CN106324247B - The application of SSBP1 albumen - Google Patents

Abstract

The invention belongs to the technical field of biomedical engineering and discloses a new application of SSBP1 protein. The application of the SSBP1 protein provided by the present invention includes: the application of the SSBP1 protein in the preparation of a kit for detecting cancer metastasis, and the application of the SSBP1 protein in the preparation of a drug for inhibiting cancer metastasis. The cancers involved in the above applications are Breast, lung, ovary, or stomach cancer. In addition, the present invention also provides an application of the SSBP1 protein in the preparation of a drug for inhibiting the TGF-β signaling pathway related to breast cancer metastasis. The above-mentioned new application of the SSBP1 protein disclosed by the present invention provides a new detection approach and inhibition method for the diagnosis or prognosis of metastasis of breast cancer, lung cancer, ovarian cancer or gastric cancer, and the SSBP1 protein is more expected to become a clinical treatment for related cancer metastasis. target molecule.

Description

Application of SSBP1 protein

technical field

The invention belongs to the technical field of biomedical engineering, and particularly relates to a new application of SSBP1 protein.

Background technique

Metastasis of cancer means that malignant tumor cells go from the original site to other parts to continue to grow through lymphatic channels, blood vessels or body cavities. Cancer metastasis is one of the characteristics of malignant tumors, and it is also considered to be the primary cause of treatment failure and patient death.

Generally speaking, the pathways of cancer metastasis mainly include the following: (1) Direct spread: With the continuous increase of tumor volume, tumor cells can directly extend along the weak points of surrounding normal tissues, invade and destroy adjacent tissues or organ. (2) Lymphatic metastasis: After the tumor cells invade the lymphatic vessels, they transfer to the lymph nodes along with the lymph fluid, and grow in the lymph nodes to form metastases, which is a common way of tumor metastasis. (3) Hematogenous metastasis: Cancer cells can enter veins through lymphatic channels, or directly invade blood circulation to cause distant metastasis. (4) Implant transfer and transfer through the paravertebral venous system, etc.

For cancer patients, early diagnosis and control of metastasis is the key to clinical cure of cancer, and finding reliable and early predictive signals related to cancer metastasis has become a hot spot in the field of clinical diagnosis and treatment of cancer.

Contents of the invention

The purpose of the present invention is to provide the application of SSBP1 protein in the preparation of kits for detecting cancer metastasis, drugs for inhibiting cancer metastasis, and drugs for inhibiting signaling pathways related to cancer metastasis. early detection and poor prognosis.

In order to solve the above technical problems, the embodiment of the present invention firstly provides the application of SSBP1 protein (single-stranded DNA binding protein 1; SSBP1 protein; mitochondrial single-strand binding protein 1) in the preparation of a kit for detecting cancer metastasis, so The cancer is breast cancer, lung cancer, ovarian cancer or gastric cancer. In the above application, the SSBP1 protein is used as a protein molecular marker for detecting cancer metastasis. Specifically, this application is to detect whether the expression of SSBP1 protein in breast, lung, ovary or gastric tissue is down-regulated: if there is down-regulation of expression, it is concluded that cancer metastasis has occurred; if there is no down-regulation of expression , it was concluded that no cancer metastasis occurred.

At the same time, the embodiment of the present invention also provides the application of SSBP1 protein in the preparation of a drug for inhibiting cancer metastasis, and the cancer is breast cancer, lung cancer, ovarian cancer or gastric cancer.

In addition, the embodiment of the present invention also provides the application of SSBP1 protein in the preparation of a drug for inhibiting TGF-β signaling pathway, which is related to breast cancer metastasis.

