Application of MISP as breast cancer diagnosis / prognosis marker and treatment target

Abstract

The invention discloses application of MISP as a breast cancer diagnosis / prognosis marker and a treatment target. The in-depth study on the expression of MISP in breast cancer cells and the influence on malignant biological behaviors such as breast cancer proliferation shows that compared with normal breast epithelial cells, the MISP is highly expressed in the breast cancer cells, the expression of the MISP is highly related to poor clinical prognosis of breast cancer patients, and the proliferation of the breast cancer cells can be inhibited by reducing the expression of the MISP. A new biomarker is provided for diagnosis and prognosis of the breast cancer, a new target spot is provided for treatment of the breast cancer, and important scientific significance is achieved in the aspects of subsequent drug research and development, clinical treatment and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to the application of MISP as a breast cancer diagnosis / prognosis marker and therapeutic target. BACKGROUND

[0002] Breast cancer (BC) is the most common malignant tumor in women worldwide and one of the leading causes of cancer death in women. In recent years, the incidence of breast cancer has shown an upward trend globally, especially in Asia and the Pacific region, with a particularly significant increase in young women. With the development of molecular biology and genomics, important progress has been made in the study of molecular markers of breast cancer, including estrogen receptor (ER), progesterone receptor (PR), HER2, BRCA1 / 2 gene mutations, etc. These markers not only provide a basis for precise diagnosis of breast cancer, but also lay the foundation for the development of individualized treatment strategies. As a highly heterogeneous disease, research and treatment of breast cancer are rapidly developing towards precision and individualization. However, despite extensive research by researchers on the etiology of breast cancer, the exact pathogenesis remains unclear, making it difficult to implement targeted prevention and treatment.

[0003] Breast cancer is the result of the combined effects of multiple gene alterations and environmental factors. In the pathogenesis of breast cancer, complex and nonlinear relationships link the cytoskeleton to various cellular life activities. The process of dynamic reorganization of the cytoskeleton is influenced by multiple factors and plays an important role in different stages of the occurrence and development of breast cancer.

[0004] MISP (Mitotic Spindle Positioning) is an actin-related protein that runs through the cell cycle and plays an important role in the occurrence and development of various tumors. As a substrate of PLK1, MISP is involved in the regulation of proper positioning and orientation of the mitotic spindle, thereby affecting the cell division process. Although the function of MISP in intestinal cancer, gastric cancer and pancreatic ductal carcinoma has been studied more, its role in breast cancer has not been fully explored. Given that MISP is a key regulatory protein of mitotic spindle positioning, it is likely to participate in the occurrence and development of breast cancer by affecting cell division and proliferation. In addition, the signaling pathways regulated by MISP in other tumors (such as JAK2-STAT3 and Wnt / β-catenin pathways) are also crucial in breast cancer, which suggests that MISP may affect breast cancer progression through similar mechanisms.

[0005] However, current research on MISP in breast cancer is still very scarce, and its specific function and molecular mechanism need to be further explored. SUMMARY

[0006] The present application aims to provide an application of MISP as a breast cancer diagnostic / prognostic marker and therapeutic target.

[0007] In order to achieve the above object, the present application adopts the following technical solution:

[0008] The first aspect of the present application provides an application of a reagent for detecting the expression of biomarker MISP in the preparation of a diagnostic product for breast cancer subjects.

[0009] The diagnostic method comprises obtaining a test sample from a subject suspected of having breast cancer, determining the expression level of MISP in the test sample, and if the expression of MISP is higher than that of a normal control, it indicates that the subject is a breast cancer patient.

[0010] The second aspect of the present application provides an application of a reagent for detecting the expression of biomarker MISP in the preparation of a product for the prognosis of breast cancer subjects.

[0011] The prognosis method comprises obtaining a test sample from a subject with breast cancer, determining the expression level of MISP in the test sample, and a patient with high expression of MISP indicates a poor prognosis.

[0012] A patient with high expression of MISP has a poorer overall survival and disease-free survival.

[0013] The third aspect of the present application provides an application of MISP as a target in the screening of drugs for treating breast cancer.

