Use of an agent with emilin1 gene inhibitory effect in the preparation of a radiosensitizer

Abstract

The present application relates to the technical field of radiotherapy and chemotherapy sensitizers, and particularly relates to application of a preparation with EMILIN1 gene inhibition in preparation of a radiotherapy and chemotherapy sensitizer. Based on deep research on the tumor microenvironment and tumor-related fibroblasts of colorectal cancer, it is found that EMILIN-1 is significantly up-regulated in patients with radiotherapy and chemotherapy resistance. By designing siRNA to specifically inhibit the expression of EMILIN1 gene, the radiotherapy and chemotherapy sensitivity of colorectal cancer can be effectively improved. The present application provides a variety of application forms such as cholesterol-modified siRNA and tumor-related fibroblasts transfected with siRNA, which significantly improve the sensitivity of colorectal cancer cells to radiotherapy and chemotherapy drugs. The technical scheme can solve the technical problem that there is no means to effectively inhibit the radiotherapy and chemotherapy resistance of colorectal cancer cells in the prior art, and provides multiple options for clinical transformation, and has good development and application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of radiotherapy and chemotherapy sensitizers, and particularly relates to application of a preparation with EMILIN1 gene inhibiting effect in preparation of a radiotherapy and chemotherapy sensitizer. BACKGROUND

[0002] Colorectal cancer (CRC) is one of the malignant tumors with high morbidity and mortality worldwide. According to epidemiological statistics, its morbidity ranks the third in the world, and its mortality ranks the second, which seriously threatens human life and health. For patients with advanced or locally advanced rectal cancer, radiotherapy and chemotherapy as standard treatment methods are widely used in preoperative neoadjuvant therapy, postoperative adjuvant therapy and radical or palliative treatment for patients who cannot be operated, which significantly improves the prognosis of some patients. However, a considerable proportion of patients show primary or acquired resistance to radiotherapy and chemotherapy in clinical practice, resulting in poor treatment effect, disease progression and even death, which has become a key bottleneck restricting the overall efficacy improvement of colorectal cancer.

[0003] At present, the mechanism of radiotherapy and chemotherapy resistance has not been fully elucidated, involving multiple intrinsic factors such as gene mutation of tumor cells, enhanced DNA damage repair ability, and up-regulation of drug efflux pump. More complex is that the tumor microenvironment (TME) plays a crucial role in mediating treatment resistance. TME is a dynamic ecosystem composed of tumor cells, stromal cells, immune cells, vascular system and extracellular matrix. Among them, cancer-associated fibroblasts (CAFs) are one of the most abundant and active stromal cell types in TME. A large number of studies have shown that CAFs not only regulate the proliferation and invasion of tumor cells by secreting various cytokines (such as TGF-β, IL-6), growth factors (such as HGF, EGF) and exosomes, but also form a physical barrier by remodeling extracellular matrix to limit drug penetration; by regulating immune cell infiltration and function, it constructs an immunosuppressive microenvironment; and by metabolic reprogramming, it provides survival advantage for tumor cells. In addition, CAFs can also promote the maintenance of tumor stem cell properties through paracrine signaling pathways, thereby enhancing the self-renewal ability of tumors and their resistance to radiotherapy and chemotherapy.

[0004] Although the multiple roles of CAFs in mediating radiochemotherapy resistance of colorectal cancer have been gradually recognized, the research on the specific mechanism of CAFs is still not in-depth, especially lacking reliable biomarkers that can be used for clinical prediction of primary radiochemotherapy resistance. More importantly, there is no effective strategy to target the function of CAFs or their interaction with tumor cells to reverse radiochemotherapy resistance. The existing treatment programs focus on tumor cells themselves, ignoring the key contribution of the microenvironment to treatment failure. Therefore, in-depth analysis of the molecular mechanism of CAFs-mediated radiochemotherapy resistance, and the exploration of new prediction markers and therapeutic targets based on TME, especially CAFs, have important theoretical significance and clinical application value for overcoming colorectal cancer treatment resistance and improving patient survival rate. SUMMARY

[0005] The present application aims to provide the use of a preparation with EMILIN1 gene inhibition in the preparation of a radiochemotherapy sensitizer, to solve the technical problem in the prior art that there is a lack of means that can effectively inhibit the radiochemotherapy resistance phenomenon of colorectal cancer cells.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] The use of a preparation with EMILIN1 gene inhibition in the preparation of a radiochemotherapy sensitizer, the preparation comprising siRNA; the siRNA is siRNA-1 or siRNA-2:

[0008] The sense strand of siRNA-1 is 5'-GAGUGGAGACAUAUGUCAA / DT / DT / -3';

[0009] The antisense strand of siRNA-1 is 5'-UUGACAUAUGUCUCCACUC / DT / DT / -3';

[0010] The sense strand of siRNA-2 is 5'-AGACAGUGACCGACAUGGA / DT / DT / -3';

[0011] The antisense strand of siRNA-2 is 5'-UCCAUGUCGGUCACUGUCU / DT / DT / -3'.

[0012] Further, the siRNA is used to reduce the expression level of EMILIN-1 protein in tumor-associated fibroblasts;

[0013] The tumor-associated fibroblasts are fibroblasts present in tumor tissue and positive for surface markers alpha smooth muscle actin and platelet-derived growth factor receptor alpha.

[0014] Further, the preparation is a cholesterol-modified siRNA; or, the preparation is a tumor-related fibroblast transfected with siRNA.

[0015] Further, the cholesterol-modified siRNA is formed by connecting cholesterol to the sense strand of siRNA through a click chemistry reaction; the cholesterol-modified siRNA is used to apply to a tumor site where tumor-related fibroblasts exist.

[0016] Further, the tumor-related fibroblast transfected with siRNA is prepared by culturing the tumor-related fibroblast using a culture medium containing an AB mixed solution to obtain the tumor-related fibroblast transfected with siRNA.

