CN111450249A

CN111450249A - Application of miRNA-320a expression promoter in preparation of tumor cell inhibiting medicine

Info

Publication number: CN111450249A
Application number: CN202010158454.7A
Authority: CN
Inventors: 杨静悦; 王楠; 李霄
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-03-09
Filing date: 2020-03-09
Publication date: 2020-07-28

Abstract

The embodiment of the invention discloses application of an expression promoter of miRNA-320a in preparation of a medicine for inhibiting tumor cells. The high expression promoter of miRNA-320a in the liver cancer tumor cells can effectively inhibit the antedisplacement and propagation of the liver cancer cells by high expression of miRNA-320a in the liver cancer cells, thereby effectively treating liver cancer.

Description

Application of miRNA-320a expression promoter in preparation of tumor cell inhibiting medicine

Technical Field

The embodiment of the invention relates to the technical field of biology, in particular to application of an expression promoter of miRNA-320a in preparation of a drug for inhibiting tumor cells.

Background

Liver cancer, a malignant tumor of the liver, can be divided into primary and secondary types. The primary liver malignant tumor originates from the epithelium or mesenchymal tissue of the liver, and the primary liver malignant tumor is called primary liver cancer and is malignant tumor with great harm; the latter is called sarcoma, and is less common than primary liver cancer. Secondary or metastatic liver cancer refers to the invasion of malignant tumors of multiple organ origins in the whole body to the liver. The individual comprehensive treatment is carried out according to different stages of the liver cancer, which is the key for improving the curative effect; the treatment method comprises operations, hepatic artery ligation, hepatic artery chemoembolization, radio frequency, freezing, laser, microwave, chemotherapy, radiotherapy and the like. Biological treatment, and traditional Chinese medicine treatment of liver cancer is also widely applied. Surgery, which is the first choice for treating liver cancer, is also the most effective method. The operation method comprises the following steps: radical hepatectomy, palliative hepatectomy, etc. The liver cancer which can not be resected can be treated by hepatic artery ligation, hepatic artery chemoembolization, radio frequency, freezing, laser, microwave and the like according to specific conditions. For patients who cannot be resected during operation, the radiation intervention treatment can be carried out, femoral artery is used for selective intubation to hepatic artery, embolic agent (often iodized oil) and anti-cancer medicine are injected for chemoembolization, and part of patients can obtain the chance of surgical resection. The radiotherapy has good general condition and liver function, is not accompanied by cirrhosis, jaundice, ascites, splenic hyperfunction and esophageal varices, has limited cancer and tumor, has no distant metastasis and is not suitable for patients with surgical excision or postoperative recurrence, and can adopt comprehensive treatment mainly by radiation. However, for patients with metastatic late-stage liver cancer, the target (ranvatinib) and immune PD-1 antibody (Pabolizumab) are treated by drugs such as chemotherapy, but the overall treatment effect is still unsatisfactory.

Disclosure of Invention

Therefore, the embodiment of the invention provides the application of the miRNA-320a expression promoter in preparing a tumor cell inhibiting drug, so as to solve the problems of poor effect and large side effect of the liver cancer treatment drug in the prior art.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

application of an expression promoter of miRNA-320a in preparing a medicament for inhibiting tumor cells.

Preferably, the tumor cell is a liver cancer cell.

Preferably, the expression promoter is L ncRNA-NEAT1, MA L AT1, OIP5-AS1 or a vector of high expression miRNA-320 a.

Preferably, the vector for highly expressing miRNA-320a is an exosome of tumor-associated fibroblasts over-expressing miRNA-320 a.

Preferably, the expression promoter is a composition comprising exosomes of the tumor-associated fibroblasts overexpressing miRNA-320a, FGK4.5, and an agonist.

Preferably, wherein the agonists are T L R4 activator L PS, T L R7 activator Gardiquimod, and T L R9 activator CpG-ODN.

