Application of FN1 gene inhibitor in preparation of product for improving far-end effect induced by tumor cell radiotherapy

Abstract

The invention relates to the technical field of biology, and particularly discloses application of an FN1 gene inhibitor in preparation of a product for improving the far-end effect induced by tumor cell radiotherapy. The research finds that when the far-end effect occurs, the FN1 gene expression in the macrophage is obviously reduced, and the growth of far-end tumors can be inhibited. The invention further provides application of the FN1 gene inhibitor in preparation of a product for improving the far-end effect induced by tumor cell radiotherapy. According to the invention, a new way is provided for converting a randomly occurring far-end effect in tumor radiotherapy into a deterministic effect, so that the radiotherapy effect of a breast cancer patient can be improved, and the radiotherapy mode of the breast cancer is changed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, to the application of FN1 gene inhibitor in the preparation of products for improving radiotherapy-induced abscopal effect of tumor cells. BACKGROUND

[0002] Breast cancer is a malignant tumor that seriously threatens women's health, with a large number of patients and high mortality. The biggest problem in the treatment of breast cancer is easy recurrence and metastasis. Lack of effective control of metastatic lesions is one of the main reasons for the death of patients with advanced cancer, including breast cancer. Radiotherapy is one of the effective means for the treatment of breast cancer, but it can only kill the tumor in the irradiation target area, and has no therapeutic effect on the metastatic lesions of breast cancer. Researchers have found that radiation can induce the occurrence of abscopal effect in clinical and experimental studies. Abscopal effect refers to the phenomenon that unirradiated tumors or normal tissues respond to radiation during radiotherapy. The discovery of abscopal effect may change radiotherapy from a local treatment method to a systemic treatment method. However, the biggest problem in current clinical application is the randomness of the occurrence of abscopal effect. Existing studies have shown that the occurrence of abscopal effect is closely related to the tumor microenvironment. Only when the unirradiated tumor is in a pro-immune microenvironment, the development of the tumor can be inhibited. Therefore, remodeling the microenvironment of the distant tumor is the determining factor for changing the randomness of the abscopal effect to a deterministic effect during radiotherapy.

[0003] FN1 is a multifunctional extracellular matrix protein and a high molecular weight glycoprotein. It can bind to various components on the cell surface and in the extracellular matrix, including collagen, fibrin, heparin, DNA and actin, to exert its functions. FN1 plays an important role in cell adhesion, growth, migration and differentiation. Initially, researchers believed that FN1 was a cancer gene, and high expression was related to the progression of non-small cell lung cancer, glioblastoma and ovarian cancer. However, recent studies have found that FN1 may be closely related to immune function, especially macrophage function. Bioinformatics analysis revealed that high expression of FN1 in diabetic nephropathy and gastric cancer was positively correlated with M2 polarization of macrophages. Experimental studies have found that FN1 promotes IL-10 secretion and reduces TNF-α production during ultraviolet-induced skin fibrosis. Another study showed that FN1 secreted by lung cancer promotes the expression of CD206 and TGF-β in macrophages. However, there is no literature reporting that FN1 has a regulatory effect on radiotherapy-induced abscopal effect of breast cancer. SUMMARY

[0004] One of the purposes of the present application is to provide a new method for improving radiotherapy-induced abscopal effect.

[0005] The present application provides the application of FN1 gene inhibitor in the preparation of products for improving radiotherapy-induced abscopal effect of tumor cells.

[0006] The discovery of the abscopal effect has the potential to revolutionize radiotherapy, changing it from a local treatment to a systemic treatment, but the randomness of the abscopal effect limits its clinical application, and how to turn the random abscopal effect into a deterministic effect is a hot research topic at present. Studies have shown that the occurrence of the abscopal effect is closely related to the change of the microenvironment of the distant tumor. The present application research finds that when the abscopal effect occurs, the expression of FN1 gene in macrophages is significantly reduced, thereby inhibiting the activity of PI3K / Akt pathway of macrophages, promoting the polarization of macrophages from M2 phenotype to M1 phenotype, and inhibiting the growth of distant tumors. By inhibiting the expression of FN1, the incidence of the abscopal effect of breast cancer radiotherapy can be improved, and the randomness problem of the abscopal effect caused by breast cancer radiotherapy is solved.

[0007] The present application specifically finds, through multi-omics sequencing, bioinformatics analysis and a large number of experiments, that when the abscopal effect of breast cancer radiotherapy occurs, the infiltration of M1 type macrophages in the microenvironment of the distant tumor increases, the infiltration of M2 type macrophages decreases, and the expression of FN1 is down-regulated. Inhibiting the expression of FN1 promotes the polarization of M1 type macrophages in the microenvironment of the distant tumor and the infiltration and activation of CD8 + T cells by inhibiting the activity of PI3K / Akt pathway, thereby improving the incidence of the abscopal effect of breast cancer radiotherapy.