[0017] The AB mixed solution is prepared by mixing the culture medium with liposome 3000 and standing to form an A system; mixing the culture medium with siRNA and standing to form a B system; mixing the A system and the B system and standing to obtain the AB mixed solution.

[0018] Further, the radiotherapy and chemotherapy sensitizer is used to increase the sensitivity of colorectal cancer cells to radiotherapy and chemotherapy.

[0019] Further, the tumor-related fibroblast is obtained by digesting the tumor tissue using collagenase type 1 and collagenase type 2, then filtering and collecting the filtrate; centrifuging the filtrate to collect cells, and culturing the cells to obtain the tumor-related fibroblast adherent cells.

[0020] The technical solution also provides a reagent for improving the sensitivity of colorectal cancer cells to radiotherapy and chemotherapy, and the reagent comprises siRNA.

[0021] The siRNA is siRNA-1 or siRNA-2.

[0022] The sense strand of siRNA-1 is 5'-GAGUGGAGACAUAUGUCAA / DT / DT / -3';

[0023] The antisense strand of siRNA-1 is 5'-UUGACAUAUGUCUCCACUC / DT / DT / -3';

[0024] The sense strand of siRNA-2 is 5'-AGACAGUGACCGACAUGGA / DT / DT / -3';

[0025] The antisense strand of siRNA-2 is 5'-UCCAUGUCGGUCACUGUCU / DT / DT / -3'.

[0026] Further, the preparation is a cholesterol-modified siRNA, and the reagent is used for application at a tumor site where tumor-associated fibroblasts exist.

[0027] Further, the preparation is a tumor-associated fibroblast transfected with siRNA.

[0028] In summary, the technical principle of the technical solution is that:

[0029] The present application is based on in-depth research on the tumor microenvironment (TME) of colorectal cancer, especially the analysis of the function of tumor-associated fibroblasts (CAFs). Through proteomic analysis of clinical samples, the inventors found that the expression of extracellular matrix (ECM) protein EMILIN-1 was significantly up-regulated in patients resistant to radiotherapy and chemotherapy, and the source was mainly CAFs. The present application proposes that by specifically inhibiting the expression of the EMILIN1 gene, the radiotherapy and chemotherapy sensitivity of colorectal cancer can be effectively improved. The present application uses small interfering RNA (siRNA) technology to design and screen siRNA molecules (siRNA-1 and siRNA-2) that can effectively silence the EMILIN1 gene. These siRNAs can specifically bind to EMILIN1 mRNA, guide its degradation, and thus significantly reduce the expression level of EMILIN-1 protein in CAFs at the post-transcriptional level. When the expression of EMILIN-1 in CAFs is inhibited, the sensitivity of tumor cells to radiotherapy and chemotherapy is significantly enhanced.

[0030] The preparation provided by the present application with EMILIN1 gene inhibition effect is used for preparing a radiotherapy and chemotherapy sensitizer, and the reagent for improving the sensitivity of colorectal cancer cells to radiotherapy and chemotherapy has the following outstanding beneficial effects:

[0031] (1) Strong targeting and clear mechanism: The present application first determines EMILIN-1 as a key microenvironment factor mediating radiotherapy and chemotherapy resistance of colorectal cancer, and provides a sensitization strategy directly targeting the factor. By inhibiting the expression of EMILIN-1 in CAFs through specific siRNA, the key interaction between tumor cells and microenvironment is precisely intervened, the mechanism is clear, and the targeting is high.

[0032] (2) Significantly enhance radiotherapy and chemotherapy sensitivity: By inhibiting EMILIN-1, the sensitivity to radiotherapy and chemotherapy drugs is significantly improved. This is expected to convert the originally insensitive "resistant" tumor into a "sensitive" tumor, and expand the benefit population of radiotherapy and chemotherapy.

[0033] (3) Overcoming treatment resistance and improving efficacy: The present application directly addresses the clinical problem of radiotherapy and chemotherapy resistance and provides a new solution. By reversing the protective effect of CAFs, it is expected to overcome primary and acquired radiotherapy and chemotherapy resistance, significantly improving local control rate of tumor and progression-free survival of patients.

[0034] (4) Potentially reducing treatment dose and side effects: Due to the significant sensitization effect, the dose of radiotherapy and chemotherapy can be reduced in theory to achieve the same therapeutic effect, thereby reducing damage to normal tissues, reducing side effects such as nausea, vomiting, and bone marrow suppression, and improving the quality of life of patients.

[0035] (5) Flexible application form and good transformation prospect: The present application provides two application forms of cholesterol-modified siRNA (which can directly target tumor sites) and CAFs transfected with siRNA (which can be used as "carrier cells"), providing multiple options for clinical transformation and having good development and application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Kaplan-Meier curves of EMILIN1 protein difference before and after treatment of neoadjuvant radiotherapy and chemotherapy resistant and sensitive patients of Example 1, and overall survival of colorectal cancer patients with high and low expression of EMILIN1 (BS: before treatment of neoadjuvant radiotherapy and chemotherapy sensitive patients, BR: before treatment of neoadjuvant radiotherapy and chemotherapy resistant patients, PR: after treatment of neoadjuvant radiotherapy and chemotherapy resistant patients; A: EMILIN1 protein expression levels before treatment of neoadjuvant radiotherapy and chemotherapy sensitive patients and before treatment of neoadjuvant radiotherapy and chemotherapy resistant patients; B: EMILIN1 protein expression levels before treatment of neoadjuvant radiotherapy and chemotherapy resistant patients and after treatment of neoadjuvant radiotherapy and chemotherapy resistant patients; C: Kaplan-Meier curves of overall survival of colorectal cancer patients with high and low expression of EMILIN1).