Preferably, the preparation method of the exosome of the tumor-associated fibroblast comprises the following steps:

washing liver tissue with PBS, and pulverizing to 1mm³Adding DMEM/F12 medium containing 0.5% collagenase IV, 1% FBS, 1% penicillin-streptomycin, and culturing at 37 deg.C and 5% CO₂Incubating in incubator for 30 min;

collecting cell suspension after the liver tissue is completely digested, centrifuging, removing supernatant, and resuspending the cell precipitate in a culture medium special for primary fibroblasts;

digesting the primary fibroblasts cultured for 2-3 generations, counting, then suspending in MACS buffer solution, adding anti-human fibroblast magnetic beads, loading onto a column, and purifying in an immunomagnetic bead cell sorter to obtain a purified exosome of tumor-related fibroblasts;

and culturing the purified exosome of the tumor-associated fibroblast in a DMEM (DMEM) culture medium containing 10% FBS (fetal bovine serum), and purifying for 4-10 generations to obtain the exosome of the tumor-associated fibroblast.

Preferably, the primary fibroblast-dedicated culture medium is a DMEM/F12 culture medium containing 10% FBS, 1% ITS cell culture additives, 1% penicillin-streptomycin and 1% glutamine.

In the embodiment of the present invention, the expression promoter of miRNA-320a is a substance capable of overexpressing miRNA-320a in cells, for example, the embodiment of the present invention utilizes bioinformatics tools such AS StarBase, ChipBase, L ncRNAdb and L ncBase to find that binding sites exist between L ncRNA-NEAT1, MA L AT1, OIP5-AS1 and the like and miR-320a, wherein NEAT1 is totally called Nuclear enriched transcript 1 (NEAT 1), has a total length of about 3.2kb, and is a key non-coding RNA for forming and maintaining the Nuclear substructure paracapkle.

The NEAT1 also plays a role in the cerRNA effect, and inhibits the molecular activity of miRNAs such as miR-129-5p, miR-613, miR-485, miR-139-5p, miR-124-3p and the like, so that downstream target genes such as NF-kappa B, DC L K1, STAT3, TGF- β, ATG L and the like are activated.

In the embodiment of the invention, CAFs-EXO + FGK4.5 are used as activated monoclonal antibodies of CD40, wherein FGK4.5 is used for inhibiting tumor cells, and the activated monoclonal antibodies are combined with different Toll-like receptor activators to be applied, so that the activators combining CD40 and Toll-like receptors (T L R) can simultaneously activate CD4+ T cells and CD8+ T cells, and the high-efficiency tumor inhibition effect is shown.

The embodiment of the invention has the following advantages:

the embodiment of the invention takes CAFs-EXO over-expressing miRNA-320a as a carrier, and over-expresses miR-320a in an animal model in combination with an immune adjuvant so as to inhibit the growth and migration of liver cancer tumor cells.

The exosome of the tumor-associated fibroblast provided by the embodiment of the invention can be combined with an activator of CD40 and a Toll-like receptor (T L R) to simultaneously activate CD4+ T cells and CD8+ T cells, and show a high-efficiency tumor inhibition effect.

In the embodiment of the invention, the exosome of the tumor-associated fibroblast has the advantages of stable property, easy storage and good induction effect as a non-cellular carrier.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a schematic diagram of a three-step separation method of L NSCs from rat fetal livers by using stem cells provided by the embodiment of the invention, wherein A is F344 rat embryos of 14-day pregnancy, B is fetal livers, C is a schematic diagram of a three-step separation method of L NSCs, D is a morphological comparison of L NSCs and mature hepatocytes, wherein F1 and F2 represent L NSCs, and F4 and F5 represent mature hepatocytes;

FIG. 2 is a graph of the stem cell characterization of L NSCs, wherein A is the flow identification of the expression rate of CD133 in each cell subset, B is the bidirectional potential identification of L NSCs, with the left side showing the mRNA level of the immature cell marker and the right side showing the mRNA level of the mature hepatocyte marker;

FIG. 3 is a diagram of liver cancer tissue isolation and liver cancer cell culture according to an embodiment of the present invention, wherein, i.e., primary cancer nodules (shown by arrows) on the liver of rat, ii.e., multiple metastatic liver cancer nodules (shown by arrows) occurring in the lung, iii.HE identification of liver cancer, primary culture of liver cancer cells (iv) for 4 days, (v) for 15 days, (vi) for 30 days, primary magnification, 100 × iii-vi;

