CN115040654B

CN115040654B - Application of exosomes for inhibiting hsa_circ_0002557 expression in treatment of esophageal cancer and medicines

Info

Publication number: CN115040654B
Application number: CN202210684857.4A
Authority: CN
Inventors: 刘冉; 孙明军; 汤颖; 高志奎
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2022-06-13
Filing date: 2022-06-13
Publication date: 2023-11-07
Anticipated expiration: 2042-06-13
Also published as: CN115040654A

Abstract

The application discloses application of an exosome for inhibiting hsa_circ_0002557 expression in treatment and medicine of esophageal cancer, wherein hsa_circ_0002557 is highly expressed in esophageal squamous cell carcinoma tissues and cancer cell source exosomes. The application inhibits invasion and migration of esophageal squamous cell carcinoma by inhibiting exosome from loading hsa_circ_0002557 medicine. The esophageal cancer microenvironment shuttle hsa_circ_002557 plays a role in promoting cancer in esophageal squamous cell carcinoma by remodelling into fibrous extracellular matrix, can be used as a treatment target for inhibiting activation of cancer-related fibroblasts to prevent migration and invasion of esophageal squamous cell carcinoma, and indicates a new direction for the cancer-related fibroblasts as potential treatment targets of esophageal cancer.

Description

Application of exosomes for inhibiting hsa_circ_0002557 expression in treatment of esophageal cancer and medicines

Technical Field

The application relates to the field of biological medicine, in particular to application of exosomes for inhibiting hsa_circ_0002557 expression in treatment of esophageal cancer and medicines.

Background

Esophageal cancer is a tumor of digestive tract with high morbidity, mortality, recurrence rate and other characteristics. According to the data report of the international cancer research institution in 2020, the incidence rate (60 ten thousand) and the death rate (54 ten thousand) of esophageal cancer are the eighth and sixth of global cancers; in China, the incidence rate (32 ten thousand) and the death rate (30 ten thousand) of esophageal cancer are the sixth and fourth cancer in China, and the incidence rate and the death rate of the esophageal cancer are more than half of those of the world. The pathological types of the esophagus cancer mainly comprise squamous cell carcinoma and adenocarcinoma, and 90% of pathological types of the esophageal cancer are esophageal squamous cell carcinoma in high-incidence areas of the esophagus cancer such as south Africa, east Asia and the like. The regions of Henan, hebei, shanxi, fujian, jiangsu and the like in China are also areas with high incidence of esophageal cancer, and become a great public health problem threatening the health of people. In recent years, although the rapid progress of medicine is improved to a certain extent in the aspect of esophageal cancer treatment, such as open surgery, minimally invasive surgery, radiotherapy and chemotherapy, immunotherapy, targeted therapy and the like, the survival rate of patients in 5 years is still lower than 15%. Poor prognosis is associated with tumor characteristics such as self-sufficient growth signals, anti-growth signal insensitivity, avoidance of apoptosis, unlimited replicative capacity, persistent angiogenesis and tissue invasion metastasis. On this basis, hanahan and Weinberg re-examined and expanded these six features, on this basis increased genomic instability, altered energy metabolism, immune killing avoidance and promotion of tumor inflammation, also covering the tumor microenvironment (Tumor microenvironment, TME).

In recent years, more and more studies have shown that TMEs, including extracellular matrix, soluble molecules, and tumor stromal cells, are the "home" for cancer cell growth, with about 50% of tumor stromal cells being composed of cancer-associated fibroblasts (cancer associated fibroblasts, CAFs). CAFs can not only inhibit immune cell functions by secreting various cytokines or metabolites, but also promote tumor development, invasion and metastasis; CAFs also have the effect of modeling the extracellular matrix of a tumor, forming a drug or therapeutic immune cell permeation barrier to prevent the deep penetration of drugs and immune cells into tumor tissue, thereby reducing the therapeutic effect of the tumor. The subject group previously found that the esophageal cancer cell-derived conditioned medium induced activation of normal fibroblasts (Normal fibroblasts, NFs) to CAFs. The exosomes are used as key components of the conditioned medium, and can modify the microenvironment of the recipient tissue to make it more suitable for the growth of tumor cells. However, at present, the mechanism how cancer cell-derived exosomes mediate NFs "traitors" has not been elucidated. Therefore, we have urgent need to verify the mechanism of action of esophageal cancer-derived exosomes in promoting NFs activation and remodeling of extracellular matrix to promote esophageal cancer progression. By elucidating the process, key targets of tumor cells for changing tumor microenvironment can be identified, biomarkers of high-risk group screening and clinical diagnosis and treatment key groups of esophageal cancer are clarified, esophageal cancer patients with high malignancy degree are found early, and novel means are provided for inhibiting tumor growth and targeted therapy by regulating and controlling CAFs or overcoming barrier function of CAFs.

