Use of a ppp1r35 inhibitor in the preparation of a medicament for the treatment of triple negative breast cancer

Abstract

The application relates to application of a PPP1R35 inhibitor in preparation of a medicine for treating triple-negative breast cancer and belongs to the technical field of biological medicine. In order to solve the problem that triple-negative breast cancer lacks effective treatment means, the application provides application of the PPP1R35 inhibitor in preparation of the medicine for treating triple-negative breast cancer. The application first proves that PPP1R35 is specifically highly expressed in triple-negative breast cancer cells and can be used as a brand-new target point for treating triple-negative breast cancer. The PPP1R35 inhibitor can significantly inhibit tumor cell proliferation and migration, promote cell apoptosis, and inhibit EMT; in the body, the PPP1R35 inhibitor can inhibit the growth and tumorigenic ability of a mouse breast orthotopic tumor, reshape a tumor immune microenvironment, increase CD8 + / CD4 + T cell infiltration, reduce the proportion of Treg cells, and enhance the anti-tumor immune response of the body, thereby providing a treatment scheme with a clear target point, accurate effect and important clinical conversion value for triple-negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of PPP1R35 inhibitors in the preparation of drugs for the treatment of triple-negative breast cancer. Background Technology

[0002] Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by earlier onset, greater metastatic potential, poorer clinical outcomes, higher recurrence rates, and lower survival. Lacking estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor (HER2), TNBC is insensitive to conventional endocrine therapy and HER2-targeted therapy, making chemotherapy the primary treatment. However, because TNBC tumors are typically poorly differentiated, distant metastasis and recurrence rates are higher than other breast cancer subtypes, and recurrent and metastatic TNBC are often more aggressive and exhibit strong resistance to chemotherapy. Furthermore, the toxicity of chemotherapy drugs and the development of tumor cell resistance also hinder treatment progress, and some patients still fail to achieve satisfactory clinical outcomes. Therefore, finding effective precision treatments for TNBC remains an important clinical challenge.

[0003] PPP1R35 is a specific regulatory subunit of the PP1 family of serine / threonine protein phosphatases. It forms a functional complex with the PP1 catalytic subunit (PPP1C) to regulate the dephosphorylation of downstream substrates. Current research indicates that PPP1R35, as a novel centriole-rich protein, is located in the centriole and drives the transition from centriole to centrosome, playing a crucial role in maintaining normal cell mitosis and genomic stability. Under pathological conditions, abnormal expression of PPP1R35 can lead to abnormal centrosome number and structure, thereby inducing mitotic errors, cell senescence, cell death, and genomic instability, resulting in aneuploidy, which is associated with the development and progression of various cancers.

[0004] Existing research has largely focused on the role of PPP1R35 in malignant tumors such as lung cancer and gastric cancer, with less attention paid to its application in breast cancer. Its association with the malignant phenotype of TNBC has not yet been verified, leaving its expression characteristics, regulatory mechanisms, and potential drug value in TNBC still unknown. Consequently, there is a lack of TNBC treatment strategies targeting PPP1R35. Summary of the Invention

[0005] To address the lack of effective treatments for triple-negative breast cancer, this invention provides the application of PPP1R35 inhibitors in the preparation of drugs for treating triple-negative breast cancer.

[0006] The technical solution of the present invention: Application of inhibitors targeting the PPP1R35 gene in the preparation of drugs for the treatment of triple-negative breast cancer.

[0007] Furthermore, the inhibitor targeting the PPP1R35 gene is siRNA or a CRISPR / Cas9 gene editing element.

[0008] Furthermore, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID No:1, and the nucleotide sequence of the antisense strand is shown in SEQ ID No:2.

[0009] Furthermore, the CRISPR / Cas9 gene editing element is a ribonucleoprotein complex formed by the Cas9 protein and sgRNA targeting the PPP1R35 gene, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0010] Furthermore, the drug for treating triple-negative breast cancer is an injectable formulation.

[0011] Furthermore, the administration method of the injectable preparation is intratumoral injection.

[0012] The beneficial effects of this invention are: This invention is the first to demonstrate that PPP1R35 is specifically and highly expressed in triple-negative breast cancer cells, making it a novel therapeutic target for triple-negative breast cancer. The provided PPP1R35 inhibitor significantly inhibits tumor cell proliferation, colony formation, and migration in vitro, blocks the epithelial-mesenchymal transition process, and promotes tumor cell apoptosis. In vivo experiments show that it significantly inhibits the growth and tumorigenicity of orthotopic breast tumors in mice, while effectively remodeling the tumor immune microenvironment and increasing CD8+. + CD4 + Effector T cells infiltrate and reduce the proportion of immunosuppressive Treg cells, thereby enhancing the body's anti-tumor immune response. Based on the above in vitro and in vivo experimental results, PPP1R35 inhibitors hold promise for development into clinically applicable dosage forms such as injectable formulations, providing a novel treatment option for triple-negative breast cancer with a clear target, definite efficacy, and significant clinical translational value. Attached Figure Description

[0013] Figure 1 This is a comparison of PPP1R35 protein expression in different mammary cell lines in Example 1. A is a Western blot electrophoresis band image, and B is a statistical graph of the relative expression levels of PPP1R35 protein.