[0037] Figure 2 Detection results of markers of tumor-associated fibroblasts of Example 2 and experimental results of inhibition effect of two siRNAs on EMILIN1 expression (A: fluorescence confocal microscopic image of tumor-associated fibroblasts; B: WB detection results of EMILIN1 protein expression of CAFs cells transfected with two siRNAs, respectively).

[0038] Figure 3Results of detection of the influence of CAFs cells transfected with two siRNAs of Example 3 on the radiosensitization of colorectal cancer cells (A: CCK-8 detection results line graph for HCT-116 cells; B: CCK-8 detection results line graph for SW480 cells; C: Clonogenic assay results line graph for HCT-116 cells; D: Representative images of clonogenic assay for HCT-116 cells; E: Clonogenic assay results line graph for SW480 cells; F: Representative images of clonogenic assay for SW480 cells; si-NC is the negative control group, using siRNA without gene targeting to transfect CAFs cells; si-EMILIN1-1, si-1 is the experimental group, using siRNA-1 to transfect CAFs cells; si-EMILIN1-2, si-2 is the experimental group, using siRNA-2 to transfect CAFs cells).

[0039] Figure 4 Results of in vivo experiments of Example 4 (A: Tumor volume dynamic change curve; B: Typical tumor tissue photo; C: Immunofluorescence staining analysis of CAFs markers and EMILIN1 in tumor microenvironment; DAPI: cell nucleus staining; αSMA: core marker of CAFs; EMILIN1: protein expression of target gene EMILIN1; NC: physiological saline control group; si-EMILIN1: siRNA treatment group of EMILIN1, specifically using cholesterol-modified siRNA-2). DETAILED DESCRIPTION

[0040] The application will be further described in detail below with examples, but the embodiments of the application are not limited thereto. If not specifically indicated, the technical means used in the following examples and experimental examples are conventional means known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels, which can be specifically referred to Table 1.

[0041] Table 1: Reagents used in the application

[0042]

[0043] For the convenience of understanding the technical solutions, the related technical terms and concepts are explained in detail as follows:

[0044] Preoperative Neoadjuvant Therapy: refers to the treatment carried out before the radical surgery of tumor, the core goal is to "create better conditions for surgery", let the tumor which originally needs large range resection be reduced, and then reduce the operation range, maximize the preservation of organ function, at the same time, it can also remove micro metastasis as soon as possible, reduce the risk of postoperative recurrence and metastasis. The commonly used treatment methods are concurrent chemoradiotherapy (such as the method adopted by the sample of embodiment 1 of the present scheme), radiotherapy, chemotherapy, targeted therapy and immunotherapy.

[0045] Postoperative Adjuvant Therapy: refers to the treatment carried out after the radical surgery of tumor, the core goal is to "consolidate the effect of surgery", to clear the micro residual cancer cells that cannot be removed by surgery, and to reduce the risk of recurrence and metastasis. The commonly used treatment methods are concurrent chemoradiotherapy and radiotherapy, chemotherapy, targeted therapy and immunotherapy.

[0046] Neoadjuvant Chemoradiotherapy Sensitivity: refers to that the tumor has a good response to preoperative chemoradiotherapy, after treatment, according to RECIST1.1 evaluation standard, it reaches partial remission (tumor shrinkage ≥30%), or even complete remission at the pathological level.

[0047] Neoadjuvant Chemoradiotherapy Resistance: refers to that the tumor has a poor response to preoperative chemoradiotherapy, after treatment, the tumor volume does not significantly shrink, or even increases, or new lesions appear, according to RECIST1.1 standard, it is evaluated as stable or progressive, and the subsequent treatment strategy needs to be adjusted.

[0048] Cancer-Associated Fibroblasts (CAFs) are a kind of activated fibroblasts in tumor microenvironment, not tumor cells themselves, and the core role is to provide support for tumor cell growth and metastasis. CAFs usually use aSMA (α-smooth muscle actin) and PDGFRa (platelet-derived growth factor receptor a) as marker molecules for identification and research in experiments.

[0049] EMILIN-1 (Elastin Microfibril Interfacing Protein 1) is a glycoprotein found in the extracellular matrix, which is involved in the maintenance of tissue structure and cell signaling. The NCBI gene ID of human EMILIN-1 is: 11117; HGNC number is 19880.

[0050] Example 1: Proteomic sequencing analysis results of rectal cancer patients

[0051] To further find the molecular characteristics of the patients with rectal cancer who are resistant to neoadjuvant chemoradiotherapy, we performed proteomic sequencing on the tumor tissues of 4 patients who were resistant to neoadjuvant chemoradiotherapy and 4 patients who were sensitive to neoadjuvant chemoradiotherapy before and after treatment. The differentially expressed proteins between the patients who were sensitive to treatment and the patients who were resistant to treatment, and the proteins that were not differentially expressed in the patients who were resistant to treatment before and after treatment were screened, so as to define the proteins that were originally resistant to chemoradiotherapy.

[0052] Figure 1 A and B show the expression of the target protein EMILIN1 in the patients who were sensitive to treatment and the patients who were resistant to treatment, and in the patients who were resistant to treatment before and after treatment. Figure 1 As shown in the experimental results of A, before treatment, the EMILIN1 protein level of the patients who were resistant to neoadjuvant chemoradiotherapy was significantly higher than that of the patients who were sensitive to neoadjuvant chemoradiotherapy; it is suggested that the high expression of EMILIN1 before treatment may be related to the resistance of the tumor to chemoradiotherapy. Figure 1 As shown in the experimental results of B, for the patients who were resistant to neoadjuvant chemoradiotherapy, after receiving treatment, the EMILIN1 protein level was not statistically different from that before treatment; it is suggested that treatment has little effect on the expression of EMILIN1 in the resistance group, which indicates that the tumor is not sensitive to the treatment and is difficult to down-regulate EMILIN1.

