CN114099685A

CN114099685A - Application of substance for inhibiting MUC1 expression and glycosylation modification in reducing drug resistance of anti-breast cancer drugs

Info

Publication number: CN114099685A
Application number: CN202210096709.0A
Authority: CN
Inventors: 战吉宬; 席晓敏; 黄卫东; 游义琳
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2022-03-01
Anticipated expiration: 2042-01-27
Also published as: CN114099685B; CN115068610B; CN115068610A; CN114796498A

Abstract

The invention discloses application of a substance for inhibiting MUC1 expression and glycosylation modification in reducing drug resistance of an anti-breast cancer drug. Cell experiments prove that the sensitivities of flavonoid compounds such as apigenin, chrysin, diosmetin, luteolin and quercetin and clinical small molecule drugs (such as cisplatin, 5-fluorouracil and burley mycin) are all dependent on the expression of MUC1 or glycosylation modification of MUC 1; animal experiments prove that the knockout of MUC1 can effectively improve the sensitivity of the medicine, promote the toxicity of the medicine in vivo and reduce the occurrence and development of tumors, thereby achieving the purpose of treating breast cancer. The invention has important application value.

Description

Application of substance for inhibiting MUC1 expression and glycosylation modification in reducing drug resistance of anti-breast cancer drugs

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of a substance for inhibiting MUC1 expression and glycosylation modification in reducing drug resistance of an anti-breast cancer drug.

Background

Breast cancer is the most commonly diagnosed cancer in women worldwide. Breast cancer tumors are highly heterogeneous, and multiple signaling pathways can mediate the development of the breast cancer tumors, so that early screening is a main means for preventing breast cancer. Although the treatment of breast cancer is increasing with the development of technology (e.g., surgical resection, chemotherapy, radiation therapy, endocrine-targeted therapy), like triple negative breast cancer, chemotherapy is becoming the most prominent method to rescue such cancers due to the lack of hormone receptors. Drug resistance is the most serious challenge in clinical treatment of breast cancer, and the toxicity sensitivity of breast cancer after recurrence to chemotherapy is reduced sharply. Moreover, some breast cancers have inherent properties that make them less responsive to chemotherapeutic drugs, even if they do not develop resistance to a particular drug. Therefore, increasing the sensitivity of breast cancer chemotherapy is a formidable challenge to overcome.

Apigenin, known as 4',5, 7-trihydroxyflavone, is insoluble in water, is yellow needle-like crystal, has high content in flos Matricariae Chamomillae, parsley, Bulbus Allii Cepae, berry and Fortunella margarita (lour.) Swingle, and is widely present in resident dietary diet. Apigenin is used as a secondary metabolite of plants for resisting stress, has extremely strong physiological activity, has excellent performances in the aspects of resisting inflammation and bacteria, resisting oxidation, stabilizing body metabolism, regulating endocrine, preventing cardiovascular and cerebrovascular diseases, protecting central nervous system and the like, and is particularly increasingly expanded and deepened in the research of inhibiting cancer cell proliferation and metastasis, namely apigenin has extremely low toxic and side effects on normal cell tissues, but has strong killing power on cancer cells. In the previous research, due to the limited bioavailability and absorptivity of the water-insoluble apigenin, the in vivo and in vitro dosage of the water-insoluble apigenin is usually high, so that the clinical wide application of the natural medicine is greatly limited. The flavonoid drugs with similar structures with the apigenin also comprise chrysin, diosmetin, luteolin, quercetin and the like, have strong cancer inhibition activity in vivo and in vitro, and have great potential for being developed into natural anticancer drugs. However, clinical drugs such as flavonoids and cisplatin belong to small molecule drugs, and the drug sensitivity and drug resistance of the drugs are always main obstacles for clinical application of cancer treatment.

MUC1 is a type I transmembrane mucin with a highly glycosylated extracellular structure. MUC1 contains a variable number of O-linked glycosylation sites, and different populations can carry over the range of about 100-750O-glycans due to genetic polymorphisms. The highly glycosylated extracellular domain of MUC1 may extend from the cell surface to 200-500 nm, and usually has a large number of sialic acid modifications in the terminal regions of its sugar chains. Specifically, the N-terminal domain of MUC1 is extensively modified with O-glycosyl groups, interweaving to cover the cell surface to form a protective barrier. Depending on the number of repeat sequences and the degree of glycosylation, O-glycosylation accounts for 50-90% of the total weight of MUC1, and thus the molecular weight varies, with higher molecular weights representing more glycosylation modifications. Typical mucin-type O-glycosylation is initiated by N-acetylgalactosamine (GalNAc), which is the first monosaccharide attached to a serine or threonine and can be extended to a variety of structures. Meanwhile, glycosylation modification of MUC1 depends on tissue-specific expression and co-coordination of various glycosyltransferases, among which the glycosyltransferase GCNT3 is one of the key transferases that modifies O-glycosylation of the core region of MUC 1.

Disclosure of Invention

The invention aims to improve the sensitivity of breast cancer cells to anti-breast cancer drugs, thereby treating breast cancer.

The invention firstly protects the application of a substance inhibiting MUC1 expression in breast cancer cells or a substance inhibiting MUC1 glycosylation modification in breast cancer cells, which can be at least one of A1) -A5):

A1) increasing the sensitivity of breast cancer cells to anti-breast cancer drugs;

A2) reducing the drug resistance of anti-breast cancer drugs;

A3) preparing a product for increasing the sensitivity of breast cancer cells to anti-breast cancer drugs;

A4) preparing a product for reducing the drug resistance of an anti-breast cancer drug;

A5) preparing a product for adjuvant therapy of breast cancer.

In the above application, the anti-breast cancer drug may be at least one of cisplatin, 5-fluorouracil, burley mycin, apigenin, chrysin, diosmetin, luteolin, and quercetin.

In the above application, the substance for inhibiting the expression of MUC1 in breast cancer cells may be oligo-nucleic acid 1, oligo-nucleic acid 2 or oligo-nucleic acid 3;

the oligomeric nucleic acid 1 consists of a nucleic acid molecule shown as SEQ ID NO. 1 and a nucleic acid molecule shown as SEQ ID NO. 2;

the oligomeric nucleic acid 2 consists of a nucleic acid molecule shown as SEQ ID NO. 3 and a nucleic acid molecule shown as SEQ ID NO. 4;

the oligo-nucleic acid 3 consists of a nucleic acid molecule shown as SEQ ID NO. 5 and a nucleic acid molecule shown as SEQ ID NO. 6.

In the above application, the substance for inhibiting the glycosylation modification of MUC1 in breast cancer cells may be BAG, sialidase or a substance for inhibiting the expression of glycosyltransferase GCNT3 in breast cancer cells.