Specifically, in the embodiment of the present invention, the mitochondrial quantitative proteomics comparative analysis was first performed on the parental breast cancer cell MDA-MB-231 and its derived highly metastatic cells MDA-MB-231HM and MDA-MB-231Bo, and found that the mitochondria Single-chain binding protein 1 (SSBP1) exhibits low expression in breast cancer cells with high metastatic potential. Secondly, through in vitro cell function experiments and nude mouse animal models, it was confirmed that silencing SSBP1 can promote the metastasis of breast cancer; while overexpression of SSBP1 can significantly inhibit its metastasis. It was further found that silencing SSBP1 can increase the transcription of TGF-β, which leads to the increase of TGF-β secretion in cells, the activation of TGF-β/SMAD signaling pathway, the induction of EMT in cells, and finally the promotion of breast cancer cell metastasis. At the same time, in lung cancer, ovarian cancer or gastric cancer cell lines, it was also found that interference with SSBP1 could significantly promote tumor migration. The above-mentioned application of the SSBP1 protein provided by the embodiments of the invention will provide a new way for the diagnosis or prognosis of breast cancer, lung cancer, ovarian cancer or gastric cancer metastasis, and the SSBP1 protein is more likely to become a target for clinical treatment of cancer metastasis molecular.

Description of drawings

Fig. 1 is the mass spectrum result of mitochondrial protein SSBP1 in the specific embodiment;

Fig. 2 is the results figure showing the differential expression of SSBP1 in MDA-MB-231, MDA-MB-231HM, MDA-MB-231Bo cell lines by mitochondrial proteomics in the specific embodiment;

Fig. 3 is the result figure of Western blot verification SSBP1 expression situation in MDA-MB-231, MDA-MB-231HM, MDA-MB-231Bo cell lines in the specific embodiment;

Fig. 4 is the result figure showing the expression of SSBP1 in breast cancer cell lines by Real-Time PCR in the specific embodiment;

Fig. 5 is the result figure of Western blot verification interference expression SSBP1 and the effect of overexpressing SSBP1 in breast cancer cells in the specific embodiment;

Fig. 6 is the result figure of Transwell verification in MDA-MB-231 interfering with the effect of SSBP1 expression on invasion and migration in the specific embodiment, wherein, Fig. 6-1 is the representative picture of experimental result, Fig. 6-2 is experimental Histogram of results;

Fig. 7 is the result figure of Transwell verification in MDA-MB-468 after interfering with the expression of SSBP1 in the embodiment of the effect on invasion and migration, wherein, Fig. 7-1 is a representative picture of the experimental results, and Fig. 7-2 is the experiment Histogram of results;

Fig. 8 is the result figure of Transwell verifying the impact on invasion after overexpressing SSBP1 in MDA-MB-231Bo in the specific embodiment, wherein, Fig. 8-1 is the representative picture of experimental result, Fig. 8-2 is the representative picture of experimental result Histogram;

Fig. 9 is the result figure of WB detecting TGF-beta signaling pathway-related molecules in the specific embodiment;

Figure 10 is a graph showing the results of reversing the effect of SSBP1 on metastasis after treatment with TGF-β signaling pathway inhibitors in the specific embodiment;

Fig. 11 is the mRNA level of TGF-beta after Real-time PCR detects SSBP1 interference and overexpression in the specific embodiment: Wherein, Fig. 11-1 is the experimental result figure of MDA-MB-231 and MDA-MB-468 cell line, Figure 11-2 is a diagram of the experimental results of the MDA-MB-231Bo cell line;

Fig. 12 is the secretion level of TGF-beta after ELISA detects SSBP1 interference and overexpression in the specific embodiment: Wherein, Fig. 12-1 is the experimental result figure of MDA-MB-231 and MDA-MB-468 cell line, Fig. 12- 2 is a diagram of the experimental results of the MDA-MB-231Bo cell line;

Fig. 13 is a result diagram of the in vivo transfer effect after verifying interference and overexpression of SSBP1 by live imaging of mice in the specific embodiment;

Fig. 14 is a result diagram of detection of SSBP1 in clinical samples and patient's survival rate in a specific embodiment;

Fig. 15 is a result diagram of detection of SSBP1 in clinical specimens and patient's tumor staging in a specific embodiment;

Fig. 16 is a result diagram of detection of SSBP1 in clinical specimens and the patient's lymph node metastasis status in a specific embodiment;

Fig. 17 is a result diagram of Real-time PCR verification of the interference effect of SSBP1 in lung cancer cell A549, gastric cancer cell SGC-7901 and ovarian cancer cell A2780 in the specific embodiment;

Figure 18 is the results of Transwell verification of the effect of silencing SSBP1 expression on invasion and migration in lung cancer cells A549;

Figure 19 is the results of Transwell verification of the effect of silencing SSBP1 expression on invasion and migration in ovarian cancer cell A2780;

Fig. 20 is a graph showing the results of Transwell verification of the effect of silencing SSBP1 expression on invasion and migration in gastric cancer cell SGC-7901.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, various embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. However, those of ordinary skill in the art can understand that, in each implementation manner of the present invention, many technical details are provided for readers to better understand the present application. However, even without these technical details and various changes and modifications based on the following implementation modes, the technical solution claimed in each claim of the present application can be realized.

