Application of gap junction protein 43 in preparation of medicine for preventing or treating triple negative breast cancer

Abstract

The invention provides application of gap junction protein 43 in preparation of a medicine for preventing or treating triple negative breast cancer. Researches find that Cx43 is highly expressed in triple negative breast cancer tissues and cell lines and is mainly positioned in cell membranes and cytoplasm; after Cx43 expression is inhibited, CCK8, scratch and Transwell experiments find that proliferation, migration and invasion capabilities of triple negative breast cancer cells are inhibited, and overexpression promotes the progress of the process of the breast cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of connexin 43 in preparing drugs for preventing or treating triple-negative breast cancer. Background Art

[0002] Breast cancer is a common, highly heterogeneous malignant tumor in women, especially triple-negative breast cancer (TNBC), which is characterized by negative expression of estrogen receptor (ER), progesterone receptor (PR), and HER2 (Human Epidermal Growth Factor Receptor 2). It is the most aggressive type of breast cancer, with the strongest metastatic potential and the worst prognosis. Therefore, the prevention and treatment of triple-negative breast cancer require greater attention and research.

[0003] Triple-negative breast cancer lacks specific targets, and clinical treatment primarily relies on chemotherapy with anthracyclines and paclitaxel. Despite comprehensive and aggressive treatment, over 50% of patients with triple-negative breast cancer experience recurrence, and over a third of these patients die within 5 years. Therefore, developing new and effective drugs to treat triple-negative breast cancer is crucial. Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides the use of gap junction protein 43 in the preparation of a drug for preventing or treating triple-negative breast cancer. Gap junction protein 43 (connexin43, Cx43) is a transmembrane protein (Bonacquisti EE, Nguyen J. Connexin43 (Cx43) in cancer: Implications for therapeutic approaches via gap junctions [J]. Cancer Letters, 2019, 442: 439-444.).

[0005] The present invention adopts the following technical solutions:

[0006] Use of connexin 43 in the preparation of drugs for preventing or treating triple-negative breast cancer.

[0007] The amino acid sequence of the gap junction protein 43 is as shown in SEQ ID NO: 1 shows: MGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVRFWVLQIIFVSVPTLL YLAHVFYVMRKEEKLNKKEEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEHGKVKMRGGLLRTYIISILFKSIFEVAFLLIQWYIYGFSLSAVYTCKR DPCPHQVDCFLSRPTEKTIFIIFMLVVSLVSLALNIIELFYVFFKGVKDRVKGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAAGHELQPLAIVDQRPSSRASSRASSRPRPDDLEI(SEQ ID NO:1).

[0008] According to one embodiment of the present invention, the application is to inhibit Cx43 expression. Further, the application is to inhibit the proliferation, migration and / or invasion of triple-negative breast cancer cells.

[0009] A method for preparing a triple-negative breast cancer model, characterized by using siRNA to knock down Cx43, wherein the sequences of the siRNA are shown in SEQ ID NO: 4 and SEQ ID NO: 5, or SEQ ID NO: 6 and SEQ ID NO: 7, or SEQ ID NO: 8 and SEQ ID NO: 9.

[0010] According to one embodiment of the present invention, the sequences of the siRNA are shown as SEQ ID NO: 6 and SEQ ID NO: 7.

[0011] Beneficial effects:

[0012] The present invention provides the use of connexin 43 in the preparation of a drug for the prevention or treatment of triple-negative breast cancer. Studies have found that Cx43 is highly expressed in triple-negative breast cancer tissues and cell lines, primarily localized to the cell membrane and cytoplasm. CCK8, scratch wound, and Transwell assays have shown that inhibition of Cx43 expression inhibits the proliferation, migration, and invasion of triple-negative breast cancer cells, while overexpression promotes these processes. This suggests that inhibiting connexin 43 expression could be an effective approach for the prevention or treatment of triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the result of immunohistochemical detection of Cx43 expression in triple-negative breast cancer and adjacent cancer tissues (200×);

[0014] Figure 2 This is the result of qRT-PCR detection of differential expression of Cx43;

[0015] Figure 3 This is the result of Western Blot detection of differential expression of Cx43;

[0016] Figure 4 This is a graph showing the interference efficiency of Cx43 at the mRNA level detected by qRT-PCR;