The substance for inhibiting the expression of glycosyltransferase GCNT3 in breast cancer cells can be oligonucleotide a, oligonucleotide b or oligonucleotide c;

the oligomeric nucleic acid a consists of a nucleic acid molecule shown as SEQ ID NO. 7 and a nucleic acid molecule shown as SEQ ID NO. 8;

the oligomeric nucleic acid b consists of a nucleic acid molecule shown as SEQ ID NO. 9 and a nucleic acid molecule shown as SEQ ID NO. 10;

the oligonucleotide c consists of a nucleic acid molecule shown as SEQ ID NO. 11 and a nucleic acid molecule shown as SEQ ID NO. 12.

The invention also provides a product which consists of any one of the anti-breast cancer drugs and a substance for inhibiting MUC 1;

the substance for inhibiting MUC1 is the substance for inhibiting MUC1 expression in the breast cancer cells or the substance for inhibiting MUC1 glycosylation modification in the breast cancer cells;

the product may function as at least one of B1) -B3):

B1) increasing the sensitivity of breast cancer cells to anti-breast cancer drugs;

B2) reducing the drug resistance of anti-breast cancer drugs;

B3) can be used for adjuvant treatment of breast cancer.

The invention also provides a sensitizer of any one of the anti-breast cancer drugs, which is a substance for inhibiting MUC1 expression in a breast cancer cell or a substance for inhibiting MUC1 glycosylation modification in the breast cancer cell.

The invention also provides a screening method of the anti-breast cancer drug, and the anti-breast cancer drug can be sensitized by any substance for inhibiting the expression of MUC1 in the breast cancer cells or any substance for inhibiting the glycosylation modification of MUC1 in the breast cancer cells.

The invention also provides a method for improving the sensitivity of the breast cancer cells to the anti-breast cancer drugs, which can be realized by inhibiting the expression of MUC1 in the breast cancer cells or inhibiting the glycosylation modification of MUC1 in the breast cancer cells; the method is useful for diagnosis and treatment of non-diseases.

The invention also provides a method for reducing drug resistance of breast cancer cells to anti-breast cancer drugs, which can be realized by inhibiting MUC1 expression in the breast cancer cells or inhibiting MUC1 glycosylation modification in the breast cancer cells; the method is useful for diagnosis and treatment of non-diseases.

In any of the above methods, the inhibition of the expression of MUC1 in the breast cancer cell can be achieved by introducing into the breast cancer cell a substance that inhibits the expression of MUC1 in the breast cancer cell. The substance for inhibiting MUC1 expression in the breast cancer cell can be oligo-nucleic acid 1, oligo-nucleic acid 2 or oligo-nucleic acid 3;

In any of the methods described above, the inhibition of the modification of MUC1 glycosylation in breast cancer cells may be achieved in one or two ways; the first mode can be treating breast cancer cells with BAG or sialidase; the second mode can be realized by introducing a substance which inhibits the glycosylation modification of MUC1 in the breast cancer cells into the breast cancer cells. The substance inhibiting modification of MUC1 glycosylation in a breast cancer cell may be a substance inhibiting expression of glycosyltransferase GCNT3 in a breast cancer cell; the substance for inhibiting the expression of glycosyltransferase GCNT3 in breast cancer cells can be oligonucleotide a, oligonucleotide b or oligonucleotide c;

In one such mode, the BAG may be treated at a concentration of 1-3mM (e.g., 1-2mM, 2-3mM, 1mM, 2mM, or 3 mM).

In the first mode, the treatment time of the sialidase can be 6-12h (such as 6-9h, 9-12h, 6h, 9h or 12 h).

In any of the above methods, the anti-breast cancer drug may be at least one of cisplatin, 5-fluorouracil, burley green mycin, apigenin, chrysin, diosmetin, luteolin, and quercetin.

Any one of the breast cancer cells is MCF-7 cell and MDA-MB-231 cell.

Cell experiments prove that the sensitivities of flavonoids such as apigenin, chrysin, diosmetin, luteolin and quercetin and clinically common small molecule drugs (such as cisplatin, 5-fluorouracil and burley mycin) depend on the expression of MUC1 or glycosylation modification of MUC1, and when MUC1 is lacked, the glycosylation inhibition of the targeted flavonoid drugs cannot synergize the intracellular toxicity of the drugs. Animal experiments prove that the knockout of MUC1 can effectively improve the drug sensitivity of apigenin, promote the toxicity of the drug in vivo and reduce the tumorigenesis and development. Therefore, the substance for inhibiting the expression of MUC1 in the breast cancer cells or the substance for inhibiting the glycosylation modification of MUC1 in the breast cancer cells can be combined with the anti-breast cancer medicine to be applied to patients, thereby achieving the aim of treating the breast cancer. The invention has important application value.

Drawings

Figure 1 is a western immunoblot and qPCR assay of a CRISPR Cas 9-mediated MUC1 monoclonal knockout cell line. Whereinp <0.05 was considered statistically significant: denotesP<0.05, denotesP<0.01, star tableDisplay deviceP<0.001, isP<0.0001, ns means no significant difference.

FIG. 2 is a screen showing the concentration of a Benzyl-N-acetyl-alpha-galactosamine (hereinafter, BAG) and the treatment time of a sialidase (hereinafter, Neu) without additional toxicity to the growth of breast cancer cells and without influence on the transcription level of MUC 1. Whereinp <0.05 was considered statistically significant: denotesP<0.05, denotesP<0.01, denotesP<0.001, isP<0.0001, ns means no significant difference.

FIG. 3 is a protein validation of the O-glycosylation inhibitor BAG for inhibiting the glycosylation of MUC 1.

FIG. 4 is a protein demonstration of inhibition of MUC1 glycosylation by sialidase Neu.

FIG. 5 is a protein validation of the glycosyltransferase GCNT3 knockout and the effect on MUC1 glycosylation.

Fig. 6 is a graph showing that BAG inhibition of glycosylation increases the intracellular toxicity sensitivity of breast cancer cells to apigenin. Whereinp <0.05 was considered statistically significant: denotesP<0.05, denotesP<0.01, denotesP<0.001, isP<0.0001, ns means no significant difference.

FIG. 7 shows that sialidase Neu hydrolyzes the glycosyl chain end to increase the intracellular toxicity sensitivity of breast cancer cells to apigenin. Whereinp <0.05 was considered statistically significant: denotesP<0.05, denotesP<0.01, denotesP<0.001, isP<0.0001, ns means no significant difference.

Figure 8 is a graph showing that glycosyltransferase GCNT3 knockdown increases the sensitivity of breast cancer cells to intracellular toxicity of apigenin. Whereinp <0.05 was considered statistically significant: denotesP<0.05, denotesP<0.01, denotesP<0.001, isP<0.0001, ns means no significant difference.

