CN110302383A - Use of TRIM11 gene and protein targets and inhibitors in breast cancer - Google Patents

Abstract

The present invention relates to the use of TRIM11 gene and protein targets and inhibitors in breast cancer, and specifically discloses the use of TRIM11 gene or TRIM11 protein as a drug target in screening and/or preparing drugs for preventing and/or treating breast cancer ; The use of TRIM11 gene or TRIM11 protein inhibitors in the preparation of drugs for the treatment or prevention of breast cancer; and the use of TRIM11 gene or TRIM11 protein as drug targets in screening to improve the sensitivity of anti-tumor drugs in the prevention and/or treatment of breast cancer The use of the medicine; the composition of TRIM11 gene or TRIM11 protein inhibitor and anti-tumor medicine, and its use in the preparation of medicine for treating or preventing breast cancer.

Description

Use of TRIM11 gene and protein targets and inhibitors in breast cancer

technical field

The invention belongs to the field of pharmacy, and specifically relates to a method for screening drugs for treating or preventing breast cancer or improving drug efficacy by targeting TRIM11 protein.

Background technique

There are various types of malignant tumors, with different properties and types, different tissues and organs involved, different disease stages, and different responses to various treatments. Therefore, most patients need comprehensive treatment. At present, surgery, chemotherapy, radiotherapy, immunotherapy, traditional Chinese medicine treatment, interventional therapy, microwave therapy and other methods are mainly used for tumor treatment in order to greatly increase the cure rate and improve the quality of life of patients. At present, more and more new anti-cancer drugs are used in clinical practice, which is extremely important for increasing the survival period of cancer patients and improving their quality of life; however, in many cases, due to the emergence of cancer drug resistance, making Anticancer drugs with excellent efficacy in the initial stage will no longer exert their effect in the later stage, which will lead to the recurrence and recurrence of cancer. At present, although tumor treatment methods are changing with each passing day, chemotherapy is still an extremely effective and realistic choice for cancer patients, and about 90% of chemotherapy failures in tumor invasion and metastasis are related to tumor drug resistance; therefore, tumor drug resistance is of great importance to the entire tumor The therapeutic area is a huge and serious problem. The process of tumor drug resistance is very complex, and the mechanism of tumor cell drug resistance is also diverse, such as inactivation of drugs, generation of multidrug resistance, inhibition of cell death/apoptosis, changes in drug metabolism pathways or drug targets. point, alter epigenetics, enhance DNA damage and target gene amplification, etc. Therefore, it is of great significance for more effective tumor treatment to discover and elucidate new mechanisms of tumor cell drug resistance, and design drugs for new targets to overcome tumor drug resistance.

Bortezomib (Bortezomib/BTZ, PS-341, Velcade), which reversibly inhibits proteasome activity through boronic acid dipeptide, is the first proteasome inhibitor to be used in clinical treatment by the US Food and Drug Administration (FDA) , mainly for the treatment of multiple myeloma (Multiple myeloma, MM) and mantle cell lymphoma.

So far, the application of bortezomib in the treatment of solid tumors is more limited. For example, the attempts to treat breast cancer, lung cancer and prostate cancer have not achieved good results. One of the important reasons is that there may be Drug resistance to bortezomib in specific or all solid tumors, and finding a new drug resistance mechanism of bortezomib in tumors is of great significance for bortezomib-based anti-tumor therapy.

Breast cancer is a common malignant tumor and the leading cause of cancer death among women worldwide. It is known that bortezomib is not effective against solid tumor breast cancer because breast cancer cells are resistant to bortezomib, but the mechanism of drug resistance involved is unclear.

TRIM11 is reported to be an oncogene that has recently been found to function in various types of cancer, including ovarian and lung and breast cancers.

At present, bortezomib is mainly used in the treatment of multiple myeloma and mantle cell lymphoma, but its efficacy on solid tumors is not high, and its application range is very limited. In view of the above, clarifying the relationship between TRIM11, bortezomib and breast cancer will provide ideas for finding the drug resistance mechanism of bortezomib, so that bortezomib can be more widely used in the treatment of tumors, rather than limited to lymphatic Tumor treatment.

The inventors believe that an application of bortezomib is limited because of a certain drug resistance mechanism in solid tumors.

Contents of the invention

In view of the above problems, the present invention finds the TRIM11 protein as the target, and its downregulation can promote cell apoptosis and increase the therapeutic effect of antitumor drugs. However, overexpression of TRIM11 will lead to a decrease in the therapeutic effect.

One aspect of the present invention provides a use of TRIM11 gene or TRIM11 protein as a drug target in screening and/or preparing drugs for preventing and/or treating breast cancer.

Another aspect of the present invention provides a use of a TRIM11 gene or TRIM11 protein inhibitor in the preparation of a drug for treating or preventing breast cancer.

