CN111206102A

CN111206102A - A serum exosomal miRNA marker related to the diagnosis of liver cancer and its application

Info

Publication number: CN111206102A
Application number: CN202010185192.3A
Authority: CN
Inventors: 施晓雷; 任昊桢; 王经琳; 丁义涛
Original assignee: Nanjing Drum Tower Hospital
Current assignee: Nanjing Drum Tower Hospital
Priority date: 2020-03-17
Filing date: 2020-03-17
Publication date: 2020-05-29

Abstract

The invention belongs to the fields of biological diagnosis and medicine, and particularly relates to a serum exosome miRNA marker related to liver cancer diagnosis and an application thereof. The marker is has-miRNA-21-5p, and has-miRNA-21-5p is used as Biomarkers of liver cancer can be used to diagnose whether a person has liver cancer or the risk of developing liver cancer by detecting its expression level. It has the characteristics of convenient detection, little damage, and accurate quantification, which can improve the sensitivity and specificity of detection. has‑miRNA ‑21‑5p and its specific amplification primers can be used to prepare liver cancer diagnostic kits, which are convenient for rapid and auxiliary diagnosis at an early stage, and provide a basis for clinicians to conduct further in-depth examinations or quickly and accurately grasp the disease state of patients.

Description

Serum exosome miRNA marker related to liver cancer diagnosis and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a serum exosome miRNA marker related to liver cancer diagnosis and application thereof.

Background

Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide, the third leading cause of cancer-related death. Currently, surgical resection is still the most effective treatment for HCC, but the high metastasis rate and high recurrence rate of HCC severely limit the long-term efficacy of HCC patients. Meanwhile, the HCC specific diagnosis indexes which are commonly used clinically are not sensitive enough, and the liver has strong compensation capacity, so that patients are in the middle and late stages of cancer when the patients have symptoms for treatment, and the deficiency of 10 percent of radical operators can be obtained. Therefore, the problems to be solved are urgent at present in order to explain the specific mechanism of HCC development, explore the clinical therapeutic targets and find reliable HCC resection prognostic indicators.

The growth of tumors, despite the subject being an unrestricted proliferation of tumor cells themselves, the interaction between tumor cells and neighboring cells is of increasing concern. In recent years, the important role of exosomes released by tumor cells in the tumor growth process is gradually known, and in the process of transporting bioactive substances to receptor cells, the substances in vesicles are prevented from being hydrolyzed by enzymes in the intercellular spaces, so that the transmission effectiveness of the bioactive substances is ensured. Exosomes thus provide good targets in elucidating tumorigenesis development, metastasis and treatment options, which play a crucial role in tumor development.

The vesicle of the exosome plays a role mainly depending on the bioactive substances of the exosome, including protein, mRNA, non-coding RNA, DNA fragments and the like, wherein miRNAs are used as nucleic acid substances with the highest expression quantity in the non-coding RNA in the exosome, and the abnormal expression of the miRNAs promotes the generation and development of tumors. Research shows that miRNA can regulate the expression of one third of human genes, and one miRNA can also regulate the expression of hundreds of genes. Thus, aberrant expression of individual miRNAs also has the potential to transform normal cells into tumor cells. There is now increasing evidence that unbalanced exosome miRNAs are important links of tumor cells to recipient cells. Therefore, the development of exosome miRNAs providing a new theoretical basis for clinical diagnosis, treatment and prognosis judgment of HCC is urgently needed.

Disclosure of Invention

The invention aims to solve the technical problem of providing a serum exosome miRNA marker related to liver cancer diagnosis and application thereof aiming at the defects of the prior art.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows: the invention provides a serum exosome miRNA marker related to liver cancer diagnosis, which is has-miRNA-21-5 p.

The invention also provides application of the has-miRNA-21-5p in preparation of a liver cancer diagnostic kit.

The invention also provides a specific amplification primer of the marker has-miRNA-21-5p, wherein the specific amplification primer is as follows: the kit comprises a forward primer has-miRNA-21-5p-F and a reverse primer has-miRNA-21-5p-R, wherein the nucleotide sequence of the forward primer has-miRNA-21-5p-F is SEQ ID NO.17, and the nucleotide sequence of the reverse primer has-miRNA-21-5p-R is SEQ ID NO. 18.

The invention also provides application of the specific amplification primer has-miRNA-21-5p-F/has-miRNA-21-5p-R in preparation of a liver cancer diagnosis kit.

The invention also provides a liver cancer diagnosis kit, which is used for detecting the expression quantity of has-miRNA-21-5p in the serum exosome miRNA.

Further, the kit comprises specific amplification primers of the markers has-miRNA-21-5p, has-miRNA-21-5p-F and has-miRNA-21-5 p-R.

Further, the kit also comprises reagents commonly used in PCR technology.

The invention also provides application of the has-miRNA-21-5p analogue in preparing a liver cancer animal model, wherein the nucleotide sequence of the has-miRNA-21-5p analogue is SEQ ID NO. 1.

The invention also provides application of the has-miRNA-21-5p inhibitor in preparation of a product for treating liver cancer, wherein the upstream sequence of the has-miRNA-21-5p inhibitor is SEQ ID NO.2, and the downstream sequence of the has-miRNA-21-5p inhibitor is SEQ ID NO. 3.