1 Materials and methods

1.1 Materials

1.1.1 Cell lines

Human breast cancer cell lines MDA-MB-231, MDA-MB-468, lung cancer cell A549, gastric cancer cell SGC-7901, and ovarian cancer cell A2780 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, MDA-MB-231HM, MDA-MB- The 231Bo series of highly metastatic cell lines were constructed by our laboratory.

1.1.2 Source, strain and breed of nude mice

BALB/c (nu/nu) nude mice were provided by Shanghai Experimental Animal Center, Chinese Academy of Sciences. Experimental animal production license number: SCXK (Shanghai) (2007-0005); experimental animal use license number and certificate number: SCXK (Shanghai) (2007-0005); age: 28-35 days; body weight: 16-20g ; Gender: female; raised in SPF grade environment.

1.1.2 Main reagents

1.1.2.1 Reagents for protein electrophoresis and western blotting

Protein lysate T-PER (Pierce)

BCA Kit (Solebo)

4× protein loading buffer, ammonium persulfate APS (Sanko)

Tris base, glycine, sodium dodecyl sulfate SDS (Amresco)

N,N,N',N'-Tetramethylethylenediamine TEMED (Sigma)

30% Polyacrylamide (Solebol)

Separating gel buffer: 1.5M Tris, pH8.8 (Bio-Rad)

Stacking gel buffer: 1.0M Tris, pH6.8 (Bio-Rad)

Primary Antibody Diluent (Biyuntian)

Chemiluminescent Reagent (Millipore)

Skimmed Milk Powder (BD)

For details of the antibodies used in the western blot (WB) experiment in this example, see Table 1 below:

Table 1 Antibodies and dilutions used in Western blot (WB) experiments

Antibody Host Dilution Company SSBP1 rabbit polyclonal 1:1000 Abcam GAPDH rabbit polyclonal 1:2000 Protein Tech

1.1.3 Main instruments

Micro vertical electrophoresis tank (Bio-Rad company)

Mini Trans-Blot micro electrophoresis transfer tank (Bio-Rad company)

_CO2 constant temperature incubator (Shanghai Jinghong Company)

Mettler PE-160 electronic balance (Mettler)

Low-temperature high-speed centrifuge (Thermo Sorvall company)

MultiSKAN MK3 microplate reader (Thermo company)

Animal Live Imager (Bruker MI)

1.1.4 Culture medium and common solution preparation

Add constant double antibody (Sigma) and 10% FBS (Gibco) to DMEM high glucose medium (Hyclone) and L15 medium (Sigma) before use;

Protein loading buffer Pack (5×Loading buffer, 20×Reducing agent, Fermentas);

Electrophoresis buffer (25mM Tris-HCl, 250mM glycine, 0.1% SDS);

1.0M Tris-HCl (pH6.8); 1.5M Tris-HCl (pH8.8); 10% ammonium persulfate;

Blocking solution: 5% skimmed milk powder or 5% BSA prepared in 0.1M PBS (pH7.4);

20× transfer buffer (48mM Tris base, 39mM glycine, 20% methanol);

The acrylamide gel formula is shown in Table 2 below:

Table 2 Acrylamide gel formula

Reagent 10ml 10% separating gel 5ml 5% stacking gel H ₂ O 4.00ml 3.40ml 30% acrylamide 3.30ml 0.83ml Tris-HCl 2.50ml (1.5M pH8.8) 0.63ml (1.0M pH 6.8) 10% SDS 0.10ml 0.05ml 10% ammonium persulfate 0.10ml 0.05ml TEMED 4.00μl 5.00μl

1.2 Method

1.2.1 Cell culture

MDA-MB-231, MDA-MB-468, and MDA-MB-231Bo cells were cultured in L15 medium (Sigma) containing 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μl/ml streptomycin at 37 °C, 5% CO ₂ and an incubator. Lung cancer cells A549, gastric cancer cells SGC-7901, and ovarian cancer cells A2780 were cultured in DMEM (Sigma) with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μl/ml streptomycin. Cells were cultured to exclude mycoplasma and fungal contamination, and all cells were discarded and revived after 3 months.