Another aspect of the present invention provides the use of TRIM11 gene or TRIM11 protein as a drug target in screening drugs for improving the sensitivity of anti-tumor drugs in the prevention and/or treatment of breast cancer.

Another aspect of the present invention provides a combination of an inhibitor of TRIM11 gene or TRIM11 protein and an antitumor drug.

Another aspect of the present invention provides an anti-tumor drug, which includes an active ingredient and an auxiliary material, the active ingredient is an inhibitor of TRIM11 gene or TRIM11 protein, or a combination of an inhibitor of TRIM11 gene or TRIM11 protein and an anti-tumor drug.

Another aspect of the present invention provides the TRIM11 gene or TRIM11 protein inhibitor and the composition of antitumor drugs, and its use in the preparation of drugs for treating or preventing breast cancer.

Another aspect of the present invention provides the use of an inhibitor of TRIM11 gene or TRIM11 protein in the treatment or prevention of breast cancer to improve the sensitivity or therapeutic effect of anti-tumor drugs.

In the technical solution of the present invention, the antitumor drug is bortezomib.

Another aspect of the present invention provides TRIM11 gene knockdown breast cancer cells, preferably, the breast cancer cells are breast cancer cells.

In the technical solution of the present invention, the TRIM11 protein inhibitor is a TRIM11 protein antibody or a TRIM11 protein binding protein.

In the technical solution of the present invention, the TRIM11 gene inhibitor is selected from one or more of TRIM11 gene-specific RNAi, TRIM11 gene-specific microRNA, or inhibitors that inhibit the TRIM11 gene promoter.

Beneficial effect

The invention provides a new target for treating breast cancer, and at the same time, the target can improve the sensitivity and therapeutic effect of other antitumor drugs, and provides a new direction for tumor treatment.

Description of drawings

Figure 1 is the Western blot identification of TRIM11 protein in MCF7 cell line; breast cancer MCF7 cell line stably overexpressing Flag-TRIM11 or knocking down TRIM11 was established. (A) The MCF7 cell line stably overexpressing the control group (Vector) or Flag-TRIM11 was constructed using retrovirus, and the protein level of Flag-TRIM11 was analyzed by western blotting. Among them, Tubulin is an internal reference protein. (B) The stable knockdown control (shNC) or TRIM11 (shTRIM11-1/2) MCF7 cell line was constructed using adenovirus, and the protein level of TRIM11 was analyzed by western blotting. Among them, GAPDH is an internal reference protein.

Figure 2 shows the apoptosis of MCF7 cells under the treatment conditions of bortezomib (BTZ); the expression of TRIM11 and the effect of bortezomib on killing tumors were analyzed, and the combined application of TRIM11 knockdown and bortezomib treatment Apoptosis of MCF7 cells. (A) The control (vector) or TRIM11 was stably overexpressed in MCF7 cells, treated with different concentrations of BTZ (+, 25nM; ++, 50nM) for 16 hours, and the percentage of apoptosis was analyzed by flow cytometry. (B) In MCF7 cells stably knocked down control (shNC) or TRIM11 (shTRIM11-1,-2), treated with BTZ drug (25nM) for 16 hours, flow cytometric analysis of cell apoptosis ratio. Data presented represent mean ± SEM (n=3). *, P<0.05, ***, P<0.001.

Figure 3 is the determination of tumor growth in nude mice; the determination of tumor growth in nude mice. Among them, Fig. 3-A is the tumor volume of the mice treated with PBS or BTZ (500ug/kg) after the cells of the control group were injected into the mice; Tumor volume of mice treated with BTZ (500ug/kg). Data presented represent mean ± SEM (n=4). *, P<0.05, n.s., no significant difference.

Fig. 4 is the determination of tumor weight in nude mice; the determination of tumor weight in nude mice. Fig. 4-A is the tumor tissue taken after sacrificing the mice; Fig. 4-B is the comparison of tumor weights in each group after weighing the tumor tissues of the mice. Data presented represent mean ± SEM (n=4). *, P<0.05; **, P<0.01; n.s., no significant difference.

Detailed ways

First construct a tumor cell line that stably overexpresses or knocks down TRIM 11, then treats the tumor cell line that stably overexpresses or knocks down TRIM 11 with bortezomib, and detects cell apoptosis by flow cytometry to see the relationship between the expression level of TRIM11 and boron To determine the effect of tezomib on killing tumors, a mouse subcutaneous tumor model was then established and treated with bortezomib to monitor the growth of the mouse tumors. Finally, the mice were sacrificed and the tumor tissues were taken and weighed.