FIG. 4 is a schematic diagram of L CSCs separation by a three-step separation method according to an embodiment of the present invention, wherein A is the culture and purification of liver cancer cells, the left picture shows the liver cancer cells before purification, the right picture shows the purified liver cancer cells, B is the stratification of the liver cancer cells and the ratio of each layer of cells;

FIG. 5 is a morphological feature diagram of liver cancer cells of each layer, the upper row represents an optical microscope and the lower row represents an electron microscope, according to an embodiment of the present invention;

fig. 6 is a surface marker detection map of each layer of hepatoma cells provided in the example of the present invention, wherein a: flow assay, top row for CD133 expression assay, bottom row for EpCAM expression assay, B: performing immunofluorescence detection on CD133, AFP and CK-19;

FIG. 7 is a diagram of invasion and migration capabilities of liver cancer cells in layers according to an embodiment of the present invention, in which the upper row represents the situation of small penetrating holes of cells in layers in a Transwell experiment, and the lower row represents the situation of closure of a scratch by migrated cells after 48 hours in a scratch experiment;

FIG. 8 is a graph of the tumorigenicity of hepatoma cells in layers according to an embodiment of the present invention, wherein the first row represents the gross case of subcutaneous tumorigenicity, the second row represents the gross case of hepatic tumorigenicity, and the third row represents the pathological examination of tumor tissue;

FIG. 9 is a graph of the differential analysis of miRNAs expression between L NSCs and L CSCs, wherein A is an electrophoresis result showing that the brightness of the total RNA 28S band is 2 times that of the 18S band, B is a L CSCs cell miRNA chip hybridization pattern, C is a L NSCs cell miRNA chip hybridization pattern, D is a SAM3.0 software statistical test performed by taking the deregulation 2 times as a threshold value to determine the effectiveness of the deregulated miRNA, red dots represent that the upregulation is more than two times, green dots represent that the downregulation is more than two times, and black colors represent nonsense deregulation;

FIG. 10 is a drawing of the extraction and identification of the CAFs and PAFs provided in the present invention, wherein A is the fusiform adherent growth of the extracted cells under the mirror, and the extracted cells have the morphological characteristics of fibroblasts, B is the marking of α -SMA, FAP and PDGFR- β fibroblasts detected in the two types of cells by Western blotting experiment, and C is the expression of α -SMA and FAP in the two types of cells detected by immunofluorescence;

fig. 11 is a graph of the differential expression of miRNAs between CAFs and PAFs provided in the present invention, wherein a: screening heatmap of 6 specimen differential miRNAs, red for up-regulated molecules, green for down-regulated molecules, black for unchanged molecules, B: taking 2 times of disorder as a threshold value, determining the effectiveness of the disorder miRNA after statistical test of SAM3.0 software, wherein red points represent that the up-regulation is more than two times, green points represent that the down-regulation is more than two times, and black points represent nonsense disorder, and finally performing statistical analysis by using results of 6 specimens, wherein the miRNAs with statistical difference comprise 2 expression up-regulation and 42 expression down-regulation;

FIG. 12 is a graph showing the expression rules of miR-320a in different tissues and cells, wherein A is the in situ hybridization for detecting the expression in liver cancer and tissues adjacent to the cancer, B is the qPCR for detecting the expression in 6 CAFs and corresponding PAFs, C is the qPCR for detecting the expression in liver cancer cell lines and liver cell lines, and D is the qPCR for detecting the expression in L NSCs and L CSCs;

fig. 13 is a graph showing the effect of up-regulating miR-320a expression on the proliferation and clonotype forming ability of hepatoma cells, provided by an embodiment of the present invention, wherein a: CCK measures cell proliferation capacity, B: clone formation experiments. C: flow-detecting the cell cycle;

fig. 14 is a graph showing the effect of up-regulating miR-320a expression on the migration ability of liver cancer cells, provided by an embodiment of the present invention, wherein a: transwell cell experiment, B: performing cell scratching experiments;

fig. 15 is a graph for detecting the effect of miRNA-320a overexpression on hepatoma cell tumorigenicity, provided by the embodiment of the present invention, wherein a: tumor mass and tumor weight of subcutaneous neoplasia, B: in vivo fluorescence imaging to detect the number of fluorescently labeled tumor cells, C: pathological examination of subcutaneous tumor body and lung metastasis, HE staining on the left side and Ki67 positive cell number on the right side in immunohistochemical detection; scale bar,100 μm;

