CN113209297A - Application of hepatitis B virus pregenomic RNA inhibitor in preparing medicine for treating hepatitis B related liver cancer - Google Patents
Application of hepatitis B virus pregenomic RNA inhibitor in preparing medicine for treating hepatitis B related liver cancer Download PDFInfo
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- CN113209297A CN113209297A CN202110393700.1A CN202110393700A CN113209297A CN 113209297 A CN113209297 A CN 113209297A CN 202110393700 A CN202110393700 A CN 202110393700A CN 113209297 A CN113209297 A CN 113209297A
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Abstract
The invention relates to the technical field of biological medicines, in particular to application of a hepatitis B virus pregenomic RNA inhibitor in preparation of a medicine for treating hepatitis B related liver cancer. The invention also provides the application of the interferon alpha in preparing the medicine for treating hepatitis B related liver cancer by reducing the stability of hepatitis B virus pregenomic RNA and inhibiting the proliferation capacity and stem cell characteristics of hepatitis B related liver cancer. The invention discovers a new mechanism and new application of interferon alpha for treating hepatitis B related liver cancer, widens the treatment indications of interferon alpha, and the medicine interferon alpha used in the invention is a classical medicine clinically applied for many years, is safe and reliable, has wide sources, and is easy to clinically use and popularize.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of Hepatitis B virus pregenomic RNA (HBV pgRNA) inhibitor interferon alpha (IFN-alpha) in preparation of a medicine for treating Hepatitis B related liver cancer.
Background
Primary Hepatocellular Carcinoma (HCC, hereinafter referred to as liver cancer) is the third leading cause of cancer mortality in the world and the second leading cause of cancer death in our country. More than 50% of HCC worldwide occurs in our country every year. Surgical resection is the preferred method for treating liver cancer at present. However, clinically, most patients have advanced to the middle and late stage when they see a doctor due to the hidden onset of liver cancer, liver cirrhosis combined and high invasiveness, and the chance of operation is missed. In addition, the survival condition of liver cancer patients is severely limited by key problems of low radical resection rate of liver cancer, high recurrence rate after resection and the like. Various non-operative treatment modes such as transcatheter arterial chemoembolization, chemotherapy, ablation treatment, radiotherapy, molecular targeted therapy, immunotherapy and the like are also important means for treating liver cancer at present, but the treatment effect of the treatment method on the liver cancer in the advanced stage is still not ideal.
Chronic Hepatitis B (CHB, hereinafter referred to as Chronic Hepatitis B) is the leading cause of liver cirrhosis in China and eventually develops into HCC. Perioperative anti-Hepatitis B Virus (HBV) treatment can significantly improve the prognosis of patients with Hepatitis B-related liver cancer. In the research of the occurrence and development of hepatitis B associated liver cancer (HBV-related HCC, hereinafter referred to as hepatitis B associated liver cancer), most of the research mainly focuses on chronic inflammation caused by HBV replication in the liver and the influence of host immune response on the HCC occurrence and development, but ignores the direct cancer promotion effect of HBV itself. Relevant researches find that the insertion and integration of HBV in a key site of a host genome and direct factors of viruses such as X protein of HBV and the like can enable liver cells to have the potential of malignant transformation, so that the liver cells are closely involved in the development process of HCC. Therefore, research on direct carcinogenesis of HBV has important significance for preventing and treating liver cancer.
The persistent risk of hepatitis b-associated liver cancer is due to the presence of nuclear HBV covalently closed circular DNA (cccDNA), which is a sustainable template for HBV replication. Since invasive liver biopsy is required for cccDNA detection, quantification of intrahepatic cccDNA as a routine diagnosis is not realistic, and therefore it is necessary to find and develop non-invasive surrogate markers to monitor the amount or activity of cccDNA. Since Hepatitis B virus pregenomic RNA (hereinafter referred to as HBV pgRNA) as a direct transcription product of cccDNA can reflect its activity to a certain extent, HBV pgRNA has gained much attention in recent years. An increasing number of studies have shown that HBV pgRNA can serve as a new serum biomarker for HBV infection, therapy and prognosis. However, what role HBV pgRNA plays in the process of developing and progressing hepatitis B related liver cancer is not clear, and it is worth further research.