[0053] Figure 1 C is the negative correlation between EMILIN1 and the overall survival of patients with colorectal cancer. According to the expression level of EMILIN1, it is divided into High EMILIN1 Group (high expression group) and Low EMILIN1 Group (low expression group). The overall survival of the high EMILIN1 expression group is significantly shorter than that of the low expression group (the curve drops faster and the survival rate is lower); it is suggested that the high expression of EMILIN1 is a potential marker of poor prognosis (shortened overall survival) of patients.

[0054] As can be seen, EMILIN1 is not only closely related to the resistance of the tumor to treatment (the resistance group has higher EMILIN1 before treatment, and it is difficult to down-regulate its expression), but also related to the poorer overall survival of the patients, and may be a key regulatory factor or biomarker of tumor treatment sensitivity and prognosis.

[0055] Example 2: siRNA for inhibiting the expression of EMILIN1

[0056] (1) Extraction and identification of CAFs (tumor-associated fibroblasts)

[0057] Penicillin-streptomycin-amphotericin B (triple antibiotic) was added to PBS at 5: 100 (v:v). Human colorectal cancer tumor tissue was washed twice in PBS containing triple antibiotic. The tumor tissue was removed and soaked in levofloxacin injection for about 5 min. Collagenase type 1 and collagenase type 2 were added to serum-free DMEM at 0.1% (w / v), triple antibiotic was added at 5: 100 (v:v), and levofloxacin sodium chloride injection was added at 5: 1000 (v:v) to prepare a digestion solution, which was filtered using a 0.22 μm filter. A small amount of digestion solution was added to the tumor tissue in a sterile EP tube, and the tissue was cut into a homogenate using tissue scissors. The tissue homogenate was transferred to the digestion solution, and about 1 ml of tumor tissue was digested using 10 ml of digestion solution in a 15 ml centrifuge tube. The centrifuge tube was placed in a 37°C shaking incubator, and digestion was performed for 1 h. After digestion, the filtrate was collected using a 70 μm filter, and the cells were collected by centrifugation at 500 g for 10 min at room temperature. The cells were resuspended in DMEM complete medium containing 5: 100 (v:v) triple antibiotic and 5: 1000 (v:v) levofloxacin injection, and inoculated into a culture vessel. The extracted CAFs cells and conventional tumor cells were cultured and stored using the same method: placed in DMEM medium containing 10% serum, cultured in a 37°C incubator containing 5% CO2, and frozen in liquid nitrogen or at -80°C. In addition, it should be noted that this protocol only involves the application of CAFs cells, and does not involve the process of taking colorectal cancer tissue. After obtaining CAFs cells, they can be cultured and expanded in vitro, and then applied. After applying siRNA to CAFs cells according to this protocol, the effect of radiotherapy and chemotherapy sensitization can be achieved.

[0058] The adherent CAFs were discarded, washed twice with PBS, and trypsin was added. The mixture was incubated at 37°C for about 3 min. An equal amount of DMEM complete medium was added to neutralize the trypsin, and the cell suspension was collected in a 15 ml centrifuge tube. The tube was centrifuged at 1000 rpm at room temperature for 3 min, and the precipitate was collected. The precipitate was resuspended in 1 ml of DMEM complete medium and mixed well. The number of cells was counted using a cell counter. About 4 x 10 4The CAFs were inoculated in a confocal dish, 1 ml of DMEM complete medium was added, and the dish was placed in a 37°C, 5% CO2 incubator for culture; the next day, after observing the cell adhesion, the culture medium in the confocal dish was discarded, and the cells were washed with PBS for 2 times. 1 ml of 4% paraformaldehyde was added for room temperature fixation for 30 min. PBS was washed for 3 times, each time for 5 min, 0.5% Triton-X-100+5% BSA mixed solution was added, and the mixture was incubated at room temperature for 30 min. TBST was washed for 3 times, each time for 5 min, the αSMA and PDGFRα antibodies were diluted by 1:1000 with the antibody diluent, and the diluted antibodies were added to the confocal dish, which was incubated at 4°C on a shaking table overnight. TBST was washed for 3 times, each time for 5 min, the secondary antibody (R488, M647) was diluted by 1:200 with the antibody diluent, and the diluted secondary antibody was added to the confocal dish, which was incubated at room temperature in the dark for 1 h. TBST was washed for 3 times, each time for 5 min, DAPI was added to the confocal dish, which was incubated at room temperature in the dark for 5 min. TBST was washed for 3 times, each time for 5 min, and the confocal microscope was observed.

[0059] According to Figure 2 A can be seen that the cells extracted from the tumor tissue are spindle-shaped and express the CAF-specific markers αSMA and PDGFRα. The obtained cells are identified as CAF cells, which will be used for subsequent experimental research. More specifically, Figure 2 A shows the localization and co-expression of different molecules in the cells, and three fluorescent markers are used: DAPI (blue fluorescence): staining the cell nucleus to show the nuclear position of the cells; αSMA (green fluorescence): marking smooth muscle actin; PDGFRα (red fluorescence): marking platelet-derived growth factor receptor α; Merge (merged image): superimposing the three fluorescent channels to show the co-localization of multiple molecules, which helps to analyze the spatial distribution correlation between cell phenotypes or molecules.

