CN111560349A

CN111560349A - Method for preventing liver cancer caused by hepatitis B virus

Info

Publication number: CN111560349A
Application number: CN202010385183.9A
Authority: CN
Inventors: 胡宗强; 李立; 尹燕锋; 颜春涛; 张熙冰; 江杰
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2020-08-21

Abstract

The invention relates to a method for preventing morbidity, and discloses a method for preventing liver cancer caused by hepatitis B virus, which comprises the following specific steps: externally culturing HBV infection positive liver cancer cells and HBV infection negative liver cancer cells, separating exosomes in the two cells, and detecting the expression level of miR-142-3p in the exosomes by adopting qRT-PCR; culturing HBV infection positive liver cancer cells, co-culturing culture supernatant and peripheral blood T lymphocytes, and detecting the expression condition of miR-142-3p by adopting qRT-PCR; co-culturing T lymphocyte and HBV infection positive liver cancer cell, and then grouping the cells. The experimental scheme of the invention shows that HBV infection positive liver cancer cell exosome has influence on the occurrence and development of liver cancer, so that the liver cancer caused by hepatitis B virus can be reasonably prevented according to the conclusion obtained by the method.

Description

Method for preventing liver cancer caused by hepatitis B virus

Technical Field

The invention relates to a method for preventing diseases, in particular to a method for preventing liver cancer caused by hepatitis B virus.

Background

Liver cancer is a common malignant tumor, which can be divided into primary and secondary types, wherein the primary liver cancer originates from liver epithelium or mesenchymal tissue; secondary or metastatic liver cancer refers to the invasion of malignant tumors of multiple organ origins in the whole body to the liver. The number of new cases and deaths is far greater in developing countries than in developed countries, and more men and women are likely to have more morbidity and mortality worldwide.

Although medical technology is continuously improved, the early diagnosis rate of liver cancer in China is only about 20%, and the survival rate of liver cancer patients after 5 years of treatment is less than 10%. Meanwhile, the prognosis of liver cancer is much worse in developing countries. Liver cancer onset is a complex process of multiple factors and steps. Liver cancer may be induced by excessive drinking, long-term exposure to carcinogens and some metabolic diseases.

Meanwhile, research has proved that Hepatitis B Virus (HBV) is the main pathogen causing liver cancer, and liver cancer caused by HBV infection accounts for about 80% of all liver cancer cases. HBV infection is a global public health problem, and in China, one hepatitis B virus carrier exists among every ten people. Research shows that the current incidence of hepatitis B virus carriers in China is 5% -10%, and how to prevent hepatitis B is very important for chronic hepatitis B virus carriers.

Disclosure of Invention

The invention provides a method for preventing hepatitis B virus from causing liver cancer, aiming at the problems of hepatitis B pathogenesis in the prior art.

In order to solve the technical problem, the invention is solved by the following technical scheme:

a method for preventing liver cancer caused by hepatitis B virus, comprising the steps of:

step one, culturing HBV infection positive liver cancer cells and HBV infection negative liver cancer cells in vitro, separating exosomes in the two cells, and detecting the expression level of miR-142-3p in the exosomes by adopting qRT-PCR;

step two, culturing HBV infection positive liver cancer cells, co-culturing culture supernatant and peripheral blood T lymphocytes, and detecting the expression condition of miR-142-3p by adopting qRT-PCR;

step three, co-culturing the T lymphocytes and HBV infection positive liver cancer cells, and then grouping the cells, wherein the steps are as follows:

group A: transfecting miR-142-3p mimic in HBV infection positive liver cancer cell exosome;

group B: transfecting miR-142-3p mimic in HBV infection positive liver cancer cell exosome, and adding an iron death inhibitor;

group C: transfecting miR-142-3p mimic in HBV infection positive liver cancer cell exosome, and adding an apoptosis inhibitor;

group D: transfecting miR-142-3p mimic in HBV infection positive liver cancer cell exosome, and adding a necrotic apoptosis inhibitor;

group E: miR-142-3p imic is transfected in HBV infection positive liver cancer cell exosome, and pcDNA3.1-SLC3A2 is added.

Preferably, the proliferation activity of T lymphocytes is tested using CCK-8 and clonogenic assays. And detecting the ROS content in the T lymphocyte by adopting an active oxygen detection kit. And (3) detecting the intake of the GSH by adopting a GSH kit.