Compared with the prior art, the invention has the beneficial effects that:

1) the invention discovers for the first time that has-miRNA-21-5p in the serum exosome can be used as a biomarker for liver cancer diagnosis, has the characteristics of convenient detection and small damage, is accurate in quantification, and can improve the sensitivity and specificity of HCC diagnosis;

2) the has-miRNA-21-5p exosome is taken from serum, so that invasive diagnosis can be avoided, extraction is easy, the amount of the serum is small, and the blood extraction amount of a detector is reduced;

3) the invention also prepares a liver cancer diagnosis kit, which comprises specific amplification primers of has-miRNA-21-5p-F and has-miRNA-21-5p-R and other reagents commonly used in qRT-PCR technology, and can carry out rapid and auxiliary diagnosis in early stage by detecting the expression level of exosome has-miRNA-21-5p in serum of a detector, thereby providing basis for further deep inspection of a clinician or rapid and accurate grasp of the disease state of a patient, helping the clinician to adopt a prevention and treatment scheme in time, and delaying and stopping the disease progress;

4) the has-miRNA-21-5p inhibitor can inhibit the growth of transplanted tumors by inhibiting the expression of the has-miRNA-21-5p in exosomes, and lays a foundation for preparing liver cancer treatment products.

Drawings

Fig. 1 is a transmission electron microscope image of exosomes from different sources in example 1, b is the diameter of exosomes from each cell source in random different fields, c is the number of exosomes from each cell source in random different fields, and d is the expression level of exosome markers CD63, CD81, CD9 identified by Western Blot method (p < 0.001).

FIG. 2 is a diagram of CCK-8 experiment for detecting the cell proliferation level of the cells after stimulation by has-miRNA-21-5p in example 1 of the present invention, wherein a is the effect of the CCK-8 experiment for detecting the has-miRNA-21-5p on the cell proliferation level of HSCs at different time points; b CCK-8 experiments to detect the effect of has-miRNA-21-5p on the proliferation level of HSCs (. p.0.01,. p.0.001).

FIG. 3 is a graph showing the migration effect of hepatoma cells in example 2 of the present invention.

Fig. 4 is a diagram of the growth state of subcutaneous transplantable tumors in example 3 of the present invention (. p <0.01,. p < 0.001).

Fig. 5 shows the results of testing the ability of HSCs to synthesize pro-angiogenic factors after stimulation with exosomes from different cell sources according to example 4 of the present invention ([ p ] p <0.05, [ p ] p <0.01, [ p ] p < 0.001).

Fig. 6 is a transmission electron microscope image of exosomes of the liver cancer group (HCC) and the control group (Normal), b is the diameter of the exosomes of the liver cancer group (HCC) and the control group (Normal) in random different fields, c is the number of the exosomes of the liver cancer group (HCC) and the control group (Normal) in random different fields, and d is the expression level of the serum exosome markers CD63, CD81 and CD9 of the liver cancer group (HCC) and the control group (Normal) identified by the Western Blot method (p < 0.001).

FIG. 7 shows the relative expression level of has-miRNA-21-5p in the serum exosomes of the liver cancer group and the control group in example 5 of the present invention (. about.p < 0.01).

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention is further described below by using specific examples. The experimental procedures used in the examples below are, unless otherwise specified, conventional procedures and the reagents, methods and equipment used are, unless otherwise specified, conventional in the art.

In the quantitative experiments in the following examples, three repeated experiments were set, and the average value was taken; the data obtained by the statistics of a t test method are used, and the statistical significance is considered to be achieved when the p value is less than 0.05 and the change multiple is more than 2 times;

in the following examples, each english abbreviation means: LM 3-EXO: secreted exosomes in HCCLM3 cell line culture supernatant; 97H-EXO: secreted exosomes in the culture supernatant of MHCC97H cell line; LO 2-EXO: secreted exosomes in the culture supernatant of the LO2 cell line; LM 3: HCCLM3 cell line; 97H: MHCC97H cell line; has-miRNA-21-5p imic: has-miRNA-21-5p mimetics; miRNA-micic-NC: has-miRNA-21-5p mimic negative control; has-miRNA-21-5p inhibitor: has-miRNA-21-5p inhibitor; LM 3-EXO-miR-21: exosomes of HCCLM3 that inhibit expression of has-miRNA-21-5 p; 97H-EXO-miR-21: an exosome of MHCC97H that inhibits expression of has-miRNA-21-5 p;

example 1: has-miRNA-21-5p in exosome promotes liver cancer cell proliferation

1.1) cell source: the human hepatoma cell line HCCLM3 (human high-transfer hepatoma cells) is purchased from a cell bank of China academy of sciences (catalog number: TCTU 94), the MHCC97H (human high-transfer hepatoma cells) is purchased from the liver cancer institute of Dandan university (Shanghai, China), the human normal hepatoma cells LO2 and the human Hepatic Stellate Cells (HSCs) are purchased from the liver cancer institute of Dandan university (Shanghai, China);

1.2) exosome extraction and identification in hepatoma cell lines

1.2.1) culture of human hepatoma cell lines HCCLM3, MHCC97H and human hepatocyte LO 2: human hepatoma cell lines HCCLM3 and MHCC97H were cultured and passaged in a 5% CO2 cell culture box at 37 ℃ in high-glucose DMEM medium (containing penicillin 100U/ml and streptomycin 100. mu.g/ml) containing 10% fetal bovine serum; culturing human hepatocyte LO2 in RPMI1640 medium containing 10% fetal calf serum (containing penicillin 100U/ml and streptomycin 100U/ml) at 37 deg.C in 5% CO2 cell culture box, and passaging;