[0014] The drug inhibits the expression of MISP, thereby inhibiting the viability, migration, proliferation and invasion ability of breast cancer cells, and further slowing down or preventing the development of breast cancer.

[0015] The fourth aspect of the present application provides an application of a MISP expression inhibitor in the preparation of a drug for treating breast cancer.

[0016] The MISP expression inhibitor includes nucleic acid molecules, proteins, and compounds.

[0017] The nucleic acid molecules include siRNA or shRNA that silences or down-regulates the expression of MISP, and the siRNA is siRNA1, siRNA2 or siRNA3.

[0018] The nucleotide sequences of the sense strand and the antisense strand of the siRNA1 are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively, the nucleotide sequences of the sense strand and the antisense strand of the siRNA2 are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively, and the nucleotide sequences of the sense strand and the antisense strand of the siRNA3 are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively.

[0019] The present application has the beneficial effect that through the in-depth research on the expression of MISP in breast cancer cells and the influence on the malignant biological behaviors such as the proliferation of breast cancer, it is found that compared with normal breast epithelial cells, MISP is highly expressed in breast cancer cells, the expression of MISP is highly related to the poor clinical prognosis of breast cancer patients, and reducing the expression of MISP can inhibit the proliferation of breast cancer cells. The present application provides a new biomarker for the diagnosis and prognosis of breast cancer, and provides a new target for the treatment of breast cancer, which has important scientific significance for subsequent drug research and development, clinical treatment and the like. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a result graph of analyzing MISP in pan-cancer by using TCGA database.

[0021] Figure 2 is an analysis of the expression of MISP by using the online analysis website GEPIA2 on 1085 breast cancer samples and 112 normal tissue samples downloaded from TCGA database.

[0022] Figure 3 is an analysis of the correlation between MISP and the prognosis of breast cancer by using the online analysis website GEPIA2: A. MISP in the overall survival of breast cancer patients; B. MISP in the overall survival and disease-free survival of breast cancer patients.

[0023] Figure 4 is a violin plot of the expression distribution of MISP gene in tumor tissue and normal tissue: the horizontal coordinate represents different sample groups, and the vertical coordinate represents the expression distribution of the gene; G1: breast cancer liver metastasis tissue sample; G2: breast cancer lymph node metastasis tissue sample; G3: normal tissue; the asterisk represents the significance p value, wherein * represents that the p value is less than 0.05, ** represents that the p value is less than 0.01, and *** represents that the p value is less than 0.001, and the number of asterisks represents the degree of significance; the significance of the three groups of samples is determined by Kruskal-Wallis test.

[0024] Figure 5 shows the expression of MISP in normal breast epithelial cells and breast cancer cell lines.

[0025] Figure 6 Showing the localization of MISP in normal mammary epithelial cells and breast cancer cell lines.

[0026] Figure 7 Showing the knockdown efficiency of three siRNAs against MISP in BT-549 and MCF-7 breast cancer cell lines by real-time quantitative fluorescent PCR.

[0027] Figure 8 Showing the knockdown efficiency of siRNAs against MISP in BT-549 and MCF-7 breast cancer cell lines by immunofluorescence.

[0028] Figure 9 Showing that knocking down the expression of MISP reduces the cell viability of BT-549 and MCF-7.

[0029] Figure 10 Showing that knocking down the expression of MISP reduces the proliferation ability of BT-549 and MCF-7. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the example figures of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

[0031] Without special instructions, the cell lines listed in the context of the present application, including breast cancer-related cell lines MCF-10A, MDA-MB-231, BT-549, MCF-7 and T47D, were purchased from the Chinese Academy of Sciences after being cultured, and were frozen in liquid nitrogen for subsequent experiments. The reagents used in the present application are commercially available. The experimental methods used in the present application, such as RNA extraction, primer design, PCR, tumor cell culture, cell proliferation and Western Blot, are conventional methods and techniques in the art.

[0032] The present application uses Graphpad Prism 8 software to process and analyze data. n represents the sample number, all experiments in the present application are done three times, the experimental data is represented by `X±S, and the rank sum test and variance analysis are used for comparison between groups. p<0.05 is determined as having statistically significant difference. Representative results in biological experiment repetition are presented in the accompanying drawings in the context.