[0014] Figure 2 This is a comparison of PPP1R35 mRNA and protein expression in 4T1 cells of each group in Example 2. A is a statistical chart of relative mRNA expression levels, and B is a Western blot electrophoresis band image.

[0015] Figure 3 This is a comparison of PPP1R35 mRNA and protein expression in MDA-MB-231 cells of each group in Example 2. A is a statistical chart of relative mRNA expression levels, and B is a Western blot electrophoresis band image.

[0016] Figure 4 The images show a comparison of the results of the plate colony formation experiment of 4T1 cells in each group in Example 2. A is a photograph of the colony formation, and B is a comparison of the number of colonies formed.

[0017] Figure 5 The images show a comparison of the results of the plate colony formation experiment of MDA-MB-231 cells in each group in Example 2. A is a photograph of the colony formation, and B is a comparison of the number of colonies formed.

[0018] Figure 6 The images show a comparison of the scratch test results of 4T1 cells in each group in Example 2. A shows the scratch healing morphology at different time points, and B shows the migration rate comparison.

[0019] Figure 7 The image shows a comparison of the scratch test results of MDA-MB-231 cells in different groups in Example 2. A is a scratch healing morphology diagram at different time points, and B is a migration rate comparison diagram.

[0020] Figure 8 The images show a comparison of the cell proliferation experiment results of 4T1 cells in each group in Example 2. A is an EdU staining fluorescence imaging image, and B is a comparison of the average fluorescence intensity of EdU staining.

[0021] Figure 9 The images show a comparison of the cell proliferation experiment results of MDA-MB-231 cells in each group in Example 2. A is an EdU staining fluorescence imaging image, and B is a comparison of the average fluorescence intensity of EdU staining.

[0022] Figure 10 This is a comparison of cell viability of MCF-10A cells in the OE group under different ATO (arsenic trioxide) concentrations in Example 3;

[0023] Figure 11 This is a comparison of the protein expression of E-cadherin, N-cadherin, and PPP1R35 in MCF-10A cells of the CON group and OE group in Example 3. A is a Western blot electrophoresis band image, B is a statistical graph of the relative expression level of E-cadherin protein, C is a statistical graph of the relative expression level of N-cadherin protein, and D is a statistical graph of the relative expression level of PPP1R35 protein.

[0024] Figure 12These are comparative photographs of tumor tissues removed from the two groups of mice at the experimental endpoint in Example 4.

[0025] Figure 13 This is a comparison of tumor volume growth curves in two groups of mice in Example 4;

[0026] Figure 14 The images show the in vivo fluorescence imaging results of two groups of mice in Example 4. A is a representative in vivo fluorescence imaging image, and B is a comparison of the total fluorescence flux of the tumor over 21 days.

[0027] Figure 15 The images show the results of immunohistochemical staining of two groups of mice in Example 4 to detect the expression level of caspase3, a protein related to tumor apoptosis. A is a representative image of caspase3 immunohistochemical staining, and B is a comparison image of the quantitative expression of caspase3 protein.

[0028] Figure 16 The images show the results of immunohistochemical staining of two groups of mice in Example 4 to detect the expression level of Ki-67, a protein related to tumor tissue proliferation. A is a representative image of Ki-67 immunohistochemical staining, and B is a comparison image of Ki-67 protein expression quantification.

[0029] Figure 17 The images show the results of multiplex immunofluorescence staining of tumor tissues from two groups of mice in Example 4. A is a representative image of multiplex immunofluorescence staining, and B is a comparison of the quantitative expression of immune cell markers. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0031] The following examples use Student's t-test to examine differences between groups. The experiment was conducted in triplicate, and the data are presented in Mean ± SD format. ns indicates no statistically significant difference. This indicates that there is a certain statistical difference. This means there is a statistically significant difference, while P<0.001(***) represents an extremely statistically significant difference.

[0032] Example 1

[0033] This study investigated the expression differences of PPP1R35 in normal breast cells, non-triple-negative breast cancer cells, and triple-negative breast cancer cells, clarifying whether PPP1R35 is specifically abnormally expressed in TNBC, and providing preliminary experimental evidence for screening potential TNBC targets.