Example 1 Preparation of MCF7 cell lines stably overexpressing or knocking down TRIM 11

A tumor cell line that stably overexpressed or knocked down TRIM 11 was constructed using lentivirus, and then the stable cell line was obtained by screening with puromycin. A part of the obtained MCF7 cell line that stably overexpressed or knocked down TRIM 11 was collected for western blot detection. Indicating TRIM11 overexpression/knockdown, so far to MCF7 cell lines that stably overexpress or knockdown TRIM11.

1. Establishment of MCF7 cell line stably overexpressing TRIM11

1. Packaging Lentivirus

(1) 293T plating, 10cm dish, 2-4*10^6cells/dish, 6ml DMEM+10%FBS medium, transfect when the cell density reaches 60%-80%

(2) Three-plasmid system transfected cells: pCMV-DR8.91: pCMV-VSVG:

TRPE-GFP-mCherry/TRPE-TRIM11-mCherry＝9:1:10

a.①25ul Lipo3000+700ulOptiMEM,②15ul P3000+700ulOptiMEM+6ug plasmid,

b. Mix ①+②, RT10-15min, 1.4ml/dish.

c. Change the medium every 4-6 hours, containing serum (20-30%), without double antibody.

d. Collect the virus supernatant after culturing for 48-72h (37°C, 5% CO2).

(3) Collection of virus supernatant

a. Centrifuge the virus supernatant at 3000 r and 4 degrees for 10 min.

b.0.22um filter to filter virus supernatant

2. Viral Infection of MCF7 Tumor Cell Line

a. The day before virus collection, digest MCF7 cells and spread 6-well plates, 1-2*10^5cells/dish,

b. 1ml virus supernatant + 1ml (DMEM + 10% FBS) medium + polybrene (8ug/ml) infection.

3. Western blot detection

Cells were collected 24-48 hours after infection for Western blot detection

The results showed that TRIM11 was overexpressed in MCF7 cells (Fig. 1A)

2. Establishment of MCF7 cell line stably knocking down TRIM11

1. Packaging Lentivirus

(1) 293T plating, 10cm dish, 2-4*10^6cells/dish, 6ml DMEM+10%FBS medium, transfect when the cell density reaches 60%-80%

(2) Three-plasmid system transfected cells: pCMV-DR8.91: pCMV-VSVG: pLKO.1 (pLKO.1-shTRIM11, pLKO.1-shTRIM11) = 9:1:10

a.①25ul Lipo3000+700ulOptiMEM,②15ul P3000+700ulOptiMEM+6ug plasmid,

b. Mix ①+②, RT10-15min, 1.4ml/dish.

c. Change the medium every 4-6 hours, containing serum (20-30%), without double antibody.

d. Collect the virus supernatant after culturing for 48-72h (37°C, 5% CO2).

(3) Collection of virus supernatant

a. Centrifuge the virus supernatant at 3000 r and 4 degrees for 10 min.

b.0.22um filter to filter virus supernatant

2. Viral Infection of MCF7 Tumor Cell Line

a. The day before virus collection, digest MCF7 cells and spread 6-well plates, 1-2*10^5cells/dish,

b. 1ml virus supernatant + 1ml (DMEM + 10% FBS) medium + polybrene (8ug/ml) infection.

c. After 24 hours, change the medium and add 1ug/ml puromycin to screen for 3-5 days

3. Western blot detection

Collect cells for Western blot detection

The results showed that TRIM11 was knocked down in MCF7 cells (Fig. 1B)

See Figure 1 for the experimental results.

Example 2 Bortezomib can antagonize BTZ anti-tumor cell experiment

The MCF7 cell line that stably overexpressed or knocked down TRIM 11 was treated with bortezomib (BTZ), the collected cells were treated with an apoptosis kit, and the apoptosis was detected by flow cytometry. The results showed that bortezomib (25nM, 50nM ) treatment, the apoptosis of the MCF7-TRIM11 experimental group was lower than that of the control group; without bortezomib treatment, the apoptosis of the TRIM11 knockdown group was higher than that of the control group; after bortezomib treatment, the TRIM11 knockdown Apoptosis in the low group was higher than that in the control group. It shows that bortezomib can antagonize the anti-tumor effect of BTZ.

1. The control (vector) or TRIM11 was stably overexpressed in MCF7 cells, treated with different concentrations of BTZ drugs (+, 25nM; ++, 50nM) for 16 hours, and the percentage of cell apoptosis was analyzed by flow cytometry.

(1) Digest the constructed MCF7-Vector and MCF7-TRIM11 cell lines into 6-well plates, 2-5*10^ ⁵ cells/well

(2) After 16-18 hours, add different concentrations of BTZ drugs (25nM, 50nM) for treatment

(3) After 16 hours, digest and collect cells according to the instructions of the apoptosis kit

(4) Flow detection

2. After stably knocking down control (shNC) or TRIM11 (shTRIM11-1, -2) in MCF7 cells, BTZ drug (25nM) was treated for 16 hours, and the percentage of cell apoptosis was analyzed by flow cytometry.