Fig. 9 shows the structure of apigenin similar to flavonoid compounds. Whereinp <0.05 is considered to have statisticsSignificance of study: denotesP<0.05, denotesP<0.01, denotesP<0.001, isP<0.0001, ns means no significant difference.

FIG. 10 shows the sensitivity of O-glycosylation inhibitors in combination with flavonoids to cytotoxicity of breast cancer. Wherein, Chr is chrysin, Dio is diosmetin, Lut is luteolin, and Que is quercetin. Whereinp <0.05 was considered statistically significant: denotesP<0.05, denotesP<0.01, denotesP<0.001, isP<0.0001, ns means no significant difference.

FIG. 11 is a graph demonstrating sensitivity to breast cancer cytotoxicity for cisplatin, 5-fluorouracil, and brevicmycin. Whereinp <0.05 was considered statistically significant: denotesP<0.05, denotesP<0.01, denotesP<0.001, isP<0.0001, ns means no significant difference.

FIG. 12 shows the experimental verification of tumor-bearing capacity of nude mice. Whereinp <0.05 was considered statistically significant: denotesP<0.05, denotesP<0.01, denotesP<0.001, isP<0.0001, ns means no significant difference.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The following examples relate to breast cancer cells that are MCF-7 cells and MDA-MB-231 cells, and the media used to culture breast cancer cells were DMEM high-glucose media purchased from Saimer Fei. Both the O-glycosylation inhibitor and the sialidase are products from Sigma. Apigenin (greater than 99% pure) is a product of medemnst bio. Cisplatin, burley mycine and 5-fluorouracil are all products of the United states Selleck company, and cisplatin, burley mycine and 5-fluorouracil are all chemotherapeutic drugs. The CCK-8 cell viability detection reagent and the crystal violet dye are all products of Shanghai Biyuntian biological company. Monoclonal antibodies are all products of ProteinTech. Both the qPCR primers and the gRNA were synthesized by Beijing Rui Bo Biotechnology, Inc.

Example 1 acquisition and characterization of a MUC1 monoclonal knockout cell line

1. Human MUC1 sg1-forward was artificially designed and synthesized: 5'-CACCGCAGCAGGAAGAAAGGAGAC-3' (SEQ ID NO: 1) and human MUC1 sg 1-reverse: 5'-AAACGTCTCCTTTCTTCCTGCTGC-3' (SEQ ID NO: 2), followed by annealing, to give an annealed product 1. The pLenti-CRISPR v2 all in one vector (Addgene) is cut by BsmI (product of NEB company) to remove a specific Filter region, and the vector framework is obtained by cutting and recovering the gel. And then connecting the annealed product 1 with a vector framework by T4 ligase (a product of Takara company) to obtain a pLenti-CRIPSR V2 all-in-one MUC1 knockout vector 1, which is called as a knockout vector No. 1 for short.

2. The human MUC1 sg1-forward was replaced with human MUC1 sg2-forward as described above: 5'-CACCGACTGGGTGCCCGGTGTCA-3' (SEQ ID NO: 3), human MUC1 sg1-reverse was replaced with human MUC1 sg 2-reverse: 5'-AAACTGACACCGGGCACCCAGTC-3' (SEQ ID NO: 4), and the other steps are not changed, thus obtaining a Lenti-CRIPSR V2 all-in-one MUC1 knockout vector 2, which is called a knockout vector No. 2 for short. The human MUC1 sg1-forward was replaced with human MUC1 sg3-forward as described above: 5'-CACCGCAGGAAGAAAGGAGACT-3' (SEQ ID NO: 5), human MUC1 sg1-reverse was replaced with human MUC1 sg 3-reverse: 5'-AAACAGTCTCCTTTCTTCCTGC-3' (SEQ ID NO: 6), and the other steps are not changed, thus obtaining a Lenti-CRIPSR V2 all-in-one MUC1 knockout vector 3, which is called a knockout vector No. 3 for short.

3. Taking 293T cells with excellent growth state, culturing to 80% fusion degree, transfecting a knockout vector (knockout vector No. 1, knockout vector No. 2 or knockout vector No. 3), collecting virus liquid after 72h, filtering with a 0.45-micrometer filter membrane, and collecting filtrate, namely the virus liquid.

4. And (2) taking breast cancer cells (MCF-7 cells or MDA-MB-231 cells) with good growth state, adding virus liquid to infect the cells for 24h when the cell fusion degree grows to 40%, killing the cells for 1-2 weeks by using puromycin with the concentration of 0.5-1 mu g/ml, collecting the surviving cells, and carrying out monoclonal sorting in a 96-well plate, wherein each surviving and well-grown monoclonal cell is an MUC1 monoclonal knockout cell line. Extracting proteins of breast cancer cells and each amplified MUC1 monoclonal knockout cell line, and carrying out immunoblot verification, wherein a MUC1 rabbit monoclonal antibody (a product of Biyuntian corporation) is used as a primary antibody (beta-actin is an internal reference protein), and horseradish peroxidase-labeled goat anti-rabbit IgG is used as a secondary antibody.

Some of the results are shown in FIGS. 1A and C (WT is a breast cancer cell). The results showed that MUC1 in the MUC1 monoclonal knockout cell line was knocked out, and 3 of the MUC1 monoclonal knockout cell lines were named MUC1 KO1, MUC1 KO2 and MUC1 KO 3.

5. Extracting mRNA of cells (MCF-7 cells, MDA-MB-231 cells, MCF-7 MUC1 KO1, MCF-7 MUC1 KO3, MDA-MB-231MUC1 KO1 or MDA-MB-231MUC1 KO 3) by using a conventional TRIZOL lysis method, and dissolving the mRNA in DEPC water; then, carrying out concentration and purity detection on mRNA by using an enzyme-labeling instrument, and ensuring that the concentration is higher than 500 ng/mu l and the OD value is between 1.8 and 2.0 to carry out subsequent reverse transcription; finally, after carrying out reverse transcription on the extracted mRNA, carrying out real-time fluorescent quantitative PCR according to the steps of the instruction book of M5 Super qPCR RT kit, and detecting the mRNA expression quantity of MUC1 in cells; the mRNA expression level of MUC1 in breast cancer cells was defined as 1, and the relative mRNA expression levels in other cells were calculated (GAPDH was defined as an internal control).

Primers for detecting MUC1 were: 5'-GTTACGGGTTCTGGTCATGC-3', and 5'-TAGTCGGTGCTGGGATCTTC-3'. Primers for detecting GAPDH were: 5'-GGGTCATCATCTCTGCACCT-3', and 5'-GGTCATAAGTCCCTCCACGA-3'.

The results are shown in FIGS. 1B and D (WT is breast cancer cell, B is MCF-7, and D is MDA-MB-231). The results show that the expression level of mRNA of MUC1 in the MUC1 monoclonal knockout cell line is remarkably reduced compared with that of the breast cancer cells.