1.2.2) extracting serum exosomes of HCCLM3, MHCC97H and LO2, comprising the following steps:

1.2.2.1) aspirate 10mL each of HCCLM3, MHCC97H, LO2 medium in 1.2.1);

1.2.2.2) filtering the culture medium by using a microporous membrane filter with the specification of 0.22 mu m, collecting filtrate, and removing cells and bacteria;

1.2.2.3) centrifuging at 300g × 10min at 4 deg.C, collecting supernatant, and removing cells;

1.2.2.4) centrifuging at 2000g × 10min at 4 deg.C, collecting the supernatant, and removing dead cells;

1.2.2.5) centrifuging at 4 deg.C 10000g × 30min, collecting supernatant, and removing cell debris;

1.2.2.6) centrifuging at 4 deg.C for 100000g × 70min, and collecting precipitate to obtain exosome;

1.2.3) detection of exosomes by transmission electron microscopy:

1.2.3.1) taking part of the exosome precipitate extracted in the step 1.2.2.6), and resuspending the precipitate with 100 μ l of PBS solution;

1.2.3.2) utilizing 2.5 percent glutaraldehyde stationary liquid (special for an electron microscope) to carry out fixation;

1.2.3.3) samples were prepared, cut to 0.12 μm and stained with 0.2% lead citrate and 1% uranyl acetate;

1.2.3.4) observing the section using JEM-2000EX II electron microscope (JEOL, Tokyo, Japan), the result of transmission electron microscope imaging of exosomes derived from HCCLM3, MHCC97H and LO2 cells as shown in FIG. 1 (a);

1.2.3.5) calculating the diameter and number of exosomes in different visual fields:

exosome diameters at random different fields: randomly selecting three fields under JEM-2000EX II electron microscope, calculating average diameters of exosomes derived from HCCLM3, MHCC97H and LO2 cells, as shown in FIG. 1(b), wherein exosomes extracted from HCCLM3 and MHCC97H cells are smaller than exosomes extracted from LO2 cells;

exosome numbers were randomized under different fields: randomly selecting three fields under a JEM-2000EX II electron microscope, and calculating the number of exosomes derived from HCCLM3, MHCC97H and LO2 cells, wherein as shown in a figure 1(c), the number of exosomes secreted in the culture supernatants of HCCLM3 and MHCC97H cell lines is obviously more than that secreted in the culture supernatants of LO2 cell lines (p is less than 0.001);

1.2.4) exosome markers CD63, CD81, CD9 expression identification:

1.2.4.1) exosome protein extraction:

a. before the experiment, the lysate is melted and mixed evenly and then placed on ice (if precipitates are separated out, the lysate can be heated to be dissolved at 37 ℃;

b. taking part of the exosome precipitate extracted in the step 1.2.2), re-suspending the exosome precipitate by using 100 mu L of PBS solution to obtain an exosome sample, and mixing the exosome sample and the lysis solution according to the volume ratio of 1: 1, adding the mixture evenly and placing the mixture on ice for cracking for 10 min;

c. centrifuging at 4 deg.C for 12000g × 5min, and collecting supernatant as exosome protein, and storing at-80 deg.C;

1.2.4.2) Western Blot assay for the exosome markers CD63, CD81, CD 9:

1.2.4.2.1) the exosome proteins of HCCLM3, MHCC97H, LO2 extracted in step 1.2.4.1) were boiled with 1 XWestern loading buffer;

1.2.4.2.2) electrophoresing equivalent amount of protein to polyvinylidene fluoride membrane by sodium dodecyl sulfate-polyacrylamide gel electrophoresis;

1.2.4.2.3) after blocking with 5% BSA in TBS-T, membranes were incubated overnight at 4 ℃ with primary antibodies (CD63, CD81, CD 9);

1.2.4.2.4) using goat anti-rabbit IgG marked by horseradish peroxidase or goat anti-mouse IgG marked by horseradish peroxidase as secondary antibody, and imaging the immunoreaction zone by using a Tanon gel imaging system.

The above-used Exosome-identifying reagent was obtained from an Exosome identification kit for Westernblot Exosome identification kit (Shanghai assist in san-Jose Biotech Co., Ltd., product No. 41203ES05), and CD81 antibody (cat No. ab109201) and CD9 antibody (cat No. ab92726) were each purchased from Abcam;

as shown in fig. 1(d), the identification results showed that CD63, CD81, and CD9 were all positive, indicating that all the substances extracted from the serum of HCCLM3, MHCC97H, and LO2 cells in step 1.2.2) were exosomes.