FIG. 16 is a graph showing the relationship between NEAT1 and miR320a, wherein A is a graph obtained by predicting 2 binding sites of NEAT1 and miR-320a through bioinformatics, 2 wild-type NEAT1 and mutant NEAT1 are designed according to the above, B is a graph obtained by detecting the expression of NEAT1 in L CSCs and L NSCs through qPCR, C is a graph obtained by detecting the expression of NEAT1 in 7702 cells and various liver cancer cells, and D is a graph obtained by detecting the expression correlation between NEAT1 and miR-320a in liver cancer tissues;

FIG. 17 shows the prediction of downstream target molecules regulated by miR-320a and preliminary verification provided by the embodiment of the invention, wherein A is intersection analysis of the results predicted by four software, B is the up-regulation of miR-320a expression of L CSCs, and the possible expression change of downstream molecules is verified;

FIG. 18 is an expression diagram for verifying regulation and control of PBX3 by miR-320a, provided by the embodiment of the invention, wherein A is a predicted binding site of miRNA-320a on 3'UTR and a design of a wild-type and mutant sequence at the 3' end of PBX3, B is a luciferase reporter gene detection result, and C-F is an inhibition effect of miR-320a on proliferation and invasion capacity of L CSCs (Circuit switched Cs) after PBX3 is over-expressed;

fig. 19 is a graph showing the identification of exosomes of tumor-associated fibroblasts provided by an embodiment of the present invention, wherein a: electron microscopy confirmed that the extracted exosomes are of membranous microcapsule structure, B: detecting an exosome-specific marker CD63 which indicates high expression of tumor-associated fibroblasts by a Western blotting experiment;

fig. 20 shows that the CAFs provided in the embodiments of the present invention introduce exogenous miR-320a into hepatoma cells by secreting exosomes, a: performing immunofluorescence detection, wherein green fluorescence is liver cancer cells marked by GFP; red fluorescence is cy 3-labeled miR-320a fragment, B: detecting the expression quantity of miR-320a in exosome after transfection by qPCR;

FIG. 21 is a graph showing that the nude mouse tumorigenicity experiment provided by the embodiment of the present invention detects the influence of miR-320a overexpression in CAFs on the tumorigenicity capacity of liver cancer cells in vivo.

Detailed Description

Other advantages and features of the present invention will become readily apparent to those skilled in the art from the following detailed description, wherein it is to be understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1 isolation, culture and identification of Normal hepatic Stem cells

(1) Isolation and culture of Normal hepatic Stem cells (liver normal stem cells, L NSCs) L NSCs were successfully isolated from fetal liver of F344 rats as shown in FIG. 1B and cultured in vitro in Stem cell serum-free Williams' E medium containing 20ng/ml HGF and 10ng/ml EGF it was found that adherent L NSCs (F1, F2) were smaller in size than mature hepatocytes (F4, F5) and had irregular morphological characteristics as shown in FIG. 1C.

(2) Identification, in order to identify the stem cell characteristics of the isolated L NSCs, the stem cell marker CD133 is detected by using flow cytometry, L NSCs (F1 and F2) are found to highly express the stem cell marker CD133(F3-F6) compared with mature hepatocytes as shown in figure 2A, in order to further detect the bidirectional differentiation potential of L NSCs, the expression conditions of immature hepatocyte markers such as AFP and CK19 are also detected, and the result proves that L NSCs both highly express two markers and have bidirectional differentiation potential, and simultaneously, the low-expression mature hepatocyte marker such as A L B, G P has the immature marker characteristics, and the results show that the method for separating L NSCs by using the three-step method is successful as shown in figure 2B.

Example 2 isolation, culture and identification of liver cancer liver Stem cells (L CSCs)

(1) Preparation of rat liver cancer model: after an F344 rat liver cancer model is successfully established by induction of diethylnitrosamine, cancer cells of primary liver cancer are isolated and cultured by a two-step collagenase digestion method. After 30 days of culture, the liver cancer cells can be subjected to first passage. This cell line is now expansion stable as shown in FIG. 3.

