A biomarker for diagnosing and predicting the prognosis of triple-negative breast cancer and its application

Abstract

The present invention relates to the field of biomedical technologies, and particularly to a biomarker for diagnosing and predicting the prognosis of triple-negative breast cancer and its applications. Through bioinformatics analysis of the gene expression of triple-negative breast cancer compared with other types of breast cancer, the present invention screens out the gene FAISL that is highly expressed in triple-negative breast cancer and is closely related to the prognosis, which can be used as a new molecular biomarker for triple-negative breast tumors. Its high expression indicates a poor prognosis of the tumor. This gene can also be used as a therapeutic target for tumors. The present invention uses a disulfide bond reduction-responsive nanomaterial to encapsulate siRNA targeting the pro-cancer gene FAISL. Due to the characteristics of the nanocarrier having a long blood circulation time and strong tumor enrichment ability, the nanoparticles loaded with siRNA can significantly inhibit the expression of the pro-cancer gene, inhibit the growth and metastasis of tumor cells, and reduce the damage to normal tissue cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a biomarker for diagnosis and prognosis of triple-negative breast cancer and its application. Background Art

[0002] Breast cancer is a common malignant tumor that seriously threatens the health of women around the world. Triple-negative breast cancer (TNBC) is the most invasive subtype of breast tumors. It is more likely to metastasize at an early stage than other subtypes of breast cancer. It has the characteristics of strong invasiveness, high recurrence rate and poor prognosis. Due to the lack of hormone receptors and human epidermal growth factor receptors, triple-negative breast cancer is insensitive to traditional endocrine and targeted drug treatments. Although great progress has been made in basic and clinical research on breast cancer in recent years, the clinical prognosis of triple-negative breast cancer is still poor. Local surgical treatment and systemic chemotherapy are still the main treatment methods. However, due to the lack of specific targeting of chemotherapy drugs, while killing triple-negative breast cancer cells, they also cause great damage to normal breast cells, which leads to some triple-negative breast cancer patients being unable to tolerate the side effects of chemotherapy and interrupting treatment. In clinical practice, many patients who have received chemotherapy are also prone to chemotherapy resistance after a period of treatment. Therefore, it is of great clinical significance to find a treatment strategy that can effectively target triple-negative breast cancer.

[0003] In recent years, antibody-drug conjugates (ADCs) have made significant progress in the field of tumor treatment, especially in the treatment of breast cancer, where ADCs have become an important treatment. ADCs form an innovative anti-tumor therapy by combining highly targeted antibodies with effective cytotoxic drugs. The design of this drug includes three main parts: the targeting antibody, the linker that connects the antibody to the drug, and the conjugated cytotoxic drug. Although ADCs have shown great potential in the treatment of breast cancer, their stability remains a challenge. In the blood circulation, ADCs may be unstable and decompose, which affects the efficacy of the drug. For example, T-DM1 (Trastuzumabemtansine) is an ADC drug for HER2-positive breast cancer, but it did not show significant efficacy advantages compared with Trastuzumab (Herceptin) alone in clinical studies. This may be due to the instability of T-DM1 in the blood circulation, which causes it to decompose into Trastuzumab and drug parts.

[0004] In addition, there are other factors that may limit the therapeutic efficacy of ADCs, including antibody-mediated drug resistance, impairment of drug delivery mechanisms, lysosomal dysfunction, and drug payload-related drug resistance. To overcome these challenges, researchers are exploring new design strategies and improved linker technologies to enhance the stability and efficacy of ADCs. Through these efforts, ADCs are expected to play a more crucial role in future breast cancer treatment.

[0005] In recent years, RNA interference (RNAi) technology has attracted significant attention due to its great potential in treating various diseases. Compared with small molecule drugs, siRNA exhibits higher selectivity, capable of precisely targeting and downregulating the expression of specific genes without interfering with the functions of other normal genes. However, the main challenge in the clinical application of RNAi technology is how to safely and efficiently deliver siRNA to the diseased site and into cells. For RNAi-based cancer treatment strategies, siRNA needs to overcome multiple physiological barriers during delivery, including precisely targeting tumor cells, penetrating tumor tissues and cell membranes, effectively escaping endosomes, and releasing siRNA in the cytoplasm. Although the use of viral vectors such as adenoviruses and retroviruses can achieve effective delivery of siRNA, these methods have drawbacks such as complex preparation, limited siRNA loading capacity, insufficient targeting, and high immunogenicity.

[0006] As mentioned above, chemotherapy drugs for triple-negative breast cancer lack targeting, and while killing tumor cells, they also damage normal cells, causing some patients to be unable to tolerate the side effects of chemotherapy and interrupt treatment. In addition, many patients develop drug resistance after receiving chemotherapy for some time. Although molecular targeted drugs are specific, they also have toxic side effects and may lead to the occurrence of drug resistance after long-term use. Although antibody-drug conjugates have improved the targeting of chemotherapy drugs, their instability in the blood circulation may result in unsatisfactory therapeutic effects.