(1) Digest the constructed MCF7-shNC, MCF7-TRIM11-1, MCF7-TRIM11-2 cell lines into 6-well plates, 2-5*10^ ⁵ cells/well

(2) After 16-18 hours, add different concentrations of BTZ drugs (25nM) for treatment

(3) After 16 hours, digest and collect cells according to the instructions of the apoptosis kit

(4) Flow detection

The experimental results are shown in Figure 2

Example 3 Bortezomib can antagonize BTZ anti-tumor animal experiments

Establish a mouse subcutaneous tumor model, treat with BTZ drug, monitor the growth of the mouse tumor, and finally kill the mouse, take the tumor tissue and weigh it.

1. BALb/c nude mice were subcutaneously injected with breast cancer cells: control: MCF7-Vector cells; experiment: MCF7-Trim11 cells;

(1) Prepare cells

The number of injected cells is 1× ¹⁰⁶ /mouse

a. Add trypsin to digest the amplified cells,

b. Combine the digested cells of the same kind, transfer to a 15mL centrifuge tube, centrifuge at 1000rpm for 2min, and remove the supernatant

c. Add 1-2mL 1×PBS to each tube to resuspend, blow off and count

d. According to the counting results, the cells are divided into tubes, and each tube is divided into 1×10 ⁶ cells

e. Centrifuge to remove the supernatant, add 75 μL serum-free resuspended DMEM, add 25 μL Matrigel, mix well, and then use for injection.

Note: Matrigel needs to be stored at -20°C. Before use, it must be taken out and stored at 4°C. It can be thawed overnight. Cell suspension: Matrigel=3:1.

(2) The right side of the mouse was injected subcutaneously near the armpit

(3) After injection, measure the tumor size once every 2-3 days

(4) Dosing starts after the tumor grows to a certain extent (this time, the administration starts on the 10th day), and the mouse tumor volume needs to be measured every day after the administration

2. BALb/c nude mice grouping (4 mice/group): a.control+PBS; b.control+BTZ (500ug/kg); c.F-Trim11+PBS; d.F-Trim11+BTZ (500ug/kg ); method: intraperitoneal injection; cycle: three times a week, administration for 1-2 weeks;

3. Indicator detection:

(1) Calculation of the volume of the subcutaneous transplanted tumor: calculated according to π/6×L×W^2 (the maximum diameter of the tumor should not exceed 2cm) (Figure 3);

(2) Tumor weighing (Figure 4)

The results showed that after administration to mice in the control group, the tumor volume and weight of the mice were significantly different from those injected with PBS, and both decreased; while mice in the experimental group had no significant difference compared with those injected with PBS.

The experimental results are shown in Fig. 3 and Fig. 4. Fig. 3 and Fig. 4 confirm that the use of BTZ alone can inhibit the growth of tumor to a certain extent, but if TRIM is overexpressed, it will inhibit the inhibitory effect of BTZ on tumor.

Claims (10)

1. A use of TRIM11 gene or TRIM11 protein as a drug target in screening and/or preparing drugs for preventing and/or treating breast cancer. 2. The use of an inhibitor of TRIM11 gene or TRIM11 protein in the preparation of medicaments for treating or preventing breast cancer. 3. Use of TRIM11 gene or TRIM11 protein as a drug target in screening drugs for improving the sensitivity of anti-tumor drugs in the prevention and/or treatment of breast cancer. 4. An anti-tumor pharmaceutical composition, which includes an active ingredient and an auxiliary material, the active ingredient is an inhibitor of TRIM11 gene or TRIM11 protein, or a combination of an inhibitor of TRIM11 gene or TRIM11 protein and an anti-tumor drug. 5. The composition of an inhibitor of TRIM11 gene or TRIM11 protein and an antitumor drug, and its use in the preparation of a drug for treating or preventing breast cancer. 6. Use of an inhibitor of TRIM11 gene or TRIM11 protein in the treatment or prevention of breast cancer to improve the sensitivity or therapeutic effect of anti-tumor drugs. 7. The composition according to claim 4, or the use according to claim 5 or 6, wherein the antineoplastic drug is bortezomib. 8. A TRIM11 knockdown breast cancer cell, preferably, the breast cancer cell is a human breast cancer cell. 9. The use according to any one of claims 2, 5-6, or the antitumor drug according to claim 4, wherein the inhibitor of TRIM11 protein is an antibody to TRIM11 protein or a binding protein of TRIM11 protein. 10. Claim 2, the use described in any one of 5-6, or the antitumor drug described in claim 4, wherein, the inhibitor of described TRIM11 gene is selected in TRIM11 gene-specific RNAi, TRIM11 gene-specific One or more of the anti-microRNA, or the inhibitor that inhibits the TRIM11 gene promoter.