transferred into 24-well plates to a confluency of 70% to 80%. 200 μL of lentiviral silencing vector was added to each well and cultured for 24 h before switching to complete culture medium.

[0095] b. Detection of killing curve:

[0096] (1) The MDA-MB-231 cells obtained in step a were prepared into a cell suspension using a serum-free medium without dual antibodies, inoculated into a 96-well plate, and cultured for 24 hours;

[0097] (2) adding different concentrations of puromycin: the concentrations were 0 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.2 μg / mL, 1.4 μg / mL, 1.6 μg / mL, 1.8 μg / mL, and 2 μg / mL;

[0098] (3) The cells were cultured for 7 days, during which the cell culture medium was changed once. On the 7th day, it was found that the cells with a puromycin concentration of 0 to 1 μg / mL survived, while the cells with a puromycin concentration of 1 to 2 μg / mL partially died.

[0099] (4) Select 1 μg / mL of puromycin as the concentration for stable transfectant screening;

[0100] c. Screening of stable transgenic strains:

[0101] (1) MDA-MB-231 cells were infected with a stably silenced circEGFR lentiviral vector and cultured for 48 h. Complete medium containing 1 μg / mL puromycin was added and cultured for 7 days, during which the medium was changed once;

[0102] (2) Observe and mark individual cell populations on the culture plate under a microscope;

[0103] (3) Digest the cell population with trypsin and inoculate it into a new 24-well plate for culture;

[0104] (4) Continue subclone screening with puromycin for 7 days;

[0105] (5) Observe under a microscope and select the normally growing cell population for subculture, and use 1 μg / mL puromycin to maintain stable culture for 30 days;

[0106] The expression of circEGFR in tissues and plasma exosomes of patients with triple-negative breast cancer was detected by RT-qPCR, and it was found that circEGFR has the function of predicting the prognosis of patients.

[0107] The present invention also provides an application of a substance for overexpressing or silencing circEGFR in preparing a product for treating triple-negative breast cancer, wherein the circBase ID of the circEGFR is: hsa_circ_0080220.

[0108] Preferably, the substance for overexpressing or silencing circEGFR includes amplification primers of circEGFR, and the specific sequence information thereof is as follows:

[0109] Upstream primer: 5′-CATGTCGATGGACTTCCAGA-3′;

[0110] Downstream primer: 5′-AAATTCCCAAGGACCACCTC-3′.

[0111] Preferably, the substance for silencing circEGFR further comprises a shRNA sequence cloned in a lentiviral silencing vector, wherein the shRNA sequence is any one or more of the following three pairs of complementary chain shRNAs designed for the cleavage site of circEGFR;

[0112] CircEGFR shRNA-1BamHI F: 5'-gatccCCTGGGCAGCTTTTGCCAAGGC TCGAGCCTTGGCAAAAGCTGCCCAGGTTTTTa-3';

[0113] CircEGFR shRNA-1Mlul R: 5'-cgcgtAAAAACCTGGGCAGCTTTTGCCAAGGCTCGAGCCTTGGCAAAAGCTGCCCAGGg-3';

[0114] CircEGFR shRNA-2BamHI F: 5'-gatccTGGGCAGCTTTTGCCAAGGCAC TCGAGTGCCTTGGCAAAAGCTGCCCATTTTTa-3';

[0115] CircEGFR shRNA-2Mlul R: 5'-cgcgtAAAAATGGGCAGCTTTTGCCAAGGCACTCGAGTGCCTTGGCAAAAGCTGCCCAg-3';

[0116] CircEGFR shRNA-3BamHI F: 5'-gatccGCAGCTTTGCCAAGGCACGAC TCGAGTCGTGCCTTGGCAAAAGCTGCTTTTTa-3';

[0117] CircEGFR shRNA-3Mlul R: 5'-cgcgtAAAAAGCAGCTTTTGCCAAGGCACGACTCGAGTCGTGCCTTGGCAAAAGCTGCg-3'.