[0007] Despite the great therapeutic promise of RNAi technology, its development is limited by the lack of effective delivery systems. Nanomedicine technology can significantly reduce toxic side effects while improving the therapeutic efficacy of cancer treatment, thus improving the quality of life of patients. However, currently used nanomedicines in clinical practice still have problems such as insufficient control of drug release and single treatment modalities. Future research needs to focus on developing novel nanocarriers to achieve efficient and targeted delivery of siRNA, thereby overcoming existing obstacles and promoting the application of RNAi technology in cancer treatment. Summary of the Invention

[0008] The object of the present invention is to overcome the deficiencies of the prior art and provide a biomarker FAISL, which can be used as a new molecular biomarker for the diagnosis and prognosis judgment of triple-negative breast cancer. Its high expression indicates a poor prognosis of the tumor. Nanoparticles loaded with siRNA targeting FAISL can significantly inhibit the expression of oncogenes.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides the use of a reagent for detecting the expression level of LncRNA FAISL in the preparation of a product for the diagnosis of triple-negative breast cancer metastasis or prognosis evaluation, and the nucleotide sequence of the LncRNA FAISL is as shown in SEQ ID NO.1.

[0011] Through bioinformatics analysis of the gene expression of triple-negative breast cancer compared with other types of breast cancer, the present invention screened out a long non-coding RNA - FAISL, whose original gene name is VPS9D1-AS1, located on chromosome 16 of the genome, with a full length of 1753 nt, consisting of 4 exons, and containing a poly A tail. The bioinformatics analysis results show that the expression of LncRNA FAISL in triple-negative breast cancer cell lines and triple-negative breast cancer tissues is higher than that in non-triple-negative breast cancer cell lines and normal breast cancer tissues. In various tumor tissues, the high expression of the long non-coding RNA FAISL is related to the tumor prognosis.

[0012] Preferably, the product includes a gene chip, a kit or a test strip.

[0013] In a second aspect, the present invention provides the use of a reagent for inhibiting the expression level of LncRNA FAISL in the preparation of a drug for treating triple-negative breast cancer.

[0014] The present invention first discovers that LncRNA FAISL is involved in the malignant progression of triple-negative breast cancer and is related to the survival prognosis of breast cancer. LncRNA FAISL affects the progression of triple-negative breast cancer by affecting cell adhesion to the extracellular matrix, anchorage-dependent proliferation, and anti-anoikis ability. By effectively silencing the target gene, the purpose of treating tumors can be achieved.

[0015] Preferably, the reagent includes a nucleic acid molecule, a small molecule compound or an interfering lentivirus.

[0016] Preferably, the reagent includes siRNA; the nucleotide sequence of the siRNA is as shown in SEQ ID NO.2 or SEQ ID NO.3.

[0017] In a third aspect, the present invention provides a nanoparticle targeting LncRNA FAISL, which encapsulates siRNA; the nucleotide sequence of the siRNA is as shown in SEQ ID NO.2 or SEQ ID NO.3; the nanoparticle further includes a cationic lipid compound and a GSH-responsive polymer material.

[0018] For the gene LncRNA FAISL highly expressed in triple-negative breast cancer, the present invention prepares a novel nanocarrier with a simple synthesis method, easy transformation, and reduction response for efficient delivery of siRNA; by utilizing the property that the nanomaterial itself can consume excessive GSH in tumor cells, the nanomaterial can effectively release siRNA at the tumor site, silence the target gene LncRNA FAISL, and significantly improve the tumor treatment effect. When the final concentration of the siRNA of the present invention is 30 nM and 50 nM, the prepared nanoparticles can knockdown LncRNA FAISL to below 50% and 20% respectively.

[0019] Preferably, the preparation method of the nanoparticle includes the following steps:

[0020] (1) Mix the cationic lipid compound with siRNA, and then add the GSH-responsive polymer material for mixing to obtain a mixed solution;

[0021] (2) Drop the mixed solution obtained in step (1) into deionized water and stir to obtain a suspension;

[0022] (3) Ultrafilter, centrifuge, and wash the suspension obtained in step (2) to obtain the nanoparticle targeting LncRNA FAISL.

[0023] Since the siRNA molecule is negatively charged, the positively charged cationic lipid G0-C14 can form a complex G0-C14 / siRNA with siRNA through electrostatic interaction. Subsequently, the GSH-responsive polymer material Meo-PEG-S-S-PLGA (methoxy-poly(ethylene glycol)-disulfide-poly(lactic acid-glycolic acid) copolymer) can encapsulate the complex G0-C14 / siRNA by the nanoprecipitation method (hydrophobic-hydrophilic interaction) to form nanoparticles.