Preferably, the MDA kit is used for detecting the content of MDA. And detecting the iron ion expression condition by adopting an iron ion kit. And detecting the expression condition of the miR-142-3p by adopting qRT-PCR. And detecting the expression condition of the SLC3A2 by Western blotting.

Due to the adoption of the technical scheme, the invention has the remarkable technical effects that:

the experimental scheme of the invention shows that HBV infection positive liver cancer cell exosomes influence the biological behavior of T lymphocytes, so as to regulate and control the liver cancer tumor microenvironment and mediate the development of liver cancer, and meanwhile, HBV infection related liver cancer cell exosomes miR-142-3p target SLC3A2 regulate and control the death of T lymphocytes, thereby promoting the generation and development of liver cancer. Therefore, the liver cancer caused by the hepatitis B virus can be reasonably prevented according to the conclusion obtained by the method.

Drawings

FIG. 1 is a schematic diagram of the expression level of miR-142-3p in HBV infection-positive hepatoma cell exosomes.

FIG. 2 is a diagram showing the qRT-PCR result of miR-142-3p expression level in HBV infection positive hepatoma cell exosome.

FIG. 3 is a graph showing the expression levels of miR-142-3p in T lymphocytes of a co-culture system.

Detailed Description

Example 1

In the embodiment, CCK-8 and clone formation experiments are adopted to detect the proliferation activity of the T lymphocyte; detecting the content of ROS in T lymphocytes by adopting an active oxygen detection kit; detecting the intake of GSH by adopting a GSH kit; detecting the content of MDA by adopting an MDA kit; detecting the iron ion expression condition by adopting an iron ion kit; detecting the expression condition of miR-142-3p by adopting qRT-PCR; and detecting the expression condition of the SLC3A2 by Western blotting.

In this example, the following experimental means were used for the experiment.

One, qRT-PCR experiment

Extracting total RNA in HBV infection positive/negative liver cancer tissue or tumor infiltration T lymphocyte by using Trizol reagent, carrying out reverse transcription on all RNA by adopting a one-step reverse transcription kit to synthesize cDNA, taking U6 as an internal reference, and carrying out qRT-PCR reaction by using a SYBR GreenReal-Time PCR Master Mix kit, wherein the amplification procedure is as follows: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 35s, and extension at 72 ℃ for 10s for 45 cycles. And calculating the expression level of the miR-142-3p by adopting a 2-delta Ct method.

Western blotting experiment

And (3) extracting the total protein of the cells of each treatment group by using RAPI protein lysate, and quantifying the protein concentration by using a BCA method. Taking 80 mu g of protein from each group, carrying out SDS-PAGE electrophoresis, transferring a target band onto a PVDF membrane, sealing with 5% skimmed milk powder at room temperature for 2h, adding primary antibody diluted according to a ratio of 1:1000, incubating overnight at 4 ℃, and washing for 3 times and 5 min/time by TBST; HRP-labeled secondary antibody diluted at a ratio of 1:5000 was added, incubated at room temperature for 2h, washed 3 times with TBST, 5 min/time, stained with ECL, and analyzed for band gray value by Image J. The experiment was repeated 3 times.

Third, immunohistochemical experiment

Incubating the tissue slices in 3% H2O2 in the dark for 20min to block endogenous peroxidase, washing with PBS, placing the slices in an autoclave, repairing with 0.01M citric acid buffer (pH 6.0) antigen for 4min, and naturally cooling; dropping goat serum, placing in a wet box, sealing at room temperature for 30min, and removing excessive liquid; adding primary antibody, and incubating overnight at 4 deg.C in a wet box; dripping secondary antibody, and incubating for 30min in a wet box; dripping DAB color development liquid, developing at room temperature for 5-10min, and flushing with running water to stop developing; counterstaining with hematoxylin for 2 min; separating with 1% hydrochloric acid alcohol for 2 s; 1% ammonia water for 10 s; alcohol gradient dehydration, transparent clearing agent and neutral gum sealing; observed under an optical microscope and photographed.