[0118] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0119] Example:

[0120] The present invention provides an application of circEGFR as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product, which specifically comprises the following steps:

[0121] 1. Construction of a stable silencing circEGFR short hairpin RNA lentiviral vector:

[0122] (1) Short hairpin RNA (shRNA) design:

[0123] CircEGFR shRNA-1BamHI F: 5'-gatccCCTGGGCAGCTTTTGCCAAGGC TCGAGCCTTGGCAAAAGCTGCCCAGGTTTTTa-3';

[0124] CircEGFR shRNA-1Mlul R: 5'-cgcgtAAAAACCTGGGCAGCTTTTGCCAAGGCTCGAGCCTTGGCAAAAGCTGCCCAGGg-3';

[0125] CircEGFR shRNA-2BamHI F: 5'-gatccTGGGCAGCTTTTGCCAAGGCAC TCGAGTGCCTTGGCAAAAGCTGCCCATTTTTa-3';

[0126] CircEGFR shRNA-2Mlul R: 5'-cgcgtAAAAATGGGCAGCTTTTGCCAAGGCACTCGAGTGCCTTGGCAAAAGCTGCCCAg-3';

[0127] CircEGFR shRNA-3BamHI F: 5'-gatccGCAGCTTTGCCAAGGCACGAC TCGAGTCGTGCCTTGGCAAAAGCTGCTTTTTa-3';

[0128] CircEGFR shRNA-3Mlul R: 5'-cgcgtAAAAAGCAGCTTTTGCCAAGGCACGACTCGAGTCGTGCCTTGGCAAAAGCTGCg-3'.

[0129] They were cloned into PLent-U6-GFP-Puro vector respectively;

[0130] (2) Double-chain preparation

[0131] CircEGFR shRNA-1BamHI F, circEGFR shRNA-2BamHI F, circEGFR shRNA-3BamHIF and circEGFR shRNA-1Mlul R, circEGFR shRNA-2Mlul R, circEGFR-3Mlul R were mixed in equal volumes, boiled in boiling water for 5 min, incubated at 72°C for 15 min, cooled naturally to room temperature, and then connected to the PLent-U6-GFP-Puro vector or stored at -20°C.

[0132] (3) Double digestion of vector

[0133] The BamHI and Mlul double enzyme digestion of the PLent-U6-GFP-Puro vector was performed in a 0.2 mL RNase-free centrifuge tube. The enzyme digestion system is as follows:

[0134]

[0135] Incubate at 37°C for 4 hours.

[0136] (4) Recovery of enzyme digestion products

[0137] ①After 1% gel electrophoresis of the PCR product, cut the gel band containing the target circRNA fragment into a 1.5mL RNase-free centrifuge tube, and add 100μL BD solution per 100mg gel;

[0138] ② Place in a 60℃ water bath for 10 min until the gel is dissolved, vortexing 3 times during this period;

[0139] ③ Transfer the solution to a DNA purification column, let it stand for 2 minutes, centrifuge at 12000r / min for 1 minute at room temperature, and discard the effluent;

[0140] ④ Add 500 μL PE solution, centrifuge at 12000 r / min for 1 min at room temperature, and discard the effluent;

[0141] ⑤Repeat ④ once;

[0142] ⑥ Centrifuge at 12000r / min for 1min and discard the effluent;

[0143] ⑦ Place the column in a new 1.5 mL RNase-free centrifuge tube, add 30 μL of RNase-free water, and centrifuge at 13400 × g for 1 min to obtain a DNA solution.

[0144] (5) Ligation of target fragment and vector

[0145] Add the following reagents to a 0.2 mL RNase-free centrifuge tube:

[0146]

[0147] Connect at 16°C for 1 h.

[0148] (6) Transformation of Ligation Product

[0149] 1) Add 10 μL of ligation product to 50 μL of DH5α competent cells, mix well and incubate on ice for 30 minutes;

[0150] 2) Heat shock in 42℃ water bath for 90s;

[0151] 3) Ice bath for 2 minutes;

[0152] 4) Add 200 μL LB medium, mix well, and culture at 37°C, 200 rpm for 1 h;

[0153] 5) In a clean bench, evenly spread the bacterial solution on an LB plate containing ampicillin (100 μg / mL) and leave it at room temperature until the liquid is absorbed;

[0154] 6) Invert the plate and transfer it to a 37°C biochemical incubator for overnight culture.