[0024] Preferably, the cationic lipid compound is G0-C14; the GSH-responsive polymer material is Meo-PEG-S-S-PLGA.

[0025] In a fourth aspect, the present invention provides the application of the above-mentioned nanoparticle targeting LncRNA FAISL in the preparation of a drug for treating triple-negative breast cancer.

[0026] Fifth aspect, the present invention provides a drug for treating triple-negative breast cancer, and the drug comprises the above-mentioned nanoparticles targeting LncRNA FAISL.

[0027] The beneficial effects of the present invention are as follows:

[0028] Through bioinformatics analysis of gene expression in triple-negative breast cancer compared with other types of breast cancer, the gene FAISL, which is highly expressed in triple-negative breast cancer and closely related to prognosis, is screened out. It can be used as a new molecular marker for breast tumors, and its high expression indicates poor prognosis of tumors. This gene can also be used as a therapeutic target for tumors. In the present invention, siRNA targeting the oncogene FAISL is encapsulated by a disulfide bond reduction-responsive nanomaterial. Due to the characteristics of long blood circulation time and strong tumor enrichment ability of the nanocarrier, the nanoparticles loaded with siRNA can significantly inhibit the expression of the oncogene, inhibit the growth and metastasis of tumor cells, and reduce the damage to normal tissue cells. Description of the Drawings

[0029] Figure 1 It is a result diagram for database analysis of the expression differences of FAISL in different subtypes of breast cancer cells or tissues; A is the differential expression of LncRNA FAISL in non-triple-negative breast cancer cell lines (non-TNBC, n = 25) and triple-negative breast cancer cell lines (TNBC, n = 24) analyzed by the CCLE database; B is the differential expression of LncRNA FAISL in normal breast tissues (normal, n = 105), non-triple-negative breast cancer tissues (non-TNBC, n = 644), and triple-negative breast cancer tissues (TNBC, n = 84) analyzed by the TCGA database; where, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0030] Figure 2 It is a diagram for database analysis of the relationship between FAISL and the prognosis of breast cancer; A is the correlation between the high expression of FAISL and the prognosis of all subtypes of breast cancer (all cohorts) analyzed by the KM-Plotter database, HR = 3.09 (1.81 - 5.27), log-rank P = 1.3e-05; B is the correlation between the high expression of FAISL and the prognosis of the triple-negative breast cancer subtype (TNBC subtype) analyzed by the KM-Plotter database, HR = 3.77 (1.12 - 12.71), log-rank P = 0.022; HR represents the hazard ratio.

[0031] Figure 3 It is a diagram for TCGA database analysis of the relationship between the expression of FAISL in tumor tissues and the prognosis of various tumors.

[0032] Figure 4Synthesis roadmap of nanoparticles loaded with siRNA.

[0033] Figure 5 Chemical structure of amphiphilic cationic lipid G0-C14.

[0034] Figure 6 Chemical structure of Meo-PEG-S-S-PLGA.

[0035] Figure 7 Size distribution diagram of nanoparticles.

[0036] Figure 8 Zeta potential analysis of nanoparticles.

[0037] Figure 9 Pharmacokinetic analysis of nanoparticles in nude mice.

[0038] Figure 10 Particle size change of nanoparticles loaded with siRNA in aqueous solutions of different concentrations of GSH.

[0039] Figure 11 Release curve of nanoparticles loaded with siRNA in aqueous solutions of different concentrations of GSH.

[0040] Figure 12 Comparison diagram of the target gene knockdown efficiency of reduction-responsive nanoparticles at different siRNA concentrations.

[0041] Figure 13 Comparison diagram of the in vitro silencing efficiency of traditional transfection (Nakid-siRNA) and reduction-responsive nanoparticles (NPs-siRNA).

[0042] Figure 14 Result diagram of the reduction-responsive nanoparticles knocking down FAISL to inhibit cell proliferation.

[0043] Figure 15 Result diagram of the reduction-responsive nanoparticles knocking down FAISL to inhibit the adhesion of cells to the extracellular matrix.

[0044] Figure 16 Result diagram of the effect of the reduction-responsive nanoparticles knocking down FAISL on anoikis apoptosis of cells at a siRNA concentration of 50 nM.

[0045] Figure 17 Fluorescence signal enrichment diagram of nanoparticles in tumors and other organs after NPs loaded with fluorescent cy5-labeled siFAISL were injected into mice via the tail vein.

[0046] Figure 18 Growth curve diagram of tumors and tumor weight analysis diagram of each treatment group.

[0047] Figure 19 These are the pathological tissue staining result diagrams of the hearts, livers, spleens, lungs, and kidneys of nude mice in each treatment group.

[0048] Figure 20 These are the HE staining and metastasis ratio statistics of in-situ tumor lung metastasis in each treatment group.