1.3) CCK-8 assay to detect CCLM3 and MHCC97H exosomes on human Hepatic Stellate Cells (HSC) at different time points_SCell line) effect of proliferation level

1.3.1) HSCS cell lines were cultured and passaged in a 5% CO2 cell culture box at 37 ℃ in high glucose DMEM medium (containing penicillin 100U/ml, streptomycin 100. mu.g/ml) containing 10% fetal bovine serum;

1.3.2) preparation of 2mL of density 4 x10⁴Adding HSCs cell suspension of each/mL into a 96-well plate at a rate of 100 mu L per well, and inoculating 20 wells in total;

1.3.3) adding PBS buffer solution (negative control), HCCLM3, exosomes of MHCC97H and LO2 into the wells added with the HSCs cell suspension, wherein each 5 wells contain 100 mu L of exosomes of HCCLM3, MHCC97H and LO2, and the exosomes of HCCLM3, MHCC97H and LO2 are obtained in the step 1.2.2) and are added with 100 mu L of PBS suspension liquid after the identification of the exosomes;

1.3.4) incubation in a 5% CO2 cell incubator at 37 ℃;

1.3.5) CCK-8 cell proliferation experiment, which comprises the following steps:

1.3.5.1) adding 10 mul of CCK-8 detection solution into each well incubated for 6h, 12h and 24h in the step 1.3.4) respectively;

1.3.5.2) incubating for 1-4 hours at 37 ℃ in a 5% CO2 cell incubator;

1.3.5.3) reading, mixing the mixture on a shaking table gently, and detecting the absorbance of each hole by an enzyme-labeling instrument at the wavelength of 460 nm;

the CCK-8 experiment shown in FIG. 2(a) examined the effect of exosomes on proliferation levels of HSCs cell lines at different time points, and found that CCLM3 exosome (LM3-EXO) and MHCC97H exosome (97H-EXO) were able to promote proliferation of hepatic stellate cells (HSCs cell lines) compared to negative control (PBS) and LO2 exosome (LO 2-EXO).

1.4) CCK-8 experiment to detect the influence of has-miRNA-21-5p mimic (has-miRNA-21-5pmimic) on the proliferation level of hepatic stellate cells (HSCs cell line) at different time points

1.4.1) sources of materials: the has-miRNA-21-5p mimic (has-miRNA-21-5p mimic) and the has-miRNA-21-5p mimic negative control (miRNA-mimic-NC) were purchased from Ruibo Biotech, Guangzhou;

the sequence of has-miRNA-21-5p mimic is as follows: 5'-AUCGAAUAGUCUGACUACAACU-3' (SEQ ID NO. 1);

1.4.2) transfecting HSCs cells by adopting has-miRNA-21-5p mimic and miRNA-mimic-NC, and specifically comprising the following steps:

1.4.2.1) HSCs cell lines were cultured and passaged in high-glucose DMEM medium (containing penicillin 100U/ml, streptomycin 100. mu.g/ml) containing 10% fetal bovine serum in a 5% CO2 cell culture box at 37 ℃;

1.4.2.2) 2mL of a density of 4 x10 were prepared⁴Adding HSCs cell suspension of each/mL into a 96-well plate at a rate of 100 mu L per well, and inoculating 10 wells in total;

1.4.2.3) adding has-miRNA-21-5p imic and miRNA-imic-NC into the holes added with the HSCs cell suspension, wherein each hole has 5 holes, each hole has 100 mu L, and the titer is 10^7 TU/mL;

1.4.2.4) incubation in a 5% CO2 cell incubator at 37 ℃;

1.4.3) CCK-8 cell proliferation assay, comprising the following steps:

1.4.3.1) adding 10 mu l of CCK-8 detection solution into each well of the has-miRNA-21-5p mimic and miRNA-mimic-NC transfected HSCs cells incubated for 6h, 12h and 24h in the step 1.4.2) respectively;

1.4.3.2) incubating for 1-4 hours at 37 ℃ in a 5% CO2 cell incubator;

1.4.3.3) reading, mixing the mixture on a shaking table gently, and detecting the absorbance of each hole by an enzyme-labeling instrument at the wavelength of 460 nm;

the CCK-8 experiment shown in FIG. 2(b) detects the influence of the proliferation level of the has-miRNA-21-5p mimic HSCs2 cell line at different time points, and finds that the has-miRNA-21-5p mimic group can promote the proliferation of hepatic stellate cells (HSCs cell line) compared with the negative control miRNA-mimic-NC, which indicates that the has-miRNA-21-5p in exosomes can promote the proliferation of liver cancer cells.

Example 2: has-miRNA-21-5p in exosome promotes liver cancer cell migration

2.1) transfecting HSCs cells by adopting has-miRNA-21-5p mimic and a negative control miRNA-mimic-NC according to the transfection step of the step 1.4.2);

2.2) preparation of cell suspension: collecting transfected HSCs cells, and preparing into 1.0x10 serum-free DMEM high-sugar medium⁶Cell suspension per ml;

2.3) scratching: after the cells adhere to the wall, scratching along a pre-marked line, selecting a 200 mu L gun head to directly scratch along the marked line in the scratching process, and manufacturing a plurality of scratches which are parallel to each other, wherein the force is based on scraping the cells without leaving marks on the culture plate; then rinsing for 2 times by PBS to remove floating cells; then, continuously incubating and culturing for 24h by using a DMEM high-sugar culture medium without FBS at 37 ℃ in an incubator with 5% CO 2;

2.4) observation of results: observing the scratch repair process of the cells under an inverted microscope, incubating and culturing each group in the step 2.3) for 0h and 24h, taking pictures, and comparing the scratch repair speed of the HSCs cells;

as can be seen from FIG. 3, the transfection of the has-miRNA-21-5p imic can promote the repair of HSCs cells, which indicates that the has-miRNA-21-5p can promote the migration of liver cancer cells.