[0034] I. Cell Culture

[0035] The MCF10A cells (human normal breast epithelial cells), SKBR3 cells (human HER2-positive breast cancer cells), T47D cells (human Luminal A breast cancer cells), BT549 cells (human triple-negative breast cancer cells), MDA-MB-231 (human triple-negative breast cancer cells, abbreviated as 231), and 4T1 (mouse triple-negative breast cancer cells) used in this embodiment were all purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0036] MCF10A cells were cultured using their dedicated medium (Pronosai); MDA-MB-231 cells, BT549 cells, and 4T1 cells were cultured using RPMI-1640 medium containing 10% fetal bovine serum; T47D cells and SKBR3 cells were cultured using DMEM medium containing 10% fetal bovine serum. All cells were routinely cultured in an incubator at 37°C and 5% CO2.

[0037] 2. Western blot

[0038] Western blot was used to detect the expression level of PPP1R35 protein in various cell lines, with β-actin as an internal control. The specific steps were as follows: Cells were lysed on ice using RIPA buffer (Beyotime, China) containing a protease inhibitor to extract total protein; the protein concentration in the cell lysate was measured using a BCA protein quantification kit (BCA, Beyotime, China), and the loading volume was adjusted accordingly; equal volumes of protein samples were separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Millipore, USA); the membrane was placed in 5% skim milk solution and blocked at room temperature for 2 hours; then diluted primary antibody was added and incubated overnight at 4°C; subsequently, the membrane was washed with TBST buffer, HRP-labeled secondary antibody was added, and incubated at room temperature for 1 hour; after washing again, the membrane was developed using an ECL chemiluminescence reagent in an imaging system, and band images were acquired.

[0039] ImageJ software was used for quantitative analysis of band gray values. The gray ratio of PPP1R35 to β-actin was used as the relative protein expression level. Each experiment was performed in triplicate. The results are shown below. Figure 1 As shown.

[0040] Figure 1 The results showed that PPP1R35 was almost not expressed in normal human breast epithelial cells MCF10A, exhibited extremely low expression in HER2-positive breast cancer cells SKBR3, and showed low expression in Luminal A type breast cancer cells T47D. However, PPP1R35 protein showed significantly high expression in triple-negative breast cancer cells BT549, MDA-MB-231, and mouse triple-negative breast cancer cells 4T1. Comparison of relative PPP1R35 protein expression levels showed that the protein expression level of PPP1R35 was significantly increased in triple-negative breast cancer cells compared with normal breast epithelial cells and non-triple-negative breast cancer cells, and the difference was statistically significant. .

[0041] Example 2

[0042] To investigate the regulatory role of PPP1R35 in the biological behavior of triple-negative breast cancer (TNBC) cells, this study knocked down the expression of PPP1R35 in TNBC cells using siRNA technology. The effects of PPP1R35 on the malignant phenotype of TNBC cells were verified from two dimensions: cell proliferation and migration, providing functional evidence for its potential as a therapeutic target.

[0043] I. Construction of MDA-MB-231 and 4T1 cell lines with low expression of PPP1R35 using small interfering RNA

[0044] MDA-MB-231 and 4T1 cells were seeded into 6-well cell culture plates. When the cell confluence reached 60-70%, the cells were divided into three groups for treatment, as follows:

[0045] ① CTR group: No transfection treatment was performed, only routine culture was performed;

[0046] ②NC group: Transfected with non-specific negative control siRNA. The sequence of the negative control siRNA is an artificially designed non-target sequence that has no homology with the nucleotide sequences of various target genes in the experimental system. After transfection, it will not have a silencing effect on the expression of any endogenous or exogenous genes in the cells. It is only used as an experimental control to exclude the non-specific effects of the transfection operation itself on the cell physiological state and subsequent detection indicators.

[0047] ③si group: Transfected with siRNA targeting the PPP1R35 gene, the expression level of the PPP1R35 gene in cells was specifically downregulated by RNA interference technology. The reference sequence accession number of the PPP1R35 gene in NCBI is NG_69871; the sense strand of the siRNA targeting the PPP1R35 gene is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.2.

[0048] Specifically:

[0049] Justice Chain Sequence: 5'-GCCCUUCAGAAGAUACCUUTT-3';

[0050] Antisense sequence: 5'-AAGGUAUCUUCUGAAGGGCTT-3'.

[0051] To comply with WIPO ST.26 standards, uracil in RNA is represented by replacing the U at positions 5, 6, 14, 18, and 19 of the 5' end in the nucleotide sequence shown in SEQ ID NO:1, and uracil in RNA is represented by replacing the U at positions 5, 7, 9, 10, and 12 of the 5' end in the nucleotide sequence shown in SEQ ID NO:2.