[0155] (8) Identification of positive clones by plasmid digestion

[0156] 1) Pick several single clones from the plate and culture them in a 3 mL LB tube on a shaking platform overnight;

[0157] 2) Plasmid extraction

[0158] ① Collect 3 μL of bacterial solution in a 1.5 mL RNase-free centrifuge tube, centrifuge at 12000 r / min for 1 min, and discard the supernatant;

[0159] ②Add 250 μL of Solution I / RNase A mixture to resuspend the bacteria;

[0160] ③ Add 250 μL of solution II, mix by inversion 6 times, and leave at room temperature for 2 min;

[0161] ④Add 350 μL of solution III and mix by inversion 6 times;

[0162] ⑤ Centrifuge at 12000r / min for 10min, transfer the supernatant to the DNA purification column and let stand for 2min;

[0163] ⑥ Centrifuge at 12000r / min for 1min and discard the effluent;

[0164] ⑦ Add 500 μL of solution PB to the purification column, centrifuge at 12000 r / min for 1 min, and discard the effluent;

[0165] ⑧ Add 500 μL of solution W to the purification column, centrifuge at 12000 r / min for 1 min, and discard the effluent;

[0166] ⑨ Centrifuge the empty purification column at 12000r / min for 3min;

[0167] ⑩ Place the purification column in a new 1.5mL RNase-free centrifuge tube, add 50μL RNase-free water, let it stand for 2min, and centrifuge at 13400r / min for 1min to elute the plasmid.

[0168] 3) Enzyme digestion and identification of extracted plasmid

[0169] The enzyme digestion reaction system is as follows:

[0170]

[0171] The enzyme digestion was carried out at 37°C for 2 h, and the digestion products were separated by 1% agarose gel electrophoresis containing ethidium bromide and imaged by UVP gel imaging system.

[0172] 4) Sequencing of positive plasmids.

[0173] 2. Stable silencing of circEGFR lentiviral vector transfection, screening of MDA-MB-231 stable expression cell lines. The specific steps are as follows:

[0174] Infect cells with lentiviral vectors:

[0175] One day before lentiviral vector infection, MDA-MB-231 cells were passaged and transferred into 24-well plates to a confluency of 70% to 80%. 200 μL of lentiviral vector was added to each well and the cells were cultured for 24 hours before being replaced with complete culture medium.

[0176] Kill curve detection:

[0177] (1) MDA-MB-231 cells were prepared into a cell suspension using a serum-free medium without dual antibodies, inoculated into a 96-well plate, and cultured for 24 h;

[0178] (2) adding different concentrations of puromycin: 0 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.2 μg / mL, 1.4 μg / mL, 1.6 μg / mL, 1.8 μg / mL, and 2 μg / mL;

[0179] (3) The cells were cultured for 7 days, during which the cell culture medium was changed once. On the 7th day, it was found that the cells with a puromycin concentration of 0 to 1 μg / mL survived, while the cells with a puromycin concentration of 1 to 2 μg / mL partially died.

[0180] (4) Select a puromycin concentration of 1 μg / mL as the stable transfectant screening concentration.

[0181] Screening of stable transfectants:

[0182] (1) MDA-MB-231 cells were infected with a stably silenced circEGFR lentiviral vector and cultured for 48 h. Complete medium containing 1 μg / mL puromycin was added and cultured for 7 days, during which the medium was changed once;

[0183] (2) Observe and mark individual cell populations on the culture plate under a microscope;

[0184] (3) Digest the cell population with trypsin and inoculate it into a new 24-well plate for culture;

[0185] (4) Continue subclone screening with puromycin for 7 days;

[0186] (5) Observe under a microscope and select the normally growing cell population for subculture, and use 1 μg / mL puromycin to maintain stable culture for 30 days;

[0187] 3. Design of specific primer sequences for circEGFR amplification The specific information is as follows:

[0188] Upstream primer: 5′-CATGTCGATGGACTTCCAGA-3′;

[0189] Downstream primer: 5′-AAATTCCCAAGGACCACCTC-3′;

[0190] The expression of circEGFR in tissues and plasma exosomes of patients with triple-negative breast cancer was detected by RT-qPCR, and it was found that circEGFR has the function of predicting the prognosis of patients.