Example 3: liver cancer cell exosome has-miRNA-21-5p for promoting growth of subcutaneous transplanted tumor

3.1) sources of materials: the has-miRNA-21-5p inhibitor (has-miRNA-21-5p inhibitor) was purchased from Ruibo Biotech, Guangzhou;

the sequence of has-miRNA-21-5p inhibitor is as follows: the upstream sequence has-miRNA-21-5p inhibitor-F: 5'-UAGCUUAUCAGACUGAUGUUGA-3' (SEQ ID NO.2), and the downstream sequence has-miRNA-21-5 pinhibitor-R: 5'-AUCGAAUAGUCUGACUACAACU-3' (SEQ ID NO. 3);

3.2) preparation of HCCLM3 and MHCC97H cells that inhibit the expression of has-miRNA-21-5 p:

3.2.1) HCCLM3, MHCC97H cells were cultured and passaged in high-glucose DMEM medium (containing penicillin 100U/ml, streptomycin 100. mu.g/ml) containing 10% fetal bovine serum at 37 ℃ in a 5% CO2 cell culture box;

3.2.2) separately 2mL of HCCLM3 and MHCC97H cell suspensions with a density of 4 x10 a 4/mL were prepared and added to a 96-well plate at 100 μ L cell suspension/well, each inoculated in 10 wells;

3.2.3) adding has-miRNA-21-5p inhibitor into each hole, wherein the titer is 10^7 TU/mL;

3.2.4) incubating in a 5% CO2 incubator at 37 ℃ for 12h to obtain HCCLM3 inhibiting expression of has-miRNA-21-5p and MHCC97H inhibiting expression of has-miRNA-21-5 p;

3.3) preparing exosomes of HCCLM3 cells, MHCC97H cells, LO2 cells, HCCLM3 cells inhibiting expression of has-miRNA-21-5p, and MHCC97H cells inhibiting expression of has-miRNA-21-5p with reference to step 1.2.2), and then resuspending to 200 μ L with PBS;

3.4) tumor implantation:

3.4.1) nude mice of 3 weeks old were inoculated subcutaneously with 200 μ L of PBS buffer (blank control), HCCLM3 exosome, MHCC97H exosome, LO2 exosome, HCCLM3 exosome inhibiting expression of has-miRNA-21-5p, MHCC97H exosome suspension inhibiting expression of has-miRNA-21-5p, respectively, at the right axillary abdominal wall;

3.4.2) killing the mice 4 weeks later, obtaining transplanted tumor tissues, measuring the sizes of the tumors and calculating the number of surface nodules;

as shown in fig. 4(a), after PBS (blank control, Ctr), HCCLM3 exosome (LM3-EXO), LO2 exosome (LO2-EXO), and HCCLM3 exosome (LM3-EXO-miR-21) inhibiting the expression of has-miRNA-21-5p were inoculated into nude mice, LM3-EXO group promoted the growth of tumors and stimulated tumors showed more tumor nodules compared to Ctr group and LO2-EXO group, while LM3-EXO-miR-21 group reduced the tumor volume and tumor nodule number;

as shown in FIG. 4(b), after the nude mice were inoculated with PBS (blank control, Ctr), MHCC97H exosome (97H-EXO), LO2 exosome (LO2-EXO), MHCC97H exosome (97H-EXO-miR-21) for inhibiting has-miRNA-21-5p expression, the 97H-EXO group promoted the growth of tumors and stimulated tumors showed more tumor nodules compared with the Ctr group and the LO2-EXO group, while the 97H-EXO-miR-21 group reduced the tumor volume and tumor nodule number.

The results of the above figures 4(a) -4 (b) show that has-miRNA-21-5p in the liver cancer cell exosome can promote the growth of subcutaneous transplanted tumor, and has-miRNA-21-5p inhibitor can inhibit the growth of transplanted tumor by inhibiting the expression of has-miRNA-21-5p in the exosome.

Example 4: capacity of Hepatic Stellate Cells (HSCs) stimulated by exosomes from different cell sources to synthesize angiogenesis promoting factors

4.1) HSCs cell lines were cultured and passaged in a 5% CO2 cell culture box at 37 ℃ in high-glucose DMEM medium containing 10% fetal bovine serum (containing penicillin 100U/mL, streptomycin 100. mu.g/mL) and seeded at a density of 10^ 5/mL in 6-well plates;

4.2) preparing HCCLM3 exosomes, MHCC97H exosomes, LO2 exosomes, HCCLM3 exosomes inhibiting expression of has-miRNA-21-5p, and MHCC97H exosomes inhibiting expression of has-miRNA-21-5p with reference to step 1.2.2);

4.3) adding the five exosomes into HSCs (HSCs) cells in 5 holes of the five exosomes in an amount of 100 mu L/hole, and adding 100 mu L DEPC buffer solution into the rest 1 hole of the five exosomes to serve as a control group (CTR);

4.4) adding 1mL of Trizol into each hole of the five differently treated cells, adding chloroform with the same volume, standing for 10min at normal temperature after vigorous oscillation for 20s, centrifuging for 15min at 12000g at 4 ℃, absorbing the upper aqueous phase, adding isopropanol with the same volume, centrifuging for 10min at 12000g at 4 ℃, removing the supernatant, adding 1mL of precooled 75% alcohol, centrifuging for 5min at 7500g at 4 ℃, removing the supernatant, standing for 10min at normal temperature, drying RNA precipitate, adding 20 mul of DEPC water to dissolve RNA, standing for 10min at 55-60 ℃, and quantifying the total RNA by an ultraviolet spectrophotometer;