1.2.2 Construction of recombinant wild-type SSBP1 expression vector

Using the cell line MDA-MB-231cDNA as a template, design primers to introduce the BamHI/EcoRI restriction site, PCR amplify the CDS region of SSBP1, purify the PCR amplified fragment and digest it with BamHI/EcoRI, and then digest it with BamHI/EcoRI The pCDH vector was ligated according to the Fermentas T4 ligase ligation procedure. The primer sequences are as follows:

Forward:

5'-CCGGAATTCGCCACCATGGACTACAAGGACGATGATGACAAGCTCGATGGAGGAATGTTTCGAAGACCTGTATT-3'

Reverse:

5'-CGCGGATCCCTACTCCTTCTCTTTCGTCT-3'

1.2.3 Transformation of competent bacteria and extraction and purification of plasmids

Use DH5α (Takara) competent bacteria to transform, and operate according to laboratory routine methods. The resulting clones were extracted with a small plasmid extraction kit, digested with enzymes to identify whether the exogenous fragments were inserted, and after confirmation, the clones with inserted exogenous fragments were sent to Shanghai Boshang Biological Co., Ltd. for sequencing. The correct sequenced clones were used to prepare plasmids according to the operating instructions of the TIANGEN plasmid mini-extraction kit.

1.2.4 Packaging viruses

In this study, a lentiviral packaging system was used, using a three-plasmid system: pCDH (overexpression carrying the target gene vector) or GV122 (carrying the interference fragment vector), psPAX2 and pMD2.G (packaging plasmid). HEK-293T cells in good growth state were inoculated into a 6 cm cell culture dish, and the transfection was carried out on the second day when the confluence was over 90%. PEI was used as the transfection reagent, the molar ratio of the amount of the three plasmids was 1:1:1, and the total amount was 8.2 μg. Mix 4μg of pCDH/GV122 or the corresponding pcdh-SSBP1/shRNA, 3μg of psPAX2 and 1.2μg of pMD2.G with 24μl of PEI, and place at room temperature for 10min to form a transfection complex; add the mixture to the connected HEK-293T Add DMEM to the cells to a total volume of 3ml; 8h after transfection, change to 3ml of DMEM containing 10% FBS to terminate the transfection; take the culture supernatant after changing the medium for 48h, filter through a 0.45μm filter membrane, aliquot, -70 ℃ frozen for later use.

1.2.5 Virus infection of tumor cells

Tumor cells in good growth state (including breast cancer cells MDA-MB-231, MDA-MB-468, lung cancer cells A549, gastric cancer cells SGC-7901 and ovarian cancer cells A2780) were placed in a 6cm culture dish, and the next day was about At 50% confluency, infection was performed. Dilute the virus solution 1:1 with L15 (or DMEM) medium containing 10% FBS, add polybrene to the diluted solution to a final concentration of 8 μg/ml; add the diluted solution to the cells, and replace with 10% after 8 hours of incubation L15 (or DMEM) medium with FBS. The corresponding target gene transfection efficiency was further confirmed by Western blot or Real time PCR.

1.2.6 Protein extraction

(1) Discard the medium for adherent cells, wash the cells twice with pre-cooled PBS and absorb the cleaning solution, place the cell culture dish on ice, add an appropriate amount of protein lysate, and scrape the cells with a cell scraper;

(2) Draw the lysate into the EP tube, place it in an ice bath at 4°C for 40 minutes, and place it on a shaker for several times during the period;

(3) After centrifugation at 14000rpm for 10min, transfer the supernatant to a new EP tube;

(4) Take 5 μl to measure the protein concentration by BCA method.