Thus, it can be seen that MUC1 in MCF-7 MUC1 KO1, MCF-7 MUC1 KO3, MDA-MB-231MUC1 KO1 and MDA-MB-231MUC1 KO3 have been successfully knocked out.

Example 2 screening of O-glycosylation inhibitor (BAG) concentration and sialidase (Neu) treatment time without additional toxicity to breast cancer cell growth and influence on transcript levels

Primers for detecting the MUC1 gene were 5'-GTTACGGGTTCTGGTCATGC-3' and 5'-TAGTCGGTGCTGGGATCTTC-3'. The primers for detecting the reference gene GAPDH are as follows: 5'-GGGTCATCATCTCTGCACCT-3', and 5'-GGTCATAAGTCCCTCCACGA-3'.

1. Breast cancer cells (MCF-7 cells or MDA-MB-231 cells) were uniformly plated in a sterile import 96-well plate 1-2 days in advance so that the density was 3000 cells/well, and 100 μ l of medium was added per well. When the degree of cell fusion reached 30-40%, a medium (as a control group) or a medium containing BAG (BAG at a concentration of 2mM or 4mM in the medium) was added and the culture was continued for 3 days at 37 ℃ in a constant temperature incubator.

OD of the daily detection System during the experiment using CCK8 method_450nmA value; using time as abscissa, OD_450nmValues are plotted as ordinate, growth curves are plotted. The results of MCF-7 cell detection are shown in FIG. 2A (CON is control). The results show that 2mM BAG is not additionally toxic to breast cancer cell growth after 3 days of continuous treatment.

After 24h of treatment, mRNA from breast cancer cells was extracted and qPCR detection of the MUC1 gene was performed. Relative expression level of MUC1 in breast cancer cells of control group was calculated as 1.0 (GAPDH as internal reference) for BAG-treated breast cancer cells.

The results are shown in B of FIG. 2 (CON is control group). The results show that 2mM BAG had no effect on the level of transcription of MUC 1.

Subsequently 2mM BAG was selected for the experiment.

2. Cells (MCF-7 cells, MDA-MB-231 cells, MCF-7 MUC1 KO1 or MDA-MB-231MUC1 KO 1) were uniformly plated in a sterile import 96-well plate 1-2 days in advance to a density of 3000 cells per well, and 100. mu.l of medium was added per well. When the cell confluence reached 30-40%, DPBS (as a control group) or DPBS containing 150 mU/mL sialidase was added and cultured continuously for 6h or 12h in a 37 ℃ incubator.

Cell viability was measured by the CCK8 method, and relative viability of sialidase-treated cells was calculated with the cell viability of the control group as 1.0.

The results of the partial assay are shown in FIG. 2C (CON is control, Neu-6h is sialidase-treated 6h, Neu-12h is sialidase-treated 12h, WT MCF-7 is MCF-7 cells, WT 231 is MDA-MB-231 cells, KO MCF-7 is MCF-7 MUC1 KO1, KO 231 is MDA-MB-231MUC1 KO 1). The results show that sialidase treatment for 6h was not additionally toxic to cell growth, either for wild-type cells (MCF-7 cells or MDA-MB-231 cells) or knock-out cells (MCF-7 MUC1 KO1 or MDA-MB-231MUC1 KO 1).

After sialidase treatment, mRNA from cells (MCF-7 cells or MDA-MB-231 cells) was extracted and subjected to qPCR detection of the MUC1 gene. The relative expression level of the sialidase-treated cells was calculated with the expression level of the control cells as 1.0.

The results are shown in D in FIG. 2 (CON is control, Neu-6h is sialidase treatment 6h, Neu-12h is sialidase treatment 12 h). The results show that sialidase treatment for 6h or 12h had no effect on the transcriptional level of MUC 1.

Subsequently, sialidase treatment was selected for 6h for the experiment.

Example 3 inhibition of MUC1 glycosylation by BAG inhibitors

MCF-7 cells were treated with a medium (as a control group) or a medium containing 2mM BAG for 24 hours, and after protein extraction, glycosylation expression thereof was detected by an immunoblotting method, and primary antibody was a membrane-whole incubated MUC1 antibody (manufactured by Proteintech) and Histone H3 (manufactured by Proteintech) was an internal control.

The results are shown in FIG. 3 (WT is the control group). The results show that MCF-7 cells treated by BAG with the concentration of 2mM for 24h remarkably inhibit the glycosylation, and particularly show that the imprinting of high molecular weight protein parts is remarkably reduced, thereby indicating that the glycosylation of MUC1 is successfully inhibited.

Example 4 inhibition of MUC1 glycosylation by sialidase

1. A6-well plate in which MCF-7 cells had grown was taken, and DPBS (as a control group) or DPBS containing 150. mu.U/mL sialidase was added thereto, and cultured at 37 ℃ for 6 hours (for desialylation).

2. After the step 1 is completed, the MCF-7 cells are carefully cleaned twice by using precooled PBS, protein is rapidly cracked on ice by using RIPA lysate, then the extracted protein is detected by using an immunoblotting method to carry out glycosylation expression, wherein the primary antibody is a full-membrane incubation MUC1 rabbit monoclonal antibody, and the Histone H3 rabbit monoclonal antibody is an internal reference.

The results are shown in FIG. 4 (WT is the control group). The results showed that sialidase treatment of MCF-7 cells for 6h significantly inhibited their glycosylation, as evidenced by a reduction in the imprinting of the high molecular weight protein fraction, indicating that MUC1 glycosylation was successfully inhibited.

Example 5 construction of glycosyltransferase GCNT3 knockout cell line using CRISPR Cas9 Gene editing technology

1. Human GCNT3 sg1-forward was designed and synthesized: 5'-CACCGGAAGTGTGACTCTGACCACT-3' (SEQ ID NO: 7) and human GCNT3 sg 1-reverse: 5'-AAACAGTGGTCAGAGTCACACTTCC-3' (SEQ ID NO: 8), followed by annealing, to give an annealed product 1. The pLenti-CRISPR v2 all in one vector (Addgene) is cut by BsmI (NEB product) to remove a specific Filter region, and the vector framework is obtained by cutting and recovering the gel. And then the annealed product 1 is connected with a vector framework through T4 ligase (a product of Takara company), so as to obtain a pLenti-CRIPSR V2 all-in-one GCNT3 knockout vector KO1, which is called a knockout vector KO1 for short.