[0017] Figure 5 This is the result of Western Blot detection of Cx43 interference efficiency at the protein level;

[0018] Figure 6 This is the result of qRT-PCR Cx43 overexpression efficiency at the mRNA level;

[0019] Figure 7 This is the result of Western Blot detection of Cx43 overexpression efficiency at the protein level;

[0020] Figure 8 This is the result of CCK8 detection of cell proliferation;

[0021] Figure 9 This is the result of the clone formation experiment to detect the proliferation ability of MDA-MB-231 cells;

[0022] Figure 10 This is a graph showing the results of scratch assay for cell migration and migration rate;

[0023] Figure 11 This is the result of Transwell assay to detect cell migration and invasion ability. DETAILED DESCRIPTION

[0024] In order to further illustrate the present invention and its advantages, the technical solution of the present invention is further described below through specific implementation methods. It should be understood that these embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0025] All breast cancer and adjacent breast cancer tissues in this study were obtained from patients undergoing surgical resection at the Second Affiliated Hospital of Chongqing Medical University. Immediately after surgical resection, the tissues were fixed and preserved in 4% paraformaldehyde fixative. Informed consent was obtained from the patients for all tissues, and this study was supported by the ethics committee of the Second Affiliated Hospital of Chongqing Medical University.

[0026] Example 1

[0027] The expression of Cx43 was analyzed in more than 60 breast cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) and Human Protein Atlas (HPA) databases. Immunohistochemistry was used to detect Cx43 expression in triple-negative breast cancer tissues and adjacent adjacent tissues. qRT-PCR and Western blot experiments were used to detect the differential expression of Cx43 in normal breast epithelial cells (MCF-10A) and two triple-negative breast cancer cell lines (MDA-MB-231 and BT-549).

[0028] Small interfering RNA was used to interfere with Cx43 expression, and a stable Cx43-overexpressing cell line was constructed using lentiviral vectors. The effect of Cx43 on the proliferation of triple-negative breast cancer cells was investigated using CCK8 and colony formation assays. The effects of Cx43 on the migration and invasion of triple-negative breast cancer cells were investigated using wound healing and Transwell assays.

[0029] Cell lines and culture conditions:

[0030] Human breast cancer cell lines BT-549 and MDA-MB-231, and normal mammary epithelial cells MCF-10A were purchased from ATCC and stored at the Institute of Life Sciences, Chongqing Medical University. MDA-MB-231 cells were cultured in DMEM medium supplemented with 10% FBS, BT-549 cells were cultured in 1640 medium supplemented with 10% FBS, and MCF-10A cells were cultured in a specialized medium. All cultures were maintained in a 37°C, 5% CO2, and saturated humidity incubator.

[0031] siRNA, cell transfection, and lentiviral infection

[0032] Three siRNAs were designed to knock down Cx43 (si1-Cx43, si2-Cx43, si3-Cx43, and siRNA-NC). Lentivirus was constructed to overexpress Cx43, and a mock vector lacking the Cx43 sequence was used as a control. Stably transfected breast cancer cell lines were then selected using puromycin. Transfection was performed using Lipo8000 transfection reagent according to the reagent manufacturer's instructions. The sequences of the four siRNAs are shown in the table below.

[0033]

[0034] qRT-PCR

[0035] Total RNA was extracted from MCF10A, BT-549, and MDA-MB-231 cells according to the RNA extraction kit instructions, and its concentration was measured. The RNA was then reverse-transcribed into cDNA. The relative expression levels of Cx43 in cells were determined using a RT-qPCR instrument according to the qRT-PCR reagent instructions. The sequences of Cx43 and GAPDH are shown in the table below.

[0036] Cx43-F AGTTCAATCACTTGGCGTGACTTC (SEQ ID NO: 10) Cx43-R GTTTGCCTAAGGCGCTCCAG (SEQ ID NO: 11) GAPDH-F GCACCGTCAAGGCTGAGAAC (SEQ ID NO: 12) GAPDH-R TGGTGAAGACGCCAGTGGA (SEQ ID NO: 13)

[0037] cDNA was used as the template, and the reaction conditions were: 95°C 30s→(95°C 5s→60°C 30s)×40→65°C 5s→95°C 10s. Three replicate wells were set up, and the relative expression level of mRNA was expressed as 2 -△△CT Methods and calculations. Transwell migration and invasion assays