Interferon alpha (IFN-. alpha.) therapy is currently considered an important antiviral therapeutic strategy for chronic hepatitis B. IFN-alpha belongs to the type I interferon cytokine family, and has various biological properties including antiviral, immunoregulatory, antiproliferative and antiangiogenic effects. However, whether IFN-alpha can play a role in preventing the development of hepatitis B related liver cancer by inhibiting HBV pgRNA or not is worth further exploration.
Disclosure of Invention
The invention aims to provide application of a hepatitis B virus pregenomic RNA (HBV pgRNA) inhibitor in preparation of a hepatitis B related liver cancer treatment drug.
The invention explores the effect of HBV pgRNA in the generation and the development of hepatitis B related liver cancer, and inhibits the expression of HBV pgRNA by using IFN-alpha, thereby achieving the effect of treating hepatitis B related liver cancer. According to the invention, researches show that the interfering HBV pgRNA can inhibit the proliferation capacity and stem cell characteristics of liver cancer cells, and the over-expression HBV pgRNA can promote the proliferation capacity and stem cell characteristics of liver cells. In addition, further research finds that IFN-alpha can increase the m6A RNA modification level of HBV pgRNA, reduce the stability of the HBV pgRNA and promote the degradation of the HBV pgRNA, so that the expression of the HBV pgRNA is inhibited, the proliferation capacity and the stem cell characteristic of liver cancer cells are reduced, and the effect of treating liver cancer is finally exerted.
The interferon alpha (IFN-alpha) reduces the stability of the interferon alpha (IFN-alpha) by increasing the modification level of N6-methyladenine (N6-methylaldenosine, m6A) of hepatitis B virus pregenomic RNA (HBV pgRNA), and inhibits the proliferation capacity and stem cell characteristics of hepatitis B related liver cancer to prepare the application of the hepatitis B related liver cancer treatment drug. The interferon alpha can inhibit the proliferation capacity and stem cell characteristics of hepatitis B related liver cancer by promoting the degradation of HBV pgRNA, thereby playing a role in treating hepatitis B related liver cancer.
The total length of HBV pgRNA (genotype A) is 3326 bases, and the sequence is shown in SEQ ID NO. 1.
In a first aspect of the present invention, there is provided an application of a hepatitis b virus pregenomic rna (hbv pgrna) inhibitor in the preparation of a medicament for treating hepatitis b-related liver cancer.
Furthermore, the HBV pgRNA inhibitor is a small interfering RNA molecule and a short hairpin RNA which specifically interfere the expression of the HBV pgRNA.
In a preferred embodiment of the present invention, the HBV pgRNA inhibitor is a specific small interfering RNA for HBV pgRNA, and the sequence thereof is shown in SEQ ID No. 14:
sequence 5 '-3': GATCAGGCAACTATTGTGG (SEQ ID NO. 14).
The invention finds the application of HBV pgRNA in the development and progress of hepatitis B related liver cancer. Through in vivo and in vitro functional tests, after the HBV pgRNA in the liver cancer cell HepG2.2.15 is interfered, the proliferation capacity and the stem cell characteristic of the liver cancer cell are obviously inhibited (figure 1); after the HBV pgRNA in the liver cancer cell hepg2.2.15 is overexpressed, the proliferation capacity and stem cell characteristics of the liver cancer cell are significantly enhanced (fig. 2).
Furthermore, the HBV pgRNA inhibitor is an antagonist, a down-regulator, a blocker and a blocker of HBV pgRNA.
Further, the HBV pgRNA inhibitor is interferon alpha (IFN-alpha).
In a second aspect of the present invention, the present invention provides an application of interferon alpha in the preparation of a medicament for treating hepatitis b-related liver cancer by inhibiting the expression of HBV pgRNA.
Furthermore, the interferon alpha can reduce the proliferation capability and stem cell characteristics of liver cancer cells by inhibiting the expression of HBV pgRNA, thereby playing a role in treating liver cancer.
Furthermore, the interferon alpha reduces the stability of HBV pgRNA and promotes the degradation of the HBV pgRNA by increasing the modification level of m6A RNA of the HBV pgRNA, thereby inhibiting the expression of the HBV pgRNA and playing a role in treating liver cancer.