[0191] Solution I: 50 mM glucose / 10 mM EDTA / 25 mM Tris-HCl, pH = 8.0, i.e., resuspension solution;

[0192] Solution II: 0.2Ml NaOH / 1% SDS, i.e., lysis solution;

[0193] Solution III: 3Ml potassium acetate / 2Ml acetic acid, i.e. neutralization precipitate solution;

[0194] Solution PB is a plasmid extraction reagent purchased from Dongsheng Biotech. Solution PB contains alkali and protein denaturants. Do not contact the skin directly. Solution W is a plasmid extraction reagent purchased from Dongsheng Biotech. Solution W is diluted with anhydrous ethanol at a ratio of 1:1.5 before first use, i.e., it contains 60% ethanol. Solution BD is a DNA gel recovery reagent purchased from Dongsheng Biotech. Solution BD contains denaturants. Do not contact the skin directly.

[0195] PE solution: 10 mM Tris-HCl (pH 7.5) and 80% ethanol.

[0196] Test and results:

[0197] 1. Identification of stable circEGFR silencing vectors:

[0198] The sequencing results were compared by Blast, see Figure 1-3 ;

[0199] like Figure 1 As shown, the gene sh-circEGFR-1 has been successfully cloned into the PLent-U6-GFP-Puro vector. The original sequence was compared with the known sequence by Blast, and the results were 100% consistent and could be used for subsequent experiments.

[0200] like Figure 2 As shown, the gene sh-circEGFR-2 has been successfully cloned into the PLent-U6-GFP-Puro vector. The original sequence was compared with the known sequence by Blast, and the results were 100% consistent and could be used for subsequent experiments.

[0201] like Figure 3 As shown, the gene sh-circEGFR-3 has been successfully cloned into the PLent-U6-GFP-Puro vector. The original sequence was compared with the known sequence by Blast, and the results were 100% consistent and could be used for subsequent experiments.

[0202] 2. Transfection efficiency evaluation

[0203] After MDA-MB-231 cells were transfected with stably overexpressing circEGFR lentivirus (circEGFR group) and stably silencing circEGFR lentivirus (sh-circEGFR group), the transfection efficiency was evaluated using a fluorescence microscope ( Figure 4 A). Figure 4 As shown in B, the transfection efficiency of MDA-MB-231 cells in the circEGFR group was 96.63±1.58%, and the transfection efficiency of MDA-MB-231 cells in the sh-circEGFR group was 95.91±2.36%. The transfection efficiency was greater than 95% and could be used for subsequent experiments.

[0204] 3. Effects of overexpression or silencing of circEGFR on proliferation, migration, invasion and EMT of MDA-MB-231 cells

[0205] (1) The results of CCK-8 and EdU experiments are as follows Figure 5As shown, compared with the CON group, there were no significant differences in the OD value and EdU positivity rate of the cells in the MOCK group and sh-NC group (p>0.05); compared with the MOCK group, the OD value and EdU positivity rate of the cells in the circEGFR group were significantly increased (p<0.01); compared with the sh-NC group, the OD value and EdU positivity rate of the cells in the sh-circEGFR group were significantly decreased (p<0.01).

[0206] (2) Effect of overexpression or silencing of circEGFR on the migration ability of MDA-MB-231 cells

[0207] The results of the scratch test and Transwell vertical migration assay are shown in Figure 6 As shown, compared with the CON group, there was no significant difference in the cell migration rate between the MOCK group and the sh-NC group (p>0.05); compared with the MOCK group, the cell migration rate in the circEGFR group was significantly increased (p<0.01); compared with the sh-NC group, the cell migration rate in the sh-circEGFR group was significantly decreased (p<0.01).

[0208] (3) Effect of overexpression or silencing of circEGFR on the invasion ability of MDA-MB-231 cells

[0209] The results of the Transwell vertical invasion assay are shown in Figure 7 As shown, compared with the CON group, there was no significant difference in the invasion ability of cells in the MOCK group and the sh-NC group (p>0.05); compared with the MOCK group, the invasion ability of cells in the circEGFR group was significantly increased (p<0.01); compared with the sh-NC group, the invasion ability of cells in the sh-circEGFR group was significantly decreased (p<0.01).

[0210] (4) Effects of overexpression or silencing of circEGFR on EMT-related proteins in MDA-MB-231 cells

[0211] Western blot results Figure 8 As shown, compared with CON group, there were no significant differences in the expressions of E-cadherin, Vimentin and Snail proteins in MOCK group and sh-NC group cells (p>0.05); compared with MOCK group, the expression of E-cadherin protein in circEGFR group cells was significantly decreased (p<0.01), and the expressions of Vimentin and Snail proteins were significantly increased (p<0.01); compared with sh-NC group, the expression of E-cadherin protein in sh-circEGFR group cells was significantly increased (p<0.01), and the expressions of Vimentin and Snail proteins were significantly decreased (p<0.01).