[0038] Migration assay: Resuspend cells from each group in serum-free medium to a cell density of 2.5 × 10^5 / mL. 200 μL of cell suspension was added to the upper chamber, and 600 μL of medium containing 10% FBS was added to the lower chamber. The cells were then incubated in the incubator. After 24 hours, the chambers were removed, washed with PBS, and fixed with 4% paraformaldehyde for 30 minutes at room temperature. The cells were then stained with crystal violet for 25 minutes. After washing with PBS, the cells in the upper chamber were wiped with a cotton swab. After air-drying, 3-5 randomly selected fields of view were photographed under a microscope to count the number of cells that had penetrated the membrane. Invasion assay: Matrigel was diluted 1:6 with serum-free medium on ice. Transwell chambers were placed in 24-well plates, forming upper and lower chambers. 60 μL of the diluted Matrigel was added to the upper chamber and the cells were incubated at 37°C for 4 hours to solidify. The remaining steps were the same as for the migration assay.

[0039] Cell scratch assay

[0040] Each group of cells was seeded into a six-well plate and allowed to grow to a cell density of 90-100% the next day. On the day of the experiment, a 200-μl pipette tip was used to scratch the bottom of the six-well plate perpendicular to the horizontal line. The scraped cells were washed with PBS and 2 ml of serum-free medium was added to each well. The plates were photographed under an inverted microscope, and the scratch distance at 0 h was recorded. The plates were then placed in a 37°C incubator and cultured. The scratch distances at 12 h and 24 h were recorded. The migration rates at 12 h and 24 h relative to the distance at 0 h were calculated based on the difference in scratch distances. The formula was: relative migration rate = (0 h - 12 h or 24 h) / 0 h distance.

[0041] CCK-8

[0042] 24 hours after transfection, cells were routinely digested and centrifuged according to pre-designed experimental groups. The cells were resuspended to a density of 2 × 10^4 / mL and 100 μl of the cell suspension was added to a 96-well culture plate. Five replicates were set up for each group. The culture plates were incubated in an incubator until the cells adhered to the wall, marking 0 h. At 24 h, 48 h, 72 h, and 96 h, 10 μl of CCK-8 solution was added to each well of cells and mixed thoroughly to prevent air bubbles. The culture plates were incubated in the incubator for another 2 hours in the dark. The absorbance at OD 450 nm was measured using a microplate reader, and growth curves were statistically plotted.

[0043] Immunohistochemistry

[0044] Tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. Prepared paraffin sections were routinely deparaffinized and hydrated in graded alcohols. Antigen retrieval was performed in a microwave oven using EDTA retrieval solution, cooled to room temperature, permeabilized with 0.3% Triton X-100 for 10 minutes, blocked with endogenous peroxidase for 30 minutes, and blocked with goat serum for 1 hour. Sections were then incubated with primary antibodies in a humidified chamber overnight at 4°C. Following three washes with PBS, sections were incubated with secondary antibodies for 30 minutes at 37°C, and washed three times with PBS for 5 minutes each. The sections were visualized under a microscope using DAB staining, rinsed with running water for 10 minutes, and counterstained with hematoxylin. Sections were then dehydrated in graded alcohols, mounted with neutral gum, and observed and photographed under a microscope.

[0045] Plate colony formation assay

[0046] 24 hours after transfection, digest and centrifuge the cells, resuspend them in culture medium, and count them. Plate 800 cells / well in a six-well plate, add 2 ml of complete culture medium, gently shake to mix, set up three replicates per well, and place in an incubator. Change the medium every three days. After 10 days, cells will form colonies and be fixed and stained. Discard the culture medium, wash three times with PBS, fix with 4% paraformaldehyde for 20 minutes, wash three times with PBS, stain with crystal violet for 20 minutes, rinse with water, air-dry, and photograph and count.

[0047] Statistical analysis

[0048] Statistical analysis was performed using GraphPad Prism 9.0. Measurement data were expressed as x ± s. The means of two samples were compared using the t-test, and the means of multiple groups were compared using analysis of variance. P < 0.05 indicated statistical significance.

[0049] Experimental results

[0050] The expression of Cx43 in more than 60 breast cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) and Human Protein Atlas (HPA) databases was analyzed, and it was found that triple-negative breast cancer accounted for more than 70% of the cell lines with high Cx43 expression, while other types of breast cancer had almost no expression or low expression.