(2) L separation and culture of CSCs, namely, separation of L CSCs by a three-step separation method, liver cancer cells are initially separated from a DEN induced F344 rat liver cancer model through PDGC in the first step, the cells have different shapes and sizes in vitro culture, and the liver cancer cells show uniform shapes after DTDC purification, as shown in figure 4A, the purified liver cancer cells are divided into four parts according to the density range of the cells in the liver cancer cells through PCGC, and the cells in the third layer are found to be the least potential L CSCs, as shown in figure 4B.

(3) L identification of CSCs

① morphological identification the third layer was observed by light and electron microscopy to show the greatest nuclear serological ratio and the least organelle, which is a morphological feature of potential L CSCs, as shown in fig. 5.

② marker identification two relatively common markers (CD133 and EpCAM) were detected for L CSCs by flow cytometry, results suggested that cells in the third layer expressed the highest of the two stem cell markers, as shown in FIG. 6A, immunofluorescence detection suggested that cells in the third layer expressed CD133 and AFP, as shown in FIG. 6B.

③ detection of invasion and migration ability, L CSCs should have stronger invasion and migration ability to distinguish common liver cancer cells through Transwell experiments and scratch experiments, it is found that the cells in the third layer can penetrate pores to the greatest extent and have the strongest ability to migrate to repair scratches, as shown in FIG. 7.

④ examination of ability to form tumor Each layer of cells was transplanted into nude mice in the same number, and as a result, it was found that the cells of the third layer formed tumors most rapidly subcutaneously and required the least number of cells, and further, the cells of the third layer could migrate to the liver to form tumors, as shown in FIG. 8.

Example 3, L establishment of differential miRNAs expression profiles of NSCs and L CSCs

By using the currently most comprehensive Exiqon miRNA gene chip, L NSCs and L CSCs were obtained respectively, as shown in fig. 9B and 11, and further data processing was performed by SAM3.0 software, it was shown that 78 miRNAs were significantly differentially expressed in L CSCs, including 68 upregulations and 10 downregulations, and some miRNAs with significantly different expressions, including miRNA-320a, as shown in fig. 9D, compared to L NSCs.

Example 4 methods for isolation and culture of tumor-associated fibroblasts (CAFs) and Paracarcinoma fibroblasts (PAFs) and obtaining differential miRNAs expression profiles

Extraction and identification of CAFs and PAFs

By adopting a method of collagenase digestion and immunomagnetic bead sorting, CAFs and PAFs are separated from surgical excision specimens of 6 primary liver cancer patients, as shown in figure 10A, and the markers of high-expression α -SMA, FAP, PDGFR- β and other fibroblasts in Western blotting experiments and immunofluorescence detection are shown in figures 10B and 12C.

Establishment of differential miRNAs expression profiles of CAFs and PAFs

The current most complete Exiqon miRNA gene chip is utilized to respectively obtain hybridization maps of 6 CAFs and PAFs, as shown in FIG. 11A, and further SAM3.0 software is used for data processing, the result shows that compared with PAFs, a plurality of miRNAs are differentially expressed in L CSCs, but statistical analysis shows that the miRNAs have statistical differences including 2 expression up-regulation and 42 expression down-regulation, and miRNA-320a exists, as shown in FIG. 11B.

Example 5 expression of miR-320a in liver cancer tissue and liver cancer cell line

Expression of miR-320a in different tissues and cells

In situ hybridization detection indicates that miR-320a is low in expression in liver cancer tissues, and is high in expression in para-cancer tissues (figure 12A). qPCR detection indicates that the expression quantity of miR-320a in 6 CAFs is higher than that of corresponding PAFs (figure 12B). qPCR detection indicates that the expression quantity of miR-320a in liver cancer cell lines MHCC97-H, SMCC-7721, Huh7 and other cells is remarkably higher than that of a liver cell line 7702, as shown in figure 12℃ qPCR detection indicates that miR-320a is high in expression in L NSCs and is hardly expressed in L CSCs, as shown in figure 12D.