[0060] (2) Design of siRNA for inhibiting EMILIN1 expression

[0061] (2.1) Sequence information

[0062] Two siRNA sequences siRNA-1 and siRNA-2 for EMILIN1 were designed, which were synthesized by GenScript Biotech Corporation, and the specific sequence information is as follows:

[0063] siRNA-1

[0064] Sense strand: 5'-GAGUGGAGACAUAUGUCAA / DT / / DT / -3' (SEQ ID NO. 1);

[0065] Sense: 5'-CUGACAUUGUCUCCACUC-3' (SEQ ID NO. 1);

[0066] siRNA-2

[0067] Sense: 5'-AGACAGUGACCGACAUGGA-3' (SEQ ID NO. 3);

[0068] Antisense: 5'-UCCAUGUCGGUCACUGUCU-3' (SEQ ID NO. 4);

[0069] si-NC is negative control siRNA

[0070] Sense: 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO. 5);

[0071] Antisense: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO. 6);

[0072] wherein / DT / in each sequence refers to a 3' terminal overhanging thymine deoxynucleotide (DNA with base thymine), and the rest of the nucleotides are ribonucleotides (RNA) except / DT / . The total length of each sequence is: 19 ribonucleotides + 2 deoxynucleotides = 21 nucleotides. The sense and antisense strands form a stable double-stranded RNA (dsRNA) core through base pairing (A-U, G-C).

[0073] (2.2) siRNA transfection

[0074] The well-conditioned CAFs cells were plated into 6-well plates at 1 x 10 5 cells / well; after adherent culture for 24 h, siRNA transfection was performed according to the following system mixing:

[0075] System A: 125 μL opti-MEM + 5 μL Lipo 3000; System B: 150 μL opti-MEM + 5 μL siRNA (264 ng / μl); after mixing and standing for 5 min, the B system was added to the A system, mixed and stood for 15 min to obtain AB mixed solution.

[0076] Discard the culture medium in the 6-well plate, add 1.75 mL of DMEM complete culture medium per well; add 250 μL of the above AB mixture per well, shake gently, and then place the 6-well plate in a 37°C, 5% CO2 cell incubator for culture. After 24 h of transfection culture, the siRNA transfection is completed, and CAFs cells transfected with the specified siRNA are obtained, which are referred to as siRNA-1 CAFs cells and siRNA-2 CAFs cells, respectively. The transfected cells are then cultured in ordinary DMEM complete culture medium, and protein extraction is performed after 48 h of culture.

[0077] (2.3) Extraction of protein from cells and WB detection

[0078] The CAFs cells transfected with two kinds of si-RNA, respectively, are taken out, the original culture medium is discarded, and the cells are washed twice with PBS. Trypsin is added, and the cells are digested at 37°C for 3 min. The cell suspension is collected and centrifuged to collect the cell pellet. RIPA 80 μl is added to lyse the cells on ice for 30 min, and the cells are blown once every 10 min. Centrifugation is performed at 14,000 rpm and 4°C for 25 min, and the supernatant is aspirated into a new EP tube for standby. The obtained protein is quantified (BCA method), and the quantified protein is placed in a 100°C electric bath for 10 min to denature the protein, and then cooled to room temperature for standby. The obtained protein is subjected to routine WB detection, and β-Actin (β-actin) is used as an internal reference. The expression of EMILIN1 protein in the cells after transfection with the two kinds of siRNA is detected. The WB results are shown in Figure 2 B. Under the condition that the expression of β-Actin is basically the same, the band signal intensity of EMILIN1 protein extracted from the siRNA-1 CAFs cells is lower than that of the si-NC group; and the expression of EMILIN1 protein in the siRNA-2 CAFs cells is particularly obviously decreased. This indicates that the siRNA-2 has a higher efficiency in inhibiting the expression of EMILIN1 protein in CAFs cells.

[0079] Example 3: In vitro radiosensitization of colorectal cancer cells by EMILIN1 siRNA

[0080] (1) CCK8 proliferation experiment

[0081] The CAFs cells transfected with siRNA are obtained in the manner of Example 2, and a CCK8 proliferation experiment is performed, and the specific process is as follows:

[0082] After 24h transfection, the well CAFs cells were seeded with HCT-116 or SW480 (two colorectal cancer cell lines) at a ratio of 1:1, 2400 cells per well (96-well plate), three identical replicates were plated for each biological replicate, and 100 μL DMEM complete medium was added to each well. Repeat plating five 96-well plates according to the same operation (divided into 0Gy, 2Gy, 4Gy, 6Gy, 8Gy groups). Incubate in a 37°C, 5% CO2 incubator.

[0083] After 48h culture, 4 μM 5-Fu (5-fluorouracil) was added to each well of the 96-well plate (0Gy group was excluded), and the corresponding group was irradiated (X-ray, dose rate 400 cGy / min) at the same time. Among them, 5-Fu is a commonly used anti-metabolic chemotherapeutic drug in clinical practice, and its core function is to interfere with the DNA synthesis and repair of tumor cells, inhibit their proliferation and induce death, and is mainly used for the treatment of various solid tumors. This study simulates the common clinical concurrent chemoradiotherapy treatment method, which is a model of colorectal cancer cells receiving concurrent chemoradiotherapy.

[0084] After 48h, the medium containing 5-Fu was removed and replaced with fresh DMEM complete medium. CCK8 was used to detect cell viability 120h after plating (avoid light treatment). CCK8 detection solution was prepared according to the medium: CCK8 reagent = 10:1. The culture medium in the 96-well plate was aspirated, and 100 μL of CCK8 detection solution was added to each well. After 2h incubation at 37°C, the absorbance value of each well at 450nm was measured using a microplate reader.