IFN-alpha can inhibit the proliferation capacity and stem cell characteristics of liver cancer cells, and after HBV pgRNA is over-expressed, the application of the inhibition effect of IFN-alpha on the proliferation capacity and dryness of liver cancer cells can be partially recovered. Through in vivo and in vitro functional tests, IFN-alpha can inhibit the proliferation capacity and stem cell characteristics of liver cancer cells (HepG2.2.15); after the HBV pgRNA is over-expressed, the inhibition effect of IFN-alpha on the proliferation capacity of the liver cancer cells and the characteristics of stem cells can be partially recovered (figure 3).
In a third aspect of the present invention, there is provided the use of interferon alpha (IFN- α) in the preparation of an HBV pgRNA inhibitor.
Furthermore, interferon alpha can reduce the stability of HBV pgRNA and promote the degradation of HBV pgRNA by up-regulating the m6A RNA modification level of HBV pgRNA, thereby inhibiting the expression of HBV pgRNA.
Calculating the half-life of the HBV pgRNA after IFN-alpha treatment on HepG2.2.15 cells by an actinomycin D transcription inhibition test, and finding that the half-life of the HBV pgRNA after IFN-alpha treatment is obviously shortened and the stability of the HBV pgRNA is obviously reduced (fig. 4B); the MeRIP experiment specific to m6A shows that after HepG2.2.15 cells are treated by interferon alpha, the m6A specific antibody can enrich more HBV pgRNA compared with a negative control IgG antibody, and the m6A modification site exists in the HBV pgRNA; furthermore, after hepg2.2.15 cells after IFN- α treatment, HBV pgRNA enriched with m 6A-specific antibody was significantly increased in the interferon α -treated group compared to the group without IFN- α treatment, suggesting that interferon α treatment could significantly up-regulate the m6A modification level of HBV pgRNA (fig. 4C).
The invention has the advantages that:
1. the invention provides application of HBV pgRNA in the occurrence and development of hepatitis B related liver cancer, and researches show that the HBV pgRNA can increase the proliferation capacity of liver cancer cells and the characteristics of stem cells.
2. The invention provides an application of IFN-alpha in reducing the stability of HBV pgRNA and promoting the degradation of the HBV pgRNA by increasing the m6A RNA modification level of the HBV pgRNA, so as to inhibit the expression of the HBV pgRNA and finally play a role in treating hepatitis B related liver cancer. The invention discovers a brand new action mechanism of a classic drug IFN-alpha for inhibiting the development of hepatitis B related liver cancer, enlarges the adaptation of the IFN-alpha for treating the hepatitis B related liver cancer, reduces the time and the economic cost for developing a brand new anti-liver cancer drug and realizes the 'new use of old drug' of the IFN-alpha.
3. The interferon alpha used in the invention is a classical medicine which is clinically applied for many years, and the medicine is safe and reliable, has wide sources and is easy to clinically use and popularize.
Drawings
FIG. 1: the interference of the expression of HBV pgRNA can inhibit the proliferation capacity and stem cell characteristics of liver cancer cells. (A) efficiency verification after constructing a hepatoma cell line with HBV pgRNA stable interference by using lentivirus-mediated short hairpin RNA; after interfering the expression of HBV pgRNA, the proliferation capacity (B), the clonogenic capacity (C), the cell balling capacity (D) and the expression of important dry markers (CD44, CD90, CD133) (E) of the liver cancer cell are all obviously inhibited; the nude mouse subcutaneous tumor formation experiment shows that after interfering the expression of HBV pgRNA, the tumor formation ability and proliferation ability of liver cancer cells are obviously reduced (F, G).
FIG. 2: after the HBV pgRNA is over-expressed, the proliferation capability and the stem cell characteristic of the liver cancer cells can be enhanced. (A) efficiency verification after construction of HBV pgRNA stable over-expressed hepatoma cell lines using over-expression plasmids; after the HBV pgRNA is over-expressed, the proliferation capacity (B), the clonogenic capacity (C), the cell balling capacity (D) and the expression of important dry markers (CD44, CD90, CD133) (E) of the liver cancer cell are obviously enhanced; the nude mouse subcutaneous tumor formation experiment shows that after over-expressing HBV pgRNA, the tumor formation ability and proliferation ability of liver cancer cells are obviously improved (F, G).