[0212] The above tests and results show that silencing circEGFR can significantly reduce the proliferation, migration, invasion and EMT ability of MDA-MB-231. circEGFR can be used as a target for regulating the proliferation, migration, invasion and EMT of MDA-MB-231, providing new molecular targets and intervention strategies for the diagnosis and treatment of TNBC.

[0213] In summary, the present invention provides a kind of circEGFR as a molecular target and prognostic biomarker in the treatment of triple-negative breast cancer. By constructing a lentiviral overexpression vector and a lentiviral silencing vector of circEGFR, the effects of its overexpression or silencing on MDA-MB-231 cell proliferation, migration, invasion and EMT are evaluated, and the use of silencing circEGFR to treat triple-negative breast cancer is achieved. The expression of circEGFR in tissues and plasma exosomes of patients with triple-negative breast cancer was detected by RT-qPCR, and it was found that circEGFR has the effect of predicting the prognosis of patients. Therefore, the present invention provides a prognostic marker and a promising therapeutic target for TNBC. The intrinsic function and mechanism of the molecular target hsa_circ_0080220 (circEGFR) in the occurrence, development and drug treatment of TNBC are clarified.

[0214] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An application of circEGFR as a molecular target and prognostic biomarker in the preparation of a product for the treatment of triple-negative breast cancer, wherein the circBase ID of the circEGFR is: hsa_circ_0080220.

2. The use of circEGFR as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product according to claim 1, characterized in that: The following steps are involved: (1) shRNA was cloned into a lentiviral silencing vector to obtain a lentiviral vector that stably silenced circEGFR; (2) transfecting MDA-MB-231 cells with the stably silencing circEGFR lentiviral vector obtained in step (1); (3) RT-qPCR was used to detect the expression level of circEGFR in tissue and plasma exosomes of patients with triple-negative breast cancer.

3. The use of circEGFR as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product according to claim 2, characterized in that: The lentiviral silencing vector in step (1) is PLent-U6-GFP-Puro.

4. The use of circEGFR as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product according to claim 3, characterized in that: The shRNA sequence in step (1) is any one or more of the following three pairs of complementary chain shRNAs designed for the cleavage site of circEGFR; CircEGFR shRNA-1BamHI F: 5'-gatccCCTGGGCAGCTTTTGCCAAGGC TCGAGCCTTGGCAAAAGCTGCCCAGGTTTTTa-3'; CircEGFR shRNA-1Mlul R: 5'-cgcgtAAAAACCTGGGCAGCTTTTGCCAAGGCTCGAGCCTTGGCAAAAGCTGCCCAGGg-3'; CircEGFR shRNA-2BamHI F: 5'-gatccTGGGCAGCTTTTGCCAAGGCAC TCGAGTGCCTTGGCAAAAGCTGCCCATTTTTa-3'; CircEGFR shRNA-2Mlul R: 5'-cgcgtAAAAATGGGCAGCTTTTGCCAAGGCACTCGAGTGCCTTGGCAAAAGCTGCCCAg-3'; CircEGFR shRNA-3BamHI F: 5'-gatccGCAGCTTTGCCAAGGCACGAC TCGAGTCGTGCCTTGGCAAAAGCTGCTTTTTa-3'; CircEGFR shRNA-3Mlul R: 5'-cgcgtAAAAAGCAGCTTTTGCCAAGGCACGACTCGAGTCGTGCCTTGGCAAAAGCTGCg-3'.

5. The use of circEGFR as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product according to claim 4, characterized in that: Step (1) The construction steps of shRNA cloned into lentiviral silencing vector are as follows: (1) Preparation of shRNA complementary strand; (2) Double restriction digestion of lentiviral silencing vector; (3) Recovering the enzyme digestion product to obtain the lentiviral silencing vector after enzyme digestion; (4) connecting the complementary strand of the target fragment shRNA to the vector obtained in step (3) to obtain a connection product; (5) transformation of the ligation product obtained in step (4); (6) Sequencing and identifying the converted ligation product obtained in step (5).