[0049] Figure 21 These are the survival curves of mice in each treatment group. Detailed implementation manners

[0050] To better illustrate the objectives, technical solutions, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0051] Example 1:

[0052] The present invention utilizes existing databases: the CCLE (Cancer Cell Line Encyclopedia) database and the TCGA database. Through bioinformatics analysis of the gene expression of triple-negative breast cancer compared with other types of breast cancer, the long non-coding RNA - FAISL was screened out.

[0053] Research has found that the long non-coding RNA FAISL is expressed higher in triple-negative breast cancer cell lines and triple-negative breast cancer tissues than in non-triple-negative breast cancer cell lines and normal breast cancer tissues ( Figure 1 ). In various tumor tissues, the high expression of the long non-coding RNA FAISL is related to tumor prognosis ( Figure 2 , Figure 3 ).

[0054] In previous studies, the long non-coding RNA FAISL was mainly related to the proliferation of colorectal cancer cells. The results of the present invention first found that this gene is related to the promotion of cancer and prognosis of triple-negative breast cancer.

[0055] Example 2: Preparation of a nanomaterial loaded with siRNA and its characterization

[0056] For the gene LncRNA FAISL highly expressed in triple-negative breast cancer, the present invention prepared a novel nanocarrier with a simple synthesis method, easy to transform, and reduction-responsive for efficient delivery of siRNA; by utilizing the property that the nanomaterial itself can consume the excessive GSH in tumor cells, the nanomaterial can effectively release siRNA at the tumor site, silence the target gene LncRNA FAISL, and significantly improve the tumor treatment effect.

[0057] 1. The present invention designed two siRNAs targeting FAISL, namely siFAISL#1 and siFAISL#2, and the sequences of the siRNAs are shown in Table 1.

[0058] Table 1 Target sequences of siRNA

[0059] siRNA Sequence (5'-3') siFAISL#1 5’-CTTGGCATGGAGCACCTCT-3’ siFAISL#2 5’-GGGATCGCCTCACCCATCT-3’

[0060] 2. Preparation of reduction-reactive siRNA nanoparticles

[0061] The NPs (Nanoparticles) loaded with siRNA described in the present invention are prepared by the nanoprecipitation method, and the synthetic roadmap is as Figure 4 shown.

[0062] The specific preparation process is as follows: Mix 50 μL of G0-C14 (concentration 5 mg / mL, dissolved in dimethylformamide; the chemical structure of G0-C14 is shown in Figure 5 ) with 10 μL of siFAISL (concentration 0.1 nmol / μL, dissolved in water), and then add 200 μL of the polymer Meo-PEG-S-S-PLGA (methoxy-poly(ethylene glycol)-disulfide-poly(lactic acid-glycolic acid) copolymer), concentration 20 mg / mL, dissolved in dimethylformamide; its chemical structure diagram is shown in Figure 6 ).

[0063] Subsequently, the mixed solution was dropped into 5 mL of deionized water, and at the same time, it was vigorously stirred at a speed of 1000 rpm to obtain an NPs suspension; the formed NPs suspension was then purified by an ultrafiltration device (EMD Millipore, molecular weight cut-off 100K) and centrifuged to remove the organic solvent (dimethylformamide); after washing twice, NPs (siRNA) loaded with siRNA were obtained, and deionized water was added according to the final concentration of siRNA being 1 nmol / mL for later use.

[0064] 3. Characterization experiments of nanoparticles

[0065] Use a dynamic light scattering (Malvern Zetasizer Nano ZS90) instrument to detect the particle size and zeta potential of the NPs nanoparticles loaded with siRNA.

[0066] The results show that the average particle size of the NPs nanoparticles loaded with siRNA is about 120 nm ( Figure 7 ), and the zeta potential is about -0.128 mW ( Figure 8 ).

[0067] Example 3: In vivo pharmacokinetics of nanoparticles

[0068] Prepare reduced-reactivity siRNA nanoparticles according to the preparation method of Example 2; wherein, the siRNA encapsulated in the nanoparticles is siCtrl, a scrambled siRNA sequence, and the prepared nanoparticles are called siRNA-loaded NPs. Cy5 label the siRNA. Additionally, set a control group: Cy5-labeled siRNA that is not encapsulated by nanoparticles, simply referred to as nakid-siRNA.

[0069] Intravenously inject 1 nmol of each of the two siRNA drugs into female BALB / C Nude SPF-grade nude mice (5 weeks old, purchased from Vital River Laboratory Animal Technology Co., Ltd., Zhejiang) respectively. At different time points after injection (5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours), extract Cy5-siRNA from the orbital blood of the nude mice, and measure the fluorescence intensity of the Cy5-labeled siRNA with an enzyme-linked immunosorbent assay (ELISA) reader (BioTek, USA). Calculate the content of Cy5-siRNA in the mouse blood according to the standard curve. The results are as Figure 9 shown.