4.5) use

RT Master Mix (Perfect Real Time) (Takara, cat # DRR036A) reverse transcribes the RNA of the liver cancer group and the control group, the experimental operation is carried out according to the product instruction, and the concrete operation is as follows: the concentration of each total RNA sample was quantified using an ultraviolet spectrophotometer, and then RT reaction solution (20 μ L) was prepared on ice as follows:

RT Master Mix (for Real Time) 4. mu.L, Total RNA 1. mu.g, plus RNase Free dH₂O to 20 μ L; after being mixed gently and evenly, the mixture is put into a PCR instrument for reverse transcription reaction, and the conditions of the reverse transcription reaction are as follows: carrying out reverse transcription at 37 ℃ for 15min and 85 ℃ for 5sec to obtain a cDNA sample;

4.6) use of ChamQ^TM

qRT-PCR amplification is carried out on qPCR Master Mix (Without ROX) (Nanjing Novowed Biotechnology Co., Ltd., Code No.: Q321-02), and the experimental operation is carried out according to the product specification and specifically comprises the following steps:

quantitative reaction system (20 μ l): 2 XChamQ SYBR qPCR Master Mix 10. mu.L, forward primer (10. mu.M) 0.4. mu.L, reverse primer (10. mu.M) 0.4. mu. L, cDNA 5. mu. L, ddH₂O 4.2μL；

The forward primer and the reverse primer are VEGF- α, MMP-2, MMP-9, bFGF and TGF β quantitative primers, the internal reference is GAPDH, the sequences of the primers are shown in Table 1, wherein the upstream and downstream primers of VEGF- α are respectively shown as SEQ ID No.4 and SEQ ID No.5, the upstream and downstream primers of MMP-2 are respectively shown as SEQ ID No.6 and SEQ ID No.7, the upstream and downstream primers of MMP-9 are respectively shown as SEQ ID No.8 and SEQ ID No.9, the upstream and downstream primers of bFGF are respectively shown as SEQ ID No.10 and SEQ ID No.11, the upstream and downstream primers of TGF β are respectively shown as SEQ ID No.12 and SEQ ID No.13, and the upstream and downstream primers of internal GAPDH are respectively shown as SEQ ID No.14(GAPDH-F) and SEQ ID No.15 (GAPDH-R);

TABLE 1 sequence listing

The quantitative reaction procedure was: pre-denaturation: a.95 ℃ for 30 s; and (3) cyclic reaction: b.95 ℃ for 10s, c.60 ℃ for 30s, and b-c circulating for 40 times; dissolution curve: d.95 ℃ 15s, e: 60 ℃ 60, f: 15s at 95 ℃;

data processing: according to RT-qPCR data, by 2^-(△△Ct)Analyzing the expression quantity of has-miRNA-21-5p in the serum exosomes of the liver cancer group and the control group;

results and analysis, as can be seen from the quantitative results shown in FIG. 5, the exosomes of HCCLM3(LM3-EXO) and MHCC97H (97H-EXO) have enhanced ability to synthesize angiogenesis promoting factors (VEGF- α, MMP-2, MMP-9, bFGF and TGF β) in HSCs cells after being stimulated by the control group (CTR) and LO2(LO2-EXO) exosomes, and the expression of has-miRNA-21-5p in the exosomes of HCCLM3(LM3-EXO-miR-21) and MHCC97H (97H-EXO-miR-21) can be interfered to inhibit the synthesis ability of the angiogenesis promoting factors of the HSCs, which indicates that the liver cancer exosomes can promote the angiogenesis promoting ability of the HSCs through has-miRNA-21-5 p.

Example 5: human serum exosome has-miRNA-21-5p can be used as biomarker for liver cancer diagnosis

5.1) study objects and groups

Experimental blood samples are collected in liver and gall surgery of Nanjing drugstore hospital, the material taking time is 2014-2016, the use of the blood samples informs the source of the sample and the approval of the ethical committee of the Nanjing drugstore hospital, 43 blood samples of liver cancer patients (with liver cancer and without other serious systemic diseases proved by pathology) are selected as a liver cancer group, 43 blood samples of healthy people (without other serious systemic diseases proved by detection) are selected as a control group, and 86 blood samples of the two groups are stored in a refrigerator at the temperature of-80 ℃; based on the fact that has-miRNA-21-5p verified in examples 1-4 has the functions of promoting liver cancer cell proliferation, promoting liver cancer cell migration and promoting subcutaneous transplantation tumor growth, blood samples of a liver cancer group and a control group are used as experimental objects for detecting has-miRNA-21-5p expression through qRT-PCR.