[0008] In the application of the present application, when the expression amount of FN1 in macrophages in the microenvironment of the distant tumor is reduced, the probability of the abscopal effect of breast cancer induced by radiation will increase.

[0009] The present application research finds that FN1 can change the immune characteristics of macrophages, change the immune characteristics of macrophages in the distant tumor, change the activity of PI3K / Akt pathway in macrophages, change the phagocytic ability of macrophages in the immune microenvironment of the distant tumor, and change the infiltration ability and activity of CD8 + T cells in the immune microenvironment of the distant tumor.

[0010] In the application of the present application, when the expression of FN1 in macrophages is reduced, the expressions of p-PI3K, p-Akt1 and p-STAT6 in macrophages will be reduced, and the macrophages will be polarized from M2 phenotype to M1 phenotype.

[0011] The present application research finds that the key mechanism of inhibiting FN1 to increase the incidence of the abscopal effect of breast cancer radiotherapy is related to the polarization remodeling of PI3K / Akt regulated macrophages. "FN1-PI3K / Akt" plays a crucial role in regulating the occurrence of the abscopal effect of breast cancer.

[0012] The application also finds that, after the FN1 expression is inhibited to promote the M1 polarization of macrophages, the phagocytosis ability of macrophages is improved, the infiltration ability and activity of CD8 + T cells in the distal tumor microenvironment are improved, so as to inhibit the growth of the distal tumor.

[0013] The method for improving the incidence of the distal effect of breast cancer in the application can be used to prepare a drug for inhibiting breast cancer metastasis in combination with radiotherapy, in particular, a targeted drug or a small molecule biological preparation.

[0014] The application also provides an application of the FN1 gene inhibitor in preparing a product for placing non-irradiated tumor cells in an immunocompatible microenvironment.

[0015] The application also provides an application of the FN1 gene inhibitor in preparing a product for slowing down the growth of distal tumor cells.

[0016] The application also provides an application of the FN1 gene inhibitor in preparing a product for slowing down the volume of distal tumor cells.

[0017] In the application, the distal tumor cells refer to tumor cells that have not received irradiation when radiotherapy is performed.

[0018] In the application, the irradiation is from X-ray irradiation.

[0019] In the application, the tumor cells are breast cancer cells.

[0020] In the application, the breast cancer cells are HER2-positive or triple-negative breast cancer cells.

[0021] In the application, the FN1 gene inhibitor is an siRNA of the FN1 gene.

[0022] In the application, the NCBI accession number of the FN1 gene is 14268.

[0023] The application has at least the following beneficial effects: The application first explicitly relates the FN1 gene expression to the distal effect, and proposes to use the FN1 gene as a target gene for improving the incidence of the distal effect. By inhibiting the FN1 gene, the radiotherapy-induced distal effect of tumor cells can be improved.

[0024] The application provides a new way for converting the random distal effect in tumor radiotherapy into a deterministic effect, so that the radiotherapy effect of breast cancer patients can be improved, and the radiotherapy mode of breast cancer can be changed. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The result of detecting differential gene expression by transcriptomics.

[0026] Figure 2 Analysis of FN1 expression in normal tissues and breast cancer in TCGA database.

[0027] Figure 3 Analysis of FN1 expression in each cell in the tumor microenvironment for single-cell sequencing results.

[0028] Figure 4 Analysis of the relationship between FN1 expression and the survival of breast cancer patients and qPCR verification of the change in FN1 expression when the abscopal effect occurs.

[0029] Figure 5 Expression of FN1 in macrophages of different polarity.

[0030] Figure 6 Changes in the expression of M1 and M2 macrophage markers after FN1 knockdown by siRNA.

[0031] Figure 7 Changes in the expression of M1 and M2 macrophage markers after overexpression of FN1.

[0032] Figure 8 Results of simulating the occurrence of the abscopal effect at the cellular level, A is the expression of FN1 after irradiation of breast cancer cells and co-culture with macrophages, B is the change in the expression of macrophage polarity markers after co-culture of breast cancer cells irradiated with BMDM cells overexpressing FN1.

[0033] Figure 9 Results of pathway research on FN1 regulation of macrophage polarity, A is the effect of FN1 inhibition or overexpression on the activity of key proteins in the PI3K / Akt pathway, B is the change in the expression of immune stimulation genes and immune inhibition genes after treatment of BMDM with knockdown and overexpression of FN1 with agonists and inhibitors, respectively.