[0070] The results show that nakid-siRNA and siRNA-loaded NPs have different half-lives in the mouse blood circulation. The half-life of siRNA-loaded NPs is longer than that of nakid-siRNA, and nakid-siRNA is more easily degraded than siRNA-loaded NPs.

[0071] Example 4: Particle size change during nanoparticle release

[0072] Dissolve the siRNA-loaded NPs and GSH (glutathione) prepared in Example 3 in PBS buffer (pH 7.4) such that the concentration of siRNA (siCtrl) encapsulated in the siRNA-loaded NPs is 1 nmol, and the concentrations of GSH are 1 mM and 10 mM respectively. Use dynamic light scattering to detect the particle size of the nanoparticles at different time periods. The results are as Figure 10 shown.

[0073] The results show that the nanoparticles encapsulating siRNA prepared in the present invention exhibit good reduction responsiveness. After adding GSH, as the concentration of GSH increases, the particle size of the nanoparticles increases, the nanostructure is destroyed, and the siRNA in the nanoparticles is rapidly released.

[0074] Example 5: Determination of nanoparticle release rate

[0075] The NPs (NPs(siFAISL#1)) loaded with siFAISL#1 were labeled with the fluorescent dye cy5. After multiple washings, they were dispersed in 1 mL of PBS solution (pH 7.4) to obtain an NPs suspension, which was transferred to a Float-a-lyzer G2 dialysis device (MWCO 100 kDa, Spectrum). The device was immersed in PBS buffer (pH 7.4, containing 1 mM and 10 mM GSH respectively) at a temperature of 37 °C. Every once in a while, 5 μL of the NPs suspension was taken out and mixed with 100 μL of dimethyl sulfoxide (DMSO). The fluorescence intensity of cy5-labeled siFAISL was measured using a multimode microplate reader (Bio TEK, USA) (the stronger the fluorescence intensity, the more the release amount). The cumulative release rate of siRNA was calculated according to formula (1), and the results are as Figure 11 shown.

[0076] Cumulative release amount (%) = (M t / M - ) × 100% —— Formula (1)

[0077] In the above formula, M t is the fluorescence intensity of siRNA released from the nanoparticles at the specified time point, and M - is the fluorescence intensity of the total amount of siRNA loaded in the nanoparticles.

[0078] The results showed that as the concentration of GSH increased, the release rate of siRNA from the nanoparticles increased.

[0079] Example 6: In vitro silencing efficiency of nanoparticles loaded with different concentrations of siRNA on the target gene FAISL

[0080] Breast cancer MDA-MB-231 cells were seeded in a 6-well plate (about 10,000 cells per well), and 2 mL of DMEM high-glucose complete medium containing 10% fetal bovine serum was added and incubated for 12 hours to allow them to adhere well. Subsequently, nanoparticles loaded with siRNA (NPs(siFAISL#1) and NPs(siFAISL#2)) with different final concentrations (0 nM, 10 nM, 30 nM, 50 nM respectively) were added. At the same time, a control group NPs(siCtrl) was set, and siCtrl was a scrambled siRNA sequence, used as a negative control for siRNA knockdown. After incubation for 24 hours, the culture medium was replaced with fresh DMEM high-glucose complete medium containing 10% fetal bovine serum and incubated for another 24 hours. After extracting RNA, RT-qPCR was performed to detect the expression changes of FAISL, and the results are as Figure 12 shown.

[0081] The results showed that when the final concentration of siRNA in the nanoparticles loaded with siRNA was 30 nM and 50 nM, the LncRNA FAISL could be knocked down to below 50% and 20% respectively.

[0082] Example 7: Comparative analysis of the in vitro silencing efficiency of traditional liposome-transfected siRNA and nanoparticles loaded with siRNA on target genes

[0083] The in vitro silencing experiment of the nanoparticles loaded with siRNA on the target gene was the same as that in Example 6, and the final concentration of siRNA was 30 nM.

[0084] The experimental procedure for the in vitro silencing of the target gene by traditional liposome-transfected siRNA was as follows:

[0085] 1. Prepare the transfection system in a 12-well plate (Table 2). Mix 5 μL of siRNA and 5 μL of the transfection reagent si-mate with 125 μL of OPTI-MEM serum-free medium in a 2 mL centrifuge tube and incubate for 25 minutes.

[0086] 2. Add the transfection system to the 12-well plate at 250 μL of the transfection system / well, and add 750 μL of fresh DMEM + 10% FBS complete medium. After incubation for 6 - 8 hours, replace it with fresh DMEM + 10% FBS complete medium.

[0087] After 48 - 72 hours of transfection, extract the RNA of both transfection methods simultaneously, synthesize cDNA using the AG one-step reverse transcription kit, and detect the knockdown efficiency of the target gene FAISL by RT-qPCR. The results are as Figure 13 shown.