[0085] The experimental results are shown in Table 1. Figure 3A and B, CCK8 proliferation experiment results fold line graph can prove that after CAFs cells were transfected with two kinds of siRNA respectively, colorectal cancer cells were more sensitive to concurrent radiotherapy and chemotherapy. Among them, the effect of siRNA-2 is better, and the sensitization effect is more obvious. For HCT-116 cells, siRNA-1 did not produce significant sensitization effect relative to the negative control group (2-8 Gy irradiation + 4 μM 5-Fu, none produced significant sensitization effect); while siRNA-2 showed more significant sensitization effect for the negative control group (2-8 Gy irradiation + 4 μM 5-Fu, all produced significant sensitization effect in the above range). For SW480 cells, siRNA-2 produced significant sensitization effect relative to the control group (2-8 Gy irradiation + 4 μM 5-Fu, all produced significant sensitization effect in the above range); while siRNA-1 only produced significant effect when 8 Gy irradiation + 4 μM 5-Fu. In the tumor staging model, HCT-116 cells often represent Duke's stage D (late colorectal cancer, high degree of invasion and metastasis); SW480 cells represent Duke's stage B (earlier than HCT-116's D stage). The references for the above tumor types are as follows: "Liu Z-P, Tang W-S, Wang G-Z, et al. Ferulic acid inhibiting colon cancer cells at different Duke’s stages. Food&Medicine Homology, 2025, 2(3): 9420063. https: / / doi.org / 10.26599 / FMH.2025.9420063". The relevant content recorded in the literature is as follows: SW-480 cells correspond to Duke's Stage B, Caco-2 cells correspond to Duke's Stage C, and HCT-116 cells correspond to Duke's Stage D, representing an increasing order of tumor progression. siRNA-2 can effectively sensitize various colorectal cancer cells to radiotherapy and chemotherapy, and has more ideal application value than siRNA-1.In addition, from the results of the 0 Gy group, it can be seen that if the cancer cells are not treated with radiotherapy and chemotherapy, whether the colorectal cancer cells are treated with CAFs cells transfected with siRNA does not cause different effects on the activity of colorectal cancer cells. This shows that CAFs cells transfected with siRNA itself does not affect the activity of colorectal cancer cells, and CAFs cells transfected with siRNA only acts as a radiotherapy and chemotherapy sensitizer to colorectal cancer cells, which is unpredictable by the prior art.

[0086] (2) Cloning formation experiment

[0087] The CAFs cells transfected with siRNA were obtained in the manner of Example 2, and then the cloning formation experiment was performed, and the specific process was as follows:

[0088] After 24 h of transfection, the CAFs cells in good condition were inoculated with HCT-116 / SW480 at a ratio of 1:1, 1200 cells per well (12-well plate), and five 12-well plates were repeated according to the same operation (divided into 0 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy groups).

[0089] After 48 h, 4 μM 5-Fu was added to each well of the 12-well plate except for the 0 Gy group, and the corresponding dose was irradiated. After 48 h, the culture medium containing 5-Fu was removed, and fresh DMEM complete culture medium was replaced. Continuous culture for 8 days or until the number of cells in most single clones was greater than 50, and the liquid was changed every 3 days and the cell state was observed in the middle. After the cloning was completed, the original culture medium was discarded, washed with PBS twice, 1 mL of 4% paraformaldehyde was added to each well for fixation for 20 min. After fixation, PBS was washed once, 1 ml of crystal violet staining solution was added to each well, and staining was performed for 10 min. PBS was washed for 3 times, air-dried, and photographed.

[0090] The experimental results are shown in Table 1. Figure 3Colony formation assays demonstrated that transfection of CAFs cells with two different siRNAs followed by concurrent chemoradiotherapy inhibited both the number and size of colony formation in colorectal cancer cells. For HCT-116 cells, siRNA-1 and siRNA-2 showed significant sensitization effects compared to the negative control group (4-8 Gy irradiation + 4 μM 5-Fu, within which range, significant sensitization was observed); and siRNA-2 showed a superior sensitization effect compared to siRNA-1. For SW480 cells, siRNA-1 only showed a significant sensitization effect compared to the control group under 6 Gy irradiation + 4 μM 5-Fu conditions (no significant effect was observed under other conditions); siRNA-2 showed a significant sensitization effect compared to the control group (4-8 Gy irradiation + 4 μM 5-Fu, within which range, significant sensitization was observed); and siRNA-2 showed a superior sensitization effect compared to siRNA-1.

[0091] The CCK8 assay focuses on the overall metabolic activity and short-term proliferation trend of the cell population; the clonogenic assay focuses on the long-term proliferation and clonogenic capacity of individual cells. These two experiments, from different perspectives, reflect the effects of siRNA-1 and siRNA-2 targeting the EMILIN1 gene in CAF cells on the chemoradiosensitivity of colorectal cancer cells. The results of the two assays are consistent. These experimental results indicate that siRNA-1 and siRNA-2 targeting the EMILIN1 gene can enhance the sensitization of concurrent chemoradiotherapy in colorectal cancer cells, with siRNA-2 showing a more significant effect. CAF cells transfected with siRNA (with downregulated EMILIN1 protein expression) can increase the chemoradiosensitivity of colorectal cancer cells.

[0092] Example 4: siRNA sensitizes the radiotherapy and chemotherapy efficacy of colorectal cancer cells in vivo.

[0093] CAFs cells and HCT-116 cells were mixed at a 1:1 ratio and then subjected to 2×10⁻⁶ ppm. 6 One injection per mouse was administered subcutaneously into the groin area of ​​nude mice. The tumor was allowed to grow to 150 mm. 3 For volume determination, nude mice were randomly divided into two groups: a control group and an interference group. After grouping, the length and width of subcutaneous tumors in the mice were measured and recorded every two days. The tumor volume was calculated using the formula: Volume (mm²) 3 ) = (width) 2 (×length) / 2.