[0034] Figure 10 Effect of FN1 inhibition on the immune function of macrophages, A is the effect of FN1 knockdown on the phagocytosis of macrophages on tumor cells, B is the effect of FN1-regulated macrophage polarization on the function of CD8 + T cells.

[0035] Figure 11 Results of in vivo experiments to verify that inhibition of FN1 improves the incidence of the abscopal effect, A is a schematic diagram of the experimental process, B is the change in the volume of the distal tumor, C is a picture of the distal tumor, and D is the survival of each group.

[0036] In each figure (if any), P <0.05, P <0.01, P<0.001, P<0.0001. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present application will be described in detail below with examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and substitutions to the present application without departing from the spirit and principles of the present application.

[0038] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially or prepared according to conventional methods in the art unless otherwise specified.

[0039] In the specific embodiments section of the present application, each detection is the average value of 3 sets of repeated sample detection.

[0040] Example 1 The present application studies the incidence of improving the occurrence of distant effects of radiotherapy for breast cancer by targeting FN1.

[0041] 1. Analysis and verification of sequencing results: Balb / c female mice were injected subcutaneously with 4T1 cells on the left and right lower limbs. After the left tumor volume reached 100 mm 3 and the right tumor volume reached 20 mm 3 , the left tumor was irradiated with 8 Gy of X-rays for 3 consecutive days (X-RAD 225, PRECISION, USA). The right tumor (non-irradiated side) was taken for transcriptome sequencing on the 3rd day after the last irradiation. DESeq2 was used for differential expression analysis between sample groups. Fold Change≥2 and FDR<0.05 were used as screening criteria. Differential gene analysis was performed on all comparison groups, and it was found that FN1 expression was reduced. Figure 1 TCGA database showed that FN1 expression was up-regulated in breast cancer. Figure 2 Using TISCH2 single-cell database, it was found that FN1 was highly expressed in macrophages in breast cancer. Figure 3 And breast cancer patients with low FN1 expression had significantly longer survival (left panel in Figure 4 ). The sequencing results were verified by qRT-PCR, and it was found that FN1 expression was significantly reduced in the irradiated group of distant tumors (right panel in Figure 4 ).

[0042] 2, FN1 regulates macrophage polarization: BMDM cells were treated with LPS+IFNy or IL-4 for 24h, RNA was extracted and FN1 expression was detected by qRT-PCR. It was found that FN1 expression was reduced in M1 macrophages (treated with LPS+IFNy) and increased in M2 macrophages (treated with IL-4) compared with the control group. The same results were obtained by Western Blotting method Figure 5 ). si-1 and si-2 (designed and synthesized by GENECARER) were used to transfect BMDM cells. It was found that after FN1 was knocked down, the expression of M1 macrophage marker Nos2, IL-1β and IL-12 was increased, while the expression of M2 macrophage marker Mrc1 was decreased Figure 6 ). FN1 overexpression plasmid was used to transfect BMDM cells, and M1 and M2 macrophage markers were detected at 24 and 48 hours. It was found that the expression of M1 markers was reduced and the expression of M2 markers was increased Figure 7 ).

[0043] si-1: GGUGGACUGUACUUGUCUATT, SEQ ID No. 1; si-2: GCAGUAGCACAGAGCUCAATT, SEQ ID No. 2.

[0044] 3, FN1 regulates macrophage polarization when remote effect occurs: At the cellular level, remote effect was simulated by irradiating 4T1 and TUBO cells with 8Gy X-rays and continuing to culture for 24 hours. The culture medium of the two kinds of cells was used to culture BMDM cells for 24 hours, and FN1 expression in BMDM was detected by qRT-PCR. It was found that after co-culturing with irradiated tumor cells, FN1 expression was significantly reduced (A in Figure 8 ). FN1 overexpression BMDM cells were cultured in the medium, and M1 and M2 markers were detected by qRT-PCR. It was found that compared with the medium transfer group, the expression of M1 markers (TNF-α, IL-1β, IL-6 and IL-12) was reduced, and the expression of M2 markers (Chil3, Fizz1, Arg1 and Mrc1) was increased in the combined treatment group (medium transfer+FN1 overexpression) Figure 8 (B in