2. Human GCNT3 sg1-forward was replaced with human GCNT3 sg2-forward as described above: 5'-CACCGGGCTATTCTGAATAACCTGG-3' (SEQ ID NO: 9), human GCNT3 sg1-reverse was replaced with human GCNT3 sg 2-reverse: 5'-AAACCCAGGTTATTCAGAATAGCCC-3' (SEQ ID NO: 10), and the other steps are not changed, thus obtaining a Lenti-CRIPSR V2 all-in-one GCNT3 knockout vector KO2, which is called knockout vector KO2 for short. Human GCNT3 sg1-forward was replaced with human GCNT3 sg3-forward as described above: 5'-CACCGGCAAGCTGACCTCAACTGCA-3' (SEQ ID NO: 11), human GCNT3 sg1-reverse was replaced with human GCNT3 sg 3-reverse: 5'-AAACTGCAGTTGAGGTCAGCTTGCC-3' (SEQ ID NO: 12), and the other steps are not changed, thus obtaining a Lenti-CRIPSR V2 all-in-one GCNT3 knockout vector KO3, which is called knockout vector KO3 for short.

3. And (3) taking 293T cells with excellent growth state, culturing to 80% fusion degree, transfecting a knockout vector (a knockout vector KO1, a knockout vector KO2 or a knockout vector KO 3), collecting virus liquid after 72h, filtering with a 0.45-micrometer filter membrane, and collecting filtrate, namely the virus liquid.

4. And (3) taking MCF-7 cells with good growth state, adding virus liquid to infect the cells for 24h when the cell fusion degree grows to 30-40%, killing the cells for 1-2 weeks by using 1mu g/ml puromycin, and carrying out monoclonal sorting on the surviving cells by using a 96-pore plate, wherein each surviving monoclonal cell with good growth state is a glycosyltransferase GCNT3 knockout cell line.

Proteins of MCF-7 cells and various glycosyltransferase GCNT3 knockout cell lines were subjected to immunoblot verification, GCNT3 rabbit polyclonal antibody (product of ABClonal) was used as a primary antibody, Histone H3 was used as an internal reference protein, and horseradish peroxidase-labeled goat anti-rabbit IgG was used as a secondary antibody. Part of the assay results are shown in FIG. 5A (WT is MCF-7 cells). The results indicate that GCNT3 was knocked out in the partial glycosyltransferase GCNT3 knock-out cell line. Among these 3 glycosyltransferase GCNT3 knock-out cell lines were designated GCNT3 KO1, GCNT3 KO2 and GCNT3 KO 3.

MCF-7 cells and the proteins of the 3 glycosyltransferase GCNT3 knockout cell lines were rapidly extracted on ice, then the glycosylation expression was detected by immunoblotting, MUC1 monoclonal antibody (product of ProteinTech) was incubated as a primary antibody in whole membrane, and Histone H3 was an internal reference (product of ProteinTech).

The partial detection results are shown in B of FIG. 5 (GCNT 3 KO is GCNT3 KO 1). The results showed that the imprinting of the portion of MUC1 high molecular weight protein of the 3 glycosyltransferase GCNT3 knock-out cell line described above was reduced, indicating that the glycosylation of MUC1 was successfully inhibited.

Example 6 inhibition of BAG glycosylation increases the sensitivity of breast cancer cells to the intracellular toxicity of apigenin

1. Uniformly paving cells (MCF-7 cells, MDA-MB-231 cells, MCF-7 MUC1 KO1 or MDA-MB-231MUC1 KO 1) in a sterile import 96-well plate 1-2 days in advance to ensure that the density is 3000 cells/well, and adding 100 mu l of culture medium into each well; when the cell fusion degree reaches 30-40%, the cells are randomly divided into five groups for treatment:

10 mu M apigenin treatment group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then, changing the culture medium into a culture medium containing 10 mu M apigenin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

50 mu M apigenin treatment group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then, changing the culture medium into a culture medium containing 50 mu M apigenin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

BAG pretreatment +10 mu M apigenin treatment group: adding a culture medium containing 2mM BAG, and culturing for 24h in a constant-temperature incubator at 37 ℃; then, changing the culture medium into a culture medium containing 10 mu M apigenin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

BAG pretreatment +50 mu M apigenin treatment group: adding a culture medium containing 2mM BAG, and culturing for 24h in a constant-temperature incubator at 37 ℃; then, changing the culture medium into a culture medium containing 50 mu M apigenin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

control group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then changing the culture solution into a fresh culture medium, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

the CCK8 detection kit detects the cell activity of five groups respectively.

The experimental results are shown in A in FIG. 6 (CON represents a control group, 10 μ M is a 10 μ M apigenin treatment group, 50 μ M is a 50 μ M apigenin treatment group, 10 μ M +2mM BAG is a BAG pretreatment +10 μ M apigenin treatment group, 50 μ M +2mM BAG is a BAG pretreatment +50 μ M apigenin treatment group, WT-MCF7 is MCF-7 cells, WT-231 is MDA-MB-231 cells, KO-MCF7 is MCF-7 MUC1 KO1, and KO-231 is MDA-MB-231MUC1 KO 1).

2. Plating cells (MCF-7 cells or MCF-7 MUC1 KO 1) in a 24-well plate 1 day before the experiment, adding 500. mu.l of culture medium to the cells per well, and culturing at 37 ℃ overnight; and when the growth state is good in the second day, the growth state is randomly divided into four groups for treatment:

api only is the apigenin single treatment group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then changing the culture medium into a culture medium containing 50 mu M apigenin, and continuously culturing for 1-2 weeks in a constant-temperature incubator at 37 ℃;

BAG only is a BAG individual treatment group: adding a culture medium containing 2mM BAG, and culturing for 24h in a constant-temperature incubator at 37 ℃; then changing the culture medium to fresh culture medium, and continuously culturing for 1-2 weeks in a constant-temperature incubator at 37 ℃;

BAG + Api is a BAG and apigenin combined treatment group: adding a culture medium containing 2mM BAG, and culturing for 24h in a constant-temperature incubator at 37 ℃; then changing the culture medium into a culture medium containing 50 mu M apigenin, and continuously culturing for 1-2 weeks in a constant-temperature incubator at 37 ℃;

DMSO is the solvent control: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then changing the culture medium containing 1% DMSO, and continuously culturing for 1-2 weeks in a constant-temperature incubator at 37 ℃;

and (5) fixing paraformaldehyde for 15 minutes, then carrying out crystal violet staining, and counting by ImageJ software.

The observed colony formation results are shown in fig. 6B. The statistical results for the clones are shown in FIG. 6C, where the combination group is the BAG + Api treatment group.

The results show that in MCF-7 cells, BAG pretreatment improves the toxicity sensitivity of apigenin, cell death is increased, the number of clone formation is reduced, but the phenomenon disappears in MCF-7 MUC1 KO1, BAG no longer has the synergistic effect, and the toxicity sensitivity of the apigenin depends on MUC1 and glycosylation expression thereof.