FIG. 3: IFN-alpha can inhibit the proliferation capacity and stem cell characteristics of liver cancer cells; while overexpression of HBV pgRNA can reverse the inhibitory effect to some extent. (A) Calculating half lethal concentration of IFN-alpha to hepatoma cell HepG2.2.15 by a cytotoxicity experiment, wherein the lethal concentration is about 2000 IU/ml; IFN-alpha can inhibit the proliferation capacity (B) of liver cancer cells, the clonogenic capacity (C), the cell balling capacity (D) and the expression (E) of important dry markers (CD44, CD90 and CD133) in vitro, and over-expression HBV pgRNA can partially reverse the inhibition effect (B-E) of IFN-alpha on the proliferation and anti-apoptosis capacity of liver cancer cells; the nude mouse subcutaneous tumor formation experiment shows that IFN-alpha can inhibit the tumor formation ability and proliferation ability of liver cancer cells in vivo, and over-expression HBV pgRNA can partially reverse the inhibition effect of IFN-alpha on the tumor formation ability and proliferation ability of liver cancer cells (F, G).
FIG. 4: IFN-alpha reduces the stability of HBV pgRNA and promotes the degradation thereof by increasing the level of m6A RNA modification of HBV pgRNA, thereby inhibiting the expression of HBV pgRNA. (A) IFN-alpha can inhibit the expression of HBV pgRNA in liver cancer cells HepG2.2.15 cells; (B) calculating the half-life of HBV pgRNA after IFN-alpha treatment on HepG2.2.15 cells by an actinomycin D transcription inhibition test, and finding that the half-life of the HBV pgRNA after IFN-alpha treatment on the cells is obviously shortened, and the IFN-alpha can obviously reduce the stability of the HBV pgRNA and promote the degradation of the HBV pgRNA; (C) through MeRIP experimental analysis method specific to m6A, it was found that after IFN-alpha treatment of HepG2.2.15 cells, the m6A specific antibody can enrich more HBV pgRNA compared with negative control IgG antibody, suggesting that HBV pgRNA has m6A modification site; furthermore, the IFN- α -treated group enriched with m 6A-specific antibody HBV pgRNA was significantly increased compared to the group not treated with IFN- α, suggesting that IFN- α treatment could significantly up-regulate the m6A modification level of HBV pgRNA.
FIG. 5: HBV pgRNA full-length plasmid map.
FIG. 6: short hairpin RNA profiles targeting HBV pgRNA.
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings.
The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), either according to conventional conditions or according to the manufacturer's recommendations. Percentages and parts are by volume unless otherwise indicated.
Example 1:
materials and methods
(ii) extraction of Total RNA
Total RNA from hepatoma cells was extracted using the RNAioso Plus (Code No.9109) from Takara. Consumables such as a centrifuge tube and a gun head used by the method, various reagents and the like do not contain RNase. The method comprises the following steps: taking cells with the density of about 80% in a 10cm cell culture dish, and adding 1ml of RNAioso Plus to fully crack the cells; standing at room temperature for 5 minutes, adding 200ul of chloroform, shaking and mixing uniformly, and standing at room temperature for 5 minutes; 12000g, centrifuging for 15 minutes at 4 ℃, transferring 500ul of supernate into a new 1.5ml centrifuge tube, adding 500ul of isopropanol, and standing for 10 minutes at room temperature; 12000g, centrifuging for 10 minutes at 4 ℃; discarding the liquid, leaving a precipitate, adding 1ml of 75% ethanol, and centrifuging at 10000g and 4 ℃ for 5 minutes; discarding the liquid, and leaving the precipitate and drying; adding a proper amount of DEPC water for dissolving; the concentration is measured, and after the concentration is adjusted to 500ng/ul by using DEPC water, the mixture is placed in a refrigerator at minus 80 ℃ for standby.
(II) reverse transcription of RNA
The present invention uses PrimeScript of Takara corporationTMRT Master Mix (Code No. RR036A) kit for RNA reverse transcription. The operating procedure was carried out according to the instructions.