Four, ROS level detection

DCFH-DA was diluted in serum-free medium at a ratio of 1:1000 to a final concentration of 10. mu. mol/L. Collecting each group of cells, centrifuging to remove cell culture solution, adding DCFH-DA solution diluted in proper volume, adjusting the cell density to 1 × 106/ml, placing in a cell culture box at 37 ℃ for incubation for 20min, and reversing and uniformly mixing every 3-5 min to ensure that the probe is fully contacted with the cells; washing the cells 3 times with serum-free medium to remove DCFH-DA not entering the cells sufficiently; add 500. mu.l serum-free medium to each centrifuge tube and blow-beat well. Adding the protein into a 96-well plate according to 100 mu l/well, detecting the fluorescence intensity before and after stimulation in real time or time points by using 488nm excitation wavelength and 525nm emission wavelength on a fluorescence microplate reader, wherein the stronger the fluorescence is, the higher the ROS content is.

Fifth, MDA content detection

The cells of each group were collected, mixed at a ratio of 106:1 to physiological saline (ml), and then subjected to ultrasonication in an ice bath under the following conditions: power 20%, ultrasound 3s, interval 10s, repeat 30 times. Centrifuging at 8000g for 10min at 4 deg.C after the completion of ultrasonication, and collecting supernatant. Accurately sucking 0.6ml of the first reagent, adding the first reagent into a 1.5ml centrifuge tube, accurately adding 0.2ml of the sample, uniformly mixing, placing the mixture into a 90 ℃ water bath kettle, incubating for 30min, placing the mixture into an ice bath, cooling, and centrifuging for 10min at 10000g of normal temperature. The supernatant was taken out in a 1ml glass cuvette, and the absorbance value (A) at 532nm and 600nm was measured for each set of samples. The experiment was repeated 3 times.

Sixthly, detecting the content of iron ions

Each set of cells was collected, following cell: saline (ml) was mixed at a ratio of 106:1, and then the cells were sonicated in an ice bath under the conditions: power 20%, ultrasound 3s, interval 10s, repeat 30 times. Centrifuging at 8000g for 10min at 4 deg.C after the completion of ultrasonication, and collecting supernatant. Accurately sucking 1.5ml of iron color developing agent, adding into a centrifuge tube, adding 0.5ml of sample into the centrifuge tube, mixing uniformly, boiling in a water bath for 5min, cooling, and centrifuging at 3500r/min for 10 min; then, 200. mu.l of the supernatant was taken, and the absorbance OD value was measured at a wavelength of 520nm with an optical path of 0.5, to calculate the iron ion content. The experiment was repeated 3 times.

Seven, GSH level detection

GSH uptake was measured using a GSH kit. Collecting each group of cells, adding a protein remover M, fully mixing, then alternately placing in a refrigerator with the temperature of minus 80 ℃ and a water bath kettle with the temperature of 37 ℃ for 2 times of quick freeze thawing, placing in a condition of 4 ℃ for incubation for 5min, centrifuging by a low-temperature centrifuge, taking supernatant, diluting according to a certain proportion, sequentially adding a sample/standard substance, the protein remover M and total glutathione detection working solution, placing in a condition of 25 ℃ for incubation for 5min, adding 50 mu l of 0.5mg/ml of NADPH, incubating for 25min at room temperature, detecting an absorbance (A) value at the wavelength of 412nm on an enzyme labeling instrument, and calculating the GSH content of a sample through a standard curve.

Eight, CCK-8 method for detecting cell proliferation activity

The cells of each group were collected, counted (1X 105 cells/ml), inoculated into a 96-well plate, 100. mu.l of cell suspension per well, and cultured in a cell culture chamber at 37 ℃ and 5% CO2 saturation humidity. And detecting the proliferation activity of the T lymphocyte by adopting a CCK-8 kit. And adding 15 mu l of CCK-8 solution into each hole during the culture for 0, 1, 2, 3 and 4 days respectively, then placing the holes in an incubator for incubation for 2 hours, and detecting the OD450 value on a microplate reader.

Ninthly, clone formation experiment

First, groups of cells in logarithmic growth phase were digested with pancreatin, then the cells were suspended by using 10% FBS-containing RPMI-1640 medium, the cell density was adjusted to 300/ml, the cells were seeded in 6-well plates at a density of 1 ml/well, and after 2 weeks of culture in a cell incubator saturated with 5% CO2 at 37 ℃, they were washed 2 times with PBS and fixed with methanol for 30min, stained with 1mg/ml crystal violet for 15min, and counted in such a manner that more than 10 cells were counted as one clone. The clone formation rate (number of clones/number of seeded cells) × 100%. Set 3 sets of replicates.