6. The use of circEGFR as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product according to claim 4, characterized in that: The specific steps of transfecting MDA-MB-231 cells with the stable silencing circEGFR lentiviral vector in step (2) are as follows: a. Transfection of MDA-MB-231 cells with lentiviral vector: The day before transfection, MDA-MB-231 cells were passaged and transferred into 24-well plates to a confluency of 70% to 80%. 200 μL of lentiviral silencing vector was added to each well and cultured for 24 h before switching to complete culture medium. b. Detection of killing curve: (1) The MDA-MB-231 cells obtained in step a were prepared into a cell suspension using a serum-free medium without dual antibodies, inoculated into a 96-well plate, and cultured for 24 hours; (2) adding different concentrations of puromycin: the concentrations were 0 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.2 μg / mL, 1.4 μg / mL, 1.6 μg / mL, 1.8 μg / mL, and 2 μg / mL; (3) The cells were cultured for 7 days, during which the cell culture medium was changed once. On the 7th day, it was found that the cells with a puromycin concentration of 0 to 1 μg / mL survived, while the cells with a puromycin concentration of 1 to 2 μg / mL partially died. (4) Select 1 μg / mL of puromycin as the concentration for stable transfectant screening; c. Screening of stable transgenic strains: (1) MDA-MB-231 cells were infected with a stably silenced circEGFR lentiviral vector and cultured for 48 h. Complete medium containing 1 μg / mL puromycin was added and cultured for 7 days, during which the medium was changed once; (2) Observe and mark individual cell populations on the culture plate under a microscope; (3) Digest the cell population with trypsin and inoculate it into a new 24-well plate for culture; (4) Continue subclone screening with puromycin for 7 days; (5) Observe under a microscope and select the normally growing cell population for subculture, and use 1 μg / mL puromycin to maintain stable culture for 30 days.

7. The use of circEGFR as a molecular target and prognostic biomarker in the preparation of a triple-negative breast cancer treatment product according to claim 2, characterized in that: The specific sequence information of the amplification primers for circEGFR is as follows: Upstream primer: 5′-CATGTCGATGGACTTCCAGA-3′; Downstream primer: 5′-AAATTCCCAAGGACCACCTC-3′.

8. Use of a substance for overexpressing or silencing circEGFR in the preparation of a product for treating triple-negative breast cancer, wherein the circBase ID of the circEGFR is: hsa_circ_0080220.

9. Use of the substance for overexpressing or silencing circEGFR according to claim 8 in preparing a product for treating triple-negative breast cancer, characterized in that: The substance for overexpressing or silencing circEGFR includes amplification primers of circEGFR, and the specific sequence information thereof is as follows: Upstream primer: 5′-CATGTCGATGGACTTCCAGA-3′; Downstream primer: 5′-AAATTCCCAAGGACCACCTC-3′.

10. Use of the substance for overexpressing or silencing circEGFR according to claim 8 in preparing a product for treating triple-negative breast cancer, characterized in that: The substance for silencing circEGFR also includes a shRNA sequence cloned in a lentiviral silencing vector, wherein the shRNA sequence is any one or more of the following three pairs of complementary chain shRNAs designed for the cleavage site of circEGFR; CircEGFR shRNA-1BamHI F: 5'-gatccCCTGGGCAGCTTTTGCCAAGGC TCGAGCCTTGGCAAAAGCTGCCCAGGTTTTTa-3'; CircEGFR shRNA-1Mlul R: 5'-cgcgtAAAAACCTGGGCAGCTTTTGCCAAGGCTCGAGCCTTGGCAAAAGCTGCCCAGGg-3'; CircEGFR shRNA-2BamHI F: 5'-gatccTGGGCAGCTTTTGCCAAGGCAC TCGAGTGCCTTGGCAAAAGCTGCCCATTTTTa-3'; CircEGFR shRNA-2Mlul R: 5'-cgcgtAAAAATGGGCAGCTTTTGCCAAGGCACTCGAGTGCCTTGGCAAAAGCTGCCCAg-3'; CircEGFR shRNA-3BamHI F: 5'-gatccGCAGCTTTGCCAAGGCACGAC TCGAGTCGTGCCTTGGCAAAAGCTGCTTTTTa-3'; CircEGFR shRNA-3Mlul R: 5'-cgcgtAAAAAGCAGCTTTTGCCAAGGCACGACTCGAGTCGTGCCTTGGCAAAAGCTGCg-3'.

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