(III) Real-time quantitative PCR (Real-time PCR)
TB produced by using Takara in the present invention
Premix Ex TaqTM(Code number RR420A), then in StepOneTM real-time PCR System (Ap) using standard PCR protocolsAttached Biosystems, CA) were subjected to real-time quantitative PCR reactions. The operating procedure was carried out according to the instructions. The present invention uses housekeeping gene GAPDH as an internal reference and uses a 2-delta- Δ ct quantification method for calculation. The sequences of the primers used in the present invention are detailed in table 1:
TABLE 1 primer names and sequences used in the present invention
(IV) construction of plasmids and lentiviruses
The present invention constructs a full-length HBV pgRNA vector using the following specific primers (the genotype of HBV used is type A). The specific primer sequences are as follows, forward direction: 5'-AAAGGTACCAACTTTTTCACCTCTGCCTA-3' (SEQ ID NO. 12); and (3) reversing: 5'-GGGCTCGAGTTTATAAGGGTCAATGTCCAT-3' (SEQ ID NO. 13).
The full-length HBV pgRNA fragment was then ligated into the empty pc DNA 3.1 vector (Invitrogen), the complete information of which is shown in FIG. 5. Specific short hairpin RNAs targeting HBV pgRNA were constructed based on small interfering RNAs targeting HBV pgRNA and purchased from biology and biology (OBiO Technology) with sequences detailed in table 2; specific short hairpin RNA information for targeting HBV pgRNA is detailed in FIG. 6.
TABLE 2 short hairpin RNA sequences targeting HBV pgRNA
(V) cell culture and transfection of lentiviruses and plasmids
The human hepatoma cell line HepG2.2.15 stably expressing HBV used in the present invention was from the Shanghai cell institute of Chinese academy of sciences. All cells were cultured in an incubator containing 5% carbon dioxide at 37 ℃ and cultured using DMEM medium supplemented with 10% fetal bovine serum, 2mM glutamine and 1% penicillin and streptomycin. The human hepatoma cell line hepg2.2.15 stably expressing HBV used in the study was identified by str (short distance repeat) and LookOut o R Mycoplasma PCR Detection Kit (Sigma-Aldrich) to exclude hela cells and Mycoplasma contamination. Both lentiviruses and plasmids used in the invention were synthesized and purchased from and Biotechnology Limited (Shanghai); both lentivirus and plasmid transfections were performed as described.
(VI) in vitro cell function test
1. CCK-8 experiment
The present invention uses Dojindo Cell Counting Kit-8 Kit (Code No. CK04) assay to evaluate the effect of HBV pgRNA on the proliferation ability of hepatoma cells, and the detailed procedures are described in the specification. In brief, the cells to be detected are spread in a 96-well plate according to 2000 cells per well, after the cells adhere to the wall, 10ul of CCK-8 reagent is added into each well, the mixture is incubated for 2 hours at 37 ℃ under the condition of 5% carbon dioxide, and the absorbance is detected by using an enzyme-linked immunosorbent assay. The above procedure was then repeated every 12 hours until 72 hours of incubation.
2. Clone formation experiments
The invention uses a clonogenic experiment to evaluate the influence of HBV pgRNA on the clonogenic capacity of hepatoma cells, and the operating steps are carried out according to the instructions. Briefly, hepatoma cells were digested with pancreatin and counted, and 2X 10^3 cells plated in 6-well plates and cultured at 37 ℃ with 5 percent carbon dioxide for 10-14 days. Cells were fixed with 4% paraformaldehyde and cell clones were stained with a staining solution containing 0.1% crystal violet and 20% methanol. Finally, the clones formed by the cells are counted and analyzed
3. Cell balling experiment
The present invention uses a balling experiment to evaluate stem cell characteristics of tumor cells. The procedures for the cell spheronization experiments were performed as described in the specification. Briefly, 1000 cells were seeded into each well of an ultra-low adhesion six-well plate (Corning), and tested for their ability to form primary spheres using serum-free DMEM medium further containing B27 (1: 50; Invitrogen), 20ng/ml epidermal growth factor (Invitrogen) and 20ng/ml basic fibroblast growth factor (Invitrogen) for 14 days. On day 14, the number of cell balls formed was counted using an inverted microscope (Olympus).