5.2) extraction and identification of serum exosomes

5.2.1) extracting serum exosomes, comprising the steps of:

5.2.1.1) blood samples of the liver cancer group and the control group were collected in 1.5ml EP tubes, respectively, and allowed to clot at 37 ℃ for 1 hour without anticoagulation treatment;

5.2.1.2) placing EP into a precooled centrifuge, and centrifuging 2000g at 4 ℃ for 10min to obtain serum;

5.2.1.3) collecting upper layer serum of EP, centrifuging at 4 deg.C for 10min at 3000g, and purifying the serum;

5.2.1.4) after collection of the serum, the ratio of 1: adding sterile PBS solution at a ratio of 1 for dilution, centrifuging at 4 deg.C for 30min at 10000g, and removing supernatant to remove macromolecular protein;

5.2.1.5), collecting precipitate, adding 10ml PBS buffer solution, washing, thoroughly blowing, mixing, filtering with 0.22 μm microporous membrane filter, and centrifuging at 100000g at 4 deg.C for 70 min;

5.2.1.6), collecting the precipitate, washing with 10ml PBS solution, centrifuging at 100000g at 4 deg.C for 70min to obtain exosome precipitate;

5.2.2) identification of serum exosomes:

5.2.2.1) transmission electron microscopy of exosomes:

5.2.2.1.1) taking part of the exosome precipitate extracted in the step 5.2.1) and re-suspending the precipitate by using 100 mu l of PBS solution;

5.2.2.1.2) fixing by using electron microscope fixing liquid (2.5% glutaraldehyde);

5.2.2.1.3) samples were prepared, cut to 0.12 μm and stained with 0.2% lead citrate and 1% uranyl acetate;

5.2.2.1.4) was observed with a JEM-2000EX II electron microscope (JEOL, Tokyo, Japan), as shown in FIG. 6 (a);

5.2.2.1.5) calculating the diameter and number of exosomes in different visual fields:

exosome diameters at random different fields: randomly selecting three fields under JEM-2000EX II electron microscope, and calculating the average diameter of exosomes, as shown in FIG. 6(b), the size of the exosomes of liver cancer group (HCC) is smaller than that of the exosomes of healthy person (Normal);

exosome numbers were randomized under different fields: randomly selecting three fields under a JEM-2000EX II electron microscope, and calculating the number of exosomes, wherein as shown in figure 6(c), the number of the exosomes in the serum of the liver cancer group (HCC) is significantly more than that of the exosomes in the serum of a healthy person (Normal) (p < 0.001);

5.2.2.2) exosome markers CD63, CD81, CD9 expression identification:

5.2.2.2.1) exosome protein extraction:

b. taking a part of the exosome precipitate extracted in the step 5.2.1), re-suspending the exosome precipitate by using 100 mu L of PBS solution to obtain an exosome sample, and mixing the exosome sample and the lysis solution according to the volume ratio of 1: 1, adding the mixture evenly and placing the mixture on ice for cracking for 10 min;

5.2.2.2.2) Western Blot detection of exosome markers CD63, CD81, CD9 identification: boiling the exosome proteins of the liver cancer group and the control group extracted in the step 5.2.2.2.1) by using a 1 XWestern sample loading buffer solution, electrically transferring the equivalent proteins to a polyvinylidene fluoride membrane by using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis method, sealing the protein by using 5% BSA in TBS-T, incubating the membrane overnight by using a primary antibody (a rabbit polyclonal antibody against CD63 or a rabbit polyclonal antibody against CD81 or a rabbit polyclonal antibody against CD9) at 4 ℃, using goat anti-rabbit IgG labeled by horseradish peroxidase or goat anti-mouse IgG labeled by horseradish peroxidase as a secondary antibody, and imaging an immunoreaction belt by using a Tanon gel imaging system;

the Western Blot detection result is shown in fig. 6(d), and exosomes CD63, CD81 and CD9 obtained from each serum sample of the liver cancer group (HCC) and the control group (Normal) are all positive, indicating that the substance extracted in step 5.2.1) is an exosome.

5.3) extraction of exosome Total RNA

Carrying out total RNA extraction on the exosome precipitates of the liver cancer group and the control group extracted in the step 2.1.6), and specifically operating as follows:

5.3.1) taking the exosomes of the liver cancer group and the control group extracted in the step 5.2.1), dissolving the residual precipitate after identification by using 100 mu L of PBS solution, and then adding 1mL of Trizol;

5.3.2) adding chloroform with the same volume, violently oscillating for 20s, and standing for 10min at normal temperature;

5.3.3) centrifuging at 12000g at 4 deg.C for 15min, then sucking the upper water phase, placing in a new EP tube, adding equal volume of isopropanol, centrifuging at 12000g at 4 deg.C for 10min, and removing the supernatant;

5.3.4) adding 1ml of pre-cooled 75% alcohol, centrifuging at 4 deg.C for 5min at 7500g, removing supernatant, and standing at room temperature for 10 min;

5.3.5) drying the RNA precipitate, adding appropriate amount of DEPC water to dissolve the RNA precipitate, and storing at-80 deg.C or immediately performing reverse transcription on ice;

5.4) qRT-PCR primers for synthesizing has-miRNA-21-5p

qRT-PCR amplification primers of has-miRNA-21-5p-F and has-miRNA-21-5p-R are designed according to a nucleotide sequence (SEQ ID NO.16) of has-miRNA-21-5p, synthesized by Shanghai Bioengineering Co., Ltd, and finally determined to have the following primer sequences after multiple debugging and verification:

has-miRNA-21-5p：5'-TGTCGGGTAGCTTATCAGACTGATGTTGACTGTTGAATCTCATGGCAACACCAGTCGATGGGCTGTCTGACA-3'(SEQ ID NO.16)

the forward primer has-miRNA-21-5 p-F: 5'-TTTTGTTTTTGCTGGTCTTAG-3' (SEQ ID NO. 17);

the reverse primer has-miRNA-21-5 p-R: 5'-AGCAGACAGTCAGGCAGGAT-3' (SEQ ID NO. 18);

the primer sequence of the reference gene U6 is as follows:

forward primer U6-F: 5'-GCTTCGGCAGCACATATACTAAAAT-3' (SEQ ID NO. 19);

reverse primer U6-R: 5'-CGCTTCACGAATTTGCGTGTCAT-3' (SEQ ID NO. 20);