[0033] Example 1, Study on the Expression Level of MISP in Breast Cancer

[0034] 1. Main reagents and sources

[0035] Table 1 Experimental reagents and sources

[0036]

[0037]

[0038] 2 Experimental methods

[0039] 2.1 Database analysis

[0040] 2.1.1 MISP is highly expressed in multiple tumors

[0041] Transcriptome sequencing data of 33 kinds of tumors were downloaded from TCGA (The Cancer Genome Atlas) database, and the expression difference of MISP in pan-cancer was analyzed using online analysis website.

[0042] 2.1.2 Study on the expression level of MISP in breast cancer tissue

[0043] The expression of MISP in 1085 breast cancer samples and 112 normal tissue samples downloaded from TCGA database was analyzed using online analysis website GEPIA2 (http: / / gepia2.cancer-pku.cn / #analysis).

[0044] The overall survival (OS) and disease-free survival (DFS) of MISP in 535 breast cancer tissue samples and 535 normal tissue samples downloaded from TCGA database were analyzed using GEPIA2 online analysis website.

[0045] The expression distribution of MISP gene in tumor tissue and normal tissue was analyzed.

[0046] 2.2 Study on the expression level of mRNA of MISP in breast cancer cells

[0047] The operation steps are as follows:

[0048] 1. Extraction of total RNA of cells

[0049] The total RNA of breast cancer cells was extracted using ABclonal AFTSpin Tissue / Cell Fast RNA Extraction Kit for Animal kit.

[0050] 2. RNA reverse transcription

[0051] Total RNA of cells was reversely transcribed into cDNA using TaKaRa PrimeScript RT reagent Kit with gDNA Eraser kit.

[0052] (1) Remove genomic DNA: reagents were thawed on ice, reaction premix was prepared according to Table 2, and Mix was premixed according to the number of cell RNA tubes + 2 during preparation, then was aliquoted into each PCR tube, and the corresponding total RNA of cells was added according to the concentration.

[0053] Table 2 Preparation of reagents for removing genomic DNA

[0054]

[0055] (2) The prepared reaction solution was placed in a metal bath at 42℃ for 2 min, and after the reaction was completed, the cDNA concentration and quality were determined using a nucleic acid concentration analyzer.

[0056] (3) Reverse transcription reaction solution was prepared according to Table 3, Mix was premixed according to the number of cell cDNA samples + 10% sample number during preparation, and was aliquoted into PCR, and finally cDNA was added.

[0057] Table 3 Preparation of reverse transcription reaction solution

[0058]

[0059] (4) High-speed transient centrifuge was used for centrifugation, and after centrifugation, reverse transcription reaction was performed, and the reaction program was shown in Table 4:

[0060] Table 4 Reverse transcription reaction program

[0061]

[0062]

[0063] The synthesized cDNA needs to be stored at -20℃.

[0064] 3. Real-time fluorescent quantitative PCR (qRT-PCR)

[0065] ABclonal 2×Universal SYBR Green Fast qPCR Mix was used for qRT-PCR reaction.

[0066] (1) qRT-PCR primers: for detecting the expression level of MISP in breast cancer, the forward primer sequence of MISP detection primer is SEQ ID NO. 1: 5'- CCTCAGCTCAGATTCCATCCTCAG-3'; the reverse primer sequence is SEQ ID NO. 2: 5'- TGCGGTTCACCTTCCTCACTTC-3'.

[0067] (2) Prepare the PCR reaction solution according to Table 5, mix the sample number + 10% sample number on ice, and use a high-speed transient centrifuge to centrifuge after adding the sample.

[0068] Table 5 qRT-PCR reaction solution preparation

[0069]

[0070] (3) Use two-step method for qRT-PCR reaction, the reaction program is shown in Table 6:

[0071] Table 6 qRT-PCR reaction program

[0072]

[0073] (4) Put the sample into the Bio-rad PCR instrument, start the reaction program, and leave after the program is started normally. Check the reaction result after the reaction is completed, and confirm that the reaction process is correct.