Disclosure of Invention

Aiming at the defects of the prior art, the application provides application of exosomes for inhibiting hsa_circ_0002557 expression in the treatment of esophageal cancer and medicines.

The aim of the application can be achieved by the following technical scheme:

a medicament for treating esophageal cancer, comprising: an hsa_circ_0002557 expression inhibitor, wherein the sequence of hsa_circ_0002557 is SEQ ID NO.1. Such agents include, but are not limited to, genetic agents and chemical agents that inhibit hsa_circ_0002557 expression, such as RNA interference agents against hsa_circ_0002557, gene editing agents, inhibitors of various drug delivery systems, inhibitors or blockers of the gene's loop-forming mechanism. Wherein, hsa_circ_0002557 is derived from a circBank database.

Optionally, the inhibitor is an siRNA.

Optionally, a delivery vehicle for the inhibitor is also included.

Optionally, the siRNA sequences are SEQ ID NO.2 and SEQ ID NO.3.

Optionally, the delivery vehicle is an exosome.

On the other hand, the application also provides an esophageal cancer screening kit which comprises a reagent for detecting the expression quantity of hsa_circ_ 0002557.

Alternatively, the reagent is an RT-qPCR reagent for detecting the expression level of hsa_circ_ 0002557.

Alternatively, the two specific primer sequences of the RT-qPCR are SEQ ID NO.4 and SEQ ID NO.5 respectively.

In yet another aspect, the application discloses the use of an hsa_circ_0002557 expression inhibitor in the manufacture of a medicament for inhibiting cancer-associated fibroblast production.

The application has the beneficial effects that:

the function of the high-load hsa_circ_0002557 exosome in esophageal squamous cell carcinoma cells is proved by preparing the high-load hsa_circ_0002557 exosome, and the high-load hsa_circ_0002557 exosome can be found to be capable of remarkably activating NFs, and the exosome which inhibits the expression of hsa_circ_0002557 can be remarkably inhibited by remodelling CAFs extracellular collagen fiber rearrangement to promote invasion and migration of esophageal squamous cell carcinoma, so that the exosome which is a very important molecular target for esophageal squamous cell carcinoma treatment. Provides a new effective treatment method for the treatment of tumor patients, breaks through the traditional view of taking tumor cells as the center, solves the problems of single esophageal squamous cell carcinoma drug treatment, poor treatment effect and the like, and has good drug development prospect.

Drawings

The application is further described below with reference to the accompanying drawings.

FIG. 1 is a schematic diagram of the biosynthesis and structure of hsa_circ_ 0002557;

FIG. 2 shows the expression levels of esophageal squamous cell carcinoma tissue, esophageal carcinoma EC109 and EC9706 cell-derived exosomes hsa_circ_ 0002557;

FIG. 3 is the expression levels of hsa_circ_0002557 in over-expressing EC109 and EC9706 cells and exosomes;

fig. 4: activating NFs cell-associated marker protein levels for overexpression of hsa_circ_0002557 exosomes;

FIG. 5 is a graph showing the results of extracellular collagen fiber arrangement for the activation NFs of an over-expressed hsa_circ_0002557 exosome;

fig. 6: regulating esophageal squamous cell carcinoma cell migration and invasion result graphs for overexpression of hsa_circ_0002557 exosomes activated to CAFs source supernatant;

fig. 7: loading si_hsa_circ_0002557 exosomes for a chemical reagent method to inhibit expression of esophageal cancer cells hsa_circ_ 0002557;

fig. 8: to interfere with hsa_circ_0002557 expression levels in EC109 and EC9706 cells and exosomes;

fig. 9: activating NFs cell-associated marker protein levels for interfering hsa_circ_0002557 exosomes;

FIG. 10 is a graph showing the results of the arrangement of extracellular collagen fibers that interfere with the activation NFs of hsa_circ_0002557 exosomes;

fig. 11: results of modulation of esophageal squamous cell carcinoma cell migration and invasion by supernatant of sources of CAFs for interfering with hsa_circ_0002557 exosomes activation.