[0052] Both the NC and Si groups were transfected using the Invitrogen transfection method, and the specific procedures are as follows:

[0053] 4 μL of siRNA (final concentration 20 μM) and 12 μL of Lipofectamine 2000 were added to 250 μL of Opti-MEM transfection medium, respectively. After incubation at room temperature for 5 min, the two were mixed thoroughly and allowed to stand at room temperature for 20 min to form a complex. The original culture medium for MDA-MB-231 or 4T1 cells was discarded, and the cells were washed twice with PBS. Then, 2 mL of medium containing the complex but without FBS and antibiotics was added, and the cells were incubated in a cell incubator for 6 hours. The medium was then replaced with complete medium. 48 h after transfection, the protein and mRNA expression levels of PPP1R35 in each group of cells were detected by Western blot and RT-qPCR to verify the knockdown efficiency.

[0054] The specific steps for RT-qPCR are as follows:

[0055] The 5×MasterMix I reagent kit was used to prepare a 20 μL system for reverse transcription. The system composition was as follows: 4 μL of 5×MasterMix I and 1 μL of dsDNase were added, and Nuclease-Free Water was added to make up to 20 μL. The mixture was gently pipetted and mixed, and after a short centrifugation, it was placed in a gradient PCR instrument at 42℃ for 10-20 minutes. The reaction was then stopped by raising the temperature to 85℃ for 5 seconds. The obtained cDNA was stored at -20℃ for subsequent PCR amplification. PCR amplification was performed using the Kingbite reagent kit. Following the kit instructions, a 20 μL system was selected. The specific composition of the system was: 2 μL cDNA, 10 μL 2×Universa SYBR Green qPCR Mix fluorescent dye, 1 μL forward primer F, 1 μL reverse primer R, and 6 μL ddH2O to a final volume of 20 μL. After vortexing, the system was placed in a RealTime PCR instrument for reaction. The reaction program was: 95℃ pre-denaturation for 30 seconds, 95℃ denaturation for 10 seconds, and 60℃ annealing / extension for 30 seconds, with 40 cycles of denaturation and annealing. The detection indicators included the target gene PPP1R35 and the internal reference gene GAPDH. At least three replicates were set for each indicator, and each experiment was repeated three times. 2- ΔΔCt The method determined relative mRNA expression, and the results were as follows: Figure 2 and Figure 3 As shown.

[0056] Figure 2 and Figure 3 The results showed that, compared with the CTR and NC groups, the protein and mRNA expression levels of PPP1R35 in the Si group were significantly reduced, indicating that the knockdown model was successfully constructed.

[0057] II. Plate Colony Formation Experiment

[0058] After transfection, cells from each group were seeded at a density of 800-1000 cells / dish in 60mm culture dishes, 4mL of complete culture medium was added, and the dishes were incubated for 14-15 days, with the medium replaced with fresh medium every 4 days. After incubation, the cells were gently washed with PBS, fixed with 4% paraformaldehyde for 15-20 minutes, and stained with crystal violet for 10 minutes. Cell colonies with more than 50 cells or a diameter greater than 0.3mm were counted under a microscope. The results are shown below. Figure 4 and Figure 5 As shown.

[0059] Figure 4 and Figure 5The results showed that, compared with the CTR group and the NC group, the clone formation ability of 4T1 and MDA-MB-231 cells knocked down by PPP1R35 in the si group was significantly reduced, with a decrease in the number of clones and a smaller cell size. The difference was statistically significant, demonstrating that low expression of PPP1R35 can significantly inhibit the long-term proliferation ability of TNBC cells.

[0060] III. Scratch Test

[0061] After transfection, cells from each group were seeded into 6-well plates. When cell confluence reached 100%, a straight line was vertically drawn at the bottom of each well using a sterile 200 μL pipette tip. Cells were washed three times with PBS to remove floating cells, and then cultured in serum-free medium. The drawn area was photographed under a microscope at 0 h, 24 h, and 48 h. The area of ​​the drawn area was quantified using ImageJ software. The results are shown below. Figure 6 and Figure 7 As shown.

[0062] Figure 6 and Figure 7 The results showed that, compared with the CTR and NC groups, the scratch healing rate of the si group cells was significantly slower and the migration rate was significantly reduced, indicating that knocking down PPP1R35 can effectively inhibit the migration ability of TNBC cells.