[0045] 4. FN1 regulates macrophage polarization through the PI3K / Akt pathway: FN1 expression in BMDM was regulated by siRNA or plasmid transfection. Western blotting revealed that inhibition of FN1 expression reduced the phosphorylation levels of PI3K, AKT1, and STAT6 proteins in macrophages, while increasing the phosphorylation level of AKT2 protein. Overexpression of FN1 increased the phosphorylation levels of PI3K and AKT1 proteins in macrophages. These results indicate that FN1 overexpression can activate the PI3K / AKT signaling pathway in macrophages. Figure 9 A in Figure 1, where the "p" in front of each protein represents phosphorylation). BMDMs with knockdown or overexpression of FN1 were treated with the PI3K-specific agonist 740 YP and inhibitor LY294002, respectively, to further verify that FN1 regulates macrophage polarization toward M2 through the PI3K / AKT signaling pathway. Figure 9 As shown in Figure B, compared with the si-FN1 control group, the expression of immunostimulatory genes decreased and the expression of immunosuppressive genes increased in the siFN1 + 740 YP treatment group. Compared with the OE-FN1 control group, the expression of immunostimulatory genes increased and the expression of immunosuppressive genes decreased in the OE-FN1 + LY294002 treatment group. Based on these results, it is concluded that FN1 regulates macrophage M2 polarization through the PI3K / AKT signaling pathway. When distal effects occur, FN1 expression is suppressed, along with PI3K / AKT pathway activity, promoting macrophage M1 polarization.

[0046] 5. Inhibition of FN1 expression enhances the phagocytic ability of macrophages and CD8 + T cell activity: EGFP-carrying TUBO cells were co-cultured with Dil-stained FN1-knockdown BMDM (BMDM transfected with the aforementioned small interfering sequence si-1) for 2 hours to evaluate the effect of FN1 on macrophage phagocytosis. The results showed that the number of macrophages co-localized with tumor cells in the si-FN1 group was more than twice that in the si-NC group, indicating that macrophages showed stronger phagocytosis of tumor cells after FN1 knockdown ( Figure 10 A). Detection of FN1-regulated macrophage polarization on CD8 + Normal and FN1 knockout macrophages were isolated from spleen CD8 + T cell co-culture, and it was found that FN1 knockout abolished macrophage-mediated CD8 + T cell suppression, characterized by CD8 + Increased expression of GzmB, IFN-γ, and CD69 in T cells ( Figure 10 B in ).

[0047] 6. In vivo experiment to verify that inhibition of FN1 increases the incidence of the abscopal effect: Construct a mouse model according to the method in item 1 above. When the left tumor volume reaches 100 mm 3 , the right (non-irradiated side) tumor volume reaches 50 mm 3 , give the left tumor 8 Gy x 3 fraction local continuous irradiation, and on the first and third days of irradiation, inject siFN1 into the tumor (RT+SI group, 5 nmol per mouse per time, siFN1 is a modified si-1 sequence, the modification is: 1, 8, 10, 11, 12, 14, 16 bases 2'F modification, the rest of the bases are all-chain 2'OMe modification, 3' end of the sense strand GalNAc modification; 2, 5' end and 3' end 1, 2 and 2, 3 base connection phosphate modification, this modification can make si-1 stable in the animal body), measure the right tumor volume every 2 days until 15 days after the end of irradiation, then euthanize the mice to obtain the right tumor for photography. Set up a CK group without irradiation and injection, a RT group with the same irradiation scheme but injecting PBS instead of siFN1, and a SI group without irradiation but with siRNA injection. The experimental flowchart is shown in A of Figure 11 . The results show that when the abscopal tumor volume reaches about 50 mm 3 , simple irradiation or siRNA injection does not cause the occurrence of the abscopal effect, the mice treated with siFN1 combined with irradiation have slower growth of the abscopal tumor, smaller tumor volume, and longer survival time (B, C, and D of Figure 11 ). This indicates that inhibition of FN1 can slow down the growth of the abscopal tumor and increase the incidence of the abscopal effect.

[0048] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, are within the scope of the present application.

Claims

1. Application of FN1 gene inhibitors in the preparation of products for enhancing abscopal effects induced by radiotherapy in tumor cells.

2. Application of FN1 gene inhibitors in the preparation of products that place non-irradiated tumor cells in an immune-promoting microenvironment.

3. Application of FN1 gene inhibitors in the preparation of products for slowing the growth of distal tumor cells.

4. Application of FN1 gene inhibitors in the preparation of products for reducing the volume of distal tumor cells.

5. The use according to claim 3 or 4, characterized in that The distant tumor cells refer to tumor cells that have not received radiation during radiotherapy.

6. The use according to claim 5, characterized in that The radiation comes from X-ray irradiation.

7. The use according to any one of claims 1 to 6, characterized in that The tumor cells are breast cancer cells.

8. The use according to claim 7, characterized in that The breast cancer cells are HER2-positive or triple-negative breast cancer cells.

9. The use according to any one of claims 1 to 8, characterized in that The FN1 gene inhibitor is siRNA of the FN1 gene.

Images