[0074] 2.3 Cell immunofluorescence detection of MISP protein expression in breast cancer cells

[0075] The steps are as follows:

[0076] (1) Prepare cell slides: use tweezers to clamp the cell slides, quickly sterilize them with an alcohol lamp, and place them flat on a 24-well plate.

[0077] (2) Cell plating: digest the cells, add 150 μL of 2 x 10 4 cells in the logarithmic growth phase to each well, and place them in a cell culture incubator for culture. After 30 min of cell adhesion, add 500 μL of culture medium to each well and incubate overnight.

[0078] (3) Fixation: aspirate the culture medium, rinse with sterile PBS twice, aspirate the sterile PBS, and add 500 μL of 4% paraformaldehyde to each well for room temperature fixation for 15 min.

[0079] (4) Permeation: discard the 4% paraformaldehyde, add an appropriate amount of PBS to the horizontal shaker and wash 3 times, each time for 5 min. Aspirate the PBS, and add 300 μL of Triton X-100 immunostaining permeation solution to each well for room temperature permeation for 15 min.

[0080] (5) Sealing: Discard the Triton X-100 immunostaining permeation solution, add an appropriate amount of PBS, and wash 3 times on a horizontal shaker, 5 min each time. Absorb the PBS, and add 300 μL of QuickBlock immunostaining blocking solution to each well, and seal at room temperature for 30 min.

[0081] (6) Primary antibody incubation: Recover 300 μL of QuickBlock immunostaining blocking solution, add 300 μL of PBS-diluted primary antibody MISP to each well, and dilute at a ratio of 1:300. Incubate in a 4°C refrigerator overnight.

[0082] (7) Secondary antibody incubation: Recover the primary antibody. Add an appropriate amount of PBS and wash 3 times on a horizontal shaker, 5 min each time. Absorb the PBS, and add 300 μL of PBS-diluted secondary antibody cy3 to each well, and dilute at a ratio of 1:300. Incubate at room temperature in the dark for 1 h.

[0083] (8) Mounting: Add 3 μL of DAPI-containing anti-fluorescence quencher to the glass slide, pick up the cell slide, wipe it dry, and then directly contact the cell surface with the DAPI-containing anti-fluorescence quencher for mounting. After air drying, observe and collect images using a fluorescence microscope.

[0084] 2.4 siRNA knockdown of MISP expression

[0085] siRNA transfection, according to the following steps:

[0086] (1) Design and synthesis of siRNA

[0087] Search for the sequence of MISP (Genebank ID: 126353) in the NCBI Gene database, and entrust Shanghai Generay Biotech Co., Ltd. to design and synthesize siRNA sequences. Verify the siRNA transfection efficiency by real-time fluorescent quantitative PCR, and the si-MISP and si-NC sequences are shown in Table 7.

[0088] Table 7 siRNA sequences

[0089]

[0090] (2) siRNA transfection of cells

[0091] a. Plate the breast cancer cells into a six-well plate 24 h before the start of the transfection experiment, so that the cell density is about 60% at the time of transfection, and the cells are in the logarithmic growth phase.

[0092] b. Add 200 μL of DMEM basic culture medium to a 1.5 ml sterile EP tube, add 6 μL of siRNA-mate transfection reagent, and then add 3 μL of siRNA solution, vortex to mix, and centrifuge in a high-speed transient centrifuge for 5 s.

[0093] c. Incubate at room temperature for 12 min to form the transfection complex. Replace the fresh 2 ml complete medium to the cells during the incubation.

[0094] d. Add 200 μL transfection complex to each well. Shake gently and incubate the cells in the incubator. Collect the cells 36 h later to detect the gene expression level.

[0095] 2.5 Study the function of MISP in breast cancer

[0096] 1. CCK-8 experiment

[0097] (1) Cell plating: digest the cells transfected with siRNA for 24 h, count the cells, and add 1 x 103 cells to each well of a 96-well plate.

[0098] (2) After the cells adhere to the wall, add 10 μL CCK8 reaction solution to each well, incubate in a 37°C cell incubator for 1.5 h, and measure the absorbance value at OD450.