Example 7 sialidase hydrolysis of glycosyl chain Ends increases the sensitivity of breast cancer cells to the intracellular toxicity of apigenin

10 mu M apigenin treatment group: firstly adding a culture medium, and culturing for 6 hours in a constant-temperature incubator at 37 ℃; then, changing the culture medium into a culture medium containing 10 mu M apigenin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

50 mu M apigenin treatment group: firstly adding a culture medium, and culturing for 6 hours in a constant-temperature incubator at 37 ℃; then, changing the culture medium into a culture medium containing 50 mu M apigenin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

sialidase pretreatment +10 μ M apigenin treatment group: firstly adding DPBS containing 150 mU/mL sialidase, and culturing for 6h in a constant-temperature incubator at 37 ℃; then, changing the culture medium into a culture medium containing 10 mu M apigenin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

sialidase pretreatment +50 μ M apigenin treatment group: firstly adding DPBS containing 150 mU/mL sialidase, and culturing for 6h in a constant-temperature incubator at 37 ℃; then, changing the culture medium into a culture medium containing 50 mu M apigenin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

control group: firstly adding a culture medium, and culturing for 6 hours in a constant-temperature incubator at 37 ℃; then changing the culture solution into a fresh culture medium, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

the CCK8 detection kit detects the cell activity of five groups respectively.

The experimental results are shown in A in FIG. 7 (CON is shown as a control group, 10 mu M is a 10 mu M apigenin treatment group, 50 mu M is a 50 mu M apigenin treatment group, Neu +10 mu M is a sialidase pretreatment +10 mu M apigenin treatment group, Neu +50 mu M is a sialidase pretreatment +50 mu M apigenin treatment group, WT MCF-7 is an MCF-7 cell, WT 231 is an MDA-MB-231 cell, KO MCF-7 is MCF-7 MUC1 KO1, and KO 231 is MDA-MB-231MUC1 KO 1).

2. 1 day before the experiment, cells (MCF-7 cells or MCF-7 MUC1 KO 1) are paved in a 24-well plate, the density is about 1200 cells/well, 500 mu l of culture medium is added for culture, and the culture is carried out overnight at 37 ℃; and when the growth state is good in the second day, the growth state is randomly divided into four groups for treatment:

neuonly is the Neu single treatment group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; adding DPBS containing 150 mU/mL sialidase, pre-culturing for 6h in a constant-temperature incubator at 37 ℃, finally changing the culture solution to be a fresh culture medium, and continuously culturing for 1-2 weeks in the constant-temperature incubator at 37 ℃;

neu + Api is the combined treatment group of Neu and apigenin: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then adding DPBS containing 150 mU/mL sialidase, and pre-culturing for 6h in a constant-temperature incubator at 37 ℃; then changing the culture medium into a culture medium containing 50 mu M apigenin, and continuously culturing for 1-2 weeks in a constant-temperature incubator at 37 ℃;

and (3) fixing paraformaldehyde for 15 minutes, then carrying out crystal violet staining, and carrying out counting statistics by ImageJ software, wherein a combination group is a Neu + Api treatment group.

The observed colony formation results are shown in fig. 7B. The statistical results for the clones are shown in FIG. 7C, where the combination group is the BAG + Api treatment group.

The result shows that the sialidase pretreatment in MCF-7 cells obviously improves the toxicity sensitivity of apigenin, increases cell death and reduces the clone formation number, but the phenomenon disappears in MCF-7 MUC1 KO1, and sialidase no longer has a synergistic effect, which indicates that the toxicity sensitivity of the apigenin depends on the glycosylation expression of MUC 1.

Example 8 glycosyltransferase GCNT3 knockdown increases the sensitivity of breast cancer cells to the intracellular toxicity of apigenin

The preparation method of the GCNT3 & MUC1 double knockout MCF-7 cell comprises the following steps: (1) taking 293T cells with excellent growth state, culturing to 80% fusion degree, transfecting a GCNT3 knockout vector (obtained by replacing Puromycin (PURO) resistance sequence of knockout vector KO1 with NEO (geneticin) resistance sequence through molecular cloning technology), collecting virus liquid after 72h, filtering with a 0.45 mu m filter membrane, and collecting filtrate, namely the virus liquid; (2) and (3) taking MCF-7 MUC1 KO1 cells with good growth state, adding virus liquid to infect the cells for 24h when the cell fusion degree grows to 30-40%, killing the cells for 1-2 weeks by using 500 mu g/ml geneticin, and carrying out monoclonal sorting on the surviving cells by using a 96-well plate, wherein each surviving monoclonal cell with good growth state is a glycosyltransferase GCNT3 and MUC1 double knockout cell line. One of the cell lines was designated GCNT3 & MUC1 double knockout MCF-7 cells.

Uniformly paving cells (MCF-7 cells, MCF-7 GCNT3 KO1, MCF-7 MUC1 KO1 or GCNT3 & MUC1 double knockout MCF-7 cells) in a sterile import 96-well plate 1-2 days in advance, so that the density is 3000 cells/well, and adding 100 mu l of culture medium into each well; adding DMSO or apigenin solution (DMSO solvent) with concentration of 50 μ M when cell fusion degree reaches 30-40%, and culturing at 37 deg.C for 48 hr; the CCK8 detection kit detects the activity of the cells.

The results are shown in FIG. 8 (WT is MCF-7 cells, GCNT3 KO is MCF-7 GCNT3 KO1, MUC1 KO is MCF-7 MUC1 KO1, Double KO is GCNT3 & MUC1 Double knockout MCF-7 cells, and Api is apigenin solution added at a concentration of 50. mu.M). The results show that compared with MCF-7 cells, MCF-7 GCNT3 KO1 has obviously up-regulated toxicity sensitivity to apigenin and reduced cell survival rate; in MCF-7 MUC1 KO1 and GCNT3 & MUC1 double knockout MCF-7 cells, further knockout of GCNT3 does not increase toxicity sensitivity of the cells to apigenin, further indicates that the toxicity sensitivity of the apigenin is dependent on expression of MUC1 and glycosylation modification thereof, and glycosylation inhibition cannot synergize intracellular toxicity of the apigenin in the absence of MUC 1.

Example 9, MUC1 deletion can significantly increase toxicity sensitivity of various flavonoids

1. 4 flavonoid compounds (Chrysin, diosmin, Luteolin and Quercetin) with similar structures to apigenin are selected for experiments.

Cells (MCF-7 cells or MCF-7 MUC1 KO 1) were uniformly plated in a sterile import 96-well plate 1-2 days in advance to a density of 3000 cells/well, and 100 μ l of medium was added per well. And when the cell fusion degree reaches 30-40%, adding a culture medium containing 50 mu M of a drug to be detected (chrysin, diosmetin, luteolin or quercetin), continuously culturing for 48h in a constant-temperature incubator at 37 ℃, and detecting the cell activity by using a CCK8 detection kit.