[0063] IV. EdU Cell Proliferation Experiment

[0064] Cell proliferation was detected using an EdU-based cell proliferation imaging assay kit (Abbkine, catalog number KTA2030). Cells were seeded in 96-well plates and cultured to an appropriate density. Medium containing 10 μM EdU was added, and the cells were incubated at 37°C for 2 h. The medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, followed by permeabilization with 0.5% Triton X-100 at room temperature for 15 min. Click-iT working solution (containing Reaction Buffer, Copper Reagent, AbFluor 488 azide, and Reducing Agent) was prepared according to the manufacturer's instructions and incubated at room temperature in the dark for 30 min. After washing with BSA Wash Solution, cell nuclei were stained with Hoechst 33342, and images were observed and acquired under a fluorescence microscope (EdU: Ex / Em = 501 / 525 nm; Hoechst 33342: Ex / Em = 360 / 460 nm). Hydroxyurea (1:1000) was used as a negative control. Results are as follows: Figure 8 and Figure 9 As shown.

[0065] Figure 8 and Figure 9The results showed that the green fluorescence signal representing DNA synthesis was strong in the CTR and NC groups, with no significant difference in fluorescence intensity between the two groups (ns, p>0.05); however, the green fluorescence intensity in the Si group was significantly reduced, and the difference was statistically significant compared with the CTR group. These results indicate that knocking down PPP1R35 significantly inhibits DNA synthesis in TNBC cells, resulting in a marked decrease in cell proliferation activity.

[0066] The above results indicate that PPP1R35 plays an important role in the proliferation and migration of triple-negative breast cancer cells, and knocking down PPP1R35 can inhibit the proliferation and migration of triple-negative breast cancer cells.

[0067] Example 3

[0068] To further investigate the role of PPP1R35 in the development and progression of triple-negative breast cancer, this experiment overexpressed PPP1R35 in normal human breast epithelial cells MCF-10A with low PPP1R35 expression. The study verified whether PPP1R35 overexpression induced normal breast epithelial cells to acquire a malignant phenotype of tumor cells from two dimensions: cellular drug sensitivity and epithelial-mesenchymal transition (EMT), thus clarifying its role as a key driver gene of TNBC.

[0069] I. Constructing the PPP1R35 overexpression model

[0070] MCF-10A cells were seeded in culture plates. When the cell confluence reached 60-70%, the cells were divided into two groups for treatment, as follows:

[0071] ①CON group: transfected with PLVX-CMV-m-Ppp1r35(NM_027242)Zsgreen-puro empty vector plasmid, without foreign gene insertion, constructed by Austech Biotechnology (Shanghai) Co., Ltd.;

[0072] ②OE group: The PPP1R35 overexpression plasmid constructed with PLVX-CMV-m-Ppp1r35(NM_027242)Zsgreen-puro as the backbone was transfected and inserted into the CDS region of the human PPP1R35 gene. It was constructed by Austech Biotechnology (Shanghai) Co., Ltd.

[0073] Transfection was performed according to the Thermo Fisher Scientific Lipo2000 manual. 48 hours after transfection, overexpression efficiency was verified by Western blot. Results are as follows: Figure 11 As shown, compared with the CON group, the protein expression level of PPP1R35 in the OE group was significantly increased, indicating that the overexpression model was successfully constructed.

[0074] II. CCK-8 cell viability assay for drug sensitivity

[0075] Transfected OE group MCF-10A cells were digested into single-cell suspensions and seeded into 96-well plates at a density of 3000 cells per well (100 μL per well). Simultaneously, the wells around the perimeter of the culture plate were filled with sterile PBS, and blank control wells without cells were included. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Different concentrations of ATO-containing medium were prepared using a serial dilution method (0, 1, 2, 4, 5, 8, 10 μM), and 100 μL of the drug-containing medium was added to each well. The plates were then returned to the incubator for another 24 h. Under light-protected conditions, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 1 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. The results are shown below. Figure 10 As shown.

[0076] Figure 10 The results showed that the viability of MCF-10A cells in the OE group decreased in a concentration-dependent manner with increasing ATO concentration: within the concentration range of 0–5 μM, cell viability did not change significantly (ns, no statistically significant difference); when the concentration reached 8 μM, cell viability decreased significantly. The inhibitory effect is further enhanced at 10 μM. This indicates that MCF-10A cells overexpressing PPP1R35 have significantly increased sensitivity to ATO, suggesting an enhanced malignant phenotype.

[0077] III. Western blot detection of EMT-related protein expression

[0078] Western blot was used to detect the expression levels of EMT-related proteins in MCF-10A cells from the CON and OE groups, including the epithelial marker E-cadherin, the mesenchymal marker N-cadherin, and the internal control protein β-actin. The experimental procedures were the same as described above, specifically protein extraction, quantification, electrophoresis, transfer to a membrane, blocking, primary antibody incubation, secondary antibody incubation, and ECL imaging. The relative expression levels of proteins were quantified by band grayscale analysis. The results are shown below. Figure 11 As shown.