(VII) in vivo experiments
In the present invention, 5-week-old male BALB/c (nu/nu) nude mice were used, purchased from the animal experiment resource center of Chinese academy of sciences, and were subject to humanistic care. The effect of HBV pgRNA on the tumorigenicity of hepatoma cells was evaluated using a nude mouse subcutaneous tumor-bearing experiment. Interfering or overexpressing by subcutaneous injection of HBV pgRNA in an amount of 1 x 10^ 6; or hepg2.2.15 cells receiving different drug treatments to the axilla of nude mice, kinetic changes in tumor formation were assessed 1 time per week and mice were continuously monitored for 8 weeks; in addition, for IFN-alpha drug treatment experiments, after a subcutaneous xenograft model is established for 10 days, IFN-alpha is intraperitoneally injected every day at a dose of 1500IU/g by a group of HBV pgRNA overexpression nude mice for 4 weeks continuously; another group of HBV pgRNA overexpression group nude mice were injected with PBS of equal volume per day in the abdominal cavity for 4 weeks. The treatment method of the negative control group of nude mice is the same as that of the HBV pgRNA overexpression group of nude mice, namely, the negative control group of nude mice is injected with IFN-alpha in the abdominal cavity every day at the dose of 1500IU/g for 4 weeks continuously; the other group of negative control nude mice were injected with an equal volume of PBS each day intraperitoneally for 4 weeks continuously. Tumor volume was measured weekly (volume V ═ length × width ^2 × 1/2). After 5 weeks from subcutaneous injection, animals were sacrificed by decapping and samples were collected for further experiments.
(VIII) IFN-alpha drug sensitivity assay
The IFN-alpha used in the invention is IFN-alpha-2 a (Code No. HY-P7022) of MCE company, and a drug sensitivity test aiming at the IFN-alpha is carried out in HepG2.2.15 cells. The specific operation method refers to the specification. Briefly, cells were seeded in 96-well plates and co-cultured with a series of IFN- α dilutions (0, 10, 100, 500, 1000, 2000, 5000, 7000 and 10000IU/ml) in multiples, covering the cells every 24 hours with fresh medium containing the indicated concentration of IFN- α. After 72 hours of culture, the median lethal dose (IC 50) of IFN-. alpha.for HepG2.2.15 cells was calculated by CCK-8 assay.
(nine) measurement of HBV pgRNA stability and half-life
The procedures for the HBV pgRNA stability test can be referred to the description. Briefly, cells were treated with 5 μ g/ml actinomycin D (Abcam) and harvested at the indicated time points (0h, 4h, 8h, 12h and 24 h). Total RNA was extracted and analyzed by q-PCR. Since actinomycin D treatment causes the transcription of cells to be arrested, the change in mRNA concentration (dC/dt) at a given time is proportional to the decay constant of mRNA (Kdecoy) and the concentration of mRNA (C), giving the following equation: dC/dt ═ Kdecay C; therefore, the following equation is derived to estimate the decay constant Kdecoy of mRNA:
ln (C/C0) ═ Kdecayt; to calculate the half-life of the mRNA (t1/2), i.e. when 50% of the mRNA is degraded (C/C0 ═ 1/2), the formula is: ln (1/2) ═ Kdecay t 1/2; the formula for the calculation of the half-life of the mRNA is thus: t1/2 ═ ln 2/Kdecay.
(Ten) Gene-specific MeRIP experiment
The gene-specific MeRIP assay uses the Magna RIP RNA binding protein immunoprecipitation kit (Millipore), the procedure of which is performed according to the instructions. Briefly, magnetic beads conjugated with anti-m 6A antibodies (synthetic Systems) or mouse IgG (Millipore) were incubated with equal amounts of cell extract treated with RIP lysate overnight at 4 degrees (10 ul of each cell lysate was used as Input). The next analysis was then performed in q-PCR using HBV pgRNA specific primers and the relative enrichment of m6A in each sample was calculated by normalized Input.
(eleven) statistical analysis
All statistical analyses of the invention were performed using GraphPad Prism 5 and SPSS version 21.0 software. The results of the experiments were statistically analyzed appropriately according to various conditions using Student's t-test, chi-square test, Mann-Whitney test, Wilcoxon rank sum test, and the like. A P value less than 0.05 indicates statistical significance. "+" indicates P < 0.05; ". indicates that P < 0.01; ". indicates that P < 0.001.