Detailed Description

The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Example 1: analysis of expression level of hsa_circ_0002557 in esophageal squamous cell carcinoma tissue and cancer cell derived exocrine

1. RNA extraction of esophageal squamous cell carcinoma tissue sample

1) After approval by the medical ethics committee of southeast university, patients/families informed consent were selected from 64 patients with esophageal squamous cell carcinoma, and 64 cancer tissues and corresponding paracancerous tissues were collected altogether;

2) 0.1cm is taken ³ Shearing the tissues by scissors, putting the crushed tissues into a 1.5mL EP tube, adding 1mL Trizol and 3 grinding steel balls, grinding for 10min at 70Hz (pre-cooling a grinding box in advance), and subsequently operating according to the conventional extraction step of the Trizol reagent;

3) Taking 1 mu L of RNA sample, detecting the concentration and purity of the RNA sample by an ultraviolet spectrophotometer, wherein the ratio of A260 to A280 of the obtained RNA sample is 1.8-2.0.

2. Esophageal cancer cell-derived exosome enrichment and RNA and protein extraction

1) Harvesting supernatant of cultured esophageal cancer cells EC109, EC9706 and normal esophageal epithelial cells Het-1A 72 h;

2) Enrichment of exosomes by ultra high speed centrifugation. That is, the supernatant was centrifuged at 300g/10min,1200g/20min and 10,000g/30min at 4℃to remove dead cells and debris; the sediment is obtained by ultra-high speed centrifugation at 200,000g/2H and 4 ℃, and the pure exosome is obtained by ultra-high speed centrifugation at 100,000g/2H and 4 ℃ after the PBS buffer solution is resuspended. After PBS is resuspended, the mixture is preserved at the temperature of minus 80 ℃ for standby;

3) The Trizol method extracts exosome RNA. Namely, 1 mLTrilzol is added after the ultrapure exosomes are obtained, and the operation is carried out according to the conventional extraction steps of Trizol reagent;

4) Extracting exosome protein by solution method. That is, after obtaining ultrapure exosomes, 200. Mu.L of IPA lysate (containing 1% protease inhibitor) was added and lysed at 4℃for 15min,12000g/10min, and centrifuged at 4℃to obtain the supernatant. Quantification was performed by BCA method and stored at-80℃after denaturation for further use.

RT-qPCR quantification of hsa_circ_0002557 expression levels

1) Reverse transcription of RNA into cDNA: reverse transcription was performed using MMLV reverse transcriptase, and the reverse transcription reaction system and conditions are as follows:

reaction conditions: the reaction was carried out at 37℃for 60min, at 90℃for 10min and at 4 ℃.

2) RT-qPCR: specific primers were designed based on hsa_circ_0002557 structure and interface sequence (see FIG. 1 for results):

the nucleotide sequence of hsa_circ_0002557-F is (SEQ ID NO. 4); the nucleotide sequence of hsa_circ_0002557-R is (SEQ ID NO. 5);

reaction system and conditions:

reaction conditions: preheating at 94 ℃ for 5min,94 ℃ for 30s,60 ℃ for 30s, reacting for 40 cycles, collecting fluorescence at 60 ℃ in each cycle, heating at 60-95 ℃, collecting fluorescence, and calculating a melting curve.

Western Blot detection of exosome marker proteins

1) SDS-PAGE electrophoresis: preparing 10% polyacrylamide gel by using a quick preparation kit of PAGE gel, solidifying, using 1 Xelectrophoresis buffer solution, loading 10 mug of sample per hole, changing the voltage into 120V after 80V electrophoresis for 30min, and stopping electrophoresis when a sample electrophoresis indication belt runs to the lower edge of a glass plate;

2) Transferring and sealing: the membrane transfer was performed at 4℃and at a constant pressure of 70V for 4H in the order of negative electrode-gel-PVDF membrane-filter paper-positive electrode. Blocking 2H with TBST solution containing 5% nonfat dry milk at room temperature;

3) Antibody hybridization: diluting primary antibodies (CD 36, GM130 and Calnexin) to proper concentrations, placing the primary antibodies on a shaking table at 4 ℃ to incubate PVDF membrane overnight, rinsing TBST for 3 times, placing the primary antibodies on a corresponding secondary antibody room temperature shaking table to incubate for 2H, and developing the primary antibodies on a chemiluminescent instrument;

4) Chemiluminescence: the chemiluminescent liquid A and the chemiluminescent liquid B are mixed in equal volume, and the mixture is covered on the surface of the film for development.