[0079] Figure 11 The results showed that, compared with the CON group, the expression of epithelial marker protein E-cadherin in MCF-10A cells of the OE group was significantly reduced, while the expression of mesenchymal marker protein N-cadherin was significantly increased, suggesting that the cells underwent epithelial-mesenchymal transition and acquired stronger migration and invasion capabilities.

[0080] In summary, overexpression of PPP1R35 can induce normal breast epithelial cells MCF-10A to exhibit malignant tendencies of tumor cells, including enhanced drug sensitivity and EMT phenotype changes, further confirming that PPP1R35 is closely related to the occurrence and development of triple-negative breast cancer.

[0081] Example 4

[0082] To investigate the regulatory role of Ppp1r35 in the tumorigenicity of TNBC in vivo, this embodiment constructs a mouse mammary in situ tumor model and compares the differences in tumorigenicity between wild-type and Ppp1r35 knockout TNBC cells in vivo, clarifying the in vivo functional evidence of Ppp1r35 as a therapeutic target for TNBC.

[0083] I. Construction of a mouse orthotopic breast cancer model

[0084] 1. Laboratory animals

[0085] Female SPF-grade BALB / c mice, weighing 16-20g and aged 6-8 weeks, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. The mice were housed in an SPF-grade, pathogen-free environment with free access to food and water. All animal experiments strictly followed the National Institutes of Health's "Guidelines for the Care and Use of Laboratory Animals" and were approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (Approval No.: YS568). All animals were housed at the Laboratory Animal Center of the First Affiliated Hospital of Harbin Medical University.

[0086] 2. Animal grouping and model construction

[0087] The experimental animals were randomly divided into two groups:

[0088] ①TNBC group: Wild-type 4T1 mouse TNBC cells were used to construct the model. After weighing the mice, 5×10⁵ cells were injected into the fourth pair of mammary pads. 5 A mouse model of in situ breast cancer was constructed using wild-type 4T1 cells in the logarithmic growth phase.

[0089] ②Ppp1r35 knockout group: A stable Ppp1r35 gene knockout 4T1 cell line was constructed using CRISPR / Cas9 gene editing technology. sgRNA-A1 and sgRNA-A2 were designed downstream of Exon 1 and Exon 4 of the mouse Ppp1r35 gene (Gene ID: 69871), respectively. The targeting sequence of sgRNA-A1 is shown in SEQ ID NO.3, and the targeting sequence of sgRNA-A2 is shown in SEQ ID NO.4. sgRNA-A1: 5'-CCCGCTGCCCGAAAGTCCGAAGG-3'; sgRNA-A2: 5'-TCAGATGAGTGGTAGTACCTGGG-3'.

[0090] sgRNA-A1 and Cas9 protein were mixed at a 1:1 molar ratio and incubated at room temperature for 20 minutes to form the RNP-A1 complex; sgRNA-A2 and Cas9 protein were mixed at a 1:1 molar ratio and incubated at room temperature for 20 minutes to form the RNP-A2 complex. The RNP-A1 and RNP-A2 mixture was then electroporated into 4T1 cells. Single clones were picked using a limiting dilution method, and the homozygous knockout of the Ppp1r35 gene was confirmed by PCR and Sanger sequencing. This cell line was then inoculated into the fourth pair of mammary pads of mice, injected with 5 × 10⁻⁶ cells / mL. 5 A mouse model of orthotopic breast cancer was established using Ppp1r35 knockout 4T1 cells in logarithmic growth phase.

[0091] After inoculation with cells, both groups of mice were kept under the same feeding conditions and underwent subsequent tumor growth observation and testing in a uniform manner.

[0092] II. Photographs of in situ tumors in mouse mammary glands

[0093] At the end of the experiment, mice in both groups were sacrificed, tumor tissue was dissected, tumor size was photographed and weighed, and the growth differences between the two groups were compared. The results are as follows: Figure 12 As shown.

[0094] Figure 12 The results showed that tumor specimens in the TNBC group were generally larger and more robust. Compared with the wild-type TNBC control group, tumor specimens in the Ppp1r35 knockout group showed significantly smaller tumor volume, and the growth of some tumors was significantly inhibited. This indicates that after Ppp1r35 knockout, the tumorigenic ability of 4T1 cells in mice was significantly weakened, and tumor growth was significantly inhibited.

[0095] III. Tumor volume growth curve

[0096] During the rearing period of both groups of mice, tumor growth was observed every other day. The major axis and the minor axis perpendicular to it of the tumor were measured using calipers. The tumor volume was calculated using the formula: major axis × minor axis × minor axis / 2. A growth curve of tumor volume over time was plotted to compare the differences in tumor growth rates between the two groups. The results are as follows: Figure 13 As shown.