(5) Add 5×Loading buffer and 20×DTT to the remaining protein supernatant, and denature it in a boiling water bath for 10 minutes;

1.2.7 Western blot

(1) Prepare 10% polyacrylamide gel separating gel and 5% stacking gel respectively;

(2) Load 30 μg of protein per well;

(3) After 70V electrophoresis for 30 minutes to reach the separation gel interface, change to 120V electrophoresis and continue electrophoresis until bromophenol blue reaches the bottom of the separation gel;

(4) After electrophoresis, the protein was transferred to PVDF membrane (Milipore) by ice bath wet transfer method, the transfer condition: 220mA constant flow for 50min;

(5) Rinse the PVDF membrane once in PBST (PBS pH=7.4, 0.1% Tween 20), immerse in 5% skimmed milk blocking solution or BSA blocking solution, room temperature for 1 hour;

(6) Dilute a certain concentration of primary antibody with primary antibody diluent and incubate the membrane overnight at 4°C;

(7) Wash the membrane 3 times with PBST, 10 minutes each time;

(8) Dilute a certain concentration of peroxidase-labeled secondary antibody with skim milk and incubate at room temperature for 1 h;

(9) Wash the membrane 3 times with PBST, 10 minutes each time;

(10) Mix liquid A and liquid B in the exposure liquid in equal amounts, drop an appropriate amount on the surface of the PVDF membrane protein, put it into the darkroom of the imager, and perform exposure detection.

1.2.8 Cell migration and invasion ability detection experiment

(1) Breast cancer cells in the logarithmic growth phase were digested with 0.25% trypsin and blown into single cells, and the cells were suspended in empty DMEM culture medium for later use.

(2) Cell concentration reaches 2.5×10 ⁵ cells/ml (migration test of breast cancer cells) or 5×10 ⁵ cells/ml (invasion test of breast cancer cells), and 200 μL of cell suspension is placed in the upper chamber (migration test ) or the upper chamber of the Matrigel chamber (invasion experiment), 600 μL of 20% FBS culture solution was placed in the lower chamber, placed in the cell culture incubator, and after 15 hours of incubation, the chamber was taken out, added 10% paraformaldehyde, and fixed for 20 minutes . Finally, remove the fixative, add an appropriate amount of crystal violet staining solution to stain for 20 minutes, then carefully wash off the staining solution, and dry at room temperature. Finally counted and photographed, each group set up three replicate samples. (For the invasion experiment of lung cancer cell A549, gastric cancer cell SGC-7901 and ovarian cancer cell A2780, select the cell number 1×10 ⁶ /ml, take 200 μL and put it in the upper chamber, and the other steps are the same)

1.2.9 Animal experiments

(1) Inject 5×10 ⁵ / MDA-MB-231 and MDA-MB-231Bo cells into 6-8 week old female nude mice through the tail vein;

(2) After 6 weeks, inject 150 mg/kg of substrate fluorescein into each nude mouse, perform imaging in an in vivo imaging system, read signal values, and perform statistical analysis.

1.2.10 Immunohistochemical steps

Dewaxing and hydration: Baked slices at 60°C overnight, then soaked in xylene (×3 times), absolute ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 minutes.

Rinse: Rinse 3 times with PBS, 5min each time.

Antigen retrieval: immerse in citrate buffer, heat in a microwave oven on high heat for 10 minutes, and keep warm for 10 minutes.

Repeat the rinse step.

Eliminate endogenous peroxidase activity: incubate with 3% H ₂ O ₂ for 30 min.

Repeat the rinse step.

Primary antibody incubation: add diluted antibodies, anti-SSBP1 (1:100), anti-p-SMAD3 (1:100), overnight at 4°C in a humid box.

Repeat the rinse step.

Secondary antibody incubation: add mouse anti-rabbit (1:3000), incubate at room temperature for 1 hour in a humid chamber.

Repeat the rinse step.

Color development: monitor the DAB color development process for 3 minutes under a microscope, and terminate the reaction.

Rinse with double distilled water for 5 minutes.

The nuclei were counterstained with hematoxylin for 1 min.

Rinse with double distilled water for 5 minutes.

Dehydration and sealing: gradient alcohol dehydration, xylene transparent (×3 times), neutral gum sealing.

Observe and record the experimental results under a microscope.