[0051] To further detect the expression of Cx43 in triple-negative breast cancer, the inventors collected triple-negative breast cancer tissues and adjacent tissues from patients and detected their expression by immunohistochemical staining. They found that Cx43 expression was mainly located in the cell membrane and cytoplasm, and that the expression in cancer tissues was higher than that in adjacent tissues. Figure 1 At the same time, the inventors used qRT-PCR and WB experiments to detect the mRNA and protein expression of Cx43 in normal breast epithelial cells (MCF-10A) and two triple-negative breast cancer cells (MDA-MB-231 and BT-549). They found that the expression of Cx43 in the two triple-negative breast cancer cells MDA-MB-231 and BT-549 was higher than that in the breast epithelial cells MCF-10A, and the difference was statistically significant ( Figure 2 and Figure 3 , P<0.05).

[0052] To this end, the inventors overexpressed Cx43 in MDA-MB-231 cells, which have a relatively low expression of Cx43, and knocked down Cx43 expression in BT-549 cells, which have a relatively high expression of Cx43.

[0053] To further explore the effects of Cx43 on the biological characteristics and functions of triple-negative breast cancer cells, the inventors designed and synthesized three knockdown Cx43 expression lines and their negative controls. The knockdown and control groups were transfected into BT-549 cells, and the cells were collected and the mRNA expression level of Cx43 was detected by qRT-PCR. The protein expression level of Cx43 was detected by WB. The results showed that compared with the control group, the knockdown group had a significantly higher mRNA and protein expression level ( Figure 4 and Figure 5 ) was significantly reduced, and the inventors further selected the one with the highest knockdown efficiency (#2) for subsequent functional experiments. At the same time, the inventors also constructed a lentivirus overexpressing Cx43 and its negative control, transfected MDA-MB-231 cells, and constructed a stable transfectant strain with puromycin selection. qRT-PCR and WB were used to detect the mRNA expression level and protein expression level of Cx43 ( Figure 6 and Figure 7 ), the results showed that compared with the control group, the expression level of Cx43 in the overexpression group was significantly increased.

[0054] To observe the effect of Cx43 on the proliferation of triple-negative breast cancer cells, the inventors used CCK8 assay and plate colony formation assay to test the proliferation ability of cells that interfered with or overexpressed Cx43. The results showed that after interfering with Cx43 expression in BT-549 cells, cell proliferation was significantly inhibited, while overexpression of Cx43 increased the proliferation of MDA-MB-231 cells ( Figure 8 and Figure 9 ).

[0055] To further verify the effect of Cx43 on the migration and invasion ability of triple-negative breast cancer cells, a cell scratch assay was first performed. The results showed that the 24-hour migration rate in BT-549 cells with Cx43 knockdown was lower than that in the control group, while the 24-hour migration rate in MDA-MB-231 cells with Cx43 overexpression was higher than that in the control group ( Figure 10 , P < 0.05); then, the cell migration and invasion were detected by Transwell assay. The results showed that compared with the control group, the number of cells passing through the filter membrane in BT-549 cells with knockdown of Cx43 expression was reduced, while the number of cells passing through the filter membrane in MDA-MB-231 cells with overexpression of Cx43 was increased ( Figure 11 , P < 0.05). The above results showed that knocking down the expression of Cx43 inhibited the migration and invasion abilities of BT-549 cells, while overexpressing Cx43 increased the migration and invasion abilities of MDA-MB-231 cells.

Claims

1. Use of connexin 43 in the preparation of a drug for preventing or treating triple-negative breast cancer; the amino acid sequence of connexin 43 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The application is to inhibit Cx43 expression.

3. The use according to claim 2, characterized in that The application is to inhibit the proliferation, migration and / or invasion ability of triple-negative breast cancer cells.

4. A method for preparing a triple-negative breast cancer model, characterized in that: The siRNA for knocking down Cx43 has a sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, or as shown in SEQ ID NO: 6 and SEQ ID NO: 7, or as shown in SEQ ID NO: 8 and SEQ ID NO:

9. The method according to claim 4 , wherein the sequences of the siRNA are shown in SEQ ID NO: 6 and SEQ ID NO: 7.

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