[0097] Figure 13The results showed that the tumor volume of both groups increased over time after inoculation with 4T1 cells, but the tumor growth rate of the TNBC group was significantly faster. Starting from day 7, the difference in volume between the two groups gradually widened, and the difference reached a highly significant level by day 21. In contrast, the tumor volume growth of the Ppp1r35 knockout group was significantly suppressed, and the overall growth rate was significantly lower than that of the TNBC group, further confirming that knocking out Ppp1r35 can significantly inhibit the proliferation and tumorigenicity of TNBC cells in vivo.

[0098] IV. In vivo fluorescence imaging for detecting tumor growth

[0099] In vivo fluorescence imaging technology was used to dynamically and non-invasively monitor the growth and proliferation of mammary tumors in situ in mice in the TNBC group and Ppp1r35 knockout group under in vivo conditions.

[0100] In vivo fluorescence imaging was performed on days 7 and 21 after inoculation of 4T1 cells in both groups of mice.

[0101] Each mouse was intraperitoneally injected with 150 mg / kg of D-fluorescein potassium salt. After 10 minutes of sufficient substrate reaction, the experimental animals were anesthetized with isoflurane. The anesthetized mice were then placed in an in vivo imaging system to collect fluorescence signals and detect the total fluorescence flux at the tumor site to assess tumor growth and proliferation levels. The results are as follows: Figure 14 As shown.

[0102] Figure 14 The results showed that the tumor fluorescence signal intensity was similar in both groups of mice on day 7; by day 21, the fluorescence signal at the tumor site in the TNBC group was significantly enhanced, while the fluorescence signal in the Ppp1r35 knockout group remained at a low level. The total fluorescence flux at the tumor site in both groups of mice on day 21 was quantitatively analyzed. The results showed that the total fluorescence flux at the tumor site in the TNBC group was significantly higher than that in the Ppp1r35 knockout group, and the difference was statistically significant. The results were completely consistent with the tumor volume growth curve and tumor specimen photographs.

[0103] V. Immunohistochemical staining to detect the expression of proteins related to tumor tissue proliferation and apoptosis

[0104] Immunohistochemical staining of mouse tumor tissues was used to detect the expression levels of proliferation-related protein Ki-67 and apoptosis-related protein Caspase3, thereby verifying from a histological perspective the inhibitory effect of Ppp1r35 knockout on the in vivo proliferation of TNBC cells and its promoting effect on tumor cell apoptosis.

[0105] Mouse tumor tissue was fixed in 4% paraformaldehyde (PFA) solution. The tissue underwent dehydration, clearing, paraffin embedding, embedding, sectioning, and baking to obtain paraffin sections with a thickness of 4 μm. The paraffin sections were baked in an oven at 65℃ for 2 hours, followed by dewaxing and rehydration in xylene I (10 min), xylene II (10 min), 100% ethanol (5 min), 95% ethanol (5 min), 85% ethanol (5 min), and 75% ethanol (5 min). The sections were washed 2-3 times with purified water. The sections were then immersed in hematoxylin staining solution for 5 min, followed by differentiation (2 s) and blueing (2 s). Next, they were immersed in eosin staining solution for 3 min, dehydrated in a gradient of 70%-80%-90%-100% ethanol (10 s each), immersed in xylene I and xylene II for 3 min, and then a suitable amount of neutral resin was added. The sections were then slowly mounted with coverslips and observed and photographed under a microscope. Tumor tissue sections underwent antigen retrieval, endogenous peroxidase inactivation, and blocking. Ki-67 and Caspase 3 primary antibodies were then added, and the sections were incubated overnight at 4°C. The following day, secondary antibodies were added, followed by DAB staining, hematoxylin counterstaining, dehydration and clearing, and mounting. The sections were then observed and photographed under a microscope. ImageJ software was used to analyze the average optical density (AOD) of each group of sections and to quantitatively assess protein expression levels. The results are as follows: Figure 15 and Figure 16 As shown.

[0106] Figure 15 The results showed that Caspase3 positive expression was stained brownish-yellow, and the Caspase3 positive staining signal was significantly enhanced in the tumor tissue of the Ppp1r35 knockout group. The expression level of Caspase3 in the tumor tissue of the Ppp1r35 knockout group was significantly higher than that in the TNBC control group, and the difference was statistically significant. This indicates that knocking out Ppp1r35 can significantly promote tumor cell apoptosis.

[0107] Figure 16 The results showed that Ki-67 positive expression was brownish-yellow in color. In the TNBC control group, Ki-67 positive staining signal was widespread and strong, while in the Ppp1r35 knockout group, the positive staining signal was significantly weakened. The expression level of Ki-67 in the Ppp1r35 knockout group was significantly lower than that in the TNBC control group, and the difference was statistically significant. This indicates that knocking out Ppp1r35 can significantly inhibit the proliferation of tumor cells.