The results are shown in A of FIG. 9 (WT is MCF-7 cells, KO is MCF-7 MUC1 KO 1). The results show that compared with MCF-7 cells, the MCF-7 MUC1 KO1 mortality rate is obviously increased, the toxicity sensitivity of the 4 flavonoids compounds is obviously increased, and the cell activity is reduced.

2. Cells (MCF-7 cells or MCF-7 MUC1 KO 1) were uniformly plated in a sterile import 96-well plate 1-2 days in advance to a density of 3000 cells/well, and 100 μ l of medium was added per well. When the cell fusion degree reaches 30-40%, adding a culture medium containing a drug to be detected (chrysin, diosmetin, luteolin or quercetin) (the concentration of the drug to be detected in the culture medium is 0 muM, 2 muM, 4 muM, 8 muM, 16 muM, 32 muM, 64 muM and 128 muM), continuously culturing for 48h in a constant-temperature incubator at 37 ℃, and detecting the cell activity by using a CCK8 detection kit.

The results are shown in B of FIG. 9 (WT is MCF-7 cells, KO is MCF-7 MUC1 KO 1). The results show the IC's of chrysin, diosmetin, luteolin and quercetin₅₀(half lethal concentration) was significantly down-regulated in MCF-7 MUC1 KO1, suggesting an increase in drug sensitivity. It can be seen that toxicity sensitivity of chrysin, diosmetin, luteolin and quercetin substances is dependent on the expression of MUC 1.

Example 10 cytotoxicity susceptibility testing of O-glycosylation inhibitors in combination with apigenin-like flavonoids

Cells (MCF-7 cells or MCF-7 MUC1 KO 1) were uniformly plated in a sterile import 96-well plate 1-2 days in advance to a density of 3000 cells/well, and 100 μ l of medium was added per well. When the cell fusion degree reaches 30-40%, the cells are randomly divided into four groups for treatment:

BAG treatment group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then changing the culture medium containing 2mM BAG, culturing for 24h in a constant-temperature incubator at 37 ℃, and then changing the culture medium to a fresh culture medium;

test drug treatment group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then changing the culture medium into a culture medium containing 50 mu M of a drug to be detected (Chr, Dio, Lut or Que), and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

combination processing group: adding a culture medium containing 2mM BAG, and pre-culturing for 24h in a constant-temperature incubator at 37 ℃; then, respectively changing the culture medium into culture mediums containing 50 mu M Chr, 50 mu M Dio, 50 mu M Lut and 50 mu M Que, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

DMSO group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then changing the culture medium containing 1% DMSO, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

the CCK8 detection kit detects the activity of the cells.

The results are shown in FIG. 10 (WT is MCF-7 cells, KO is MCF-7 MUC1 KO 1). The result shows that BAG pretreatment obviously increases the sensitivity to chrysin, diosmetin, luteolin and quercetin in MCF-7 cells, and the cell activity is reduced; however, in MCF-7 MUC1 KO1, the phenomenon disappears, BAG can not synergize, and the toxicity sensitivity of chrysin, diosmetin, luteolin and quercetin depends on glycosylation modification of MUC1, and in the absence of MUC1, the targeted glycosylation inhibits the intracellular toxicity of the drug which can not synergize.

Example 11 cytotoxicity susceptibility testing of O-glycosylation inhibitors in combination with clinically common small molecule drugs (e.g., cisplatin, 5-fluorouracil, brevicmycin)

3 clinically common small-molecule drugs (Cisplatin (Cisplatin), 5-fluorouracil (5-FU) and Bleomycin (Bleomycin)) are selected for experiments.

Cells (MCF-7 cells or MCF-7 MUC1 KO 1) were uniformly plated in a sterile import 96-well plate 1-2 days in advance to a density of 3000 cells/well, and 100 μ l of medium was added per well. When the cell fusion degree reaches 30-40%, the cells are randomly divided into eight groups for treatment:

BAG treatment group: adding a culture medium containing 2mM BAG, pre-culturing for 24h in a constant-temperature incubator at 37 ℃, finally changing the culture medium to be a fresh culture medium, and continuously culturing for 48h in the constant-temperature incubator at 37 ℃;

cissplatin treatment group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then changing the culture medium into a culture medium containing 20 mu M Cisplatin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

BAG + Cis treatment group: adding a culture medium containing 2mM BAG, and culturing for 24h in a constant-temperature incubator at 37 ℃; then changing the culture medium into a culture medium containing 20 mu M Cisplatin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

5-FU treatment group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then changing the culture medium into a culture medium containing 10 mu M5-FU, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

BAG +5-FU treatment group: adding a culture medium containing 2mM BAG, and culturing for 24h in a constant-temperature incubator at 37 ℃; then changing the culture medium into a culture medium containing 10 mu M5-FU, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

bleomycin treatment group: firstly adding a culture medium, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; then changing the culture medium into a culture medium containing 20 mu M Bleomycin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

BAG + Bleo treatment group: adding a culture medium containing 2mM BAG, and culturing for 24h in a constant-temperature incubator at 37 ℃; then changing the culture medium into a culture medium containing 20 mu M Bleomycin, and continuously culturing for 48h in a constant-temperature incubator at 37 ℃;

the CCK8 detection kit detects the activity of the cells.

The results are shown in FIG. 11 (WT is MCF-7 cells, KO is MCF-7 MUC1 KO 1). The results show that BAG pretreatment obviously increases the sensitivity to cisplatin, 5-fluorouracil and burley mycin in MCF-7 cells, and the cell activity is reduced; however, in MCF-7 MUC1 KO1, the phenomenon disappears, BAG can not synergize, and the indication that the toxicity sensitivity of the small molecule drug depends on the expression of MUC1 and glycosylation modification thereof is shown, and in the absence of MUC1, the intracellular toxicity of the drug can not be synergized by the targeted glycosylation inhibition.

Example 12 verification of nude mice tumor-bearing experiment

SPF-grade female BALB/c nude mice are products of Beijing Charles River and are bred in SPF-grade animal breeding center of Chinese agriculture university; the SPF-grade female BALB/c nude mice are abbreviated as nude mice.

1. Nude mice grown to 5 weeks of age were inoculated subcutaneously with cells (MCF-7 cells or MCF-7 MUC1 KO 1) at a concentration of 1X 10 per right armpit⁶One/100. mu.l (PBS: Matrigel =1:1, Matrigel is Matrigel, available from Saimer fly) and the inoculum size was 100. mu.l/tube.

2. After completion of step 11-2 weeks, tumor-transplanted nude mice were randomly divided into a drug-treated group (n = 8) and a control group (n = 8), the control group was given a treatment with a solvent DMSO (1%), the drug-treated group was given an apigenin treatment (dose 40 mg/kg/day), and the treatment was continued for 16 days in each of intraperitoneal injections. During the experiment, tumor weight, tumor volume and nude mouse body weight were measured every 1 day.