5.5) reverse transcription Synthesis of cDNA samples

Use of

Master Mix (Perfect Real Time) (Takara, cat # DRR036A) reverse transcribes RNA from the liver cancer group and the control group, and the experimental procedures were performed according to the product instructions and specifically as follows:

the concentration of each total RNA sample was quantified using an ultraviolet spectrophotometer, and then RT reaction solution (20 μ L) was prepared on ice as follows: 5 is prepared from

RT Master Mix (for Real Time) 4. mu.L, Total RNA 1. mu.g, plus RNaseFreedH₂O to 20 μ L; after being mixed gently and evenly, the mixture is put into a PCR instrument for reverse transcription reaction, and the conditions of the reverse transcription reaction are as follows: carrying out reverse transcription at 37 ℃ for 15min and 85 ℃ for 5sec to obtain a cDNA sample;

5.6) qRT-PCR detection of expression level of has-miRNA-21-5 p:

using a ChamQ^TM

qPCR Master Mix (Without ROX) (Nanjing Novozan Biotechnology Co., Ltd., Code No.: Q321-02) was subjected to RT-qPCR amplification, and the experimental procedures were performed according to the product instructions, and were as follows:

Wherein the forward primer and the reverse downstream primer are has-miRNA-21-5p-F/has-miRNA-21-5p-R or U6-F/U6-R;

the quantitative reaction procedure was: pre-denaturation: a.95 ℃ for 30 s; and (3) cyclic reaction: b.95 ℃ for 10s, c.60 ℃ for 30s, and b-c circulating for 40 times; dissolution curve: d.95 ℃ 15s, e: 60 ℃ 60, f: s95 deg.C for 15 s;

results and analysis: as can be seen from the quantitative results shown in FIG. 7, the expression of has-miRNA-21-5p in the liver cancer group and the control group is significantly different, the relative expression level of has-miRNA-21-5p in the liver cancer group serum exosomes is significantly higher than the relative expression level (p <0.01) of has-miRNA-21-5p in the control group serum exosomes, and the human serum exosomes-miRNA-21-5 p can be used as a biomarker for liver cancer diagnosis.

Example 6: liver cancer diagnostic kit

The liver cancer diagnostic kit comprises: the quantitative amplification primers of the serum exosomes has-miRNA-21-5p-F related to liver cancer diagnosis described in example 1, have-miRNA-21-5 p-F (shown in SEQ ID NO.17) and has-miRNA-21-5p-R (shown in SEQ ID NO.18), forward and reverse primers U6-F (shown in SEQ ID NO.19) and U6-R (shown in SEQ ID NO.20) of the internal reference U6, each primer is 100 muM and 10 muL, and the quantitative amplification primers further comprise reagents commonly used in PCR technology, such as Trizol, chloroform, ddH2O, DNA Polymerase Chain Reaction (PCR),

RT Master Mix (for Real Time) consisting in particular of: 10mL of trizol, 10mL of chloroform, 10mL of ddH2O, 1mL of

RT Master Mix(forReal Time)；

The reagents commonly used in the PCR technology can also adopt corresponding commercial products;

the liver cancer diagnosis kit diagnoses liver cancer by detecting the expression level of has-miRNA-21-5p in serum exosome miRNA.

The above is the preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiment, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.

Sequence listing

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Claims

1. A serum exosome miRNA marker related to liver cancer diagnosis is characterized in that the marker is has-miRNA-21-5 p.

2. The use of the miRNA marker of claim 1 in the preparation of a liver cancer diagnostic kit.

3. The miRNA marker specific amplification primer of claim 1, wherein the specific amplification primer is: the kit comprises a forward primer has-miRNA-21-5p-F and a reverse primer has-miRNA-21-5p-R, wherein the nucleotide sequence of the forward primer has-miRNA-21-5p-F is SEQ ID NO.17, and the nucleotide sequence of the reverse primer has-miRNA-21-5p-R is SEQ ID NO. 18.

4. The use of the specific amplification primer for the miRNA marker of claim 3 in the preparation of a liver cancer diagnostic kit.

5. A liver cancer diagnosis kit is characterized in that the kit is used for detecting the expression quantity of has-miRNA-21-5p in serum exosome miRNA.

6. The liver cancer diagnostic kit according to claim 5, characterized in that: the kit comprises specific amplification primers of the miRNA marker of claim 3, namely has-miRNA-21-5p-F and has-miRNA-21-5 p-R.

7. The liver cancer diagnostic kit according to claim 6, characterized in that: the kit also comprises reagents commonly used in PCR technology.

The application of the has-miRNA-21-5p mimic in the preparation of a liver cancer animal model is characterized in that the nucleotide sequence of the has-miRNA-21-5p mimic is SEQ ID NO. 1.

The application of the has-miRNA-21-5p inhibitor in preparing a product for treating liver cancer is characterized in that the upstream sequence of the has-miRNA-21-5p inhibitor is SEQ ID NO.2, and the downstream sequence of the has-miRNA-21-5p inhibitor is SEQ ID NO. 3.