[0108] In summary, immunohistochemical results confirmed that Ppp1r35 knockout significantly reduced the expression of the proliferation-related protein Ki-67 in TNBC tumor tissues, while promoting the expression of the apoptosis-related protein Caspase3, thus verifying the inhibitory effect of Ppp1r35 knockout on the in vivo growth of TNBC at the histological level.

[0109] VI. Multiplex Immunofluorescence Analysis

[0110] Multiplex immunofluorescence staining was used to simultaneously detect the expression of various immune cell markers in tumor tissues, and the effects of Ppp1r35 knockout on T cell subsets (CD3+) in the TNBC tumor microenvironment were analyzed. + CD4 + CD8 + T cells and immunosuppressive Treg cells (Foxp3) + The study aimed to elucidate the mechanism by which Ppp1r35 regulates tumor immune escape and inhibits antitumor immune responses.

[0111] Mouse tumor tissue was fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin to prepare 4μm thick paraffin sections. After dewaxing in xylene and hydration with graded ethanol, antigen retrieval was performed (sodium citrate buffer, pH 6.0). The sections were incubated with 3% H2O2 at room temperature in the dark for 10 min to block endogenous peroxidase, followed by washing three times with PBST. After blocking with 5% BSA at room temperature for 30 min, primary antibody working solution was added and incubated overnight at 4°C. The next day, the sections were washed three times with PBST, and HRP-labeled polymer secondary antibody was added and incubated at room temperature for 30 min. After washing with PBST, TSA fluorescent dye (BNKTSA® kit) was added and incubated at room temperature in the dark for 10 min. Multiple staining was performed, repeating the antigen retrieval → primary antibody → secondary antibody → TSA steps, using a different wavelength of fluorescent dye each time, and adhering to the principle of spectral avoidance to prevent cross-contamination. After the final staining, the nuclei were stained with DAPI, mounted with anti-fluorescence quenching mounting medium, and images were acquired under a confocal microscope. Image analysis software was used to quantify the positive signals of each fluorescence channel, calculate the expression levels of different immune cell markers, and compare the differences in immune cell infiltration between the two groups of tumor tissues. The results are as follows: Figure 17 As shown.

[0112] Figure 17 The image shows single-channel fluorescence images and overlays of CD3 (cyan), CD4 (green), CD8 (orange), and Foxp3 (red), and the CD3 concentration in tumor tissue from the Ppp1r35 knockout group. + CD4 + CD8 + T cell infiltration signals were significantly enhanced, CD3 + CD4 + CD8 + The number of T cells increased significantly, including CD8 cells. + The infiltration of cytotoxic T cells was particularly enhanced; while Foxp3 + The signaling of immunosuppressive Treg cells was significantly weakened, and their numbers were significantly reduced.

[0113] In summary, multiplex immunofluorescence results confirmed that Ppp1r35 knockout can remodel the tumor immune microenvironment of TNBC and significantly increase effector T cells (CD3+). + CD4 + CD8 + It can reduce the infiltration of tumor cells, decrease the proportion of immunosuppressive Treg cells, relieve tumor immunosuppression, enhance the body's anti-tumor immune response, and thus inhibit tumor growth in vivo.

[0114] The results of the aforementioned cell and animal experiments fully demonstrate that PPP1R35 is a key molecule driving the malignant progression of triple-negative breast cancer. Inhibiting PPP1R35 can not only directly suppress tumor cell proliferation, invasion, and EMT processes, but also reshape the tumor immune microenvironment, relieve immunosuppression, and enhance the anti-tumor immune response. This provides a solid theoretical basis and functional evidence for the development of specific inhibitors targeting PPP1R35, suggesting that PPP1R35 inhibitors could serve as a novel therapeutic agent, used alone or in combination with immunotherapy for the clinical intervention of triple-negative breast cancer.

Claims

1. The application of an inhibitor targeting the PPP1R35 gene in the preparation of a drug for treating triple-negative breast cancer, characterized in that, The inhibitor targeting the PPP1R35 gene is siRNA or a CRISPR / Cas9 gene editing element. The sense strand nucleotide sequence of the siRNA is shown in SEQ ID No:1, and the antisense strand nucleotide sequence is shown in SEQ ID No:

2. The CRISPR / Cas9 gene editing element is a ribonucleoprotein complex formed by the Cas9 protein and sgRNA targeting the PPP1R35 gene, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO:3 and SEQ ID NO:

4.

2. The application according to claim 1, characterized in that, The drug used to treat triple-negative breast cancer is an injectable formulation.

3. The application according to claim 2, characterized in that, The injectable preparation is administered via intratumoral injection.

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