5. Data processing and analysis

The expression level of the to-be-detected sample has_circ_002557 is calculated by a CT value and an internal reference GAPDH CT value method to obtain delta CT, and the larger the delta CT value is, the lower the actual expression quantity is.

6. Experimental results and analysis

In this cohort esophageal squamous cell carcinoma patient cancer tissue hsa_circ_0002557 expression levels were significantly higher than in the paracancerous tissue (fig. 2A); successful enrichment of EC109, EC9706 and Het-1A cell-derived exosomes (fig. 2B-D) with hsa_circ_0002557 expression in cancer cell-derived exosomes higher than normal esophageal epithelial cell-derived exosomes levels (fig. 2E), the differences being statistically significant, suggesting hsa_circ_0002557 is an important molecular target for the treatment of esophageal squamous cell carcinoma against the tumor microenvironment.

Example 2 high load hsa_circ_0002557 exosome weight-loss plastic CAFs extracellular collagen fibers

1. Preparation of highly loaded hsa_circ_0002557 exosomes

1) Construction of hsa_circ_0002557 overexpression vector

(1) The plasmid and the target gene were digested simultaneously, and the reaction system (100. Mu.L) was as follows: plasmid vector (5. Mu.g), 10 XBuffer (10. Mu.L), sfaAI (20U), MLuI (20U); cleavage reaction conditions: reacting at 37 ℃ for 2H and at 65 ℃ for 15min;

(2) performing horizontal gel verification and recovery of enzyme digestion products, taking 5 mu L of enzyme digestion products, performing horizontal gel electrophoresis by using 1.5% agarose, and cutting and recovering required double enzyme digestion strips by using DL10000 DNA markers as references;

(3) the ligation of the target gene fragment to the vector was carried out as follows (20. Mu.L): linear vector (100 ng), fragment of interest (30 ng), T4 DNA library (1. Mu.L), 10 XT 4 library Buffer (2. Mu.L);

(4) purifying the connection product, adding 2 mu L of 3M sodium acetate (PH=5.2) into the reaction solution after the reaction is finished, adding 50 mu L of precooled absolute ethyl alcohol, standing for 1H at minus 20 ℃, centrifuging to recover precipitate, washing twice with precooled 75% ethanol, drying the precipitate, and adding 25 mu LTE solution for dissolution;

(5) transformation of recombinant plasmid, dissolving the prepared competent DH 5. Alpha (50. Mu.L) cells at 4℃and mixing the ligation product (2. Mu.L) with a 1.5mL EP tube, placing on ice for 30min, heat-shocking for 90s at 42℃and then adding the cell mixture to 500. Mu.L LB liquid medium after 5min ice-bath, shaking the culture at 37℃and 150rpm in an air bath for 1H, coating 100. Mu.L of the mixture on a plate containing 100. Mu.g/mL ampicillin, and culturing overnight at 37 ℃.

(6) Plasmid extraction and identification

Single DH5 alpha colony is selected and inoculated in LB liquid medium containing 100 mug/mL of ampicillin, shaking culture is carried out for 8-12H by a shaking table at 37 ℃ and 200rpm, the plasmid is extracted by using a DP117 endotoxin-free plasmid large extraction kit, double enzyme digestion is carried out on the extraction quality by using SfaAI and MLuI, and agarose gel electrophoresis verification is carried out on enzyme digestion products. The gel electrophoresis insert DNA fragment bands were recovered and sent to Sanger sequencing, the sequencing primers were as follows:

5 'sequencing primer (CMV-F, SEQ ID NO. 6) and 3' sequencing primer (V2, SEQ ID NO. 7). And (3) performing amplification culture on the bacterial liquid qualified in plasmid sequencing, and extracting plasmids by using a DP117 endotoxin-free plasmid large extraction kit for later-stage lentivirus packaging and corresponding experiments.