[0088] Table 2 Traditional siRNA transfection reaction system

[0089] Reagent Volume siFAISL 5μL si-mate 5μL OPTI-MEM 250μL

[0090] The results showed that the efficiency of knocking down the FAISL gene by the transfection of nanoparticles loaded with siRNA and the traditional liposome transfection of siRNA was comparable, proving the feasibility of nanoparticles loaded with siRNA in in vitro knockdown experiments.

[0091] Example 8: Verification of the gene function of long non-coding RNA FAISL in triple-negative breast cancer

[0092] Transfect the siRNA-loaded nanoparticles into triple-negative breast cancer cells MDA-MB-231 according to the method of Example 6. Divide into four groups for transfection, namely:

[0093] A: Mock group, perform the transfection process without adding siRNA;

[0094] Group B: control group, siRNA was siCtrl (NPs(siCtrl)), and the concentration was 50 nM;

[0095] Group C: transfected with FAISL siRNA 1 group, siRNA was siFAISL#1 (NPs(siFAISL#1)), and the concentration was 50 nM;

[0096] Group D: transfected with FAISL siRNA 2 group, siRNA was siFAISL#1 (NPs(siFAISL#2)), and the concentration was 50 nM.

[0097] The following experiments were conducted 48 hours after transfection:

[0098] 1. Cell proliferation assay: 1000 transfected cells were seeded into a 96-well cell culture plate, and 3 replicate wells were set up for each group. The cell viability was detected using the Cell Titer-Glo kit according to the instructions at 24, 48, and 72 hours after plating. The results are as Figure 14 shown.

[0099] 2. Adhesion assay:

[0100] The experimental steps are as follows:

[0101] (1) Coating of extracellular matrix: Fibronectin (integrin) was added to a 96-well culture plate at 50 μL (2 μg) / well and placed in a refrigerator at 4 °C overnight for later use;

[0102] (2) Rehydrate by washing twice with PBS before use and set aside;

[0103] (3) Prepare the cells to be tested into a single-cell suspension with a concentration of 8×10 4 cells / mL, and add 200 μL / well to the 96-well plate coated with the matrix Fibronectin, and incubate at 37 °C and 5% CO 2 for 30 - 60 minutes;

[0104] (4) Gently aspirate the culture medium, add PBS, and gently wash away the non-adherent cells, repeating the washing 3 times;

[0105] (5) Add 100 μL of Cell Titer-Glo reagent to each well and let it stand for lysis for 10 minutes;

[0106] (6) Aspirate the supernatant, transfer it to a white microplate, measure the luminescence value of each well with a microplate reader, and calculate the adhesion rate according to formula (2). The results are as Figure 15 shown.

[0107] Adhesion rate (%) = (OD value of adherent cells / OD value of inoculated cells) * 100% —— Equation (2)

[0108] 3. Anoikis induction and flow cytometry detection:

[0109] The specific experimental steps are as follows:

[0110] Digest the cells after the above experimental treatment, collect them by centrifugation, and refer to the method of anoikis induction in the literature. Inoculate the cells in a low-adhesion coated culture dish. After culturing for 24 hours, collect the cells and perform the following experimental operations according to the instructions of the apoptosis detection kit:

[0111] (1) After washing the cells twice with PBS, resuspend 1×10 5 / mL cells with 1*Binding Buffer;

[0112] (2) Pipette 500 μL into a 5 mL flow cytometry special centrifuge tube, and at the same time add 3 μL of FITC-Annexin V (a dye that can bind to annexin) and 6 μL of PI dye, and set the following controls:

[0113] A: Double-negative group, without adding FITC and PI dyes;

[0114] B: FITC single-positive group, adding FITC dye but no PI cells;

[0115] C: PI single-positive group, only adding PI but no FITC; Incubate in the dark for 10 - 20 minutes;

[0116] (3) Perform on-machine detection, and the results are as Figure 16 shown.

[0117] The results of cell proliferation detection showed ( Figure 14 ), compared with the Mock group and the control group (NPs(siCtrl)), the cell proliferation ability of the transfection FAISL siRNA 1 group (NPs(siFAISL#1)) and the transfection FAISL siRNA 2 group (NPs(siFAISL#2)) was significantly reduced at the transfection concentration of 50 nM; comparing the NPs(siFAISL#1) and NPs(siFAISL#2) groups with the NPs(siCtrl) group respectively, the differences were statistically significant (P < 0.05), proving that after silencing the expression of the FAISL gene, the anchorage-dependent proliferation ability of triple-negative breast cancer cells MDA-MB-231 was inhibited.

[0118] The results of the adhesion experiment showed ( Figure 15), compared with the Mock group and the control group (NPs(siCtrl)), the cell adhesion ability of the FAISL siRNA1-transfected group (NPs(siFAISL#1)) and the FAISL siRNA 2-transfected group (NPs(siFAISL#2)) was significantly reduced at a transfection concentration of 50 nM; when comparing the NPs(siFAISL#1) and NPs(siFAISL#2) groups with the NPs(siCtrl) group respectively, the differences were all statistically significant (P < 0.05), indicating that after silencing the expression of the FAISL gene, the adhesion ability of triple-negative breast cancer cells MDA-MB-231 to the extracellular matrix was inhibited.