[0099] (3) Perform step (2) at 24 h, 48 h, 72 h, and 96 h after the first time of adding the reaction solution, collect the data, and plot a line graph to calculate the difference.

[0100] 2. EdU experiment

[0101] (1) Cell plating: digest the cells transfected with siRNA for 24 h, count the cells, and add 500 μL (3 x 104 cells in total) to each well of a 24-well plate, and place it back in the incubator overnight to wait for the cells to adhere.

[0102] (2) Dilute the EdU working solution: add 500 μL of the diluted EdU working solution (the working solution has a concentration of 20 μM, and the concentration is 10 μM when used) to each well. The original concentration of EdU is 10 mM, which is diluted with DMEM complete medium. After dilution, place the EdU working solution in the cell incubator for preheating.

[0103] (3) EdU-labeled cells: change the solution in each well, add 250 μL of the EdU working solution and 250 μL of DMEM complete medium, and place it back in the incubator for 2 h of incubation.

[0104] (4) Fixation: after the EdU-labeled cells are completed, aspirate the culture solution, and add 500 μL of the fixing solution (4% paraformaldehyde) for room temperature fixation for 15 min.

[0105] (5) Permeation: aspirate the 4% paraformaldehyde, add 500 μL of the washing solution (immunostaining fixing solution) to each well, and wash on a horizontal shaker for 3 times, 5 min each time. Aspirate the washing solution, and add 300 μL of Triton X-100 immunostaining permeation solution to each well for room temperature permeation for 15 min.

[0106] (6) Wash: aspirate the Triton X-100 permeabilization solution, add 500 μL of wash solution (immunostaining fixative) per well, and wash horizontally on a shaker for 3 times, 5 min each time.

[0107] (7) EdU detection: prepare the Click reaction solution.

[0108] (8) Nucleus staining: dilute the Hoechst 33342 solution with the wash solution, the dilution ratio is wash solution: Hoechst 33342 = 1:1000. Add 300 μL of diluted 1x Hoechst 33342 solution per well, and incubate at room temperature for 10 min in the dark.

[0109] (9) Wash: add 500 μL of wash solution (immunostaining fixative) per well, and wash horizontally on a shaker for 3 times, 3 min each time.

[0110] (10) Take pictures using a fluorescence microscope, calculate the EdU positive rate, and make a statistical plot.

[0111] 3 Results

[0112] 3.1 MISP is highly expressed in multiple tumors and highly expressed in breast cancer

[0113] The MISP transcriptome sequencing data in the TCGA database was downloaded for analysis of MISP expression differences. First, the online analysis website was used to analyze the expression of MISP in pan-cancer, and it was found that MISP had expression differences in cancers including breast cancer, renal clear cell carcinoma, lung squamous cell carcinoma, and glioblastoma multiforme, and the differences were statistically significant, and the expression of MISP in breast cancer, cholangiocarcinoma, colon cancer, and head and neck squamous cell carcinoma was higher than that in the cancer-adjacent tissue Figure 1 ).

[0114] The expression of MISP in 1085 breast cancer samples and 112 normal tissue samples downloaded from the TCGA database was analyzed using the GEPIA2 online analysis website. The results showed that the RNA level of MISP was increased in all breast cancer tissue samples compared to normal tissue Figure 2 ).

[0115] 3.2 MISP is related to the prognosis of breast cancer, and MISP is more highly expressed in breast cancer liver metastasis samples and breast cancer lymph node metastasis samples

[0116] First, the GEPIA2 online analysis website was used to analyze the MISP in the overall survival (Overall survival, OS) of breast cancer patients, and the disease-free survival (Disease Free Survival, DFS) of 535 breast cancer tissue samples and 535 normal tissue samples downloaded from the TCGA database. The results showed thatFigure 3 )MISP high expression patients have worse OS, DFS, and the difference in DFS is statistically significant.