Regulation and control function of miR-320a in liver cancer

① influence on proliferation and clonogenic capacity of hepatoma carcinoma cells

By adopting a lentivirus overexpression technology to up-regulate miR-320a expression in MHCC97-H and SMMC-7721 cells, CCK-8 detects that cell proliferation is obviously inhibited, as shown in figure 13A, and the clone forming capability is weakened, as shown in figure 13B. After flow assay up-regulation of miR-320a expression, the number of cells at G1 phase increased significantly, as shown in fig. 13C.

② on migration capacity of liver cancer cells, and detection of Transwell chamber and cell scratch experiments suggest that after miR-320a expression is up-regulated, the migration capacity of MHCC97-H and SMMC-7721 cells is remarkably reduced as shown in FIG. 14.

③ nude mouse tumorigenesis experiment

MHCC97-H cells before and after over-expression of miRNA-320a are subjected to in vitro immunofluorescence labeling, and then are inoculated into 6 nude mice subcutaneously respectively, and the conditions of tumor formation and lung metastasis are observed after 28 days. The result shows that the tumor formation rate of MHCC97-H cells is obviously reduced after the miRNA-320a is up-regulated, as shown in FIG. 15A, the fluorescence intensity is obviously weaker than that of a control group, as shown in FIG. 15B. All of 6 nude mice in the control group showed lung metastases, while 4 of the over-expressed groups showed lung metastases. Immunohistochemical staining of subcutaneous cancer foci and metastases gave a significant reduction in the number of Ki67 positive cells in rat tissues from the over-expression group, as shown in fig. 15C.

Example 6 miR-320a upstream and downstream regulatory molecules

1. Bioinformatics tools such as StarBase, ChipBase, L ncRNAdb and L ncBase are utilized to find that 2 binding sites exist between L ncRNA-NEAT1 and miR-320a, as shown in FIG. 16A. qPCR shows that the expression level of NEAT1 in L CSCs is significantly higher than that of L NSCs, as shown in FIG. 16B, and the expression level in liver cancer cell line MHCC97H and other cells is significantly higher than that of normal liver cell line 7702, as shown in FIG. 16C, while qPCR detection in liver cancer tissues proves that the expression of NEAT1 and miR-320a is significantly negatively correlated, as shown in FIG. 16D.

2. Software such as TargetScan, C L IP-Seq, mirDB, mirandda and the like is used for predicting PBX3, C-Myc and a downstream target gene regulated by miR-320a, as shown in figure 17A, after exogenous miR-320a is introduced into L CSCs, a Westernblotting experiment detects that the protein expression quantity of PBX3 and C-Myc in L CSCs is remarkably reduced, as shown in figure 17B.

3. Luciferase reporter gene detection indicates that miR-320a can reduce the fluorescence activity of wild-type PBX3 in liver cancer cells, and has no influence on 3' UTR mutant PBX3, as shown in figure 18A, B; after the antagonistic overexpression of PBX3, the effect of miR-320a on inhibiting the proliferation and invasion of the liver cancer is obviously weakened, as shown in FIGS. 18C-F.

Example 7 preparation and identification of CAFs-EXO overexpressing miRNA-320a

Extraction and characterization of CAFs

The separation of CAFs (Cancer-associated f-wbolases) was carried out by immunomagnetic bead cell sorting. Cleaning liver tissue specimen with PBS, and cutting into 1mm³The pieces were added with DMEM/F12 medium containing 0.5% collagenase IV, 1% FBS and 1% penicillin-streptomycin (DMEM/F12 medium containing 0.5% collagenase IV, 1% FBS and 1% penicillin-streptomycin) at 37 ℃ and 5% CO₂Incubate 30 min. After the tissue is completely digested, collecting cell suspension, centrifuging, discarding supernatant, and resuspending the cell pellet in a primary fibroblast-dedicated culture medium (DMEM/F12 medium containing 10% FBS, 1% ITS cell culture additive, 1% penicillin-streptomycin, and 1% glutamine). Digesting and counting the primary fibroblasts cultured for 2-3 generations, then suspending the primary fibroblasts in a MACS buffer solution (carried in an auto MACSR Pro full-automatic immunomagnetic bead cell sorting system), adding anti-human fibroblast magnetic beads, loading the beads on a column, and purifying the beads in an immunomagnetic bead cell sorting instrument. And culturing the purified CAFs in a DMEM medium containing 10% FBS, and taking 4-10 purified CAFs for subsequent experiments. Electron microscopy revealed that exosomes were membranous microcapsules with diameters of about 50-100 μm, as shown in FIG. 19A. Western blotting experiments detected exosomes expressing the specific marker CD63, as shown in FIG. 19B.