[0119] Results of anoikis induction and flow cytometry analysis showed ( Figure 16 ), compared with the Mock group and the control group (NPs(siCtrl)), the proportion of anoikis of the FAISL siRNA1-transfected group (NPs(siFAISL#1)) and the FAISL siRNA 2-transfected group (NPs(siFAISL#2)) was significantly increased at a transfection concentration of 50 nM; when comparing the NPs(siFAISL#1) and NPs(siFAISL#2) groups with the NPs(siCtrl) group respectively, the differences were all statistically significant (P < 0.05), indicating that after silencing the expression of the FAISL gene, the anti-anoikis ability of triple-negative breast cancer cells MDA-MB-231 was inhibited.

[0120] Example 9: Evaluation of the tumor enrichment ability of nanoparticles

[0121] Tumor-bearing mice with a tumor volume of about 300 mm 3 were selected, and 1 nmol of two siRNA drugs (the same as in Example 3) were injected into the nude mice via the tail vein. Subsequently, 24 hours after the tail vein injection of the siRNA drug, the mice were euthanized and dissected to isolate muscle, heart, liver, spleen, lung, kidney and tumor, and the fluorescence intensity of Cy5-siRNA in each organ tissue and tumor was detected by a small animal in vivo imaging analyzer (Xenogen IVIS Luminasystem), and the results are as Figure 17 shown.

[0122] The results showed that nakid-siRNA and siRNA-loaded NPS were both enriched to a certain extent in the liver, lung and kidney of nude mice, and the enrichment degrees were comparable; however, in breast cancer xenografts, the enrichment level of siRNA-loaded NPS was higher than that of nakid-siRNA.

[0123] Example 10: Experiment to explore the effect of nanoparticles on inhibiting tumor growth in vivo

[0124] The BALB / C Nude SPF female nude mice used in the present invention were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., with an age of 3-5 weeks and a weight of 14-16 g when purchased; after purchase, they were raised in the barrier environment of the Experimental Animal Center of Sun Yat-sen University (University Town), and the experimental animal use qualification certificate number is (East) 2017-42-25. The technical indicators of the experimental environment and animal management strictly comply with the requirements of GB 14925-2010.

[0125] After nude mice were purchased, they were cultured for one week to allow them to adapt to the feeding environment. Then, MDA-MB-321 cells in the logarithmic growth phase were collected, resuspended in serum-free DMEM medium, and mixed with matrix gel at a ratio of 1:1 on ice to form a uniform mixture, so that the cell concentration in the mixture was 1×10 7 Tumor cells were inoculated subcutaneously on the back of nude mice at a volume of 100 μL per mouse.

[0126] When the nude mouse tumors could be clearly palpated, the tumor-bearing nude mice were randomly divided into 4 groups: i) PBS, ii) NPs NPs (siCtrl), iii) NPs (siFAISL#1), iv) NPs (siFAISL#2); 8 nude mice (n=8) were injected into the tail vein every other day, for a total of 3 rounds of treatment. After the start of treatment, the growth of the tumor was measured every two days using a vernier caliper, and the tumor volume was calculated according to formula (3), and the weight of the nude mice was monitored using an electronic balance.

[0127] Tumor volume = long diameter × short diameter × short diameter / 2 - Formula (3)

[0128] The experimental endpoints and humane endpoints of nude mice are determined according to the tumor growth in nude mice and the physical condition of the mice. When nude mice have difficulty eating, severe weight loss, and the maximum diameter of the tumor exceeds 2cm, the nude mice are humanely killed, the tumor tissue is separated and photographed, and the tumor weight is measured. Half of it is then frozen at -80°C for subsequent RNA and protein extraction, and the other half is fixed with 4% paraformaldehyde, embedded in paraffin, and sliced ​​for immunohistochemical analysis. Finally, the nude mouse corpses are collected and disposed of harmlessly by the Animal Experiment Center of the East Campus of Sun Yat-sen University.

[0129] The results showed that after three consecutive tail vein nanoparticle injections, the growth curve showed that the nanoparticles loaded with siFAISL could significantly inhibit tumor growth. Compared with nude mice injected with PBS or siCtrl, the size and weight of nude mice tumors were inhibited by more than half after injection of siFAISL-loaded nanoparticles, which proved that FAISL could increase the malignant progression of triple-negative breast cancer ( Figure 18 ).

[0130] In addition, organ damage is also one of the toxic reactions of drugs. In the present invention, the organs (muscles, heart, liver, spleen, lungs and kidneys) of nude mice at the end of treatment were collected for HE staining to observe the tissue morphology, and it was found that the siRNA nanodrug targeting FAISL had no obvious damage to important organs ( Figure 19 ).