Ten, establishment of xenograft mouse model

A xenograft mouse model was established using BALB/c mice of about 5 weeks of age. Stably transfecting miR-142-3pinhibitor plasmids into HBV infection positive liver cancer cells, transfecting miR-142-3p mimic plasmids into HBV infection negative HBV liver cancer cells, respectively taking cells in logarithmic growth phase, suspending the cells in serum-free culture medium after trypsinization, adjusting the cell concentration to be 1 × 107/ml, inoculating the cells to the armpit of a mouse according to the cell amount of 1 × 106, measuring the tumor diameter of the mouse every week, killing the mouse after 5 weeks according to the V ═ shortest diameter × longest diameter × 0.5, measuring the tumor size of the mouse, and detecting various indexes. The same method was used for the verification recovery experiment.

The experimental result verifies that miR-142-3p expresses the level in the exosomes of HBV infection positive liver cancer cells to culture the HBV infection positive liver cancer cells and negative liver cancer cells, the exosomes are respectively separated, total RNA in the exosomes is extracted to carry out gene chip sequencing, and the analysis result shows that:

as shown in figure 1, compared with HBV infection negative liver cancer cell exosome, miR-142-3p is highly expressed in HBV infection positive liver cancer cell exosome;

in addition, as shown in FIG. 2, the qRT-PCR result shows that miR-142-3p is low expressed in HBV infection negative liver cancer cells and normal liver epithelial cell exosomes.

In fig. 1: a1-13: HBV infection positive hepatoma cell exosomes; B1-B3: HBV infects negative liver cancer cell exosome. In fig. 2: positive: HBV infection positive hepatoma cell exosomes; negative: HBV infection negative hepatoma cell exosomes; normal: normal liver epithelial cell exosomes.

The experimental result verifies the expression level of miR-142-3p in the T lymphocytes of the co-culture system. As shown in figure 3, the HBV infection positive liver cancer cell exosome and the T lymphocyte are co-cultured, and then the expression level of miR-142-3p in the T lymphocyte of the co-culture system is detected by adopting qRT-PCR (quantitative reverse transcription-polymerase chain reaction), and the result shows that compared with the separately cultured T lymphocyte, the expression level of miR-142-3p in the T lymphocyte is obviously increased after the HBV infection positive liver cancer cell exosome and the T lymphocyte are co-cultured.

In fig. 3: a: t lymphocytes cultured alone; b: HBV infection positive hepatoma cell exosome is co-cultured with T lymphocyte.

Experiments and related researches in this embodiment prove that miR-142-3p is highly expressed in HBV infection positive hepatoma cell exosomes (hereinafter referred to as exosome A), and miR-142-3p in T lymphocytes co-cultured with exosome A is also highly expressed; miR-142-3p is highly expressed in exosome A, and miR-142-3p in T lymphocytes co-cultured with exosome A is also highly expressed.

miRNAs have important regulation and control functions on lymphocyte biological functions; the over-expression of the exosome A can inhibit the proliferation of T lymphocytes and induce the death of T lymphocyte iron.

The dual-luciferase reporter gene proves that miR-142-3p is targeted to regulate and control the iron death key gene SLC3A 2.

Through in vivo and in vitro functional experiments, the miR-142-3/SLC3A2 molecular axis promotes the molecular mechanism of the development process of liver cancer by regulating the death of T lymphocyte iron, provides a new thought and a molecular target for HBV (hepatitis B virus) infected liver cancer patients, and then plays a role in preventing liver cancer caused by hepatitis B virus.

In summary, the above-mentioned embodiments are only preferred embodiments of the present invention, and all equivalent changes and modifications made in the claims of the present invention should be covered by the claims of the present invention.

Claims

1. The method for preventing liver cancer caused by hepatitis B virus is characterized by comprising the following steps:

2. The method of claim 1, wherein the hepatitis B virus-induced liver cancer is: detecting the proliferation activity of the T lymphocyte by adopting a CCK-8 and clone formation experiment; detecting the content of ROS in T lymphocytes by adopting an active oxygen detection kit; detecting the intake of GSH by adopting a GSH kit; detecting the content of MDA by adopting an MDA kit; detecting the iron ion expression condition by adopting an iron ion kit; detecting the expression condition of miR-142-3p by adopting qRT-PCR; and detecting the expression condition of the SLC3A2 by Western blotting.