Second, experimental results
The interference of the expression of HBV pgRNA can inhibit the proliferation capacity and stem cell characteristics of liver cancer cells.
In the present invention, firstly, by constructing and transfecting HBV pgRNA specific short hairpin RNA, a hepatoma cell line that stably interferes with HBV pgRNA was constructed, and the interference efficiency was verified by q-PCR experiment (FIG. 1A). Next, through CCK-8 experiment, colony formation experiment, cell balling experiment, detection of important dry markers (CD44, CD90, CD133) and nude mouse subcutaneous tumor-bearing experiment, it was found that interfering with the expression of HBV pgRNA could inhibit the proliferation ability and stem cell characteristics of hepatoma cells (FIG. 1B-G).
And (II) the expression of over-expressed HBV pgRNA can enhance the proliferation capacity and stem cell characteristics of liver cancer cells.
In the present invention, firstly, by constructing and transfecting HBV pgRNA overexpression plasmids, a hepatoma cell line stably overexpressing HBV pgRNA was constructed, and the overexpression efficiency was verified by q-PCR experiments (FIG. 2A). Next, through CCK-8 experiment, colony formation experiment, cell balling experiment, detection of important dry markers (CD44, CD90, CD133) and nude mouse subcutaneous tumor-bearing experiment, it was found that over-expression of HBV pgRNA can enhance the proliferation capacity and stem cell characteristics of hepatoma cells (FIG. 2B-G).
IFN-alpha can inhibit the proliferation capacity and stem cell characteristics of liver cancer cells; while overexpression of HBV pgRNA can reverse the inhibitory effect to some extent.
In the invention, the liver cancer cells transfected with HBV pgRNA overexpression plasmids and the liver cancer cells transfected with unloaded plasmids are respectively treated by using IFN-alpha and PBS with the same volume, and then a CCK-8 experiment, a clone formation experiment, a cell balling experiment, the detection of important dry markers (CD44, CD90 and CD133) and a nude mouse subcutaneous tumor-bearing experiment are respectively carried out. The results show that: the half lethal concentration of IFN-alpha against liver cancer cell HepG2.2.15 is about 2000IU/ml (FIG. 3A); IFN-alpha can obviously inhibit the proliferation capacity of hepatoma carcinoma cells (figure 3B), the clonogenic capacity (figure 3C), the cell balling capacity (figure 3D) and the expression of important dry markers (CD44, CD90, CD133) (figure 3E) at the in vitro level; at the in vivo level, IFN-alpha can also obviously inhibit the proliferation ability and the tumorigenic ability of the hepatoma cells (fig. 3F, G); the over-expression of HBV pgRNA can reverse the inhibition effect of IFN-alpha on the proliferation ability, clonogenic ability, cell balling ability, stem cell characteristics and tumorigenic ability of liver cancer cells to a certain extent (FIG. 3B-G).
(IV) IFN-alpha decreases the stability of HBV pgRNA and promotes its degradation by increasing the level of modification of m6A RNA of HBV pgRNA, thereby inhibiting the expression of HBV pgRNA.
In the present invention, IFN-alpha was found to significantly inhibit the expression of HBV pgRNA by treating hepatoma cells for 48 hours with IFN-alpha (FIG. 4A); then, by actinomycin D transcription inhibition test, the half-life of HBV pgRNA after IFN-alpha treatment of hepatoma cells is further calculated, and the half-life of HBV pgRNA after IFN-alpha treatment of cells is found to be obviously shortened, and IFN-alpha can obviously reduce the stability of HBV pgRNA and promote the degradation thereof (FIG. 4B); next, through the m 6A-specific MeRIP experimental analysis method, it was found that after IFN- α treatment of hepatoma cells, the m 6A-specific antibody can enrich more HBV pgRNA than the negative control IgG antibody, suggesting that HBV pgRNA has m6A modification site; furthermore, the IFN- α treated group enriched with m 6A-specific antibody HBV pgRNA significantly increased compared to the group without IFN- α treatment, suggesting that IFN- α treatment could significantly up-regulate the m6A modification level of HBV pgRNA (fig. 4C).