[0131] The above results prove that siRNA nanodrug therapy targeting FAISL is relatively safe for nude mice.

[0132] Example 11: Experimental study on the inhibition of in situ lung metastasis of triple-negative breast cancer by nanoparticles in vivo

[0133] After purchasing NOD / SCID immunodeficient mice, they were cultured for one week to allow the mice to adapt to the feeding environment. Then, the MDA-MB-321 cells in the logarithmic growth phase were collected, resuspended in serum-free DMEM medium, and mixed with matrix gel at a ratio of 1:1 on ice to form a uniform mixture, so that the cell concentration in the mixture was 1×10 7 Tumor cells were inoculated into the mammary fat pad of nude mice at a volume of 100 μL per mouse to establish a mammary orthotopic lung metastasis treatment model.

[0134] When the nude mouse tumors could be clearly palpated, the tumor-bearing nude mice were randomly divided into 4 groups: i) PBS, ii) NPs NPs (siCtrl), iii) NPs (siFAISL#1), iv) NPs (siFAISL#2); 10 nude mice (n=10) were injected into the tail vein every other day for a total of 5 rounds of treatment. After the start of treatment, the lung metastasis of the mice was observed weekly using a small animal live imaging device, and the weight of the nude mice was monitored using an electronic balance.

[0135] The experimental endpoints and humane endpoints of nude mice were determined based on the tumor growth in nude mice and the physical condition of the mice. When nude mice had difficulty eating, severe weight loss, and the maximum diameter of the tumor exceeded 2 cm, the nude mice were humanely killed, and the lung tissue was separated and photographed. After being fixed with 4% paraformaldehyde and embedded in paraffin, the sections were sliced ​​and analyzed by HE immunohistochemical staining. Finally, the nude mouse corpses were collected and disposed of harmlessly by the Nanhai Experimental Animal Experiment Center of Sun Yat-sen Memorial Hospital of Sun Yat-sen University.

[0136] The final results showed that after 5 consecutive tail vein nanoparticle injections, HE staining of nude mouse lung tissue showed that compared with nude mice injected with PBS or siCtrl, the incidence and pulmonary metastasis of mice injected with siFAISL nanoparticles were significantly inhibited ( Figure 20 ), and the nanoparticle treatment targeting siFAISL significantly increased the overall survival rate of nude mice ( Figure 21), which proves that the nanoparticles loaded with siFAISL have a certain inhibitory effect on the lung metastasis of triple-negative breast cancer in nude mice.

[0137] In summary, the present invention first discovered that LncRNA FAISL is involved in the malignant progression of triple-negative breast cancer and is related to the survival prognosis of breast cancer. LncRNA FAISL affects the progression of triple-negative breast cancer by influencing cell adhesion to the extracellular matrix, anchorage-dependent proliferation, and anti-anoikis ability.

[0138] Based on this discovery, the present invention designed a new generation of reduction-responsive nanoplatform, which can be synthesized by an easy-to-operate polymer synthesis technology, can efficiently deliver siRNA into tumor cells, and reducing substances such as reduced glutathione (GSH) in the tumor cytoplasm can shear the hydrophobic groups of the nanoparticles to release the siRNA drug, achieving the purpose of treating tumors by effectively silencing the target gene.

[0139] The nanoparticles loaded with siRNA prepared by the present invention have the following characteristics: 1. Longer circulation time in the blood; 2. High enrichment in tumors; 3. Can efficiently silence pro-cancer target genes; 4. Can increase the killing of tumor cells. In addition, loading siFAISL into the nanoplatform shows good tumor-killing effects both in vitro and in vivo and has no obvious damage to important organs such as the heart and liver.

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of nanoparticles targeting LncRNA FAISL in the preparation of a drug for treating triple-negative breast cancer, characterized in that: The nanoparticles are loaded with siRNA; the nucleotide sequence of the siRNA is shown in SEQ ID NO.2 or SEQ ID NO.3; the nanoparticles also include cationic lipid compounds and GSH-responsive polymer materials; the GSH-responsive polymer material is a methoxy-poly(ethylene glycol)-disulfide bond-poly(lactic acid-glycolic acid) copolymer.

2. The use according to claim 1, characterized in that The method for preparing the nanoparticles comprises the following steps: (1) mixing a cationic lipid compound with siRNA, and then adding methoxy-poly(ethylene glycol)-disulfide bond-poly(lactic acid-glycolic acid) copolymer to obtain a mixed solution; (2) adding the mixed solution obtained in step (1) dropwise into deionized water and stirring to obtain a suspension; (3) The suspension obtained in step (2) is ultrafiltered, centrifuged, and washed to obtain the nanoparticles targeting LncRNA FAISL.

3. The use according to claim 1, characterized in that The cationic lipid compound is G0-C14.

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