2 Experimental results

2.1 Low expression of SSBP1 in breast cancer cells with high metastatic potential

Mitochondrial proteins were extracted from three cell lines MDA-MB-231, MDA-MB-231HM, and MDA-MB-231Bo, and analyzed by iTRAQ quantitative proteomics to screen differentially expressed proteins. The expression level of SSBP1 was significantly downregulated in MDA-MB-231HM, MDA-MB-231Bo. Figure 1 is the results of iTRAQ mass spectrometry of the mitochondrial protein SSBP1; Figure 2 is the results of mitochondrial proteomics showing the differential expression of SSBP1 in MDA-MB-231, MDA-MB-231HM and MDA-MB-231Bo cell lines.

Subsequently, Western Blot verification was performed on the mitochondrial proteins of the three cell lines, which confirmed the results of the above differential expression. Figure 3 is a graph showing the results of Western blot verification of the expression of SSBP1 in the three cell lines MDA-MB-231, MDA-MB-231HM and MDA-MB-231Bo.

We further detected the mRNA expression level of SSBP1 in breast cancer cell lines, and the results showed that the expression level of SSBP1 was significantly lower in basal-like cell lines with strong metastatic ability (BT549, MDA-MB-468, MDA-MB-231) For Luminal cell lines with poor metastatic ability (MCF-7, T47D, ZR-75-30). Accompanying drawing 4 is the result graph showing the expression of SSBP1 in breast cancer cell lines by Real-Time PCR.

2.2 SSBP1 regulates tumor cell migration and invasion in vitro

We selected breast cancer cells MDA-MB-231 and MDA-MB-468, lung cancer cell A549, gastric cancer cell SGC-7901 and ovarian cancer cell A2780 as the cell models for interfering with SSBP1 expression, and MDA-MB-231Bo as the Cell model overexpressing SSBP1. The interference effect of two independent shRNA fragments and the effect of overexpression of SSBP1 were verified by WesternBlot or Real-time PCR. Accompanying drawing 5 is the result diagram of Western blot verification of the effect of interference expression of SSBP1 and overexpression of SSBP1 in breast cancer cells. Accompanying drawing 17 is the real-time PCR verification SSBP1 interference effect diagram in lung cancer cell A549, gastric cancer cell SGC-7901 and ovarian cancer cell A2780.

Then we carried out in vitro Transwell migration and invasion experiments. The results showed that in the MDA-MB-231 and MDA-MB-468 migration and invasion experiments, compared with the shCon group, the migration and invasion abilities of SSBP1-down-regulated cells were significantly enhanced. Conversely, after overexpressing SSBP1 in MDA-MB-231Bo highly metastatic cells, the in vitro metastatic ability of MDA-MB-231Bo was significantly decreased. Figure 6 is the results of Transwell's verification of the impact on invasion and migration after interfering with the expression of SSBP1 in MDA-MB-231, where Figure 6-1 is a representative picture of the experimental results, and Figure 6-2 is the columnar statistics of the experimental results Figure; Figure 7 is the result figure of Transwell verifying the effect on invasion and migration after interfering with the expression of SSBP1 in MDA-MB-468, wherein Figure 7-1 is a representative picture of the experimental results, and Figure 7-2 is a representative picture of the experimental results Histogram; Figure 8 is the result of the Transwell verification of the effect of overexpressing SSBP1 on invasion in MDA-MB-231Bo, where Figure 8-1 is a representative picture of the experimental results, and Figure 8-2 is the experimental results Histogram of . Figure 18 is a graph of the results of Transwell verifying the effect on invasion and migration of interfering with SSBP1 expression in lung cancer cell A549; Figure 19 is a graph of the results of Transwell verifying the effect of interfering with SSBP1 expression in ovarian cancer cell line A2780 on invasion and migration; Fig. 20 is a graph showing the effect of Transwell verification on the invasion and migration of SSBP1 expression interference in gastric cancer cell SGC-7901.