2) Construction of Hsa_circ_0002557 overexpressing cell model

On the basis of constructing Hsa_circ_0002557 over-expression vector in early stage, taking cell count in logarithmic growth phase, and then taking cell count in 9×10 ⁴ The cells were plated in 6-well plates, each well was supplemented with no-double antibody cell culture medium to 4mL in an incubator for 19H, and transfection was performed according to Lipofectamine 2000 transfection reagent protocol, as follows:

(1) 12. Mu.L of Lipofectamine 2000 transfection reagent was diluted in 2.0mL EP tubes containing 300. Mu.L 1640-medium (without FBS and double antibody), 100. Mu.L 1640 medium was added to each, and 2. Mu.L Lv_circ_0002557 and NC_circ_0002557 were added, respectively, and incubated in a greenhouse for 5min;

(2) after 5min incubation, the Lv_circ_0002557 and NC_circ_0002557 dilutions (100. Mu.L) were mixed with Lipofectamine 2000 dilutions (100. Mu.L) respectively and incubated in the greenhouse for 20min;

(3) discarding the original culture solution of the 6-hole cell culture plate, and adding 800 mu L of 1640 complete culture solution into each hole;

(4) adding 200 mu L of the mixed solution in the step (2) into a 6-hole cell culture plate, wherein the total volume is 1000 mu L, and continuously culturing in a cell culture box for 24H after uniformly mixing;

(5) after 24H, the culture was continued with a 1640 complete medium, and hsa_circ_0002557 overexpression efficiency was detected by RT-qPCR.

3) High load hsa_circ_0002557 exosome enrichment

The Lv_circ_0002557 and NC_circ_0002557 supernatants of culture 72H were harvested and enriched for exosomes according to the method of example 2.2).

3. High load hsa_circ_0002557 exosomes activate NFs to remodel extracellular collagen fiber arrangement

1) High-load hsa_circ_0002557 exosomes convert NFs to CAFs

Lv_circ_0002557 and NC_circ_0002557EC109 cell-derived exosomes were enriched. NFs cells in logarithmic growth phase were collected at 1.0X10 ⁵ The wells were plated in six well plates, 24H followed by 500ng/mL exosomes for NFs cells 72H. Cells were harvested for total protein extraction to assess cancer-associated fibroblast activation markers α -SMA, FAP and Vimentin levels.

2) Visual analysis of extracellular collagen fiber arrangement by multiphoton fiber imaging technology

(1) Preparation of cell-containing three-dimensional collagen: CAFs co-cultured with 72H loaded with Lv_circ_0002557 and NC_circ_0002557 exosomes were harvested to prepare single cell suspensions (6.6X10) ⁶ /mL) and placed in an ice bath. 200. Mu.L of rat tail collagen type I (5 mg/mL) was added to 12. Mu.L of 0.1mol/L NaOH and immediately mixed, followed by 23. Mu.L of 10 XPBS. After adjusting the pH to 7.0, 760. Mu.L of the cell suspension was added, and immediately after mixing, the mixture was added to a culture vessel. The culture vessel is placed at room temperature for 20min to be gelled and fixed, then a proper volume of cell culture solution is added, and the cell culture solution is transferred into an incubator for culturing for 24H.

(2) Multiphoton fiber imaging: this is done with a custom vertical laser scanning microscope. The sample was stored between two coverslips and imaged without any staining. A 1045nm (green-collagen fiber) wavelength laser and 880nm (red-fibroblast) wavelength were set for photographing. 3 typical z-stacks of 270 μm by 100 μm-150 μm3 were recorded for each sample, with a z-order of 2 μm, a pixel size of 0.4 μm, and a pixel rate of 100kHz. The two-dimensional images are merged using Image j.