[0094] For the interference group, the expression of EMILIN1 in the CAF cells of the nude mice was knocked down. The control group was injected with normal saline intratumorally, and the interference group was injected with cholesterol-modified siRNA for knocking down EMILIN1 (5 nmol / kg, siRNA-2) intratumorally, once every 2 days until the end of the experiment (to the 14th day). The method of using cholesterol-modified siRNA is as follows: the 3' end of the sense strand of siRNA is covalently connected to cholesterol through a click chemistry (azido-alkynyl cycloaddition reaction), which is a conventional method in the prior art. Cholesterol modification can improve its cell penetration and in vivo stability. Cholesterol-modified siRNA can be commissioned from a biotechnology company. The general preparation method is as follows: take a clean RNase-free 1.5 mL centrifuge tube and place it in an ice box. First, add 50 μL DEPC water-dissolved alkyne-modified siRNA (final concentration 1 μM, the sense strand of siRNA is alkynylated at the 3' end), then slowly add 10 μL cholesterol-azide derivative DMSO solution (10 mM), 5 μL CuSO4 aqueous solution (10 mM), 10 μL TBTA ligand (10 mM, previously dissolved in DMSO), and finally quickly inject 5 μL of freshly prepared sodium ascorbate (100 mM). Tighten the tube cap, tap the tube wall three times to make the liquid surface rotate slightly, and place it in a 37°C metal bath after instantaneous centrifugation. Avoid light and stand for 2 h. Shake it every 30 min during this period. The originally clear solution gradually turns blue-green, indicating the formation of a Cu(I)-catalyzed triazole ring. Add 10 μL of 0.5 M EDTA to terminate the reaction, mix well, then add 3 times the volume of -20°C pre-cooled ethanol, gently invert several times, and place it in a -80°C refrigerator to precipitate overnight. The next day, centrifuge at 12 000 rpm and 4°C for 15 min. A white cholesterol-siRNA precipitate can be seen at the bottom of the tube. Discard the supernatant and air dry at room temperature for 5 min. Resuspend the precipitate with 200 μL of 0.1 M TEAA buffer, filter it through a 0.22 μm filter membrane, inject it into a C18 reverse-phase column, elute it with a 5-95% acetonitrile gradient, monitor it at 260 nm, collect the main peak around 22 min, desalt it with an NAP-10 column, and freeze-dry it to obtain cholesterol-modified siRNA.

[0095] Synchronous radiotherapy and chemotherapy began on the day of siRNA injection. The nude mice were injected with 5-Fu (15 mg / kg / d) intraperitoneally, and the tumor was irradiated with X-rays at 2 Gy / d. Radiotherapy and chemotherapy were performed daily for a total of 5 days. After the experiment was terminated, the tumor was measured for volume and photographed. Part of the tumor tissue was subjected to multicolor immunofluorescence detection to verify the siRNA knockdown efficiency.

[0096] Figure 4As shown in AB, after knocking down EMILIN1, the tumor of mice was significantly reduced compared with the control group after synchronization radiotherapy and chemotherapy. The tumor volume of the NC group (negative control) continued to grow rapidly over time; the tumor volume growth of the si-EMILIN1 group, which used siRNA to interfere with the expression of EMILIN1 gene, was significantly slowed down. Therefore, reducing the expression of EMILIN1 can significantly inhibit the growth of tumor under the condition of radiotherapy and chemotherapy, and increase the sensitivity of tumor to radiotherapy and chemotherapy. Figure 4 As shown in C, the expression of EMILIN1 (green fluorescence) in the knockdown group was significantly lower than that in the control group, and the expression of αSMA (white fluorescence) in the knockdown group was significantly lower than that in the control group. It is proved that si-EMILIN1 can enhance the sensitivity of colorectal cancer to radiotherapy and chemotherapy.

[0097] Based on the above experimental results, it is proved that the use of siRNA to knock down EMILIN1 can enhance the radiotherapy and chemotherapy effect of colorectal cancer, and has the effect of improving the radiotherapy and chemotherapy resistance of colorectal cancer.

[0098] Comparative Example 1

[0099] The prior art document “Samanta Muzzin; “Unraveling EMILIN-1: A Multifunctional ECM Protein with Tumor-Suppressive Roles” Mechanistic Insights into Cancer Protection Through Signaling Modulation and Lymphangiogenesis Control; Cells. 2025 Jun 20; 14(13): 946. doi: 10.3390 / cells14130946” mentions that in the colon, the EMILIN-1 tumor-suppressive mechanism during chemically-induced carcinogenesis involves the suppression of AKT and ERK activation. EMILIN-1 deficiency results in increased tumor burden, particularly high-grade adenomas, and enhanced tumor growth. It is also mentioned that EMILIN-1 plays a key regulatory role in the tumor microenvironment (TME), preventing pathological lymphangiogenesis while maintaining lymphatic vessel (LV) integrity. Its functional importance is particularly evident in the context of inflammation, as impaired lymphangiogenesis—typically crucial for inflammation resolution and immune cell clearance—continuously triggers chronic inflammation, thus promoting the progression of malignant tumors.This relationship is particularly evident in the colon, where EMILIN-1 deficiency exacerbates tumor development through combined effects of lymphatic dysfunction and chronic inflammation (Original text: EMILIN-1 serves as a critical regulator in the TME, maintaining LV integrity while preventing pathological lymphangiogenesis. Its functional importance is particularly evident in inflammatory contexts, where impaired lymphangiogenesis—normally essential for inflammation resolution and immune cell clearance—can perpetuate chronic inflammation that promotes malignant progression. This relationship is exemplified in the colon, where EMILIN-1 deficiency exacerbates tumor development through combined effects of lymphatic dysfunction and chronic inflammation.).

[0100] In addition to the above, this document mainly expresses that EMILIN-1 is mainly a tumor suppressor factor:

[0101] Core mechanism: EMILIN-1 binds to cell surface α4β1 / α9β1 integrin through its C-terminal gC1q domain.

[0102] Signaling pathway: This binding activates the PTEN protein, thereby inhibiting the pro-proliferative and pro-survival signaling pathways such as ERK and AKT. At the same time, it can also inhibit cell proliferation by ubiquitination and degradation of HRasGTP, a proto-oncogene protein.