2.3 SSBP1 regulates migration and invasion of breast cancer cells in vivo

In order to further clarify the ability of SSBP1 to regulate breast cancer cell metastasis in vivo, we labeled four stably transfected cells with MDA-MB-231shCon, MDA-MB-231shSSBP1#1, MDA-MB-231Bo Con, and MDA-MB-231Bo SSBP1 After the Luciference fluorescent plasmid was injected into the tail vein of BALB/c nude mice at a concentration of 5×10 ⁵ cells, live imaging of nude mice was performed 6 weeks later. The results showed that the SSBP1 silencing group had significantly more lung metastases than the control group, while the SSBP1 overexpression group had significantly fewer lung metastases than the control group. The results showed that SSBP1 had a significant inhibitory effect on the metastasis of breast cancer cells in vivo. Fig. 13 is a graph showing the results of live imaging of mice to verify the in vivo metastasis effect after interference expression and overexpression of SSBP1.

2.4 Silencing SSBP1 can activate the TGF-β signaling pathway in breast cancer cells

We detected important nodes in the TGF-β/SMAD signaling pathway. Both Real-time PCR and Elisa experiments showed that silencing SSBP1 could promote the transcription and secretion of TGF-β in MDA-MB-231 and MDA-MB-468 cells, while overexpression of SSBP1 could inhibit the secretion of TGF-β in MDA-MB-231Bo cells.

Figure 11 is the mRNA level of TGF-β after Real-time PCR detection of SSBP1 interference and overexpression: among them, Figure 11-1 is the experimental result figure of MDA-MB-231 and MDA-MB-468 cell lines, Figure 11-2 It is the experimental result figure of MDA-MB-231Bo cell line;

Figure 12 is the secretion level of TGF-β after ELISA detection of SSBP1 interference and overexpression: Among them, Figure 12-1 is the experimental result figure of MDA-MB-231 and MDA-MB-468 cell lines, and Figure 12-2 is the figure of MDA-MB-468 Experimental results of MB-231Bo cell line;

At the same time, Western Blot was used to detect the expression of p-SMAD3, SMAD3, SMAD7 and TβR1 and other important node molecules in the TGF-β signaling pathway. Figure 9 is the result of WB detection of molecules related to the TGF-β signaling pathway; After the increase of secreted TGF-β, the phosphorylation level of SMAD3 was significantly increased, while the TGF-β receptor TβR1 and the TβR1 inhibitory molecule SMAD7 were not significantly changed. Overexpression of SSBP1 in MDA-MB-231Bo cells observed opposite changes in related molecules. After treatment with TGF-β signaling pathway inhibitors, the biological effects produced by SSBP1 interference can be significantly inhibited. Figure 10 is a graph showing the results of reversing the effect of SSBP1 on metastasis after treatment with TGF-β signaling pathway inhibitors.

2.5 The relationship between the expression of SSBP1 and the prognosis, tumor stage and lymph node metastasis of breast cancer patients

Next, this conclusion was further verified in clinical samples. According to the expression of SSBP1, 250 samples were divided into high expression group and low expression group. The results of Kaplan-Meier analysis showed that patients with low expression of SSBP1 were more likely to have disease recurrence, and the Log-rank test indicated that P<0.001; the above results suggested that SSBP1 could be used as an independent prognostic factor for breast cancer patients. Spearman correlation analysis showed that the expression of SSBP1 was negatively correlated with lymph node metastasis (P=0.009). The results of univariate survival analysis showed that the expression of SSBP1 (P<0.001), lymph node metastasis status (P<0.001) and tumor histological grade (P=0.038) were all influencing factors of disease-free survival. Cox risk model was used to analyze the survival data. The results showed that the positive lymph node metastasis (HR=2.23, P=0.007) and the low expression of SSBP1 (HR=0.385, P=0.002) were risk factors. Increase.

Figure 14 is the results of detecting SSBP1 in clinical samples and the patient's survival rate; Figure 15 is the results of detecting SSBP1 in clinical samples and the patient's tumor stage; Figure 16 is the result of detecting SSBP1 in clinical samples and the patient's lymph node metastasis status picture.

Those of ordinary skill in the art can understand that the above-mentioned embodiments are specific examples for realizing the present invention, and in practical applications, various changes can be made to it in form and details without departing from the spirit and spirit of the present invention. scope.

Claims (2)

1. Application of the 1.SSBP1 albumen in the drug for inhibiting cancer metastasis is prepared, which is characterized in that the cancer is breast cancer.
2. 2. application according to claim 1, which is characterized in that the drug is the drug for inhibiting TGF-β signal path.