Expression of miR-320a in CAFs

CAFs transfected cy 3-labeled miR-320a (miR-320a-cy3) in vitro, and its secreted exosome was isolated and co-cultured with GFP-labeled MHCC97-H cells for 24H. Fluorescence microscopy showed significant red fluorescence in MHCC97-H cells, as shown in FIG. 20A, and qPCR detection showed significant increase in miR-320A expression in exosomes after transfection, as shown in FIG. 20B. CAFs can introduce exogenous miR-320a into liver cancer cells in a mode of secreting exosomes.

3. Experiment grouping

Based on CAFs-EXO + FGK4.5 over-expressing miRNA-320a, the CAFs-EXO + FGK is combined with T L R3 activator poly (I: C), T L R4 activator L PS, T L R7 activator Gardiquimod (GDQ) and T L R9 activator CpG-ODN respectively, and a CAFs-EXO control group not expressing miRNA-320a is set at the same time.

4. Tumor inhibition experiment in vivo

L uciferase is used for marking MHCC97-H cells in vitro, then the MHCC97-H cells, the MHCC97-H cells + CAFs, the MHCC97-H cells + CAFs-320a are respectively inoculated on the lower limbs of nude mice, and after 28 days, the living fluorescence imaging detection proves that the number of the fluorescent cells in the experiment group of MHCC97-H cells + CAFs-320a is the minimum, and as shown in figure 21, the miR-320a is over-expressed in the CAFs to inhibit the in-vivo tumor forming capacity of the liver cancer cells.

Exosomes (CAFs-EXO) secreted by the CAFs can transport exogenous miR-320a into liver cancer cells, and obviously reduce the in-vivo tumor formation effect and the lung metastasis rate in a nude mouse tumor formation experiment.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

Application of an expression promoter of miRNA-320a in preparing a medicament for inhibiting tumor cells.
2. The use of claim 1, wherein the tumor cell is a liver cancer cell.
3. The use of claim 1, wherein the expression promoter is L ncRNA-NEAT1, MA L AT1, OIP5-AS1 or a vector highly expressing miRNA-320 a.
4. The use of claim 3, wherein the miRNA-320a high-expression vector is an exosome of tumor-associated fibroblasts over-expressing miRNA-320 a.
5. The use according to claim 4, wherein the expression promoter is a composition comprising exosomes of the tumor-associated fibroblasts overexpressing miRNA-320a, FGK4.5, and an agonist.
6. The use of claim 5, wherein the agonists are T L R4 activator L PS, T L R7 activator Gardiquimod, and T L R9 activator CpG-ODN.
7. The use according to claim 3, wherein the exosomes of tumor-associated fibroblasts are prepared by a method comprising:

washing liver tissue with PBS, and pulverizing to 1mm³Adding DMEM/F12 medium containing 0.5% collagenase IV, 1% FBS, 1% penicillin-streptomycin, and culturing at 37 deg.C and 5% CO₂Incubating in incubator for 30 min;

collecting cell suspension after the liver tissue is completely digested, centrifuging, removing supernatant, and resuspending the cell precipitate in a culture medium special for primary fibroblasts;

digesting the primary fibroblasts cultured for 2-3 generations, counting, then suspending in MACS buffer solution, adding anti-human fibroblast magnetic beads, loading onto a column, and purifying in an immunomagnetic bead cell sorter to obtain a purified exosome of tumor-related fibroblasts;

and culturing the purified exosome of the tumor-associated fibroblast in a DMEM (DMEM) culture medium containing 10% FBS (fetal bovine serum), and purifying for 4-10 generations to obtain the exosome of the tumor-associated fibroblast.
8. The use of claim 7, wherein the primary fibroblast specific culture medium is DMEM/F12 medium containing 10% FBS, 1% ITS cell culture additives, 1% penicillin-streptomycin, 1% glutamine.