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Sequence listing
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<210> 8
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 8
atcgctctcc tgctaacagt c 21
<210> 9
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 9
ctcgtactgg atgggtgaac t 21
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 10
agtcggaaac tggcagatag c 21
<210> 11
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 11
ggtagtgttg tactgggcca at 22
<210> 12
<211> 29
<212> DNA
<213> Artificial sequence (Artificial)
<400> 12
aaaggtacca actttttcac ctctgccta 29
<210> 13
<211> 30
<212> DNA
<213> Artificial sequence (Artificial)
<400> 13
gggctcgagt ttataagggt caatgtccat 30
<210> 14
<211> 19
<212> DNA
<213> Artificial sequence (Artificial)
<400> 14
gatcaggcaa ctattgtgg 19
Claims (10)
- Application of HBV pgRNA inhibitor in preparing medicine for treating hepatitis B related liver cancer.
- 2. The use of the HBV pgRNA inhibitor of claim 1 in the preparation of a medicament for the treatment of hepatitis b-related liver cancer, wherein said HBV pgRNA inhibitor is a small interfering RNA molecule, short hairpin RNA, that specifically interferes with HBV pgRNA expression.
- 3. The application of the HBV pgRNA inhibitor in the preparation of medicines for treating hepatitis B related liver cancer according to claim 2, wherein the HBV pgRNA inhibitor is a specific small interfering RNA for HBV pgRNA, and the sequence of the HBV pgRNA inhibitor is shown in SEQ ID No. 14.
- 4. The use of the HBV pgRNA inhibitor of claim 1 in the preparation of a medicament for the treatment of hepatitis b-related liver cancer, wherein the HBV pgRNA inhibitor is an antagonist, a down-regulator, a blocker, or a blocker of HBV pgRNA.
- 5. The use of the HBV pgRNA inhibitor of claim 4 in the preparation of a medicament for the treatment of hepatitis B related liver cancer, wherein the HBV pgRNA inhibitor is interferon alpha.
- 6. The interferon alpha is applied to the preparation of the hepatitis B related liver cancer treatment medicine by inhibiting the expression of HBV pgRNA.
- 7. The use of interferon alpha in the preparation of a medicament for treating hepatitis B related liver cancer by inhibiting the expression of HBV pgRNA according to claim 6, wherein said interferon alpha exerts the effect of treating liver cancer by inhibiting the expression of HBV pgRNA and by down-regulating the proliferation ability and stem cell characteristics of liver cancer cells.
- 8. The use of interferon alpha in the preparation of a medicament for treating hepatitis B related liver cancer by inhibiting the expression of HBV pgRNA according to claim 7, wherein interferon alpha reduces the stability of HBV pgRNA and promotes its degradation by increasing the level of modification of m6A RNA of HBV pgRNA, thereby inhibiting the expression of HBV pgRNA and exerting the effect of treating liver cancer.
- 9. Application of interferon alpha in preparing HBV pgRNA inhibitor.
- 10. The use of interferon α in the preparation of an HBV pgRNA inhibitor according to claim 9, wherein said interferon α decreases the stability of HBV pgRNA and promotes its degradation by up-regulating the m6A RNA modification level of HBV pgRNA, thereby inhibiting the expression of HBV pgRNA.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109381698A (en) * | 2017-08-06 | 2019-02-26 | 复旦大学 | Human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109381698A (en) * | 2017-08-06 | 2019-02-26 | 复旦大学 | Human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine |
Non-Patent Citations (4)
| Title |
|---|
| AMIR SHLOMAI ET AL.: "Inhibition of Hepatitis B Virus Expression and Replication by RNA Interference", 《HEPATOLOGY》 * |
| LAURA BELLONI ET AL.: "IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome", 《THE JOURNAL OF CLINICAL INVESTIGATION》 * |
| SENTHIL K. RADHAKRISHNAN ET AL.: "RNA interference as a new strategy against viral hepatitis", 《VIROLOGY》 * |
| 丁文斌: "乙肝病毒前基因组 RNA 在肝细胞癌发生和进展中的作用及机制研究", 《中国优秀博士学位论文全文数据库 医药卫生科技缉》 * |
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