The results show that: the expression level of hsa_circ_0002557 was 185.96 and 20.89 times that of control cells (FIG. 3A), corresponding to in vivo hsa_circ_0002557 expression in cell-derived exosomes being 16.62 and 4.84 times higher than that of control cells, when transiently transfected with the Lv_hsa_circ_0002557 plasmid in EC109 and EC9706 cells (FIG. 3B). 500ng/mL of exosome co-cultured NFs cells showed that: high load hsa_circ_0002557 exosomes significantly elevated CAFs marker protein α -SMA, FAP, and Vimentin levels (fig. 4). Importantly, the collagen fibers of the high-load hsa_circ_0002557 exosome group are surrounded into fiber cells to form linear radial shapes, the fibers are orderly arranged, the angle of the collagen fibers relative to the fiber cells is 90.8 degrees on average, and the collagen fibers are approximately perpendicular to the fiber cells; in contrast, in the control group, the morphology of the collagen fibers was crimped, the fiber distribution was disturbed, and the angle of the collagen fibers relative to the fibroblasts was 179.5 ° on average, approximately parallel to the fibroblasts (fig. 5). It is suggested that the highly loaded hsa_circ_0002557 exosomes, after promoting fibroblast activation, can change the morphology of secreted collagen fibers, the fiber arrangement becomes ordered, and the relative angle of collagen fibers is changed.

Example 3CAFs cells promote the biotypic transformation of esophageal squamous cell carcinoma cells

1) Cell migration ability assay: cell migration capacity assays were performed using an 8 μm pore size Transwell chamber. EC109 and EC9706 cells incubated with CAFs supernatant for 24H were taken, and single cell suspensions were prepared using FBS-free medium after pancreatin digestion, and each chamber was inoculated with 5×10 cells ⁴ Cells, 200. Mu.L of FBS-free medium was supplemented to the upper chamber of the cell, and 600. Mu.L of 20% FBS-containing complete medium was added to the lower chamber. Taking out the cell after conventional culture for 24H, sucking residual liquid in the cell by using a gun head, wiping off cells penetrating through the inner side of the cell by using a cotton swab in a rotating way, soaking the cell in methanol for 10min, taking out the cell, dying the cell by using 0.1% crystal violet dye after the membrane is air-dried, dying the cell for 15min at normal temperature, leaching the cell by using PBS, and placing the cell back to a 24-hole plate after natural air-drying. And 5 visual fields are randomly selected from each cell for photographing under the observation of a microscope, and the number of the cells penetrating the membrane is counted.

2) Cell invasiveness detection: dissolving Matrigel matrix gel at-20deg.C in refrigerator at 4deg.C, and pre-cooling the Transwell chamber in refrigerator at 4deg.C; diluting matrigel with FBS-free medium according to the ratio of 1:9, rapidly adding diluted matrigel into the cells, and adding 50 mu L of matrigel into each cell; heating the small chamber in a 37 ℃ incubator for 1H to solidify the matrigel, taking out the small chamber, and sucking the upper layer of non-solidified liquid; digestion and collection of 24H EC109 and EC9706 cells cultured on CAFs supernatant, single cell suspensions were prepared using FBS-free medium, according to 5X 10 ⁵ Well, cells were added to the chamber, and 600. Mu.L of complete medium containing 50% FBS was added to the lower chamber; after culturing for 24H, taking out the cell, wiping off cells and matrigel which do not penetrate through the membrane by using a cotton swab, fixing with methanol, airing, and dyeing by using 0.1% crystal violet; and 5 visual fields are randomly selected from each cell for photographing under the observation of a microscope, and the number of the cells penetrating the membrane is counted.

The results show that: high-load hsa_circ_0002557 exosome-activated CAFs can promote the ability of esophageal squamous cell carcinoma EC109 and EC9706 to migrate (fig. 6A) and invasion (fig. 6B) cells, suggesting that tumor microenvironment CAFs are important in promoting the progression of esophageal cancer.

Example 4 inhibition of exosome load hsa_circ_0002557 exosome drugs inhibit esophageal squamous cell carcinoma cell invasion and migration by modulating extracellular matrix remodeling

1) Loading si_hsa_circ_0002557 exosomes inhibits expression of esophageal cancer cells hsa_circ_0002557

(1) Preparation of si_hsa_circ_0002557 exosomes by chemical reagent method

Mu.l of si_Hsa_circ_0002557 and 4. Mu.l of Lipofectamine 2000 were incubated in 100. Mu.l of 1640 medium at room temperature for 5min, mixed for 20min and incubated with 300. Mu.l (15. Mu.g) of EC 109-derived exosome suspension for 30min. The si-lipid complexes that did not bind to exosomes were removed 4 times through Millipore 0.5ml 100kd ultrafiltration tube to obtain loaded si_hsa_circ_0002557 exosomes.