The results are shown in FIG. 12 (WT + DMSO was MCF-7 cell seeded and DMSO treated, KO + DMSO was MCF-7 MUC1 KO1 seeded and DMSO treated, WT + Api was MCF-7 cell seeded and 40 mg/kg apigenin treated, KO + Api was MCF-7 MUC1 KO1 seeded and 40 mg/kg apigenin treated). The results show that the tumor volumes and tumor weights of the MCF-7 cells and MCF-7 MUC1 KO1 inoculated in the control group are not significantly different; in the drug treatment group, the tumor volume and tumor weight of the inoculated MCF-7 MUC1 KO1 are significantly smaller than those of MCF-7 cells; the body weights of the four groups of nude mice have no significant difference, which indicates that the nude mice grow normally. Therefore, the knockout of MUC1 can effectively improve the drug sensitivity of apigenin, promote the toxicity of the drug in vivo and reduce the occurrence and development of tumors.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> university of agriculture in China

<120> application of substance for inhibiting MUC1 expression and glycosylation modification in reducing drug resistance of anti-breast cancer drugs

<160> 12

<170>PatentIn version 3.5

<210> 1

<211> 24

<212> DNA

<213> Artificial sequence

<400> 1

caccgcagca ggaagaaagg agac 24

<210>2

<211> 24

<212> DNA

<213> Artificial sequence

<400>2

aaacgtctcc tttcttcctg ctgc 24

<210>3

<211> 23

<212> DNA

<213> Artificial sequence

<400>3

caccgactgg gtgcccggtg tca 23

<210>4

<211> 23

<212> DNA

<213> Artificial sequence

<400>4

aaactgacac cgggcaccca gtc 23

<210>5

<211> 22

<212> DNA

<213> Artificial sequence

<400>5

caccgcagga agaaaggaga ct 22

<210>6

<211> 22

<212> DNA

<213> Artificial sequence

<400>6

aaacagtctc ctttcttcct gc 22

<210>7

<211> 25

<212> DNA

<213> Artificial sequence

<400>7

caccggaagt gtgactctga ccact 25

<210>8

<211> 25

<212> DNA

<213> Artificial sequence

<400>8

aaacagtggt cagagtcaca cttcc 25

<210>9

<211> 25

<212> DNA

<213> Artificial sequence

<400>9

caccgggcta ttctgaataa cctgg 25

<210>10

<211> 25

<212> DNA

<213> Artificial sequence

<400>10

aaacccaggt tattcagaat agccc 25

<210>11

<211> 25

<212> DNA

<213> Artificial sequence

<400>11

caccggcaag ctgacctcaa ctgca 25

<210>12

<211> 25

<212> DNA

<213> Artificial sequence

<400>12

aaactgcagt tgaggtcagc ttgcc 25

Claims

1. The substance inhibiting the expression of MUC1 in breast cancer cells or the substance inhibiting the glycosylation modification of MUC1 in breast cancer cells is at least one of A1) -A5):

A2) reducing the drug resistance of anti-breast cancer drugs;

A5) preparing a product for adjuvant therapy of breast cancer.

2. Use according to claim 1, characterized in that: the anti-breast cancer drug is at least one of cisplatin, 5-fluorouracil, burley mycin, apigenin, chrysin, diosmetin, luteolin and quercetin.

3. Use according to claim 1, characterized in that: the substance for inhibiting MUC1 expression in the breast cancer cells is oligo-nucleic acid 1, oligo-nucleic acid 2 or oligo-nucleic acid 3;

4. Use according to claim 1, characterized in that: the substance for inhibiting the glycosylation modification of MUC1 in the breast cancer cells is BAG, sialidase or a substance for inhibiting the expression of glycosyltransferase GCNT3 in the breast cancer cells;

the substance for inhibiting the expression of glycosyltransferase GCNT3 in the breast cancer cell is oligonucleotide a, oligonucleotide b or oligonucleotide c;

5. A product comprising the anti-breast cancer agent of claim 1 or 2 and a MUC 1-inhibiting substance;

the substance inhibiting MUC1 is the substance inhibiting MUC1 expression in breast cancer cells as claimed in claim 1 or 3 or the substance inhibiting MUC1 glycosylation modification in breast cancer cells as claimed in claim 1 or 4;

the function of the product is at least one of B1) -B3):

B2) reducing the drug resistance of anti-breast cancer drugs;

B3) can be used for adjuvant treatment of breast cancer.

6. A sensitizer for the anti-breast cancer drug according to claim 1 or 2, which is the substance according to claim 1 or 3 for inhibiting the expression of MUC1 in breast cancer cells or the substance according to claim 1 or 4 for inhibiting the glycosylation modification of MUC1 in breast cancer cells.

7. A screening method of an anti-breast cancer drug is characterized in that: the anti-breast cancer drug may be sensitized with the substance for inhibiting the expression of MUC1 in a breast cancer cell according to claim 1 or 3 or the substance for inhibiting the glycosylation modification of MUC1 in a breast cancer cell according to claim 1 or 4.

8. A method for improving the sensitivity of breast cancer cells to anti-breast cancer drugs by inhibiting the expression of MUC1 in breast cancer cells or inhibiting the modification of MUC1 glycosylation in breast cancer cells; the method is useful for diagnosis and treatment of non-diseases.

9. A method for reducing drug resistance of breast cancer cells to anti-breast cancer drugs by inhibiting MUC1 expression in breast cancer cells or inhibiting MUC1 glycosylation modification in breast cancer cells; the method is useful for diagnosis and treatment of non-diseases.

10. The method according to claim 8 or 9, characterized in that:

the inhibition of MUC1 expression in the breast cancer cell is realized by introducing a substance for inhibiting MUC1 expression in the breast cancer cell into the breast cancer cell; the substance for inhibiting MUC1 expression in the breast cancer cells is oligo-nucleic acid 1, oligo-nucleic acid 2 or oligo-nucleic acid 3;

the oligomeric nucleic acid 3 consists of a nucleic acid molecule shown as SEQ ID NO. 5 and a nucleic acid molecule shown as SEQ ID NO. 6;

the inhibition of MUC1 glycosylation modification in breast cancer cells is achieved by one or two ways;

treating breast cancer cells with BAG or sialidase; the treatment concentration of BAG is 1-3 mM; the treatment time of the sialidase is 6-12 h;

the second mode is realized by introducing a substance for inhibiting the glycosylation modification of MUC1 in the breast cancer cells into the breast cancer cells; the substance for inhibiting the glycosylation modification of MUC1 in the breast cancer cells is a substance for inhibiting the expression of glycosyltransferase GCNT3 in the breast cancer cells; the substance for inhibiting the expression of glycosyltransferase GCNT3 in the breast cancer cell is oligonucleotide a, oligonucleotide b or oligonucleotide c;