[0117] RNAseq data and corresponding clinical information of breast cancer were obtained from GEO dataset (https: / / portal.gdc.com). 48 triple negative breast cancer samples (16 breast cancer liver metastasis tissues and 32 breast cancer lymph node metastasis tissues) and 10 normal breast tissue samples were collected and subjected to expression analysis. The results showed that MISP was highly expressed in breast cancer liver metastasis and lymph node metastasis tissues compared with normal breast tissue. Figure 4 )。

[0118] 3.3 Expression of MISP in breast cancer cell lines

[0119] Compared with normal breast epithelial cell line MCF-10A and breast cancer cell lines MDA-MB-231, MCF-7, BT-549 and T47D, it was found that MISP was highly expressed in each breast cancer cell line, especially in luminal A cell line MCF-7. Therefore, we mainly used MCF-7 for MISP functional verification in subsequent experiments. Figure 5 )。

[0120] Immunofluorescence was used to detect the localization of MISP in breast cancer cell lines, and it was found that MISP was expressed in both cytoplasm and nucleus. Figure 6 )。

[0121] 3.4 Knockdown of MISP expression in MCF-7 and BT-549 breast cancer cell lines

[0122] In MCF-7 and BT-549 breast cancer cell lines with the highest MISP expression, siRNA was transfected, and the knockdown efficiency was verified by real-time quantitative PCR and cell immunofluorescence experiments. Real-time fluorescent quantitative PCR results showed that the expression of MISP was significantly lower than that of the control group after 48h of transient transfection of siMISP-2 and siMISP-3 Figure 7 ) In later experiments, we selected siMISP-2 and siMISP-3 sequences for experiments. Immunofluorescence results showed that the MISP protein level of MCF-7 and BT-549 cells was significantly reduced compared with the control group Figure 8 )。

[0123] 3.5 Knockdown of MISP can inhibit breast cancer cell viability

[0124] By CCK-8 experiment, we verify whether knockdown of MISP can affect the cell viability of breast cancer cells. The experimental results show that knockdown of MISP can significantly inhibit the cell viability of MCF-7 and BT-549 cells, and the results are statistically significant Figure 9 ).

[0125] 3.6 Knockdown of MISP can inhibit the proliferation of breast cancer cells

[0126] By EdU experiment, we verify whether knockdown of MISP can affect the proliferation ability of breast cancer cells. The EdU results show that compared with the siNC group, the number of EdU positive cells in the breast cancer cell lines transfected with siMISP-2 and siMISP-3 is significantly reduced( Figure 10 ). The results show that interfering with the expression of MISP can inhibit the proliferation ability of breast cancer cell lines.

Claims

1. Application of reagents for detecting the expression of the biomarker MISP in the preparation of products for the diagnosis of breast cancer.

2. The application according to claim 1, characterized in that, The diagnostic method includes: obtaining test samples from individuals suspected of having breast cancer, determining the expression level of MISP in the test samples, and if the expression level is higher than that of normal controls, it suggests that the individual is a breast cancer patient.

3. Application of reagents for detecting the expression of the biomarker MISP in the preparation of products for prognostic assessment of breast cancer patients.

4. The application according to claim 3, characterized in that, The prognostic method includes obtaining test samples from subjects with breast cancer, determining the expression level of MISP in the test samples, and patients with high MISP expression are indicated to have a poorer prognosis.

5. The application according to claim 4, characterized in that, Patients with high MISP expression have worse overall survival and disease-free survival.

6. Application of MISP as a target in screening drugs for the treatment of breast cancer.

7. The application according to claim 6, characterized in that: The drug inhibits the expression of MISP, thereby suppressing the vitality, migration, proliferation and invasion of breast cancer cells, and thus slowing down or preventing the development of breast cancer.

8. Application of MISP expression inhibitors in the preparation of drugs for treating breast cancer.

9. The application according to claim 8, characterized in that: MISP expression inhibitors include nucleic acid molecules, proteins, and compounds.

10. The application according to claim 9, characterized in that: The nucleic acid molecule includes siRNA or shRNA that silences or downregulates MISP expression, wherein the siRNA is siRNA1, siRNA2 or siRNA3; The nucleotide sequences of the sense and antisense strands of siRNA1 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively; the nucleotide sequences of the sense and antisense strands of siRNA2 are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively; and the nucleotide sequences of the sense and antisense strands of siRNA3 are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

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