(2) siRNA exosomes are loaded to inhibit expression of esophageal cancer cells hsa_circ_0002557

Taking 5.0X10 ⁴ Well EC109 cells were plated in 12-well plates, 24H followed by co-incubation with 500ng/ml of the above exosomes at 0H,6H,12H,24H and 48H. Inhibition of hsa_circ_0002557 on EC109 cells was examined by RT-qPCR.

2) Low-load hsa_circ_0002557 exosomes reprofile fibroblast extracellular matrix

(1) Low expression hsa_circ_0002557 esophageal squamous cell carcinoma cell construction

The specificity si_hsa_circ_0002557 (SEQ ID No.2, SEQ ID No. 3) and si_nc (SEQ ID No.8, SEQ ID No. 9) were designed and their sequences were obtained through the Invitrogen website. Transfection was performed according to example 2.1.2), 24H followed by a change to 1640-complete medium for further incubation for 72H, and hsa_circ_0002557 interference efficiency was detected using RT-qPCR.

(2) Exosome enrichment to inhibit circ 0002557 expression

The supernatants of si_circ_0002557 and nc_circ_0002557 of culture 72H were harvested and enriched for exosomes according to the method of example one 2.2).

(3) Low-load hsa_circ_0002557 exosomes inhibit fibroblast activation

Enrichment of si_circ_0002557 and si_nc EC109 cell-derived exosomes. NFs cells in logarithmic growth phase were collected at 1.0X10 ⁵ The wells were plated in six well plates, 24H followed by 500ng/mL exosomes for NFs cells 72H. Harvesting cells for extraction of total protein for assessment of cancer-relatedFibroblast activation markers α -SMA, FAP and Vimentin levels.

(4) Low-load hsa_circ_0002557 exosomes reprofile fibroblast extracellular matrix

Observations and analyses were performed according to example two 3.

3) Low-load hsa_circ_0002557 exosomes prevent NFs activation inhibiting esophageal squamous cell carcinoma progression were manipulated and analyzed according to the methods related to example 3.

The results show that: chemical reagent loading of the exosomes of si_hsa_circ_0002557 significantly inhibited EC109 cell hsa_circ_0002557 expression, with inhibition efficiencies of 28.6%, 31.7% and 42.6% at 12H,24H and 48H, respectively (fig. 7). Meanwhile, si_hsa_circ_0002557 transiently transformed EC109 and EC9706 cells significantly interfered with hsa_circ_0002557 expression levels in the cells, 0.29 and 0.61 fold that of the control cells, respectively (fig. 8). Further, si_hsa_circ_0002557 treatment reduced the degree of activation of constituent fibroblasts, reduced the collagen fiber signal, and an average number of collagen fibers in a single field of view of 96.67; whereas the si-NC control group had an enhanced collagen fiber signal, a clear green fiber signal was seen, and the average number of collagen fibers in a single field was 983. t-test showed that the number of collagen fibers in the si_hsa_circ_0002557 treated group was significantly lower than that in the si-NC control group (P <0.001, fig. 9-10), and that si_hsa_circ_0002557 treated constituent fibroblasts significantly inhibited esophageal squamous cell carcinoma EC109 and EC9706 cell migration capacity (fig. 11A) and cell invasion (fig. 11B). The suggestion that the siA_hsa_circ_ 0002557 exosomes can be used as a molecular drug to inhibit the extracellular matrix formation of cancer-related fibroblasts by inhibiting the loading of the hsA_circ_0002557 exosomes of esophageal cancer, so as to inhibit tumor progression, and can be used as a new molecular target in drug development and esophageal cancer treatment taking microenvironment as a guide.

In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing has shown and described the basic principles, principal features and advantages of the application. It will be understood by those skilled in the art that the present application is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present application, and various changes and modifications may be made without departing from the spirit and scope of the application, which is defined in the appended claims.

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Claims

Use of an inhibitor of hsa_circ_0002557 expression in the manufacture of a medicament for inhibiting oesophageal fibroblast activation to cancer associated fibroblasts, hsa_circ_0002557 having the sequence SEQ ID No.1.