[0103] Animal model evidence: Mice lacking EMILIN-1 (Emilin1 - / - ) or whose function is specifically disrupted (E1-E955A) show faster, heavier, and earlier tumor growth in various cancer models such as skin cancer, breast cancer, gastric cancer, and colon cancer.

[0104] The above shows that the prior art research shows that the absence of EMILIN-1 promotes the progression of cancer including colorectal cancer, and the overexpression of EMILIN-1 has the effect of inhibiting cancer. The trend of the effect of EMILIN-1 reported by the prior art and the trend of the effect of EMILIN-1 found in the present scheme are opposite. The main reason for the research results of the present scheme is that the present scheme adjusts the expression of EMILIN-1 in CAFs cells (inhibits expression), thereby achieving the enhancement of the radiosensitivity and chemosensitivity of colorectal cancer cells or tumor tissues. The effect of achieving the effect of the present scheme includes two key technical points: using siRNA to inhibit the expression of EMILIN-1 in CAFs cells, and at the same time, performing radiotherapy and chemotherapy on colorectal cancer cells or tumor tissues.

[0105] Comparative Example 2

[0106] The prior art document “Alessandra Capuano; Abrogation of EMILIN1-β1 integrin interaction promotes experimental colitis and colon carcinogenesis; Matrix Biol. 2019 Oct: 83: 97-115. doi: 10.1016 / j.matbio.2019.08.006. Epub 2019 Aug 31.” mentions: Colorectal cancer is one of the first tumor types for which a functional link between its development and inflammation was described; however, microenvironmental signals that influence colorectal cancer progression are still poorly understood. Here, we demonstrate that the expression of the extracellular matrix (ECM) molecule EMILIN-1 is able to prevent AOM-DSS-induced tumor development. In fact, after AOM-DSS treatment, EMILIN-1 knockout (Emilin1 - / - , E1 - / -) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar resultswere obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model,expressing a mutant EMILIN-1 unable to interact with α4 / α9β1 integrins.)。 - / - (E1 - / - ) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar resultswere obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model,expressing a mutant EMILIN-1 unable to interact with α4 / α9β1 integrins.)。

[0107] As reported in the literature, EMILIN-1 protein is a molecule that inhibits colorectal cancer, and upon inhibition of the expression of EMILIN-1 protein, the tumor further worsens. In the present protocol, however, upon inhibition of EMILIN-1 protein in CAFs cells using siRNA, the further development of the tumor was inhibited (by increasing the sensitivity of the tumor to radiotherapy and chemotherapy). Thus, in the application mode of the present protocol, the EMILIN-1 protein and the corresponding siRNA exerted an unexpected technical effect.

[0108] The above-mentioned are only embodiments of the present application, and common technical solutions and / or common knowledge of the scheme are not described in detail. It should be pointed out that, for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, and these will not affect the effect and practicality of the patent. The protection scope claimed in the present application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. The application of an EMILIN1 gene-inhibiting agent in the preparation of a radiotherapy and chemotherapy sensitizer, characterized in that: The formulation includes siRNA; the siRNA is siRNA-1 or siRNA-2. The positive strand of siRNA-1 is: 5'-GAGUGGAGACAUAUGUCAA / DT / / DT / -3'; The antisense strand of siRNA-1 is: 5'-UUGACAUAUGUCUCCACUC / DT / / DT / -3'; The positive strand of siRNA-2 is: 5'-AGACAGUGACCGACAUGGA / DT / / DT / -3'; The antisense strand of siRNA-2 is: 5'-UCCAUGUCGGUCACUGUCU / DT / / DT / -3'; The radiotherapy and chemotherapy sensitizer is used to increase the sensitivity of colorectal cancer cells to radiotherapy and chemotherapy.

2. The application of the EMILIN1 gene-inhibiting agent according to claim 1 in the preparation of radiotherapy and chemotherapy sensitizers, characterized in that, The siRNA is cholesterol-modified siRNA; or, the formulation is tumor-associated fibroblasts transfected with the siRNA.

3. The application of the EMILIN1 gene-inhibiting agent according to claim 2 in the preparation of radiotherapy and chemotherapy sensitizers, characterized in that, Cholesterol-modified siRNA is formed by linking cholesterol to the positive strand of siRNA via a click chemical reaction; cholesterol-modified siRNA is used to apply to tumor sites where tumor-associated fibroblasts are present.

4. The application of the EMILIN1 gene-inhibiting agent according to claim 2 in the preparation of radiotherapy and chemotherapy sensitizers, characterized in that, Tumor-associated fibroblasts transfected with the siRNA were prepared by culturing tumor-associated fibroblasts in a culture medium containing a mixture of A and B to obtain tumor-associated fibroblasts transfected with the siRNA. The AB mixture was prepared as follows: the culture medium was mixed with liposome 3000 and allowed to stand to form system A; the culture medium was mixed with the siRNA and allowed to stand to form system B; system A and system B were mixed and allowed to stand to obtain the AB mixture.

5. A product characterized in that, Including siRNA; The siRNA is either siRNA-1 or siRNA-2: The positive strand of siRNA-1 is: 5'-GAGUGGAGACAUAUGUCAA / DT / / DT / -3'; The antisense strand of siRNA-1 is: 5'-UUGACAUAUGUCUCCACUC / DT / / DT / -3'; The positive strand of siRNA-2 is: 5'-AGACAGUGACCGACAUGGA / DT / / DT / -3'; The antisense strand of siRNA-2 is: 5'-UCCAUGUCGGUCACUGUCU / DT / / DT / -3'.

6. A product according to claim 5, characterized in that, The siRNA is cholesterol-modified siRNA, and the product is intended to be applied to tumor sites where tumor-associated fibroblasts are present.

7. The product according to claim 5, characterized in that: The product is tumor-associated fibroblasts transfected with the siRNA.

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